Thomas Sudhof

All Publications

• A brain circuit that cements the memory of socially learnt food preferences NATURE Liu, Z., Sudhof, T. C. 2024

  View details for DOI 10.1038/d41586-024-03012-1

  View details for Web of Science ID 001326921400007

  View details for PubMedID 39294284

• A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events. Neuron Hao, Y. A., Lee, S., Roth, R. H., Natale, S., Gomez, L., Taxidis, J., O'Neill, P. S., Villette, V., Bradley, J., Wang, Z., Jiang, D., Zhang, G., Sheng, M., Lu, D., Boyden, E., Delvendahl, I., Golshani, P., Wernig, M., Feldman, D. E., Ji, N., Ding, J., Südhof, T. C., Clandinin, T. R., Lin, M. Z. 2024

  Abstract

  A remaining challenge for genetically encoded voltage indicators (GEVIs) is the reliable detection of excitatory postsynaptic potentials (EPSPs). Here, we developed ASAP5 as a GEVI with enhanced activation kinetics and responsivity near resting membrane potentials for improved detection of both spiking and subthreshold activity. ASAP5 reported action potentials (APs) in vivo with higher signal-to-noise ratios than previous GEVIs and successfully detected graded and subthreshold responses to sensory stimuli in single two-photon trials. In cultured rat or human neurons, somatic ASAP5 reported synaptic events propagating centripetally and could detect ∼1-mV EPSPs. By imaging spontaneous EPSPs throughout dendrites, we found that EPSP amplitudes decay exponentially during propagation and that amplitude at the initiation site generally increases with distance from the soma. These results extend the applications of voltage imaging to the quantal response domain, including in human neurons, opening up the possibility of high-throughput, high-content characterization of neuronal dysfunction in disease.

  View details for DOI 10.1016/j.neuron.2024.08.019

  View details for PubMedID 39305894

• Nanometer-resolution tracking of single cargo reveals dynein motor mechanisms. Nature chemical biology Peng, C. S., Zhang, Y., Liu, Q., Marti, G. E., Huang, Y. A., Sudhof, T. C., Cui, B., Chu, S. 2024

  Abstract

  Cytoplasmic dynein is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors transport vesicles by processive steps in live cells remains unclear. To dissect the molecular mechanisms of dynein, we develop optical probes that enable long-term single-particle tracking in live cells with high spatiotemporal resolution. We find that the number of active dynein motors transporting cargo switches stochastically between one and five dynein motors during long-range transport in neuronal axons. Our very bright optical probes allow the observation of individual molecular steps. Strikingly, these measurements reveal that the dwell times between steps are controlled by two temperature-dependent rate constants in which two ATP molecules are hydrolyzed sequentially during each dynein step. Thus, our observations uncover a previously unknown chemomechanical cycle of dynein-mediated cargo transport in living cells.

  View details for DOI 10.1038/s41589-024-01694-2

  View details for PubMedID 39090313

• Astrocytic Neuroligin-3 influences gene expression and social behavior, but is dispensable for synapse number. Molecular psychiatry Qin, L., Liu, Z., Guo, S., Han, Y., Wang, X., Ren, W., Chen, J., Zhen, H., Nie, C., Xing, K. K., Chen, T., Südhof, T. C., Sun, Y., Zhang, B. 2024

  Abstract

  Neuroligin-3 (Nlgn3) is an autism-associated cell-adhesion molecule that interacts with neurexins and is robustly expressed in both neurons and astrocytes. Neuronal Nlgn3 is an essential regulator of synaptic transmission but the function of astrocytic Nlgn3 is largely unknown. Given the high penetrance of Nlgn3 mutations in autism and the emerging role of astrocytes in neuropsychiatric disorders, we here asked whether astrocytic Nlgn3 might shape neural circuit properties in the cerebellum similar to neuronal Nlgn3. Imaging of tagged Nlgn3 protein produced by CRISPR/Cas9-mediated genome editing showed that Nlgn3 is enriched in the cell body but not the fine processes of cerebellar astrocytes (Bergmann glia). Astrocyte-specific knockout of Nlgn3 did not detectably alter the number of synapses, synaptic transmission, or astrocyte morphology in mouse cerebellum. However, spatial transcriptomic analyses revealed a significant shift in gene expression among multiple cerebellar cell types after the deletion of astrocytic Nlgn3. Hence, in contrast to neuronal Nlgn3, astrocytic Nlgn3 in the cerebellum is not involved in shaping synapses but may modulate gene expression in specific brain areas.

  View details for DOI 10.1038/s41380-024-02659-6

  View details for PubMedID 39003414

  View details for PubMedCentralID 5694349

• The cortical amygdala consolidates a socially transmitted long-term memory. Nature Liu, Z., Sun, W., Ng, Y. H., Dong, H., Quake, S. R., Südhof, T. C. 2024

  Abstract

  Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.

  View details for DOI 10.1038/s41586-024-07632-5

  View details for PubMedID 38961294

  View details for PubMedCentralID 2958925

• Author Correction: Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates. Nature Mall, M., Kareta, M. S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S. D., Ge, X., Drake, S., Ang, C. E., Walker, B. M., Vierbuchen, T., Fuentes, D. R., Brennecke, P., Nitta, K. R., Jolma, A., Steinmetz, L. M., Taipale, J., Südhof, T. C., Wernig, M. 2024

  View details for DOI 10.1038/s41586-024-07594-8

  View details for PubMedID 38834754

• Cartography of teneurin and latrophilin expression reveals spatiotemporal axis heterogeneity in the mouse hippocampus during development. PLoS biology Liakath-Ali, K., Refaee, R., Südhof, T. C. 2024; 22 (5): e3002599

  Abstract

  Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.

  View details for DOI 10.1371/journal.pbio.3002599

  View details for PubMedID 38713721

• Essential Role of Latrophilin-1 Adhesion GPCR Nanoclusters in Inhibitory Synapses. The Journal of neuroscience : the official journal of the Society for Neuroscience Matúš, D., Lopez, J. M., Sando, R. C., Südhof, T. C. 2024

  Abstract

  Latrophilin-1 (Lphn1, a.k.a. CIRL1 and CL1; gene symbol Adgrl1) is an Adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knockin/knockout mice for Lphn1 that contain an extracellular myc-epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but contributes to inhibitory synaptic connections.Significance Statement Previously, the Adhesion-GPCRs Latrophilin-2/Latrophilin-3 have been shown to mediate excitatory, but not inhibitory, synapse assembly onto discreet dendritic compartments of hippocampal pyramidal neurons. Here we now show that Latrophilin-1 is targeted to both excitatory and inhibitory hippocampal synapses. Unexpectedly, we find that Latrophilin-1 is selectively essential for directing inhibitory synaptic connections to the neuronal soma. Our work supports a model by which Latrophilins are widely used as organizers of synaptic connectivity that act on a subcellular level. In the light of recent findings connecting haploinsufficiency of Latrophilin-1 to a plethora of neurodevelopmental and neuropsychiatric disorders, our study contributes to our understanding of the molecular mechanisms of Latrophilins that need to be targeted in order to address various pathologies.

  View details for DOI 10.1523/JNEUROSCI.1978-23.2024

  View details for PubMedID 38684366

• Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1. Proceedings of the National Academy of Sciences of the United States of America Ang, C. E., Olmos, V. H., Vodehnal, K., Zhou, B., Lee, Q. Y., Sinha, R., Narayanaswamy, A., Mall, M., Chesnov, K., Dominicus, C. S., Sudhof, T., Wernig, M. 2024; 121 (11): e2308401121

  Abstract

  Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

  View details for DOI 10.1073/pnas.2308401121

  View details for PubMedID 38446849

• Neurexin-2 restricts synapse numbers and restrains the presynaptic release probability by an alternative splicing-dependent mechanism (Retraction of Vol 120, art no E2300363120, 2023) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lin, P., Chen, L. Y., Zhou, P., Lee, S., Trotter, J. H., Sudhof, T. C. 2024; 121 (11)

  View details for DOI 10.1073/pnas.2403021121

  View details for Web of Science ID 001207811900002

• Spatial transcriptomics reveal neuron-astrocyte synergy in long-term memory. Nature Sun, W., Liu, Z., Jiang, X., Chen, M. B., Dong, H., Liu, J., Südhof, T. C., Quake, S. R. 2024

  Abstract

  Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.

  View details for DOI 10.1038/s41586-023-07011-6

  View details for PubMedID 38326616

  View details for PubMedCentralID 4292167

• Alternative splicing of latrophilin-3 controls synapse formation. Nature Wang, S., DeLeon, C., Sun, W., Quake, S. R., Roth, B. L., Südhof, T. C. 2024

  Abstract

  The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.

  View details for DOI 10.1038/s41586-023-06913-9

  View details for PubMedID 38233523

  View details for PubMedCentralID 8186004

• The Swiss Brain Health Plan 2023-2033 CLINICAL AND TRANSLATIONAL NEUROSCIENCE Bassetti, C. A., Heldner, M. R., Adorjan, K., Albanese, E., Allali, G., Arnold, M., Begue, I., Bochud, M., Chan, A., do Cuenod, K. Q., Du Pasquier, R., Draganski, B., Eshmawey, M., Felbecker, A., Fischer, U., Frahsa, A., Frisoni, G. B., Grossmann, H., Guzman, R., Hackenberg, A., Hatzinger, M., Herdener, M., Hofman, A., Humm, A. M., Jung, S., Kaess, M., Katterer, C., Kesselring, J., Klein, A., Kleinschmidt, A., Kloppel, S., Kronig, N., Lovblad, K., Luthi, A., Lyrer, P., Penner, I., Pot, C., Rafferty, Q., Sandor, P. S., Sarikaya, H., Seifritz, E., Smith, S., Sveikata, L., Sudhof, T. P., Tettenborn, B., Unschuld, P. G., Cabrera, A., Walitza, S., Walther, S., Wancke, I., Weller, M., Wegener, S., Zalud, P., Zeltner, T., Zutter, D., Remonda, L. 2023; 7 (4)

  View details for DOI 10.3390/ctn7040038

  View details for Web of Science ID 001182201500001

• Hippocampal place code plasticity in CA1 requires postsynaptic membrane fusion. bioRxiv : the preprint server for biology Plitt, M. H., Kaganovsky, K., Südhof, T. C., Giocomo, L. M. 2023

  Abstract

  Rapid delivery of glutamate receptors to the postsynaptic membrane via vesicle fusion is a central component of synaptic plasticity. However, it is unknown how this process supports specific neural computations during behavior. To bridge this gap, we combined conditional genetic deletion of a component of the postsynaptic membrane fusion machinery, Syntaxin3 (Stx3), in hippocampal CA1 neurons of mice with population in vivo calcium imaging. This approach revealed that Stx3 is necessary for forming the neural dynamics that support novelty processing, spatial reward memory and offline memory consolidation. In contrast, CA1 Stx3 was dispensable for maintaining aspects of the neural code that exist presynaptic to CA1 such as representations of context and space. Thus, manipulating postsynaptic membrane fusion identified computations that specifically require synaptic restructuring via membrane trafficking in CA1 and distinguished them from neural representation that could be inherited from upstream brain regions or learned through other mechanisms.

  View details for DOI 10.1101/2023.11.20.567978

  View details for PubMedID 38045362

  View details for PubMedCentralID PMC10690209

• Neutral lysophosphatidylcholine mediates alpha-synuclein-induced synaptic vesicle clustering. Proceedings of the National Academy of Sciences of the United States of America Lai, Y., Zhao, C., Tian, Z., Wang, C., Fan, J., Hu, X., Tu, J., Li, T., Leitz, J., Pfuetzner, R. A., Liu, Z., Zhang, S., Su, Z., Burre, J., Li, D., Sudhof, T. C., Zhu, Z., Liu, C., Brunger, A. T., Diao, J. 2023; 120 (44): e2310174120

  Abstract

  alpha-synuclein (alpha-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that alpha-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of alpha-Syn with anionic phospholipids. Here, we report that alpha-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates alpha-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of alpha-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the alpha-Syn-lysoPC interaction may play a role in alpha-Syn function.

  View details for DOI 10.1073/pnas.2310174120

  View details for PubMedID 37883437

• Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR. Nano letters Zhong, B. L., Lee, C. E., Vachharajani, V. T., Bauer, M. S., Südhof, T. C., Dunn, A. R. 2023

  Abstract

  Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

  View details for DOI 10.1021/acs.nanolett.3c03171

  View details for PubMedID 37831891

• Efficient generation of functional neurons from mouse embryonic stem cells via neurogenin-2 expression. Nature protocols Liu, Y., Wang, J., Südhof, T. C., Wernig, M. 2023

  Abstract

  The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.

  View details for DOI 10.1038/s41596-023-00863-2

  View details for PubMedID 37596357

  View details for PubMedCentralID 3032267

• Combinatorial expression of neurexins and LAR-type phosphotyrosine phosphatase receptors instructs assembly of a cerebellar circuit. Nature communications Sclip, A., Südhof, T. C. 2023; 14 (1): 4976

  Abstract

  Synaptic adhesion molecules (SAMs) shape the structural and functional properties of synapses and thereby control the information processing power of neural circuits. SAMs are broadly expressed in the brain, suggesting that they may instruct synapse formation and specification via a combinatorial logic. Here, we generate sextuple conditional knockout mice targeting all members of the two major families of presynaptic SAMs, Neurexins and leukocyte common antigen-related-type receptor phospho-tyrosine phosphatases (LAR-PTPRs), which together account for the majority of known trans-synaptic complexes. Using synapses formed by cerebellar Purkinje cells onto deep cerebellar nuclei as a model system, we confirm that Neurexins and LAR-PTPRs themselves are not essential for synapse assembly. The combinatorial deletion of both neurexins and LAR-PTPRs, however, decreases Purkinje-cell synapses on deep cerebellar nuclei, the major output pathway of cerebellar circuits. Consistent with this finding, combined but not separate deletions of neurexins and LAR-PTPRs impair motor behaviors. Thus, Neurexins and LAR-PTPRs are together required for the assembly of a functional cerebellar circuit.

  View details for DOI 10.1038/s41467-023-40526-0

  View details for PubMedID 37591863

  View details for PubMedCentralID 9513053

• Neuronal gamma-secretase regulates lipid metabolism, linking cholesterol to synaptic dysfunction in Alzheimer's disease. Neuron Essayan-Perez, S., Sudhof, T. C. 2023

  Abstract

  Presenilin mutations that alter gamma-secretase activity cause familial Alzheimer's disease (AD), whereas ApoE4, an apolipoprotein for cholesterol transport, predisposes to sporadic AD. Both sporadic and familial AD feature synaptic dysfunction. Whether gamma-secretase is involved in cholesterol metabolism and whether such involvement impacts synaptic function remains unknown. Here, we show that in human neurons, chronic pharmacological or genetic suppression of gamma-secretase increases synapse numbers but decreases synaptic transmission by lowering the presynaptic release probability without altering dendritic or axonal arborizations. In search of a mechanism underlying these synaptic impairments, we discovered that chronic gamma-secretase suppression robustly decreases cholesterol levels in neurons but not in glia, which in turn stimulates neuron-specific cholesterol-synthesis gene expression. Suppression of cholesterol levels by HMG-CoA reductase inhibitors (statins) impaired synaptic function similar to gamma-secretase inhibition. Thus, gamma-secretase enables synaptic function by maintaining cholesterol levels, whereas the chronic suppression of gamma-secretase impairs synapses by lowering cholesterol levels.

  View details for DOI 10.1016/j.neuron.2023.07.005

  View details for PubMedID 37543038

• Postsynaptic synucleins mediate endocannabinoid signaling. Nature neuroscience Albarran, E., Sun, Y., Liu, Y., Raju, K., Dong, A., Li, Y., Wang, S., Sudhof, T. C., Ding, J. B. 2023

  Abstract

  Endocannabinoids are among the most powerful modulators of synaptic transmission throughout the nervous system, and yet little is understood about the release of endocannabinoids from postsynaptic compartments. Here we report an unexpected finding that endocannabinoid release requires synucleins, key contributors to Parkinson's disease. We show that endocannabinoids are released postsynaptically by a synuclein-dependent and SNARE-dependent mechanism. Specifically, we found that synuclein deletion blocks endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wild-type but not of mutant alpha-synuclein. Whole-cell recordings and direct optical monitoring of endocannabinoid signaling suggest that the synuclein deletion specifically blocks endocannabinoid release. Given the presynaptic role of synucleins in regulating vesicle lifecycle, we hypothesize that endocannabinoids are released via a membrane interaction mechanism. Consistent with this hypothesis, postsynaptic expression of tetanus toxin light chain, which cleaves synaptobrevin SNAREs, also blocks endocannabinoid-dependent signaling. The unexpected finding that endocannabinoids are released via a synuclein-dependent mechanism is consistent with a general function of synucleins in membrane trafficking and adds a piece to the longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

  View details for DOI 10.1038/s41593-023-01345-0

  View details for PubMedID 37248337

• Author Correction: Teneurins assemble into presynaptic nanoclusters that promote synapse formation via postsynaptic non-teneurin ligands. Nature communications Zhang, X., Lin, P. Y., Liakath-Ali, K., Südhof, T. C. 2023; 14 (1): 2957

  View details for DOI 10.1038/s41467-023-38713-0

  View details for PubMedID 37221212

  View details for PubMedCentralID PMC10205713

• Cerebellin-neurexin complexes instructing synapse properties. Current opinion in neurobiology Südhof, T. C. 2023; 81: 102727

  Abstract

  Cerebellins (Cbln1-4) are secreted adaptor proteins that connect presynaptic neurexins (Nrxn1-3) to postsynaptic ligands (GluD1/2 for Cbln1-3 vs. DCC and Neogenin-1 for Cbln4). Classical studies demonstrated that neurexin-Cbln1-GluD2 complexes organize cerebellar parallel-fiber synapses, but the role of cerebellins outside of the cerebellum has only recently been clarified. In synapses of the hippocampal subiculum and prefrontal cortex, Nrxn1-Cbln2-GluD1 complexes strikingly upregulate postsynaptic NMDA-receptors, whereas Nrxn3-Cbln2-GluD1 complexes conversely downregulate postsynaptic AMPA-receptors. At perforant-path synapses in the dentate gyrus, in contrast, neurexin/Cbln4/Neogenin-1 complexes are essential for LTP without affecting basal synaptic transmission or NMDA- or AMPA-receptors. None of these signaling pathways are required for synapse formation. Thus, outside of the cerebellum neurexin/cerebellin complexes regulate synapse properties by activating specific downstream receptors.

  View details for DOI 10.1016/j.conb.2023.102727

  View details for PubMedID 37209532

• Astrocytic Neuroligins Are Not Required for Synapse Formation or a Normal Astrocyte Cytoarchitecture. bioRxiv : the preprint server for biology Golf, S. R., Trotter, J. H., Nakahara, G., Südhof, T. C. 2023

  Abstract

  Astrocytes exert multifarious roles in the formation, regulation, and function of synapses in the brain, but the mechanisms involved remain unclear. Interestingly, astrocytes abundantly express neuroligins, postsynaptic adhesion molecules that bind to presynaptic neurexins. A pioneering recent study reported that loss-of-function of neuroligins in astrocytes impairs excitatory synapse formation and astrocyte morphogenesis. This study suggested a crucial synaptic function for astrocytic neuroligins but was puzzling given that constitutive neuroligin deletions do not decrease excitatory synapse numbers. Thus, we here examined the function of astrocytic neuroligins using a rigorous conditional genetic approach with deletion of all major neuroligins (Nlgn1-3) in astrocytes. Our results show that early postnatal deletion of neuroligins from astrocytes has no effect on cortical or hippocampal synapses and does not alter the cytoarchitecture of astrocytes. Thus, astrocytic neuroligins are unlikely to shape synapse formation or astrocyte development but may perform other important functions in astrocytes.

  View details for DOI 10.1101/2023.04.10.536254

  View details for PubMedID 37090508

  View details for PubMedCentralID PMC10120619

• A combinatorial code of neurexin-3 alternative splicing controls inhibitory synapses via a trans-synaptic dystroglycan signaling loop. Nature communications Trotter, J. H., Wang, C. Y., Zhou, P., Nakahara, G., Südhof, T. C. 2023; 14 (1): 1771

  Abstract

  Disrupted synaptic inhibition is implicated in neuropsychiatric disorders, yet the molecular mechanisms that shape and sustain inhibitory synapses are poorly understood. Here, we show through rescue experiments performed using Neurexin-3 conditional knockout mice that alternative splicing at SS2 and SS4 regulates the release probability, but not the number, of inhibitory synapses in the olfactory bulb and prefrontal cortex independent of sex. Neurexin-3 splice variants that mediate Neurexin-3 binding to dystroglycan enable inhibitory synapse function, whereas splice variants that don't allow dystroglycan binding do not. Furthermore, a minimal Neurexin-3 protein that binds to dystroglycan fully sustains inhibitory synaptic function, indicating that trans-synaptic dystroglycan binding is necessary and sufficient for Neurexin-3 function in inhibitory synaptic transmission. Thus, Neurexin-3 enables a normal release probability at inhibitory synapses via a trans-synaptic feedback signaling loop consisting of presynaptic Neurexin-3 and postsynaptic dystroglycan.

  View details for DOI 10.1038/s41467-023-36872-8

  View details for PubMedID 36997523

  View details for PubMedCentralID PMC10063607

• Neurexin-2 restricts synapse numbers and restrains the presynaptic release probability by an alternative splicing-dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America Lin, P. Y., Chen, L. Y., Zhou, P., Lee, S. J., Trotter, J. H., Südhof, T. C. 2023; 120 (13): e2300363120

  Abstract

  α- and β-neurexins are extensively alternatively spliced, presynaptic cell-adhesion molecules that are thought to organize synapse assembly. However, recent data revealed that, in the hippocampus in vivo, the deletion of one neurexin isoform, Nrxn2, surprisingly increased excitatory synapse numbers and enhanced their presynaptic release probability, suggesting that Nrxn2 restricts, instead of enabling, synapse assembly. To delineate the synaptic function and mechanism of action of Nrxn2, we examined cultured hippocampal neurons as a reduced system. In heterologous synapse formation assays, different alternatively spliced Nrxn2β isoforms robustly promoted synapse assembly similar to Nrxn1β and Nrxn3β, consistent with a general synaptogenic function of neurexins. Deletion of Nrxn2 from cultured hippocampal neurons, however, caused a significant increase in synapse density and release probability, replicating the in vivo data that suggested a synapse-restricting function. Rescue experiments revealed that two of the four Nrxn2β splice variants (Nrxn2β-SS4+/SS5- and Nrxn2β-SS4+/SS5+) reversed the increase in synapse density in Nrxn2-deficient neurons, whereas only one of the four Nrxn2β splice variants (Nrxn2β-SS4+/SS5+) normalized the increase in release probability in Nrxn2-deficient neurons. Thus, a subset of Nrxn2 splice variants restricts synapse numbers and restrains their release probability in cultured neurons.

  View details for DOI 10.1073/pnas.2300363120

  View details for PubMedID 36961922

• Single piconewton forces regulate dissociation of the Latrophilin-3 gain domain Zhong, B. L., Lee, C. E., Vachharajani, V. T., Sudhof, T. C., Dunn, A. R. CELL PRESS. 2023: 92A

  View details for Web of Science ID 000989629700459

• Single piconewton forces regulate dissociation of the Latrophilin-3 gain domain. Biophysical journal Zhong, B. L., Lee, C. E., Vachharajani, V. T., Sudhof, T. C., Dunn, A. R. 2023; 122 (3S1): 92a

  View details for DOI 10.1016/j.bpj.2022.11.696

  View details for PubMedID 36785091

• Engineered adhesion molecules drive synapse organization. Proceedings of the National Academy of Sciences of the United States of America Hale, W. D., Südhof, T. C., Huganir, R. L. 2023; 120 (3): e2215905120

  Abstract

  In multicellular organisms, cell-adhesion molecules connect cells into tissues and mediate intercellular signaling between these cells. In vertebrate brains, synaptic cell-adhesion molecules (SAMs) guide the formation, specification, and plasticity of synapses. Some SAMs, when overexpressed in cultured neurons or in heterologous cells co-cultured with neurons, drive formation of synaptic specializations onto the overexpressing cells. However, genetic deletion of the same SAMs from neurons often has no effect on synapse numbers, but frequently severely impairs synaptic transmission, suggesting that most SAMs control the function and plasticity of synapses (i.e., organize synapses) instead of driving their initial establishment (i.e., make synapses). Since few SAMs were identified that mediate initial synapse formation, it is difficult to develop methods that enable experimental control of synaptic connections by targeted expression of these SAMs. To overcome this difficulty, we engineered novel SAMs from bacterial proteins with no eukaryotic homologues that drive synapse formation. We named these engineered adhesion proteins "Barnoligin" and "Starexin" because they were assembled from parts of Barnase and Neuroligin-1 or of Barstar and Neurexin3β, respectively. Barnoligin and Starexin robustly induce the formation of synaptic specializations in a specific and directional manner in cultured neurons. Synapse formation by Barnoligin and Starexin requires both their extracellular Barnase- and Barstar-derived interaction domains and their Neuroligin- and Neurexin-derived intracellular signaling domains. Our findings support a model of synapse formation whereby trans-synaptic interactions by SAMs drive synapse organization via adhesive interactions that activate signaling cascades.

  View details for DOI 10.1073/pnas.2215905120

  View details for PubMedID 36638214

• Neurexin-2: An inhibitory neurexin that restricts excitatory synapse formation in the hippocampus. Science advances Lin, P., Chen, L. Y., Jiang, M., Trotter, J. H., Seigneur, E., Sudhof, T. C. 2023; 9 (1): eadd8856

  Abstract

  Neurexins are widely thought to promote synapse formation and to organize synapse properties. Here we found that in contrast to neurexin-1 and neurexin-3, neurexin-2 unexpectedly restricts synapse formation. In the hippocampus, constitutive or neuron-specific deletions of neurexin-2 nearly doubled the strength of excitatory CA3➔CA1 region synaptic connections and markedly increased their release probability. No effect on inhibitory synapses was detected. Stochastic optical reconstruction microscopy (STORM) superresolution microscopy revealed that the neuron-specific neurexin-2 deletion elevated the density of excitatory CA1 region synapses nearly twofold. Moreover, hippocampal neurexin-2 deletions also increased synaptic connectivity in the CA1 region when induced in mature mice and impaired the cognitive flexibility of spatial memory. Thus, neurexin-2 controls the dynamics of hippocampal synaptic circuits by repressing synapse assembly throughout life, a restrictive function that markedly differs from that of neurexin-1 and neurexin-3 and of other synaptic adhesion molecules, suggesting that neurexins evolutionarily diverged into opposing pro- and antisynaptogenic organizers.

  View details for DOI 10.1126/sciadv.add8856

  View details for PubMedID 36608123

• The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1. Scientific reports Haddad Derafshi, B., Danko, T., Chanda, S., Batista, P. J., Litzenburger, U., Lee, Q. Y., Ng, Y. H., Sebin, A., Chang, H. Y., Sudhof, T. C., Wernig, M. 2022; 12 (1): 22425

  Abstract

  The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

  View details for DOI 10.1038/s41598-022-23614-x

  View details for PubMedID 36575212

• Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism. Molecular psychiatry Wang, L., Mirabella, V. R., Dai, R., Su, X., Xu, R., Jadali, A., Bernabucci, M., Singh, I., Chen, Y., Tian, J., Jiang, P., Kwan, K. Y., Pak, C., Liu, C., Comoletti, D., Hart, R. P., Chen, C., Sudhof, T. C., Pang, Z. P. 2022

  Abstract

  Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

  View details for DOI 10.1038/s41380-022-01834-x

  View details for PubMedID 36280753

• Synaptogenic effect of APP-Swedish mutation in familial Alzheimer's disease. Science translational medicine Zhou, B., Lu, J. G., Siddu, A., Wernig, M., Sudhof, T. C. 2022; 14 (667): eabn9380

  Abstract

  Mutations in beta-amyloid (Abeta) precursor protein (APP) cause familial Alzheimer's disease (AD) probably by enhancing Abeta peptides production from APP. An antibody targeting Abeta (aducanumab) was approved as an AD treatment; however, some Abeta antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the APP-Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the APP deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Abeta production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of APP-Swedish-mutant neurons. Modest elevations of Abeta, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked APP-Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Abeta. These data are consistent with the relative inefficacy of BACE1 and anti-Abeta treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Abeta but rather by a long-term effect of elevated Abeta concentrations.

  View details for DOI 10.1126/scitranslmed.abn9380

  View details for PubMedID 36260691

• Distinct neurexin-cerebellin complexes control AMPA- and NMDA-receptor responses in a circuit-dependent manner. eLife Dai, J., Liakath-Ali, K., Golf, S. R., Sudhof, T. C. 2022; 11

  Abstract

  At CA1subiculum synapses, alternatively spliced neurexin-1 (Nrxn1SS4+) and neurexin-3 (Nrxn3SS4+) enhance NMDA-receptors and suppress AMPA-receptors, respectively, without affecting synapse formation. Nrxn1SS4+ and Nrxn3SS4+ act by binding to secreted cerebellin-2 (Cbln2) that in turn activates postsynaptic GluD1 receptors. Whether neurexin-Cbln2-GluD1 signaling has additional functions besides regulating NMDA- and AMPA-receptors, and whether such signaling performs similar roles at other synapses, however, remains unknown. Here, we demonstrate using constitutive Cbln2 deletions in mice that at CA1subiculum synapses, Cbln2 performs no additional developmental roles besides regulating AMPA- and NMDA-receptors. Moreover, low-level expression of functionally redundant Cbln1 did not compensate for a possible synapse-formation function of Cbln2 at CA1subiculum synapses. In exploring the generality of these findings, we examined the prefrontal cortex where Cbln2 was recently implicated in spinogenesis, and the cerebellum where Cbln1 is known to regulate parallel-fiber synapses. In the prefrontal cortex, Nrxn1SS4+-Cbln2 signaling selectively controlled NMDA-receptors without affecting spine or synapse numbers, whereas Nrxn3SS4+-Cbln2 signaling had no apparent role. In the cerebellum, conversely, Nrxn3SS4+-Cbln1 signaling regulated AMPA-receptors, whereas now Nrxn1SS4+-Cbln1 signaling had no manifest effect. Thus, Nrxn1SS4+- and Nrxn3SS4+-Cbln1/2 signaling complexes differentially control NMDA- and AMPA-receptors in different synapses in diverse neural circuits without regulating synapse or spine formation.

  View details for DOI 10.7554/eLife.78649

  View details for PubMedID 36205393

• Endocytosis in the axon initial segment maintains neuronal polarity. Nature Eichel, K., Uenaka, T., Belapurkar, V., Lu, R., Cheng, S., Pak, J. S., Taylor, C. A., Sudhof, T. C., Malenka, R., Wernig, M., Ozkan, E., Perrais, D., Shen, K. 2022

  Abstract

  Neurons are highly polarized cells that face the fundamental challenge of compartmentalizing a vast and diverse repertoire of proteins in order to function properly1. The axon initial segment (AIS) is a specialized domain that separates a neuron's morphologically, biochemically and functionally distinct axon and dendrite compartments2,3. How the AIS maintains polarity between these compartments is not fully understood. Here we find that in Caenorhabditis elegans, mouse, rat and human neurons, dendritically and axonally polarized transmembrane proteins are recognized by endocytic machinery in the AIS, robustly endocytosed and targeted to late endosomes for degradation. Forcing receptor interaction with the AIS master organizer, ankyrinG, antagonizes receptor endocytosis in the AIS, causes receptor accumulation in the AIS, and leads to polarity deficits with subsequent morphological and behavioural defects. Therefore, endocytic removal of polarized receptors that diffuse into the AIS serves as a membrane-clearance mechanism thatis likely to work in conjunction with the known AIS diffusion-barrier mechanism to maintain neuronal polarity on the plasma membrane. Our results reveal a conserved endocytic clearance mechanism in the AIS to maintain neuronal polarity by reinforcing axonal and dendritic compartment membrane boundaries.

  View details for DOI 10.1038/s41586-022-05074-5

  View details for PubMedID 35978188

• Neuroligin-3 confines AMPA receptors into nanoclusters, thereby controlling synaptic strength at the calyx of Held synapses. Science advances Han, Y., Cao, R., Qin, L., Chen, L. Y., Tang, A. H., Südhof, T. C., Zhang, B. 2022; 8 (24): eabo4173

  Abstract

  The subsynaptic organization of postsynaptic neurotransmitter receptors into nanoclusters that are aligned with presynaptic release sites is essential for the high fidelity of synaptic transmission. However, the mechanisms controlling the nanoscale organization of neurotransmitter receptors in vivo remain incompletely understood. Here, we deconstructed the role of neuroligin-3 (Nlgn3), a postsynaptic adhesion molecule linked to autism, in organizing AMPA-type glutamate receptors in the calyx of Held synapse. Deletion of Nlgn3 lowered the amplitude and slowed the kinetics of AMPA receptor-mediated synaptic responses. Super-resolution microscopy revealed that, unexpectedly, these impairments in synaptic transmission were associated with an increase in the size of postsynaptic PSD-95 and AMPA receptor nanoclusters but a decrease of the densities in these clusters. Modeling showed that a dilution of AMPA receptors into larger nanocluster volumes decreases synaptic strength. Nlgn3, likely by binding to presynaptic neurexins, thus is a key organizer of AMPA receptor nanoclusters that likely acts via PSD-95 adaptors to optimize the fidelity of synaptic transmission.

  View details for DOI 10.1126/sciadv.abo4173

  View details for PubMedID 35704570

• Induction of synapse formation by de novo neurotransmitter synthesis. Nature communications Burlingham, S. R., Wong, N. F., Peterkin, L., Lubow, L., Dos Santos Passos, C., Benner, O., Ghebrial, M., Cast, T. P., Xu-Friedman, M. A., Südhof, T. C., Chanda, S. 2022; 13 (1): 3060

  Abstract

  A vital question in neuroscience is how neurons align their postsynaptic structures with presynaptic release sites. Although synaptic adhesion proteins are known to contribute in this process, the role of neurotransmitters remains unclear. Here we inquire whether de novo biosynthesis and vesicular release of a noncanonical transmitter can facilitate the assembly of its corresponding postsynapses. We demonstrate that, in both stem cell-derived human neurons as well as in vivo mouse neurons of purely glutamatergic identity, ectopic expression of GABA-synthesis enzymes and vesicular transporters is sufficient to both produce GABA from ambient glutamate and transmit it from presynaptic terminals. This enables efficient accumulation and consistent activation of postsynaptic GABAA receptors, and generates fully functional GABAergic synapses that operate in parallel but independently of their glutamatergic counterparts. These findings suggest that presynaptic release of a neurotransmitter itself can signal the organization of relevant postsynaptic apparatus, which could be directly modified to reprogram the synapse identity of neurons.

  View details for DOI 10.1038/s41467-022-30756-z

  View details for PubMedID 35650274

• Transsynaptic cerebellin 4-neogenin 1 signaling mediates LTP in the mouse dentate gyrus. Proceedings of the National Academy of Sciences of the United States of America Liakath-Ali, K., Polepalli, J. S., Lee, S. J., Cloutier, J. F., Südhof, T. C. 2022; 119 (20): e2123421119

  Abstract

  SignificanceSynapses are controlled by transsynaptic adhesion complexes that mediate bidirectional signaling between pre- and postsynaptic compartments. Long-term potentiation (LTP) of synaptic transmission is thought to enable synaptic modifications during memory formation, but the signaling mechanisms involved remain poorly understood. We show that binding of cerebellin-4 (Cbln4), a secreted ligand of presynaptic neurexin adhesion molecules, to neogenin-1, a postsynaptic surface protein known as a developmental netrin receptor, is essential for normal LTP at entorhinal cortex→dentate gyrus synapses in mice. Cbln4 and neogenin-1 are dispensable for basal synaptic transmission and not involved in establishing synaptic connections as such. Our data identify a netrin receptor as a postsynaptic organizer of synaptic plasticity that collaborates specifically with the presynaptic neurexin-ligand Cbln4.

  View details for DOI 10.1073/pnas.2123421119

  View details for PubMedID 35544694

• Myt1l haploinsufficiency leads to obesity and multifaceted behavioral alterations in mice. Molecular autism Wohr, M., Fong, W. M., Janas, J. A., Mall, M., Thome, C., Vangipuram, M., Meng, L., Sudhof, T. C., Wernig, M. 2022; 13 (1): 19

  Abstract

  BACKGROUND: The zinc finger domain containing transcription factor Myt1l is tightly associated with neuronal identity and is the only transcription factor known that is both neuron-specific and expressed in all neuronal subtypes. We identified Myt1l as a powerful reprogramming factor that, in combination with the proneural bHLH factor Ascl1, could induce neuronal fate in fibroblasts. Molecularly, we found it to repress many non-neuronal gene programs, explaining its supportive role to induce and safeguard neuronal identity in combination with proneural bHLH transcriptional activators. Moreover, human genetics studies found MYT1L mutations to cause intellectual disability and autism spectrum disorder often coupled with obesity.METHODS: Here, we generated and characterized Myt1l-deficient mice. A comprehensive, longitudinal behavioral phenotyping approach was applied.RESULTS: Myt1l was necessary for survival beyond 24h but not for overall histological brain organization. Myt1l heterozygous mice became increasingly overweight and exhibited multifaceted behavioral alterations. In mouse pups, Myt1l haploinsufficiency caused mild alterations in early socio-affective communication through ultrasonic vocalizations. In adulthood, Myt1l heterozygous mice displayed hyperactivity due to impaired habituation learning. Motor performance was reduced in Myt1l heterozygous mice despite intact motor learning, possibly due to muscular hypotonia. While anxiety-related behavior was reduced, acoustic startle reactivity was enhanced, in line with higher sensitivity to loud sound. Finally, Myt1l haploinsufficiency had a negative impact on contextual fear memory retrieval, while cued fear memory retrieval appeared to be intact.LIMITATIONS: In future studies, additional phenotypes might be identified and a detailed characterization of direct reciprocal social interaction behavior might help to reveal effects of Myt1l haploinsufficiency on social behavior in juvenile and adult mice.CONCLUSIONS: Behavioral alterations in Myt1l haploinsufficient mice recapitulate several clinical phenotypes observed in humans carrying heterozygous MYT1L mutations and thus serve as an informative model of the human MYT1L syndrome.

  View details for DOI 10.1186/s13229-022-00497-3

  View details for PubMedID 35538503

• Teneurins assemble into presynaptic nanoclusters that promote synapse formation via postsynaptic non-teneurin ligands. Nature communications Zhang, X., Lin, P., Liakath-Ali, K., Sudhof, T. C. 2022; 13 (1): 2297

  Abstract

  Extensive studies concluded that homophilic interactions between pre- and postsynaptic teneurins, evolutionarily conserved cell-adhesion molecules, encode the specificity of synaptic connections. However, no direct evidence is available to demonstrate that teneurins are actually required on both pre- and postsynaptic neurons for establishing synaptic connections, nor is it known whether teneurins are localized to synapses. Using super-resolution microscopy, we demonstrate that Teneurin-3 assembles into presynaptic nanoclusters of approximately 80nm in most excitatory synapses of the hippocampus. Presynaptic deletions of Teneurin-3 and Teneurin-4 in the medial entorhinal cortex revealed that they are required for assembly of entorhinal cortex-CA1, entorhinal cortex-subiculum, and entorhinal cortex-dentate gyrus synapses. Postsynaptic deletions of teneurins in the CA1 region, however, had no effect on synaptic connectionsfrom any presynaptic input. Our data suggest that different from the current prevailing view, teneurins promote the establishment of synaptic connections exclusively as presynaptic cell-adhesion molecules, most likely via their nanomolar-affinity binding to postsynaptic latrophilins.

  View details for DOI 10.1038/s41467-022-29751-1

  View details for PubMedID 35484136

• Calsyntenin-3, an atypical cadherin, suppresses inhibitory synapses but increases excitatory parallel-fiber synapses in cerebellum. eLife Liu, Z., Jiang, M., Liakath-Ali, K., Sclip, A., Ko, J., Zhang, R. S., Sudhof, T. C. 2022; 11

  Abstract

  Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by promoting formation of excitatory parallel-fiber synapses and decreasing formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells.

  View details for DOI 10.7554/eLife.70664

  View details for PubMedID 35420982

• Mapping genomic loci implicates genes and synaptic biology in schizophrenia. Nature Trubetskoy, V., Pardinas, A. F., Qi, T., Panagiotaropoulou, G., Awasthi, S., Bigdeli, T. B., Bryois, J., Chen, C., Dennison, C. A., Hall, L. S., Lam, M., Watanabe, K., Frei, O., Ge, T., Harwood, J. C., Koopmans, F., Magnusson, S., Richards, A. L., Sidorenko, J., Wu, Y., Zeng, J., Grove, J., Kim, M., Li, Z., Voloudakis, G., Zhang, W., Adams, M., Agartz, I., Atkinson, E. G., Agerbo, E., Al Eissa, M., Albus, M., Alexander, M., Alizadeh, B. Z., Alptekin, K., Als, T. D., Amin, F., Arolt, V., Arrojo, M., Athanasiu, L., Azevedo, M. H., Bacanu, S. A., Bass, N. J., Begemann, M., Belliveau, R. A., Bene, J., Benyamin, B., Bergen, S. E., Blasi, G., Bobes, J., Bonassi, S., Braun, A., Bressan, R. A., Bromet, E. J., Bruggeman, R., Buckley, P. F., Buckner, R. L., Bybjerg-Grauholm, J., Cahn, W., Cairns, M. J., Calkins, M. E., Carr, V. J., Castle, D., Catts, S. V., Chambert, K. D., Chan, R. C., Chaumette, B., Cheng, W., Cheung, E. F., Chong, S. A., Cohen, D., Consoli, A., Cordeiro, Q., Costas, J., Curtis, C., Davidson, M., Davis, K. L., de Haan, L., Degenhardt, F., DeLisi, L. E., Demontis, D., Dickerson, F., Dikeos, D., Dinan, T., Djurovic, S., Duan, J., Ducci, G., Dudbridge, F., Eriksson, J. G., Fananas, L., Faraone, S. V., Fiorentino, A., Forstner, A., Frank, J., Freimer, N. B., Fromer, M., Frustaci, A., Gadelha, A., Genovese, G., Gershon, E. S., Giannitelli, M., Giegling, I., Giusti-Rodriguez, P., Godard, S., Goldstein, J. I., Gonzalez Penas, J., Gonzalez-Pinto, A., Gopal, S., Gratten, J., Green, M. F., Greenwood, T. A., Guillin, O., Guloksuz, S., Gur, R. E., Gur, R. C., Gutierrez, B., Hahn, E., Hakonarson, H., Haroutunian, V., Hartmann, A. M., Harvey, C., Hayward, C., Henskens, F. A., Herms, S., Hoffmann, P., Howrigan, D. P., Ikeda, M., Iyegbe, C., Joa, I., Julia, A., Kahler, A. K., Kam-Thong, T., Kamatani, Y., Karachanak-Yankova, S., Kebir, O., Keller, M. C., Kelly, B. J., Khrunin, A., Kim, S., Klovins, J., Kondratiev, N., Konte, B., Kraft, J., Kubo, M., Kucinskas, V., Kucinskiene, Z. A., Kusumawardhani, A., Kuzelova-Ptackova, H., Landi, S., Lazzeroni, L. C., Lee, P. H., Legge, S. E., Lehrer, D. S., Lencer, R., Lerer, B., Li, M., Lieberman, J., Light, G. A., Limborska, S., Liu, C., Lonnqvist, J., Loughland, C. M., Lubinski, J., Luykx, J. J., Lynham, A., Macek, M. J., Mackinnon, A., Magnusson, P. K., Maher, B. S., Maier, W., Malaspina, D., Mallet, J., Marder, S. R., Marsal, S., Martin, A. R., Martorell, L., Mattheisen, M., McCarley, R. W., McDonald, C., McGrath, J. J., Medeiros, H., Meier, S., Melegh, B., Melle, I., Mesholam-Gately, R. I., Metspalu, A., Michie, P. T., Milani, L., Milanova, V., Mitjans, M., Molden, E., Molina, E., Molto, M. D., Mondelli, V., Moreno, C., Morley, C. P., Muntane, G., Murphy, K. C., Myin-Germeys, I., Nenadic, I., Nestadt, G., Nikitina-Zake, L., Noto, C., Nuechterlein, K. H., O'Brien, N. L., O'Neill, F. A., Oh, S., Olincy, A., Ota, V. K., Pantelis, C., Papadimitriou, G. N., Parellada, M., Paunio, T., Pellegrino, R., Periyasamy, S., Perkins, D. O., Pfuhlmann, B., Pietilainen, O., Pimm, J., Porteous, D., Powell, J., Quattrone, D., Quested, D., Radant, A. D., Rampino, A., Rapaport, M. H., Rautanen, A., Reichenberg, A., Roe, C., Roffman, J. L., Roth, J., Rothermundt, M., Rutten, B. P., Saker-Delye, S., Salomaa, V., Sanjuan, J., Santoro, M. L., Savitz, A., Schall, U., Scott, R. J., Seidman, L. J., Sharp, S. I., Shi, J., Siever, L. J., Sigurdsson, E., Sim, K., Skarabis, N., Slominsky, P., So, H., Sobell, J. L., Soderman, E., Stain, H. J., Steen, N. E., Steixner-Kumar, A. A., Stogmann, E., Stone, W. S., Straub, R. E., Streit, F., Strengman, E., Stroup, T. S., Subramaniam, M., Sugar, C. A., Suvisaari, J., Svrakic, D. M., Swerdlow, N. R., Szatkiewicz, J. P., Ta, T. M., Takahashi, A., Terao, C., Thibaut, F., Toncheva, D., Tooney, P. A., Torretta, S., Tosato, S., Tura, G. B., Turetsky, B. I., Ucok, A., Vaaler, A., van Amelsvoort, T., van Winkel, R., Veijola, J., Waddington, J., Walter, H., Waterreus, A., Webb, B. T., Weiser, M., Williams, N. M., Witt, S. H., Wormley, B. K., Wu, J. Q., Xu, Z., Yolken, R., Zai, C. C., Zhou, W., Zhu, F., Zimprich, F., Atbasoglu, E. C., Ayub, M., Benner, C., Bertolino, A., Black, D. W., Bray, N. J., Breen, G., Buccola, N. G., Byerley, W. F., Chen, W. J., Cloninger, C. R., Crespo-Facorro, B., Donohoe, G., Freedman, R., Galletly, C., Gandal, M. J., Gennarelli, M., Hougaard, D. M., Hwu, H., Jablensky, A. V., McCarroll, S. A., Moran, J. L., Mors, O., Mortensen, P. B., Muller-Myhsok, B., Neil, A. L., Nordentoft, M., Pato, M. T., Petryshen, T. L., Pirinen, M., Pulver, A. E., Schulze, T. G., Silverman, J. M., Smoller, J. W., Stahl, E. A., Tsuang, D. W., Vilella, E., Wang, S., Xu, S., Indonesia Schizophrenia Consortium, PsychENCODE, Psychosis Endophenotypes International Consortium, SynGO Consortium, Adolfsson, R., Arango, C., Baune, B. T., Belangero, S. I., Borglum, A. D., Braff, D., Bramon, E., Buxbaum, J. D., Campion, D., Cervilla, J. A., Cichon, S., Collier, D. A., Corvin, A., Curtis, D., Forti, M. D., Domenici, E., Ehrenreich, H., Escott-Price, V., Esko, T., Fanous, A. H., Gareeva, A., Gawlik, M., Gejman, P. V., Gill, M., Glatt, S. J., Golimbet, V., Hong, K. S., Hultman, C. M., Hyman, S. E., Iwata, N., Jonsson, E. G., Kahn, R. S., Kennedy, J. L., Khusnutdinova, E., Kirov, G., Knowles, J. A., Krebs, M., Laurent-Levinson, C., Lee, J., Lencz, T., Levinson, D. F., Li, Q. S., Liu, J., Malhotra, A. K., Malhotra, D., McIntosh, A., McQuillin, A., Menezes, P. R., Morgan, V. A., Morris, D. W., Mowry, B. J., Murray, R. M., Nimgaonkar, V., Nothen, M. M., Ophoff, R. A., Paciga, S. A., Palotie, A., Pato, C. N., Qin, S., Rietschel, M., Riley, B. P., Rivera, M., Rujescu, D., Saka, M. C., Sanders, A. R., Schwab, S. G., Serretti, A., Sham, P. C., Shi, Y., St Clair, D., Stefansson, H., Stefansson, K., Tsuang, M. T., van Os, J., Vawter, M. P., Weinberger, D. R., Werge, T., Wildenauer, D. B., Yu, X., Yue, W., Holmans, P. A., Pocklington, A. J., Roussos, P., Vassos, E., Verhage, M., Visscher, P. M., Yang, J., Posthuma, D., Andreassen, O. A., Kendler, K. S., Owen, M. J., Wray, N. R., Daly, M. J., Huang, H., Neale, B. M., Sullivan, P. F., Ripke, S., Walters, J. T., O'Donovan, M. C., Schizophrenia Working Group of the Psychiatric Genomics Consortium, Dai, N., Wenwen, Q., Wildenauer, D. B., Agiananda, F., Amir, N., Antoni, R., Arsianti, T., Asmarahadi, A., Diatri, H., Djatmiko, P., Irmansyah, I., Khalimah, S., Kusumadewi, I., Kusumaningrum, P., Lukman, P. R., Nasrun, M. W., Safyuni, N. S., Prasetyawan, P., Semen, G., Siste, K., Tobing, H., Widiasih, N., Wiguna, T., Wulandari, D., Evalina, N., Hananto, A. J., Ismoyo, J. H., Marini, T. M., Henuhili, S., Reza, M., Yusnadewi, S., Abyzov, A., Akbarian, S., Ashley-Koch, A., van Bakel, H., Breen, M., Brown, M., Bryois, J., Carlyle, B., Charney, A., Coetzee, G., Crawford, G., Dracheva, S., Emani, P., Farnham, P., Fromer, M., Galeev, T., Gandal, M., Gerstein, M., Giase, G., Girdhar, K., Goes, F., Grennan, K., Gu, M., Guerra, B., Gursoy, G., Hoffman, G., Hyde, T., Jaffe, A., Jiang, S., Jiang, Y., Kefi, A., Kim, Y., Kitchen, R., Knowles, J. A., Lay, F., Lee, D., Li, M., Liu, C., Liu, S., Mattei, E., Navarro, F., Pan, X., Peters, M. A., Pinto, D., Pochareddy, S., Polioudakis, D., Purcaro, M., Purcell, S., Pratt, H., Reddy, T., Rhie, S., Roussos, P., Rozowsky, J., Sanders, S., Sestan, N., Sethi, A., Shi, X., Shieh, A., Swarup, V., Szekely, A., Wang, D., Warrell, J., Weissman, S., Weng, Z., White, K., Wiseman, J., Witt, H., Won, H., Wood, S., Wu, F., Xu, X., Yao, L., Zandi, P., Arranz, M. J., Bakker, S., Bender, S., Bramon, E., Collier, D. A., Crepo-Facorro, B., Hall, J., Iyegbe, C., Kahn, R., Lawrie, S., Lewis, C., Lin, K., Linszen, D. H., Mata, I., McIntosh, A., Murray, R. M., Ophoff, R. A., van Os, J., Powell, J., Rujescu, D., Walshe, M., Weisbrod, M., Achsel, T., Andres-Alonso, M., Bagni, C., Bayes, A., Biederer, T., Brose, N., Brown, T. C., Chua, J. J., Coba, M. P., Cornelisse, L. N., de Jong, A. P., de Juan-Sanz, J., Dieterich, D. C., Feng, G., Goldschmidt, H. L., Gundelfinger, E. D., Hoogenraad, C., Huganir, R. L., Hyman, S. E., Imig, C., Jahn, R., Jung, H., Kaeser, P. S., Kim, E., Koopmans, F., Kreutz, M. R., Lipstein, N., MacGillavry, H. D., Malenka, R., McPherson, P. S., O'Connor, V., Pielot, R., Ryan, T. A., Sahasrabudhe, D., Sala, C., Sheng, M., Smalla, K., Smit, A. B., Sudhof, T. C., Thomas, P. D., Toonen, R. F., van Weering, J. R., Verhage, M., Verpelli, C., de Haan, L., van Amelsvoort, T., van Winkel, R., Gareeva, A., Sham, P. C., Shi, Y., St Clair, D., van Os, J. 2022

  Abstract

  Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.

  View details for DOI 10.1038/s41586-022-04434-5

  View details for PubMedID 35396580

• Engineered synaptic tools reveal localized cAMP signaling in synapse assembly. The Journal of cell biology Sando, R., Ho, M. L., Liu, X., Sudhof, T. C. 1800; 221 (2)

  Abstract

  The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as "SynTAMs" for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.

  View details for DOI 10.1083/jcb.202109111

  View details for PubMedID 34913963

• Proteolytic regulation of calcium channels - avoiding controversy. Faculty reviews Alberts, B., Colbran, R. J., Dolphin, A. C., Pitt, G. S., Sudhof, T. C. 2022; 11: 5

  Abstract

  The publication of papers containing data obtained with suboptimal rigor in the experimental design and choice of key reagents, such as antibodies, can result in a lack of reproducibility and generate controversy that can both needlessly divert resources and, in some cases, damage public perception of the scientific enterprise. This exemplary paper by Buonarati et al. (2018)1 shows how a previously published, potentially important paper on calcium channel regulation falls short of the necessary mark, and aims to resolve the resulting controversy.

  View details for DOI 10.12703/r-01-000006

  View details for PubMedID 35373215

• Treatment of a genetic brain disease by CNS-wide microglia replacement. Science translational medicine Shibuya, Y., Kumar, K. K., Mader, M. M., Yoo, Y., Ayala, L. A., Zhou, M., Mohr, M. A., Neumayer, G., Kumar, I., Yamamoto, R., Marcoux, P., Liou, B., Bennett, F. C., Nakauchi, H., Sun, Y., Chen, X., Heppner, F. L., Wyss-Coray, T., Südhof, T. C., Wernig, M. 2022; 14 (636): eabl9945

  Abstract

  Hematopoietic cell transplantation after myeloablative conditioning has been used to treat various genetic metabolic syndromes but is largely ineffective in diseases affecting the brain presumably due to poor and variable myeloid cell incorporation into the central nervous system. Here, we developed and characterized a near-complete and homogeneous replacement of microglia with bone marrow cells in mice without the need for genetic manipulation of donor or host. The high chimerism resulted from a competitive advantage of scarce donor cells during microglia repopulation rather than enhanced recruitment from the periphery. Hematopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained full microglia replacement potency equivalent to whole bone marrow. To explore its therapeutic potential, we applied microglia replacement to a mouse model for Prosaposin deficiency, which is characterized by a progressive neurodegeneration phenotype. We found a reduction of cerebellar neurodegeneration and gliosis in treated brains, improvement of motor and balance impairment, and life span extension even with treatment started in young adulthood. This proof-of-concept study suggests that efficient microglia replacement may have therapeutic efficacy for a variety of neurological diseases.

  View details for DOI 10.1126/scitranslmed.abl9945

  View details for PubMedID 35294256

• RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons. Frontiers in molecular neuroscience Shankhwar, S., Schwarz, K., Katiyar, R., Jung, M., Maxeiner, S., Sudhof, T. C., Schmitz, F. 2022; 15: 838311

  Abstract

  Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons.

  View details for DOI 10.3389/fnmol.2022.838311

  View details for PubMedID 35153673

• Molecular self-avoidance in synaptic neurexin complexes. Science advances Wang, C. Y., Trotter, J. H., Liakath-Ali, K., Lee, S., Liu, X., Sudhof, T. C. 1800; 7 (51): eabk1924

  Abstract

  [Figure: see text].

  View details for DOI 10.1126/sciadv.abk1924

  View details for PubMedID 34919427

• RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development. Cell Wang, J., Miao, Y., Wicklein, R., Sun, Z., Wang, J., Jude, K. M., Fernandes, R. A., Merrill, S. A., Wernig, M., Garcia, K. C., Sudhof, T. C. 2021

  Abstract

  RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65A crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

  View details for DOI 10.1016/j.cell.2021.10.016

  View details for PubMedID 34758294

• CB1 receptor activation rapidly alters synaptic vesicle numbers in mouse hippocampal synapses. Molecular psychiatry Patzke, C., Dai, J., Brockmann, M. M., Sun, Z., Fenske, P., Rosenmund, C., Sudhof, T. C. 1800; 26 (11): 6103

  View details for DOI 10.1038/s41380-021-01426-1

  View details for PubMedID 35031784

• The cell biology of synapse formation JOURNAL OF CELL BIOLOGY Sudhof, T. C. 2021; 220 (7)

  Abstract

  In a neural circuit, synapses transfer information rapidly between neurons and transform this information during transfer. The diverse computational properties of synapses are shaped by the interactions between pre- and postsynaptic neurons. How synapses are assembled to form a neural circuit, and how the specificity of synaptic connections is achieved, is largely unknown. Here, I posit that synaptic adhesion molecules (SAMs) organize synapse formation. Diverse SAMs collaborate to achieve the astounding specificity and plasticity of synapses, with each SAM contributing different facets. In orchestrating synapse assembly, SAMs likely act as signal transduction devices. Although many candidate SAMs are known, only a few SAMs appear to have a major impact on synapse formation. Thus, a limited set of collaborating SAMs likely suffices to account for synapse formation. Strikingly, several SAMs are genetically linked to neuropsychiatric disorders, suggesting that impairments in synapse assembly are instrumental in the pathogenesis of neuropsychiatric disorders.

  View details for DOI 10.1083/jcb.202103052

  View details for Web of Science ID 000670874800001

  View details for PubMedID 34086051

  View details for PubMedCentralID PMC8186004

• The molecular logic of synapse formation: From structure to function Sudhof, T. C. SPRINGER. 2021: 39

  View details for Web of Science ID 000671622300004

• Cerebellin-2 regulates a serotonergic dorsal raphe circuit that controls compulsive behaviors. Molecular psychiatry Seigneur, E., Wang, J., Dai, J., Polepalli, J., Sudhof, T. C. 2021

  Abstract

  Cerebellin-1 (Cbln1) and cerebellin-2 (Cbln2) are secreted glycoproteins that are expressed in distinct subsets of neurons throughout the brain. Cbln1 and Cbln2 simultaneously bind to presynaptic neurexins and postsynaptic GluD1 and GluD2, thereby forming trans-synaptic adhesion complexes. Genetic associations link cerebellins, neurexins and GluD's to neuropsychiatric disorders involving compulsive behaviors, such as Tourette syndrome, attention-deficit hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD). Extensive evidence implicates dysfunction of serotonergic signaling in these neuropsychiatric disorders. Here, we report that constitutive Cbln2 KO mice, but not Cbln1 KO mice, display robust compulsive behaviors, including stereotypic pattern running, marble burying, explosive jumping, and excessive nest building, and exhibit decreased brain serotonin levels. Strikingly, treatment of Cbln2 KO mice with the serotonin precursor 5-hydroxytryptophan or the serotonin reuptake-inhibitor fluoxetine alleviated compulsive behaviors. Conditional deletion of Cbln2 both from dorsal raphe neurons and from presynaptic neurons synapsing onto dorsal raphe neurons reproduced the compulsive behaviors of Cbln2 KO mice. Finally, injection of recombinant Cbln2 protein into the dorsal raphe of Cbln2 KO mice largely reversed their compulsive behaviors. Taken together, our results show that Cbln2 controls compulsive behaviors by regulating serotonergic circuits in the dorsal raphe.

  View details for DOI 10.1038/s41380-021-01187-x

  View details for PubMedID 34158618

• GluD1 is a signal transduction device disguised as an ionotropic receptor NATURE Dai, J., Patzke, C., Liakath-Ali, K., Seigneur, E., Sudhof, T. C. 2021

  Abstract

  Ionotropic glutamate delta receptors 1 (GluD1) and 2 (GluD2) exhibit the molecular architecture of postsynaptic ionotropic glutamate receptors, but assemble into trans-synaptic adhesion complexes by binding to secreted cerebellins that in turn interact with presynaptic neurexins1-4. It is unclear whether neurexin-cerebellin-GluD1/2 assemblies serve an adhesive synapse-formation function or mediate trans-synaptic signalling. Here we show in hippocampal synapses, that binding of presynaptic neurexin-cerebellin complexes to postsynaptic GluD1 controls glutamate receptor activity without affecting synapse numbers. Specifically, neurexin-1-cerebellin-2 and neurexin-3-cerebellin-2 complexes differentially regulate NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors by activating distinct postsynaptic GluD1 effector signals. Of note, minimal GluD1 and GluD2 constructs containing only their N-terminal cerebellin-binding and C-terminal cytoplasmic domains, joined by an unrelated transmembrane region, fully control the levels of NMDA and AMPA receptors. The distinct signalling specificity of presynaptic neurexin-1 and neurexin-35,6 is encoded by their alternatively spliced splice site 4 sequences, whereas the regulatory functions of postsynaptic GluD1 are mediated by conserved cytoplasmic sequence motifs spanning 5-13 residues. Thus, GluDs are signalling molecules that regulate NMDA and AMPA receptors by an unexpected transduction mechanism that bypasses their ionotropic receptor architecture and directly converts extracellular neurexin-cerebellin signals into postsynaptic receptor responses.

  View details for DOI 10.1038/s41586-021-03661-6

  View details for Web of Science ID 000662164200002

  View details for PubMedID 34135511

• Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics. Stem cell reports Ng, Y. H., Chanda, S., Janas, J. A., Yang, N., Kokubu, Y., Sudhof, T. C., Wernig, M. 2021

  Abstract

  The differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We foundthat the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction.

  View details for DOI 10.1016/j.stemcr.2021.05.017

  View details for PubMedID 34171286

• Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons. Proceedings of the National Academy of Sciences of the United States of America Pak, C., Danko, T., Mirabella, V. R., Wang, J., Liu, Y., Vangipuram, M., Grieder, S., Zhang, X., Ward, T., Huang, Y. A., Jin, K., Dexheimer, P., Bardes, E., Mitelpunkt, A., Ma, J., McLachlan, M., Moore, J. C., Qu, P., Purmann, C., Dage, J. L., Swanson, B. J., Urban, A. E., Aronow, B. J., Pang, Z. P., Levinson, D. F., Wernig, M., Sudhof, T. C. 2021; 118 (22)

  Abstract

  Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

  View details for DOI 10.1073/pnas.2025598118

  View details for PubMedID 34035170

• Cannabinoid receptor activation acutely increases synaptic vesicle numbers by activating synapsins in human synapses. Molecular psychiatry Patzke, C., Dai, J., Brockmann, M. M., Sun, Z., Fenske, P., Rosenmund, C., Sudhof, T. C. 2021

  Abstract

  Cannabis and cannabinoid drugs are central agents that are used widely recreationally and are employed broadly for treating psychiatric conditions. Cannabinoids primarily act by stimulating presynaptic CB1 receptors (CB1Rs), the most abundant G-protein-coupled receptors in brain. CB1R activation decreases neurotransmitter release by inhibiting presynaptic Ca2+ channels and induces long-term plasticity by decreasing cellular cAMP levels. Here we identified an unanticipated additional mechanism of acute cannabinoid signaling in presynaptic terminals that regulates the size of synaptic vesicle pools available for neurotransmitter release. Specifically, we show that activation of CB1Rs in human and mouse neurons rapidly recruits vesicles to nerve terminals by suppressing the cAMP-dependent phosphorylation of synapsins. We confirmed this unanticipated mechanism using conditional deletion of synapsin-1, the predominant synapsin isoform in human neurons, demonstrating that synapsin-1 significantly contributes to the CB1R-dependent regulation of neurotransmission. Interestingly, acute activation of the Gi-DREADD hM4D mimics the effect of CB1R activation in a synapsin-1-dependent manner, suggesting that the control of synaptic vesicle numbers by synapsin-1 phosphorylation is a general presynaptic mechanism of neuromodulation. Thus, we uncovered a CB1R-dependent presynaptic mechanism that rapidly regulates the organization and neurotransmitter release properties of synapses.

  View details for DOI 10.1038/s41380-021-01095-0

  View details for PubMedID 33931733

• Neurexins regulate presynaptic GABAB-receptors at central synapses. Nature communications Luo, F., Sclip, A., Merrill, S., Sudhof, T. C. 2021; 12 (1): 2380

  Abstract

  Diverse signaling complexes are precisely assembled at the presynaptic active zone for dynamic modulation of synaptic transmission and synaptic plasticity. Presynaptic GABAB-receptors nucleate critical signaling complexes regulating neurotransmitter release at most synapses. However, the molecular mechanisms underlying assembly of GABAB-receptor signaling complexes remain unclear. Here we show that neurexins are required for the localization and function of presynaptic GABAB-receptor signaling complexes. At four model synapses, excitatory calyx of Held synapses in the brainstem, excitatory and inhibitory synapses on hippocampal CA1-region pyramidal neurons, and inhibitory basket cell synapses in the cerebellum, deletion of neurexins rendered neurotransmitter release significantly less sensitive to GABAB-receptor activation. Moreover, deletion of neurexins caused a loss of GABAB-receptors from the presynaptic active zone of the calyx synapse. These findings extend the role of neurexins at the presynaptic active zone to enabling GABAB-receptor signaling, supporting the notion that neurexins function as central organizers of active zone signaling complexes.

  View details for DOI 10.1038/s41467-021-22753-5

  View details for PubMedID 33888718

• The Perils of Navigating Activity-Dependent Alternative Splicing of Neurexins FRONTIERS IN MOLECULAR NEUROSCIENCE Liakath-Ali, K., Sudhof, T. C. 2021; 14: 659681

  Abstract

  Neurexins are presynaptic cell-adhesion molecules essential for synaptic function that are expressed in thousands of alternatively spliced isoforms. Recent studies suggested that alternative splicing at splice site 4 (SS4) of Nrxn1 is tightly regulated by an activity-dependent mechanism. Given that Nrxn1 alternative splicing at SS4 controls NMDA-receptor-mediated synaptic responses, activity-dependent SS4 alternative splicing would suggest a new synaptic plasticity mechanism. However, conflicting results confound the assessment of neurexin alternative splicing, prompting us to re-evaluate this issue. We find that in cortical cultures, membrane depolarization by elevated extracellular K+-concentrations produced an apparent shift in Nrxn1-SS4 alternative splicing by inducing neuronal but not astroglial cell death, resulting in persistent astroglial Nrxn1-SS4+ expression and decreased neuronal Nrxn1-SS4- expression. in vivo, systemic kainate-induced activation of neurons in the hippocampus produced no changes in Nrxn1-SS4 alternative splicing. Moreover, focal kainate injections into the mouse cerebellum induced small changes in Nrxn1-SS4 alternative splicing that, however, were associated with large decreases in Nrxn1 expression and widespread DNA damage. Our results suggest that although Nrxn1-SS4 alternative splicing may represent a mechanism of activity-dependent synaptic plasticity, common procedures for testing this hypothesis are prone to artifacts, and more sophisticated approaches will be necessary to test this important question.

  View details for DOI 10.3389/fnmol.2021.659681

  View details for Web of Science ID 000631420600001

  View details for PubMedID 33767611

  View details for PubMedCentralID PMC7985251

• Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecules. Proceedings of the National Academy of Sciences of the United States of America Jiang, X., Sando, R., Sudhof, T. C. 2021; 118 (3)

  Abstract

  Little is known about the cellular signals that organize synapse formation. To explore what signaling pathways may be involved, we employed heterologous synapse formation assays in which a synaptic adhesion molecule expressed in a nonneuronal cell induces pre- or postsynaptic specializations in cocultured neurons. We found that interfering pharmacologically with microtubules or actin filaments impaired heterologous synapse formation, whereas blocking protein synthesis had no effect. Unexpectedly, pharmacological inhibition of c-jun N-terminal kinases (JNKs), protein kinase-A (PKA), or AKT kinases also suppressed heterologous synapse formation, while inhibition of other tested signaling pathways-such as MAP kinases or protein kinase C-did not alter heterologous synapse formation. JNK and PKA inhibitors suppressed formation of both pre- and postsynaptic specializations, whereas AKT inhibitors impaired formation of post- but not presynaptic specializations. To independently test whether heterologous synapse formation depends on AKT signaling, we targeted PTEN, an enzyme that hydrolyzes phosphatidylinositol 3-phosphate and thereby prevents AKT kinase activation, to postsynaptic sites by fusing PTEN to Homer1. Targeting PTEN to postsynaptic specializations impaired heterologous postsynaptic synapse formation induced by presynaptic adhesion molecules, such as neurexins and additionally decreased excitatory synapse function in cultured neurons. Taken together, our results suggest that heterologous synapse formation is driven via a multifaceted and multistage kinase network, with diverse signals organizing pre- and postsynaptic specializations.

  View details for DOI 10.1073/pnas.2000173118

  View details for PubMedID 33431662

• Latrophilin GPCR signaling mediates synapse formation. eLife Sando, R. n., Südhof, T. C. 2021; 10

  Abstract

  Neural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In mouse CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.

  View details for DOI 10.7554/eLife.65717

  View details for PubMedID 33646123

• Bi-allelic variants in TSPOAP1, encoding the active zone protein RIMBP1, cause autosomal recessive dystonia. The Journal of clinical investigation Mencacci, N. E., Brockmann, M. M., Dai, J. n., Pajusalu, S. n., Atasu, B. n., Campos, J. n., Pino, G. n., Gonzalez-Latapi, P. n., Patzke, C. n., Schwake, M. n., Tucci, A. n., Pittman, A. n., Simon-Sanchez, J. n., Carvill, G. L., Balint, B. n., Wiethoff, S. n., Warner, T. T., Papandreou, A. n., Soo, A. K., Rein, R. n., Kadastik-Eerme, L. n., Puusepp, S. n., Reinson, K. n., Tomberg, T. n., Hanagasi, H. n., Gasser, T. n., Bhatia, K. P., Kurian, M. A., Lohmann, E. n., Õunap, K. n., Rosenmund, C. n., Südhof, T. n., Wood, N. n., Krainc, D. n., Acuna, C. n. 2021

  Abstract

  Dystonia is a debilitating hyperkinetic movement disorder, which can be transmitted as a monogenic trait. Here, we describe homozygous frameshift, nonsense and missense variants in TSPOAP1, encoding the active zone RIM-binding protein 1 (RIMBP1), as a novel genetic cause of autosomal recessive dystonia in seven subjects from three unrelated families. Subjects carrying loss-of-function variants presented with juvenile-onset progressive generalized dystonia, associated with intellectual disability and cerebellar atrophy. Conversely, subjects carrying a pathogenic missense variant (p.Gly1808Ser) presented with isolated adult-onset focal dystonia. In mice, complete loss of RIMBP1, known to reduce neurotransmission, led to motor abnormalities reminiscent of dystonia, decreased Purkinje cell dendritic arborization, and reduced numbers of cerebellar synapses. In vitro analysis of the p.Gly1808Ser variant showed larger spike-evoked calcium transients and enhanced neurotransmission, suggesting that RIMBP1-linked dystonia can be caused by either reduced or enhanced rates of spike-evoked release in relevant neural networks. Our findings establish a direct link between dysfunction of the presynaptic active zone and dystonia and highlight the critical role played by well-balanced neurotransmission in motor control and disease pathogenesis.  .

  View details for DOI 10.1172/JCI140625

  View details for PubMedID 33539324

• Persistent transcriptional programmes are associated with remote memory. Nature Chen, M. B., Jiang, X., Quake, S. R., Sudhof, T. C. 2020

  Abstract

  The role of gene expression during learning and in short-term memories has been studied extensively1-3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage.

  View details for DOI 10.1038/s41586-020-2905-5

  View details for PubMedID 33177708

• SPARCL1 promotes excitatory but not inhibitory synapse formation and function independent of neurexins and neuroligins. The Journal of neuroscience : the official journal of the Society for Neuroscience Gan, K. J., Sudhof, T. C. 2020

  Abstract

  Emerging evidence supports roles for secreted extracellular matrix proteins in boosting synaptogenesis, synaptic transmission, and synaptic plasticity. SPARCL1 (a.k.a. Hevin), a secreted non-neuronal protein, was reported to increase synaptogenesis by simultaneously binding to presynaptic neurexin-1alpha and to postsynaptic neuroligin-1B, thereby catalyzing formation of trans-synaptic neurexin/neuroligin complexes. However, neurexins and neuroligins do not themselves mediate synaptogenesis, raising the question of how SPARCL1 enhances synapse formation by binding to these molecules. Moreover, it remained unclear whether SPARCL1 acts on all synapses containing neurexins and neuroligins or only on a subset of synapses, and whether it enhances synaptic transmission in addition to boosting synaptogenesis or induces silent synapses. To explore these questions, we examined the synaptic effects of SPARCL1 and their dependence on neurexins and neuroligins. Using mixed neuronal and glial cultures from neonatal mouse cortex of both sexes, we show that SPARCL1 selectively increases excitatory but not inhibitory synapse numbers, enhances excitatory but not inhibitory synaptic transmission, and augments NMDA-receptor-mediated synaptic responses more than AMPAR-mediated synaptic responses. None of these effects were mediated by SPARCL1-binding to neurexins or neuroligins. Neurons from triple neurexin-1/2/3 or from quadruple neuroligin-1/2/3/4 conditional knockout mice that lacked all neurexins or all neuroligins were fully responsive to SPARCL1. Taken together, our results reveal that SPARCL1 selectively boosts excitatory but not inhibitory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.SIGNIFICANCE STATEMENT:Emerging evidence supports roles for extracellular matrix proteins in boosting synapse formation and function. Previous studies demonstrated that SPARCL1, a secreted non-neuronal protein, promotes synapse formation in rodent and human neurons. However, it remained unclear whether SPARCL1 acts on all or on only a subset of synapses, induces functional or largely inactive synapses, and generates synapses by bridging presynaptic neurexins and postsynaptic neuroligins. Here, we report that SPARCL1 selectively induces excitatory synapses, increases their efficacy, and enhances their NMDA receptor content. Moreover, using rigorous genetic manipulations, we show that SPARCL1 does not require neurexins and neuroligins for its activity. Thus, SPARCL1 selectively boosts excitatory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.

  View details for DOI 10.1523/JNEUROSCI.0454-20.2020

  View details for PubMedID 32973045

• Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism. The Journal of cell biology Khalaj, A. J., Sterky, F. H., Sclip, A., Schwenk, J., Brunger, A. T., Fakler, B., Sudhof, T. C. 2020; 219 (9)

  Abstract

  Neurexins are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. Here we identify FAM19A/TAFA proteins, "orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1gamma, via an unusual mechanism. Specifically, we show that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type-specific regulators of neurexin modifications.

  View details for DOI 10.1083/jcb.202004164

  View details for PubMedID 32706374

• A Trio of Active Zone Proteins Comprised of RIM-BPs, RIMs, and Munc13s Governs Neurotransmitter Release. Cell reports Brockmann, M. M., Zarebidaki, F., Camacho, M., Grauel, M. K., Trimbuch, T., Sudhof, T. C., Rosenmund, C. 2020; 32 (5): 107960

  Abstract

  At the presynaptic active zone, action-potential-triggered neurotransmitter release requires that fusion-competent synaptic vesicles are placed next to Ca2+ channels. The active zone resident proteins RIM, RBP, and Munc13 are essential contributors for vesicle priming and Ca2+-channel recruitment. Although the individual contributions of these scaffolds have been extensively studied, their respective functions in neurotransmission are still incompletely understood. Here, we analyze the functional interactions of RIMs, RBPs, and Munc13s at the genetic, molecular, functional, and ultrastructural levels in a mammalian synapse. We find that RBP, together with Munc13, promotes vesicle priming at the expense of RBP's role in recruiting presynaptic Ca2+ channels, suggesting that the support of RBP for vesicle priming and Ca2+-secretion coupling is mutually exclusive. Our results demonstrate that the functional interaction of RIM, RBP, and Munc13 is more profound than previously envisioned, acting as a functional trio that govern basic and short-term plasticity properties of neurotransmission.

  View details for DOI 10.1016/j.celrep.2020.107960

  View details for PubMedID 32755572

• Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism. Nature communications Li, J., Xie, Y., Cornelius, S., Jiang, X., Sando, R., Kordon, S. P., Pan, M., Leon, K., Sudhof, T. C., Zhao, M., Arac, D. 2020; 11 (1): 2140

  Abstract

  The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9A resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.

  View details for DOI 10.1038/s41467-020-16029-7

  View details for PubMedID 32358586

• Homozygous BZRAP1 Mutations Cause Autosomal Recessive Dystonia Mencacci, N., Brockmann, M., Pajusalu, S., Atasu, B., Latapi, P., Schwake, M., Balint, B., Papandreou, A., Pittman, A., Simon-Sanchez, J., Wiethoff, S., Warner, T., Kurian, M., Gasser, T., Lohmann, E., Ounap, K., Bhatia, K., Rosenmund, C., Sudhof, T., Wood, N., Krainc, D., Acuna, C. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for Web of Science ID 000536058007112

• Neurexins cluster Ca2+ channels within the presynaptic active zone EMBO JOURNAL Luo, F., Sclip, A., Jiang, M., Sudhof, T. C. 2020; 39 (7)

  View details for Web of Science ID 000523427200007

• Pro-neuronal activity of Myod1 due to promiscuous binding to neuronal genes. Nature cell biology Lee, Q. Y., Mall, M., Chanda, S., Zhou, B., Sharma, K. S., Schaukowitch, K., Adrian-Segarra, J. M., Grieder, S. D., Kareta, M. S., Wapinski, O. L., Ang, C. E., Li, R., Sudhof, T. C., Chang, H. Y., Wernig, M. 2020

  Abstract

  The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.

  View details for DOI 10.1038/s41556-020-0490-3

  View details for PubMedID 32231311

• Latrophilin-2 and latrophilin-3 are redundantly essential for parallel-fiber synapse function in cerebellum. eLife Zhang, R. S., Liakath-Ali, K., Sudhof, T. C. 2020; 9

  Abstract

  Latrophilin-2 (Lphn2) and latrophilin-3 (Lphn3) are adhesion GPCRs that serve as postsynaptic recognition molecules in CA1 pyramidal neurons of the hippocampus, where they are localized to distinct dendritic domains and are essential for different sets of excitatory synapses. Here, we studied Lphn2 and Lphn3 in the cerebellum. We show that latrophilins are abundantly and differentially expressed in the cerebellar cortex. Using conditional KO mice, we demonstrate that the Lphn2/3 double-deletion but not the deletion of Lphn2 or Lphn3 alone suppresses parallel-fiber synapses and reduces parallel-fiber synaptic transmission by ~50% without altering release probability. Climbing-fiber synapses, conversely, were unaffected. Even though ~50% of total cerebellar Lphn3 protein is expressed in Bergmann glia, Lphn3 deletion from Bergmann glia did not detectably impair excitatory or inhibitory synaptic transmission. Our studies demonstrate that Lphn2 and Lphn3 are selectively but redundantly required in Purkinje cells for parallel-fiber synapses.

  View details for DOI 10.7554/eLife.54443

  View details for PubMedID 32202499

• Neurexins cluster Ca2+ channels within the presynaptic active zone. The EMBO journal Luo, F., Sclip, A., Jiang, M., Sudhof, T. C. 2020: e103208

  Abstract

  To achieve ultrafast neurotransmission, neurons assemble synapses with highly organized presynaptic and postsynaptic nanomachines that are aligned by synaptic adhesion molecules. How functional assembly of presynaptic active zones is controlled via trans-synaptic interactions remains unknown. Here, we conditionally deleted all three neurexin adhesion molecules from presynaptic neurons of the calyx of Held in the mouse auditory system, a model synapse that allows precise biophysical analyses of synaptic properties. The pan-neurexin deletion had no effect on synapse development or the basic release machinery, but dramatically impaired fast neurotransmitter release. The overall properties of presynaptic calcium ion channels appeared normal, as reflected by the similar characteristics of calcium currents recorded at the nerve terminals. However, the pan-neurexin deletion significantly impaired the tight coupling of calcium influx to exocytosis, thereby suppressing neurotransmitter release. Furthermore, the pan-neurexin deletion reduced the function of calcium-activated BK potassium channels, whose activation depends on their tight association with presynaptic calcium channels. Together, these results suggest that neurexins perform a major function at the calyx synapse in coupling presynaptic calcium channels to release sites.

  View details for DOI 10.15252/embj.2019103208

  View details for PubMedID 32134527

• Evolution of the Autism-Associated Neuroligin-4 Gene Reveals Broad Erosion of Pseudoautosomal Regions in Rodents. Molecular biology and evolution Maxeiner, S., Benseler, F., Krasteva-Christ, G., Brose, N., Sudhof, T. C. 2020

  Abstract

  Variants in genes encoding synaptic adhesion proteins of the neuroligin family, most notably neuroligin-4, are a significant cause of autism spectrum disorders in humans. While human neuroligin-4 is encoded by two genes, NLGN4X and NLGN4Y, that are localized on the X-specific and male-specific regions of the two sex chromosomes, the chromosomal localization and full genomic sequence of the mouse Nlgn4 gene remain elusive. Here, we analyzed the neuroligin-4 genes of numerous rodent species by direct sequencing and bioinformatics, generated complete drafts of multiple rodent neuroligin-4 genes, and examined their evolution. Surprisingly, we find that the murine Nlgn4 gene is localized to the pseudoautosomal region (PAR) of the sex chromosomes, different from its human orthologues. We show that the sequence differences between various neuroligin-4 proteins are restricted to hotspots in which rodent neuroligin-4 proteins contain short repetitive sequence insertions compared to neuroligin-4 proteins from other species, whereas all other protein sequences are highly conserved. Evolutionarily, these sequence insertions initiate in the clade eumuroidea of the infra-order myomorpha, and are additionally associated with dramatic changes in non-coding sequences and gene size. Importantly, these changes are not exclusively restricted to neuroligin-4 genes but reflect major evolutionary changes that substantially altered or even deleted genes from the PARs of both sex chromosomes. Our results show that despite the fact that the PAR in rodents and the neuroligin-4 genes within the rodent PAR underwent massive evolutionary changes, neuroligin-4 proteins maintained a highly conserved core structure, consistent with a substantial evolutionary pressure preserving its physiological function.

  View details for DOI 10.1093/molbev/msaa014

  View details for PubMedID 32011705

• LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses. eLife Sclip, A. n., Südhof, T. C. 2020; 9

  Abstract

  LAR-type receptor phosphotyrosine-phosphatases (LAR-RPTPs) are presynaptic adhesion molecules that interact trans-synaptically with multitudinous postsynaptic adhesion molecules, including SliTrks, SALMs, and TrkC. Via these interactions, LAR-RPTPs are thought to function as synaptogenic wiring molecules that promote neural circuit formation by mediating the establishment of synapses. To test the synaptogenic functions of LAR-RPTPs, we conditionally deleted the genes encoding all three LAR-RPTPs, singly or in combination, in mice before synapse formation. Strikingly, deletion of LAR-RPTPs had no effect on synaptic connectivity in cultured neurons or in vivo, but impaired NMDA-receptor-mediated responses. Deletion of LAR-RPTPs decreased NMDA-receptor-mediated responses by a trans-synaptic mechanism. In cultured neurons, deletion of all LAR-RPTPs led to a reduction in synaptic NMDA-receptor EPSCs, without changing the subunit composition or the protein levels of NMDA-receptors. In vivo, deletion of all LAR-RPTPs in the hippocampus at birth also did not alter synaptic connectivity as measured via AMPA-receptor-mediated synaptic responses at Schaffer-collateral synapses monitored in juvenile mice, but again decreased NMDA-receptor mediated synaptic transmission. Thus, LAR-RPTPs are not essential for synapse formation, but control synapse properties by regulating postsynaptic NMDA-receptors via a trans-synaptic mechanism that likely involves binding to one or multiple postsynaptic ligands.

  View details for DOI 10.7554/eLife.53406

  View details for PubMedID 31985401

• A Synaptic Circuit Required for Acquisition but Not Recall of Social Transmission of Food Preference. Neuron Wang, C. Y., Liu, Z. n., Ng, Y. H., Südhof, T. C. 2020

  Abstract

  During social transmission of food preference (STFP), the combination of an olfactory sensory input with a social cue induces long-term memory of a food odor. How a social cue produces long-term learning of an olfactory input, however, remains unknown. Here we show that the neurons of the anterior olfactory nucleus (AON), which form abundant synaptic projections onto granule cells in the olfactory bulb (OB), express the synaptogenic molecule C1ql3. Deletion of C1ql3 in the dorsolateral AON impaired synaptic AON→OB connections and abolished acquisition, but not recall, of STFP memory without significantly affecting basal olfaction. Moreover, deletion in granule cells of the OB of Bai3, a postsynaptic GPCR that binds C1ql3, similarly suppressed synaptic transmission at AON→OB projections and abolished acquisition, but not recall, of STFP memory. Thus, synaptic AON→OB connections are selectively required for STFP memory acquisition and are formed by an essential interaction of presynaptic C1ql3 with postsynaptic Bai3.

  View details for DOI 10.1016/j.neuron.2020.04.004

  View details for PubMedID 32369733

• A simple Ca2+-imaging approach to neural network analyses in cultured neurons. Journal of neuroscience methods Sun, Z. n., Südhof, T. C. 2020: 109041

  Abstract

  Ca2+-imaging is a powerful tool to measure neuronal dynamics and network activity. To monitor network-level changes in cultured neurons, neuronal activity is often evoked by electrical or optogenetic stimulation and assessed using multi-electrode arrays or sophisticated imaging. Although such approaches allow detailed network analyses, multi-electrode arrays lack single-cell precision, whereas optical physiology generally requires advanced instrumentation that may not be universally available.Here we developed a simple, stimulation-free protocol with associated Matlab algorithms that enables scalable analyses of spontaneous network activity in cultured human and mouse neurons. The approach allows analysis of the overall network activity and of single-neuron dynamics, and is amenable to screening purposes.We validated the new protocol by assessing human neurons with a heterozygous conditional deletion of Munc18-1, and mouse neurons with a homozygous conditional deletion of neurexins. The approach described enabled identification of differential changes in these mutant neurons, allowing quantifications of the synchronous firing rate at the network level and of the amplitude and frequency of Ca2+-spikes at the single-neuron level. These results demonstrate the utility of the approach.Compared with current imaging platforms, our method is simple, scalable, accessible, and easy to implement. It enables quantification of more detailed parameters than multi-electrode arrays, but does not have the resolution and depth of more sophisticated yet labour-intensive methods, such as patch-clamp electrophysiology.The method reported here is scalable for a rapid direct assessment of neuronal function in culture, and can be applied to both human and mouse neurons. Thus, the method can serve as a basis for phenotypical analysis of mutations and for drug discovery efforts.

  View details for DOI 10.1016/j.jneumeth.2020.109041

  View details for PubMedID 33340555

• Dysfunction of parvalbumin neurons in the cerebellar nuclei produces an action tremor. The Journal of clinical investigation Zhou, M. n., Melin, M. D., Xu, W. n., Sudhof, T. C. 2020

  Abstract

  Essential tremor is a common brain disorder affecting millions of people, yet the neuronal mechanisms underlying this prevalent disease remain elusive. Here, we show that conditional deletion of synaptotagmin-2, the fastest Ca2+-sensor for synaptic neurotransmitter release, from parvalbumin neurons in mice causes an action tremor syndrome resembling the core symptom of essential tremor patients. Combining brain region-specific and cell type-specific genetic manipulation methods, we found that deletion of synaptotagmin-2 from excitatory parvalbumin-positive neurons in cerebellar nuclei was sufficient to generate an action tremor. The synaptotagmin-2 deletion converted synchronous into asynchronous neurotransmitter release in projections from cerebellar nuclei neurons onto gigantocellular reticular nucleus neurons, which might produce an action tremor by causing signal oscillations during movement. The tremor was rescued by completely blocking synaptic transmission with tetanus toxin in cerebellar nuclei, which also reversed the tremor phenotype in the traditional harmaline-induced essential tremor model. Using a promising animal model for action tremor, our results thus characterize a synaptic circuit mechanism that may underlie the prevalent essential tremor disorder.

  View details for DOI 10.1172/JCI135802

  View details for PubMedID 32634124

• Continuous and Discrete Neuron Types of the Adult Murine Striatum. Neuron Stanley, G., Gokce, O., Malenka, R. C., Sudhof, T. C., Quake, S. R. 2019

  Abstract

  The mammalian striatum is involved in many complex behaviors and yet is composed largely of a single neuron class: the spiny projection neuron (SPN). It is unclear to what extent the functional specialization of the striatum is due to the molecular specialization of SPN subtypes. We sought to define the molecular and anatomical diversity of adult SPNs using single-cell RNA sequencing (scRNA-seq) and quantitative RNA in situ hybridization (ISH). We computationally distinguished discrete versus continuous heterogeneity in scRNA-seq data and found that SPNs in the striatum can be classified into four major discrete types with no implied spatial relationship between them. Within these discrete types, we find continuous heterogeneity encoding spatial gradients of gene expression and defining anatomical location in a combinatorial mechanism. Our results suggest that neuronal circuitry has a substructure at far higher resolution than is typically interrogated, which is defined by the precise identity and location of a neuron.

  View details for DOI 10.1016/j.neuron.2019.11.004

  View details for PubMedID 31813651

• A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in brain neurons. eLife Dong, J., Lee, Y., Kirmiz, M., Palacio, S., Dumitras, C., Moreno, C. M., Sando, R., Santana, L. F., Sudhof, T. C., Gong, B., Murray, K. D., Trimmer, J. S. 2019; 8

  Abstract

  Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

  View details for DOI 10.7554/eLife.48750

  View details for PubMedID 31566565

• Differential Signaling Mediated by ApoE2, ApoE3, and ApoE4 in Human Neurons Parallels Alzheimer's Disease Risk. The Journal of neuroscience : the official journal of the Society for Neuroscience Huang, Y. A., Zhou, B., Nabet, A. M., Wernig, M., Sudhof, T. C. 2019

  Abstract

  In blood, apolipoprotein E (ApoE) is a component of circulating lipoproteins and mediates the clearance of these lipoproteins from blood by binding to ApoE receptors. Humans express three genetic ApoE variants, ApoE2, ApoE3, and ApoE4, that exhibit distinct ApoE receptor-binding properties and differentially affect Alzheimer's disease (AD), such that ApoE2 protects against, and ApoE4 predisposes to AD. In brain, ApoE-containing lipoproteins are secreted by activated astrocytes and microglia, but their functions and role in AD pathogenesis are largely unknown. Ample evidence suggests that ApoE4 induces microglial dysregulation and impedes Abeta clearance in AD, but the direct neuronal effects of ApoE variants are poorly studied. Extending previous studies, we here demonstrate that the three ApoE variants differentially activate multiple neuronal signaling pathways and regulate synaptogenesis. Specifically, using human neurons (male embryonic stem cell-derived) cultured in the absence of glia to exclude indirect glial mechanisms, we show that ApoE broadly stimulates signal transduction cascades. Among others, such stimulation enhances APP synthesis and synapse formation with an ApoE4>ApoE3>ApoE2 potency rank order, paralleling the relative risk for AD conferred by these ApoE variants. Unlike the previously described induction of APP transcription, however, ApoE-induced synaptogenesis involves CREB activation rather than cFos activation. We thus propose that in brain, ApoE acts as a glia-secreted signal that activates neuronal signaling pathways. The parallel potency rank order of ApoE4>ApoE3>ApoE2 in AD risk and neuronal signaling suggests that ApoE4 may in an apparent paradox promote AD pathogenesis by causing a chronic increase in signaling, possibly via enhancing APP expression.SIGNIFICANCE STATEMENTHumans express three genetic variants of apolipoprotein E (ApoE), ApoE2, ApoE3, and ApoE4. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), whereas ApoE2 protects against AD. Significant evidence suggests that ApoE4 impairs microglial function and impedes astrocytic Abeta clearance in brain, but the direct neuronal effects of ApoE are poorly understood, and the differences between ApoE variants in these effects are unclear. Here, we report that ApoE acts on neurons as a glia-secreted signaling molecule that, among others, enhances synapse formation. In activating neuronal signaling, the three ApoE variants exhibit a differential potency of ApoE4>ApoE3>ApoE2, which mirrors their relative effects on AD risk, suggesting that differential signaling by ApoE variants may contribute to AD pathogenesis.

  View details for DOI 10.1523/JNEUROSCI.2994-18.2019

  View details for PubMedID 31331998

• Synaptic neurexin-1 assembles into dynamically regulated active zone nanoclusters. The Journal of cell biology Trotter, J. H., Hao, J., Maxeiner, S., Tsetsenis, T., Liu, Z., Zhuang, X., Sudhof, T. C. 2019

  Abstract

  Neurexins are well-characterized presynaptic cell adhesion molecules that engage multifarious postsynaptic ligands and organize diverse synapse properties. However, the precise synaptic localization of neurexins remains enigmatic. Using super-resolution microscopy, we demonstrate that neurexin-1 forms discrete nanoclusters at excitatory synapses, revealing a novel organizational feature of synaptic architecture. Synapses generally contain a single nanocluster that comprises more than four neurexin-1 molecules and that also includes neurexin-2 and/or neurexin-3 isoforms. Moreover, we find that neurexin-1 is physiologically cleaved by ADAM10 similar to its ligand neuroligin-1, with 4-6% of neurexin-1 and 2-3% of neuroligin-1 present in the adult brain as soluble ectodomain proteins. Blocking ADAM10-mediated neurexin-1 cleavage dramatically increased the synaptic neurexin-1 content, thereby elevating the percentage of Homer1(+) excitatory synapses containing neurexin-1 nanoclusters from 40-50% to 80%, and doubling the number of neurexin-1 molecules per nanocluster. Taken together, our results reveal an unexpected nanodomain organization of synapses in which neurexin-1 is assembled into discrete presynaptic nanoclusters that are dynamically regulated via ectodomain cleavage.

  View details for DOI 10.1083/jcb.201812076

  View details for PubMedID 31262725

• Neuroligin-4 Regulates Excitatory Synaptic Transmission in Human Neurons. Neuron Marro, S. G., Chanda, S., Yang, N., Janas, J. A., Valperga, G., Trotter, J., Zhou, B., Merrill, S., Yousif, I., Shelby, H., Vogel, H., Kalani, M. Y., Sudhof, T. C., Wernig, M. 2019

  Abstract

  The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human embryonic stem cell-derived neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change-of-function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting that human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved.

  View details for DOI 10.1016/j.neuron.2019.05.043

  View details for PubMedID 31257103

• Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses NEURON Dai, J., Aoto, J., Sudhof, T. C. 2019; 102 (5): 993-+

  View details for DOI 10.1016/j.neuron.2019.03.032

  View details for Web of Science ID 000470104900012

• Neuroligin-1 Signaling Controls LTP and NMDA Receptors by Distinct Molecular Pathways NEURON Wu, X., Morishita, W. K., Riley, A. M., Hale, W. D., Sudhof, T. C., Malenka, R. C. 2019; 102 (3): 621-+

  View details for DOI 10.1016/j.neuron.2019.02.013

  View details for Web of Science ID 000467225800015

• Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses. Neuron Dai, J., Aoto, J., Sudhof, T. C. 2019

  Abstract

  AMPA- and NMDA-type glutamate receptors mediate distinct postsynaptic signals that differ characteristically among synapses. How postsynaptic AMPA- and NMDA-receptor levels are regulated, however, remains unclear. Using newly generated conditional knockin mice that enable genetic control of neurexin alternative splicing, we show that in hippocampal synapses, alternative splicing of presynaptic neurexin-1 at splice site 4 (SS4) dramatically enhanced postsynaptic NMDA-receptor-mediated, but not AMPA-receptor-mediated, synaptic responses without altering synapse density. In contrast, alternative splicing of neurexin-3 at SS4 suppressed AMPA-receptor-mediated,but not NMDA-receptor-mediated, synaptic responses, while alternative splicing of neurexin-2 at SS4 had no effect on NMDA- or AMPA-receptor-mediated responses. Presynaptic overexpression of the neurexin-1beta and neurexin-3beta SS4+ splice variants, but not of their SS4- splice variants, replicated the respective SS4+ knockin phenotypes. Thus, different neurexins perform distinct nonoverlapping functions at hippocampal synapses that are independently regulated by alternative splicing. These functions transsynaptically control NMDA and AMPA receptors, thereby mediating presynaptic control of postsynaptic responses.

  View details for PubMedID 31005376

• Synaptic retinoic acid receptor signaling mediates mTOR-dependent metaplasticity that controls hippocampal learning PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hsu, Y., Li, J., Wu, D., Sudhof, T. C., Chen, L. 2019; 116 (14): 7113–22

  View details for DOI 10.1073/pnas.1820690116

  View details for Web of Science ID 000463069900094

• Neuroligin-1 Signaling Controls LTP and NMDA Receptors by Distinct Molecular Pathways. Neuron Wu, X., Morishita, W. K., Riley, A. M., Hale, W. D., Sudhof, T. C., Malenka, R. C. 2019

  Abstract

  Neuroligins, postsynaptic cell adhesion molecules that are linked to neuropsychiatric disorders, are extensively studied, but fundamental questions about their functions remain. Using invivo replacement strategies in quadruple conditional knockout mice of all neuroligins to avoid heterodimerization artifacts, we show, in hippocampal CA1 pyramidal neurons, that neuroligin-1 performs two key functions in excitatory synapses by distinct molecular mechanisms. N-methyl-D-aspartate (NMDA) receptor-dependent LTP requires trans-synaptic binding of postsynaptic neuroligin-1 to presynaptic beta-neurexins but not the cytoplasmic sequences of neuroligins. In contrast, postsynaptic NMDA receptor (NMDAR)-mediated responses involve a neurexin-independent mechanism that requires the neuroligin-1 cytoplasmic sequences. Strikingly, deletion of neuroligins blocked the spine expansion associated with LTP, as monitored by two-photon imaging; this block involved a mechanism identical to that of therole of neuroligin-1 in NMDAR-dependent LTP. Our data suggest that neuroligin-1 performs two mechanistically distinct signaling functions and that neurolign-1-mediated trans-synaptic cell adhesion signaling critically regulates LTP.

  View details for PubMedID 30871858

• Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity GENES & DEVELOPMENT Shimojo, M., Madara, J., Pankow, S., Liu, X., Yates, J., Sudhof, T. C., Maximov, A. 2019; 33 (5-6): 365–76

  View details for DOI 10.1101/gad.320077.118

  View details for Web of Science ID 000460112800010

• Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity. Genes & development Shimojo, M., Madara, J., Pankow, S., Liu, X., Yates, J. 3., Sudhof, T. C., Maximov, A. 2019

  Abstract

  Synaptotagmin-11 (Syt11) is a Synaptotagmin isoform that lacks an apparent ability to bind calcium, phospholipids, or SNARE proteins. While human genetic studies have linked mutations in the Syt11 gene to schizophrenia and Parkinson's disease, the localization or physiological role of Syt11 remain unclear. We found that in neurons, Syt11 resides on abundant vesicles that differ from synaptic vesicles and resemble trafficking endosomes. These vesicles recycle via the plasma membrane in an activity-dependent manner, but their exocytosis is slow and desynchronized. Constitutive knockout mice lacking Syt11 died shortly after birth, suggesting Syt11-mediated membrane transport is required for survival. In contrast, selective ablation of Syt11 in excitatory forebrain neurons using a conditional knockout did not affect life span but impaired synaptic plasticity and memory. Syt11-deficient neurons displayed normal secretion of fast neurotransmitters and peptides but exhibited a reduction of long-term synaptic potentiation. Hence, Syt11 is an essential component of a neuronal vesicular trafficking pathway that differs from the well-characterized synaptic vesicle trafficking pathway but is also essential for life.

  View details for PubMedID 30808661

• Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science (New York, N.Y.) Sando, R., Jiang, X., Sudhof, T. C. 2019; 363 (6429)

  Abstract

  Bidirectional signaling by cell adhesion molecules is thought to mediate synapse formation, but the mechanisms involved remain elusive. We found that the adhesion G protein-coupled receptors latrophilin-2 and latrophilin-3 selectively direct formation of perforant-path and Schaffer-collateral synapses, respectively, to hippocampal CA1-region neurons. Latrophilin-3 binds to two transcellular ligands: fibronectin leucine-rich repeat transmembrane proteins (FLRTs) and teneurins. In transgenic mice in vivo, both binding activities were required for input-specific synapse formation, which suggests that coincident binding of both ligands is necessary for synapse formation. In cultured neurons in vitro, teneurin or FLRT alone did not induce excitatory synapse formation, whereas together they potently did so. Thus, postsynaptic latrophilins promote excitatory synapse formation by simultaneous binding of two unrelated presynaptic ligands, which is required for formation of synaptic inputs at specific dendritic localizations.

  View details for PubMedID 30792275

• Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins SCIENCE Sando, R., Jiang, X., Sudhof, T. C. 2019; 363 (6429): 837-+

  View details for DOI 10.1126/science.aav7969

  View details for Web of Science ID 000459387100036

• Synaptic retinoic acid receptor signaling mediates mTOR-dependent metaplasticity that controls hippocampal learning. Proceedings of the National Academy of Sciences of the United States of America Hsu, Y., Li, J., Wu, D., Sudhof, T. C., Chen, L. 2019

  Abstract

  Homeostatic synaptic plasticity is a stabilizing mechanism engaged by neural circuits in response to prolonged perturbation of network activity. The non-Hebbian nature of homeostatic synaptic plasticity is thought to contribute to network stability by preventing "runaway" Hebbian plasticity at individual synapses. However, whether blocking homeostatic synaptic plasticity indeed induces runaway Hebbian plasticity in an intact neural circuit has not been explored. Furthermore, how compromised homeostatic synaptic plasticity impacts animal learning remains unclear. Here, we show in mice that the experience of an enriched environment (EE) engaged homeostatic synaptic plasticity in hippocampal circuits, thereby reducing excitatory synaptic transmission. This process required RARalpha, a nuclear retinoic acid receptor that doubles as a cytoplasmic retinoic acid-induced postsynaptic regulator of protein synthesis. Blocking RARalpha-dependent homeostatic synaptic plasticity during an EE experience by ablating RARalpha signaling induced runaway Hebbian plasticity, as evidenced by greatly enhanced long-term potentiation (LTP). As a consequence, RARalpha deletion in hippocampal circuits during an EE experience resulted in enhanced spatial learning but suppressed learning flexibility. In the absence of RARalpha, moreover, EE experience superactivated mammalian target of rapamycin (mTOR) signaling, causing a shift in protein translation that enhanced the expression levels of AMPA-type glutamate receptors. Treatment of mice with the mTOR inhibitor rapamycin during an EE experience not only restored normal AMPA-receptor expression levels but also reversed the increases in runaway Hebbian plasticity and learning after hippocampal RARalpha deletion. Thus, our findings reveal an RARalpha- and mTOR-dependent mechanism by which homeostatic plasticity controls Hebbian plasticity and learning.

  View details for PubMedID 30782829

• SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse. Neuron Koopmans, F. n., van Nierop, P. n., Andres-Alonso, M. n., Byrnes, A. n., Cijsouw, T. n., Coba, M. P., Cornelisse, L. N., Farrell, R. J., Goldschmidt, H. L., Howrigan, D. P., Hussain, N. K., Imig, C. n., de Jong, A. P., Jung, H. n., Kohansalnodehi, M. n., Kramarz, B. n., Lipstein, N. n., Lovering, R. C., MacGillavry, H. n., Mariano, V. n., Mi, H. n., Ninov, M. n., Osumi-Sutherland, D. n., Pielot, R. n., Smalla, K. H., Tang, H. n., Tashman, K. n., Toonen, R. F., Verpelli, C. n., Reig-Viader, R. n., Watanabe, K. n., van Weering, J. n., Achsel, T. n., Ashrafi, G. n., Asi, N. n., Brown, T. C., De Camilli, P. n., Feuermann, M. n., Foulger, R. E., Gaudet, P. n., Joglekar, A. n., Kanellopoulos, A. n., Malenka, R. n., Nicoll, R. A., Pulido, C. n., de Juan-Sanz, J. n., Sheng, M. n., Südhof, T. C., Tilgner, H. U., Bagni, C. n., Bayés, À. n., Biederer, T. n., Brose, N. n., Chua, J. J., Dieterich, D. C., Gundelfinger, E. D., Hoogenraad, C. n., Huganir, R. L., Jahn, R. n., Kaeser, P. S., Kim, E. n., Kreutz, M. R., McPherson, P. S., Neale, B. M., O'Connor, V. n., Posthuma, D. n., Ryan, T. A., Sala, C. n., Feng, G. n., Hyman, S. E., Thomas, P. D., Smit, A. B., Verhage, M. n. 2019

  Abstract

  Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).

  View details for DOI 10.1016/j.neuron.2019.05.002

  View details for PubMedID 31171447

• Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing. The EMBO journal Wilson, S. C., White, K. I., Zhou, Q. n., Pfuetzner, R. A., Choi, U. B., Südhof, T. C., Brunger, A. T. 2019: e101603

  Abstract

  Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll β-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.

  View details for DOI 10.15252/embj.2019101603

  View details for PubMedID 31566781

• Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses. Cell Patzke, C. n., Brockmann, M. M., Dai, J. n., Gan, K. J., Grauel, M. K., Fenske, P. n., Liu, Y. n., Acuna, C. n., Rosenmund, C. n., Südhof, T. C. 2019; 179 (2): 498–513.e22

  Abstract

  Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 "latches" synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.

  View details for DOI 10.1016/j.cell.2019.09.011

  View details for PubMedID 31585084

• Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity. Cell stem cell Chanda, S. n., Ang, C. E., Lee, Q. Y., Ghebrial, M. n., Haag, D. n., Shibuya, Y. n., Wernig, M. n., Südhof, T. C. 2019

  Abstract

  Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.

  View details for DOI 10.1016/j.stem.2019.04.021

  View details for PubMedID 31155484

• Specific factors in blood from young but not old mice directly promote synapse formation and NMDA-receptor recruitment. Proceedings of the National Academy of Sciences of the United States of America Gan, K. J., Südhof, T. C. 2019

  Abstract

  Aging drives a progressive decline in cognition and decreases synapse numbers and synaptic function in the brain, thereby increasing the risk for neurodegenerative disease. Pioneering studies showed that introduction of blood from young mice into aged mice reversed age-associated cognitive impairments and increased synaptic connectivity in brain, suggesting that young blood contains specific factors that remediate age-associated decreases in brain function. However, whether such factors in blood from young animals act directly on neurons to enhance synaptic connectivity, or whether they act by an indirect mechanism remains unknown. Moreover, which factors in young blood mediate cognitive improvements in old mice is incompletely understood. Here, we show that serum extracted from the blood of young but not old mice, when applied to neurons transdifferentiated from human embryonic stem cells, directly increased dendritic arborization, augmented synapse numbers, doubled dendritic spine-like structures, and elevated synaptic N-methyl-d-aspartate (NMDA) receptors, thereby increasing synaptic connectivity. Mass spectrometry revealed that thrombospondin-4 (THBS4) and SPARC-like protein 1 (SPARCL1) were enriched in serum from young mice. Strikingly, recombinant THBS4 and SPARCL1 both increased dendritic arborization and doubled synapse numbers in cultured neurons. In addition, SPARCL1 but not THBS4 tripled NMDA receptor-mediated synaptic responses. Thus, at least two proteins enriched in young blood, THBS4 and SPARCL1, directly act on neurons as synaptogenic factors. These proteins may represent rejuvenation factors that enhance synaptic connectivity by increasing dendritic arborization, synapse formation, and synaptic transmission.

  View details for DOI 10.1073/pnas.1902672116

  View details for PubMedID 31160442

• Ablation of all synaptobrevin vSNAREs blocks evoked but not spontaneous neurotransmitter release at neuromuscular synapses. The Journal of neuroscience : the official journal of the Society for Neuroscience Liu, Y. n., Sugiura, Y. n., Südhof, T. C., Lin, W. n. 2019

  Abstract

  Synaptic transmission occurs when an action potential triggers neurotransmitter release via the fusion of synaptic vesicles with the pre-synaptic membrane, driven by the formation of SNARE complexes composed of the vesicular (v)-SNARE synaptobrevin and the target (t)-SNAREs Snap-25 and syntaxin-1. Neurotransmitters are also released spontaneously, independent of an action potential, through the fusion of synaptic vesicles with the pre-synaptic membrane. The major neuronal vSNAREs, synaptobrevin-1 and synaptobrevin-2, are expressed at the developing neuromuscular junction (NMJ) in mice, but their specific roles in NMJ formation and function remain unclear. Here, we examine the NMJs in mutant mouse embryos lacking either synaptobrevin 1 (Syb1lew/lew ) or synaptobrevin 2 (Syb2-/-), and those lacking both (Syb1lew/lewSyb2-/-). We found that compared with controls: (1) the number and size of NMJs was markedly increased in Syb2-/- and Syb1lew/lewSyb2-/- mice, but not in Syb1lew/lew mice; (2) synaptic vesicle density was markedly reduced in Syb1lew/lewSyb2-/- NMJs, and (3) evoked neurotransmission was markedly reduced in Syb2-/- NMJs and completely abolished in Syb1lew/lewSyb2-/- NMJs. Surprisingly, however, spontaneous neurotransmission persists in the absence of both Syb1 and Syb2. Furthermore, spontaneous neurotransmission remains constant in Syb1lew/lewSyb2-/- NMJs despite changing Ca2+ levels. These findings reveal an overlapping role for Syb1 and Syb2 (with Syb2 being dominant) in developing NMJs in mice. Moreover, because spontaneous release becomes Ca2+ insensitive in Syb1lew/lewSyb2-/- NMJs, our findings suggest that synaptobrevin-based SNARE complexes play a critical role in conferring Ca2+ sensitivity during spontaneous release.SIGNIFICANCE STATEMENTNeurotransmitters can be released at synapses with (evoked) or without (spontaneous) the influence of action potentials. Whereas evoked neurotransmission requires Ca2+ influx, those underlying the spontaneous neurotransmission may occur with or without Ca2+ Our findings show that, in the absence neuronal vSNARE synaptobrevin-1 and synaptobrevin-2, evoked neurotransmission is completely abolished; however, spontaneous synaptic transmission not only persists but even increased. Furthermore, spontaneous synaptic transmission that is normally highly Ca2+-sensitive became Ca2+-independent upon deletion of vSNARE synaptobrevin-1 and synaptobrevin-2. These findings reveal distinct mechanisms for evoked and spontaneous neurotransmitter release. Moreover, these findings suggest that synaptobrevin-based SNARE complexes play critical roles in conferring Ca2+ sensitivity during spontaneous neurotransmission at developing neuromuscular synapses in mice.

  View details for DOI 10.1523/JNEUROSCI.0403-19.2019

  View details for PubMedID 31160536

• Retinoic Acid Receptor RAR alpha-Dependent Synaptic Signaling Mediates Homeostatic Synaptic Plasticity at the Inhibitory Synapses of Mouse Visual Cortex JOURNAL OF NEUROSCIENCE Zhong, L. R., Chen, X., Park, E., Sudhof, T. C., Chen, L. 2018; 38 (49): 10454–66

  View details for DOI 10.1523/JNEUROSCI.1133-18.2018

  View details for Web of Science ID 000452156200006

• A central amygdala to zona incerta projection is required for acquisition and remote recall of conditioned fear memory NATURE NEUROSCIENCE Zhou, M., Liu, Z., Melin, M. D., Ng, Y., Xu, W., Sudhof, T. C. 2018; 21 (11): 1515-+

  View details for DOI 10.1038/s41593-018-0248-4

  View details for Web of Science ID 000448319500005

• Retinoic acid receptor RARalpha-dependent synaptic signaling mediates homeostatic synaptic plasticity at the inhibitory synapses of mouse visual cortex. The Journal of neuroscience : the official journal of the Society for Neuroscience Zhong, L., Chen, X., Park, E., Sudhof, T. C., Chen, L. 2018

  Abstract

  Homeostatic synaptic plasticity is a synaptic mechanism through which the nervous system adjusts synaptic excitation and inhibition to maintain network stability. Retinoic acid (RA) and its receptor RARalpha have been established as critical mediators of homeostatic synaptic plasticity. In vitro studies reveal that RA signaling enhances excitatory synaptic strength and decreases inhibitory synaptic strength. However, it is unclear whether RA-mediated homeostatic synaptic plasticity occurs in vivo, and if so, whether it operates at specific types of synapses. Here, we examine the impact of RA/RARalpha signaling in the monocular zone of primary visual cortex (V1m) in mice of either sex. Exogenous RA treatment in acute cortical slices resulted in a reduction in miniature inhibitory post-synaptic currents (mIPSCs) of layer 2/3 pyramidal neurons (PNs), an effect mimicked by visual deprivation induced by binocular enucleation in post-critical period animals. Postnatal deletion of RARalpha blocked RA's effect on mIPSCs. Cell type-specific deletion of RARalpha revealed that RA acted specifically on parvalbumin (PV)-expressing interneurons. RARalpha deletion in PV+ interneurons blocked visual deprivation-induced changes in mIPSCs, demonstrating the critical involvement of RA signaling in PV+ interneurons in vivo Moreover, visual deprivation- or RA-induced downregulation of synaptic inhibition was absent in the visual cortical circuit of constitutive and PV-specific Fmr1 KO mice, strongly suggesting a functional interaction between FMRP and RA signaling pathways. Taken together, our results demonstrate that RA/RARalpha signaling acts as a key component for homeostatic regulation of synaptic transmission at the inhibitory synapses of the visual cortex.SIGNIFICANCE STATEMENT In vitro studies established that retinoic acid (RA) and its receptor RARalpha play key roles in homeostatic synaptic plasticity, a mechanism by which synaptic excitation/inhibition balance and network stability are maintained. However, whether synaptic RA signaling operates in vivo remains undetermined. Here, using a conditional RARalpha knockout mouse and cell type-specific Cre-driver lines, we showed that RARalpha signaling in parvalbumin-expressing interneurons is crucial for visual deprivation-induced homeostatic synaptic plasticity at inhibitory synapses in visual cortical circuits. Importantly, this form of synaptic plasticity is absent when FMRP is selectively deleted in parvalbumin-expressing interneurons, suggesting a functional connection between RARalpha and FMRP signaling pathways in vivo Thus, dysfunction of RA-dependent homeostatic plasticity may contribute to cortical circuit abnormalities in fragile X syndrome.

  View details for PubMedID 30355624

• Towards an Understanding of Synapse Formation. Neuron Sudhof, T. C. 2018; 100 (2): 276–93

  Abstract

  Synapses are intercellular junctions specialized for fast, point-to-point information transfer from a presynaptic neuron to a postsynaptic cell. At a synapse, a presynaptic terminal secretes neurotransmitters via a canonical release machinery, while a postsynaptic specialization senses neurotransmitters via diverse receptors. Synaptic junctions are likely organized by trans-synaptic cell-adhesion molecules (CAMs) that bidirectionally orchestrate synapse formation, restructuring, and elimination. Many candidate synaptic CAMs were described, but which CAMs are central actors and which are bystanders remains unclear. Moreover, multiple genes encoding synaptic CAMs were linked to neuropsychiatric disorders, but the mechanisms involved are unresolved. Here, I propose that engagement of multifarious synaptic CAMs produces parallel trans-synaptic signals that mediate the establishment, organization, and plasticity of synapses, thereby controlling information processing by neural circuits. Among others, this hypothesis implies that synapse formation can be understood in terms of inter- and intracellular signaling, and that neuropsychiatric disorders involve an impairment in such signaling.

  View details for PubMedID 30359597

• Towards an Understanding of Synapse Formation NEURON Sudhof, T. C. 2018; 100 (2): 276–93

  View details for DOI 10.1016/j.neuron.2018.09.040

  View details for Web of Science ID 000448288900002

• Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Seigneur, E., Polepalli, J. S., Sudhof, T. C. 2018; 115 (43): E10235–E10244

  View details for DOI 10.1073/pnas.1811086115

  View details for Web of Science ID 000448040500026

• A central amygdala to zona incerta projection is required for acquisition and remote recall of conditioned fear memory. Nature neuroscience Zhou, M., Liu, Z., Melin, M. D., Ng, Y. H., Xu, W., Sudhof, T. C. 2018

  Abstract

  The formation and retrieval of conditioned fear memories critically depend on the amygdala. Here we identify an inhibitory projection from somatostatin-positive neurons in the central amygdala to parvalbumin-positive neurons in the zona incerta that is required for both recent and remote fear memories. Thus, the amygdala inhibitory input to parvalbumin-positive neurons in the zona incerta, a nucleus not previously implicated in fear memory, is an essential component of the fear memory circuitry.

  View details for PubMedID 30349111

• Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs. Proceedings of the National Academy of Sciences of the United States of America Seigneur, E., Polepalli, J. S., Sudhof, T. C. 2018

  Abstract

  Cerebellins are important neurexin ligands that remain incompletely understood. Two critical questions in particular remain unanswered: do different cerebellins perform distinct functions, and do these functions act in the initial establishment of synapses or in rendering nascent synapses capable of normal synaptic transmission? Here we show that in mice, Cbln2 and Cbln4 are expressed in the medial habenula (MHb) nucleus in different types of neurons that project to distinct target neurons in the interpeduncular nucleus. Conditional genetic deletion of Cbln2 in the MHb impaired synaptic transmission at Cbln2+ synapses in the interpeduncular neurons within 3 wk, but decreased synapse numbers only after 3 mo, suggesting a functional, but not a structural, requirement for Cbln2 in synapses formed by Cbln2-expressing neurons. In contrast, genetic deletions of Cbln4 in the MHb had no major effect on synaptic transmission or synapse numbers in interpeduncular target neurons. Nevertheless, MHb ablation of both Cbln2 and Cbln4 significantly impaired behavioral responses in mice, but affected different types of behaviors. Specifically, Cbln2 MHb deletions decreased spatial learning, as measured in the water T-maze, whereas Cbln4 MHb deletions increased anxiety levels, as monitored in the open field test and elevated plus maze. Thus, Cbln2 and Cbln4 are expressed in distinct MHb neurons that contribute to different behaviors.

  View details for PubMedID 30287486

• RIM-binding proteins recruit BK-channels to presynaptic release sites adjacent to voltage-gated Ca2+-channels EMBO JOURNAL Sclip, A., Acuna, C., Luo, F., Suedhof, T. C. 2018; 37 (16)

  View details for DOI 10.15252/embj.201798637

  View details for Web of Science ID 000441707100002

• The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling. Science translational medicine Zhang, Z., Marro, S. G., Zhang, Y., Arendt, K. L., Patzke, C., Zhou, B., Fair, T., Yang, N., Sudhof, T. C., Wernig, M., Chen, L. 2018; 10 (452)

  Abstract

  Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.

  View details for PubMedID 30068571

• The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling SCIENCE TRANSLATIONAL MEDICINE Zhang, Z., Marro, S. G., Zhang, Y., Arendt, K. L., Patzke, C., Zhou, B., Fair, T., Yang, N., Sudhof, T. C., Wernig, M., Chen, L. 2018; 10 (452)

  View details for DOI 10.1126/scitranslmed.aar4338

  View details for Web of Science ID 000440494900004

• RIM-binding proteins recruit BK-channels to presynaptic release sites adjacent to voltage-gated Ca2+-channels. The EMBO journal Sclip, A., Acuna, C., Luo, F., Sudhof, T. C. 2018

  Abstract

  The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca2+-triggered synaptic vesicle exocytosis. BK-channels are Ca2+-activated large-conductance K+-channels that require close proximity to Ca2+-channels for activation and control Ca2+-triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK-channels are recruited to presynaptic Ca2+-channels, however, is unknown. Here, we show that RBPs (for RIM-binding proteins), which are evolutionarily conserved active zone proteins containing SH3- and FN3-domains, directly bind to BK-channels. We find that RBPs interact with RIMs and Ca2+-channels via their SH3-domains, but to BK-channels via their FN3-domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK-currents and depleted BK-channels from active zones. Our data suggest that RBPs recruit BK-channels into a RIM-based macromolecular active zone complex that includes Ca2+-channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio-temporal coupling of Ca2+-influx to Ca2+-triggered neurotransmitter release in a presynaptic terminal.

  View details for PubMedID 29967030

• Transdifferentiation of human adult peripheral blood T cells into neurons PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tanabe, K., Ang, C., Chanda, S., Olmos, V., Haag, D., Levinson, D. F., Sudhof, T. C., Wernig, M. 2018; 115 (25): 6470–75

  View details for DOI 10.1073/pnas.1720273115

  View details for Web of Science ID 000435585200056

• Deletion of LRRTM1 and LRRTM2 in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons. Proceedings of the National Academy of Sciences of the United States of America Bhouri, M., Morishita, W., Temkin, P., Goswami, D., Kawabe, H., Brose, N., Sudhof, T. C., Craig, A. M., Siddiqui, T. J., Malenka, R. 2018; 115 (23): E5382–E5389

  Abstract

  Leucine-rich repeat transmembrane (LRRTM) proteins are synaptic cell adhesion molecules that influence synapse formation and function. They are genetically associated with neuropsychiatric disorders, and via their synaptic actions likely regulate the establishment and function of neural circuits in the mammalian brain. Here, we take advantage of the generation of a LRRTM1 and LRRTM2 double conditional knockout mouse (LRRTM1,2 cKO) to examine the role of LRRTM1,2 at mature excitatory synapses in hippocampal CA1 pyramidal neurons. Genetic deletion of LRRTM1,2 in vivo in CA1 neurons using Cre recombinase-expressing lentiviruses dramatically impaired long-term potentiation (LTP), an impairment that was rescued by simultaneous expression of LRRTM2, but not LRRTM4. Mutation or deletion of the intracellular tail of LRRTM2 did not affect its ability to rescue LTP, while point mutations designed to impair its binding to presynaptic neurexins prevented rescue of LTP. In contrast to previous work using shRNA-mediated knockdown of LRRTM1,2, KO of these proteins at mature synapses also caused a decrease in AMPA receptor-mediated, but not NMDA receptor-mediated, synaptic transmission and had no detectable effect on presynaptic function. Imaging of recombinant photoactivatable AMPA receptor subunit GluA1 in the dendritic spines of cultured neurons revealed that it was less stable in the absence of LRRTM1,2. These results illustrate the advantages of conditional genetic deletion experiments for elucidating the function of endogenous synaptic proteins and suggest that LRRTM1,2 proteins help stabilize synaptic AMPA receptors at mature spines during basal synaptic transmission and LTP.

  View details for PubMedID 29784826

• Deletion of LRRTM1 and LRRTM2 in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bhouri, M., Morishita, W., Temkin, P., Goswami, D., Kawabe, H., Brose, N., Sudhof, T. C., Craig, A., Siddiqui, T. J., Malenka, R. 2018; 115 (23): E5382–E5389

  View details for DOI 10.1073/pnas.1803280115

  View details for Web of Science ID 000434114900018

• Transdifferentiation of human adult peripheral blood T cells into neurons. Proceedings of the National Academy of Sciences of the United States of America Tanabe, K., Ang, C. E., Chanda, S., Olmos, V. H., Haag, D., Levinson, D. F., Sudhof, T. C., Wernig, M. 2018

  Abstract

  Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.

  View details for PubMedID 29866841

• Autism-associated neuroligin-4 mutation selectively impairs glycinergic synaptic transmission in mouse brainstem synapses JOURNAL OF EXPERIMENTAL MEDICINE Zhang, B., Gokce, O., Hale, W., Brose, N., Sudhof, T. C. 2018; 215 (6): 1543–53

  View details for DOI 10.1084/jem.20172162

  View details for Web of Science ID 000440821300005

• The Neurobiology of Opioid Addiction and the Potential for Prevention Strategies JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Peltz, G., Sudhof, T. C. 2018; 319 (20): 2071–72

  View details for DOI 10.1001/jama.2018.3394

  View details for Web of Science ID 000432679400013

• Genetic Ablation of All Cerebellins Reveals Synapse Organizer Functions in Multiple Regions Throughout the Brain JOURNAL OF NEUROSCIENCE Seigneur, E., Sudhof, T. C. 2018; 38 (20): 4774–90

  View details for DOI 10.1523/JNEUROSCI.0360-18.2018

  View details for Web of Science ID 000432347200012

• Autism-associated neuroligin-4 mutation selectively impairs glycinergic synaptic transmission in mouse brainstem synapses. The Journal of experimental medicine Zhang, B., Gokce, O., Hale, W. D., Brose, N., Sudhof, T. C. 2018

  Abstract

  In human patients, loss-of-function mutations of the postsynaptic cell-adhesion molecule neuroligin-4 were repeatedly identified as monogenetic causes of autism. In mice, neuroligin-4 deletions caused autism-related behavioral impairments and subtle changes in synaptic transmission, and neuroligin-4 was found, at least in part, at glycinergic synapses. However, low expression levels precluded a comprehensive analysis of neuroligin-4 localization, and overexpression of neuroligin-4 puzzlingly impaired excitatory but not inhibitory synaptic function. As a result, the function of neuroligin-4 remains unclear, as does its relation to other neuroligins. To clarify these issues, we systematically examined the function of neuroligin-4, focusing on excitatory and inhibitory inputs to defined projection neurons of the mouse brainstem as central model synapses. We show that loss of neuroligin-4 causes a profound impairment of glycinergic but not glutamatergic synaptic transmission and a decrease in glycinergic synapse numbers. Thus, neuroligin-4 is essential for the organization and/or maintenance of glycinergic synapses.

  View details for PubMedID 29724786

• The Neurobiology of Opioid Addiction and the Potential for Prevention Strategies. JAMA Peltz, G., Sudhof, T. C. 2018

  View details for PubMedID 29710202

• Genetic ablation of all cerebellins reveals synapse organizer functions in multiple regions throughout the brain. The Journal of neuroscience : the official journal of the Society for Neuroscience Seigneur, E., Sudhof, T. C. 2018

  Abstract

  Cerebellins are synaptic organizer molecules that bind to presynaptic neurexins and postsynaptic receptors. Cerebellins are well studied in cerebellum, but three of the four cerebellins (Cbln1, Cbln2, and Cbln4) are also broadly expressed outside of the cerebellum, suggesting that they perform general functions throughout the brain. Here we generated male and female single, double, and triple constitutive KO mice of Cbln1, Cbln2, and Cbln4. We found that all constitutive cerebellin-deficient mice are viable and fertile, suggesting that cerebellins are not essential for survival. Cbln1/2 double KO mice exhibited salience-induced seizures that were aggravated in Cbln1/2/4 triple KO mice, suggesting that all cerebellins contribute to brain function. Cbln1 KO mice displayed major motor impairments as described previously that were aggravated by additional KO of Cbln2. Strikingly, the Cbln1/2 double KO did not cause alterations in synapse density in the hippocampus of young adult, 1- and 2-month old mice, but produced a selective 50% decrease in hippocampal synapse density in the S. lacunosum-moleculare of the CA1 region and in the dentate gyrus of aging, 6-month old mice. A similar decrease in excitatory synapse density was observed in the striatum and retrosplenial cortex. Behaviorally, the Cbln1 KO produced dramatic changes in motor behaviors that were partly aggravated by additional deletion of Cbln2 and/or Cbln4. Our results show that cerebellins are not essential for survival and do not contribute to initial synapse formation, but perform multiple functions throughout the brain whose ablation results in a delayed loss of synapses and behavioral impairments.SIGNIFICANCE STATEMENTCerebellins (Cbln1-4) are trans-synaptic cell-adhesion molecules. In the cerebellum, Cbln1 functions as a bidirectional organizer of parallel fiber-Purkinje cell synapses by binding to presynaptic neurexins and postsynaptic GluRdelta2. Little is known about the function of cerebellins outside of the cerebellum; therefore, the present study uses single, double, and triple constitutive KO mice of Cbln1, Cbln2, and Cbln4 to analyze the overall function of cerebellins. We show that cerebellins act as important synaptic organizers in specific subsets of neurons and likely contribute to many different brain functions, and that cerebellins are not initially required for synapse formation, but for specification and long-term synapse maintenance. We also demonstrate for the first time that all cerebellins, not only Cbln1, contribute to brain function.

  View details for PubMedID 29691328

• Structural Basis for Teneurin Function in Circuit-Wiring: A Toxin Motif at the Synapse CELL Li, J., Shalev-Benami, M., Sando, R., Jiang, X., Kibrom, A., Wang, J., Leon, K., Katanski, C., Nazarko, O., Lu, Y. C., Sudhof, T. C., Skiniotis, G., Arac, D. 2018; 173 (3): 735-+

  Abstract

  Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-Å cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins. The ECR includes a large β barrel that partially encapsulates a C-terminal domain, which emerges to the solvent through an opening in the mid-barrel region. An immunoglobulin (Ig)-like domain seals the bottom of the barrel while a β propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the β propeller acts as a switch to regulate trans-cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans-cellular signaling in a LPHN-dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.

  View details for PubMedID 29677516

• Molecular Mechanisms Controlling Neurotransmitter Release by the Primed SNARE-complexin-Synaptotagmin Complex Zhou, Q., Zhou, P., Sudhof, T. C., Brunger, A. T. CELL PRESS. 2018: 554A–555A

  View details for Web of Science ID 000430563200523

• Building Bridges through Science NEURON Lissek, T., Adams, M., Adelman, J., Ahissar, E., Akaaboune, M., Akil, H., a'Absi, M., Arain, F., Carlos Arango-Lasprilla, J., Atasoy, D., Avila, J., Badawi, A., Bading, H., Baig, A., Baleriola, J., Belmonte, C., Bertocchi, I., Betz, H., Blakemore, C., Blanke, O., Boehm-Sturm, P., Bonhoeffer, T., Bonifazi, P., Brose, N., Campolongo, P., Celikel, T., Chang, C. C., Chang, T., Citri, A., Cline, H. T., Cortes, J. M., Cullen, K., Dean, K., M. Delgado-Garcia, J., Desroches, M., Disterhoft, J. F., Dowling, J. E., Draguhn, A., El-Khamisy, S. F., El Manira, A., Enam, S., Encinas, J. M., Erramuzpe, A., Esteban, J. A., Farinas, I., Fischer, E., Fukunaga, I., Gabilondo, I., Ganten, D., Gidon, A., Carlos Gomez-Esteban, J., Greengard, P., Grinevich, V., Gruart, A., Guillemin, R., Hariri, A. R., Hassan, B., Hausser, M., Hayashi, Y., Hussain, N. K., Jabbar, A., Jaber, M., Jahn, R., Janahi, E., Kabbaj, M., Kettenmann, H., Kindt, M., Knafo, S., Koehr, G., Komai, S., Krugers, H., Kuhn, B., Lakhdar Ghazal, N., Larkum, M. E., London, M., Lutz, B., Matute, C., Martinez-Millan, L., Maroun, M., McGaugh, J., Moustafa, A. A., Nasim, A., Nave, K., Neher, E., Nikolich, K., Outeiro, T., Palmer, L. M., Penagarikano, O., Perez-Otano, I., Pfaff, D. W., Poucet, B., Rahman, A., Ramos-Cabrer, P., Rashidy-Pour, A., Roberts, R. J., Rodrigues, S., Sanes, J. R., Schaefer, A. T., Segal, M., Segev, I., Shafqat, S., Siddiqui, N., Soreq, H., Soriano-Garcia, E., Spanagel, R., Sprengel, R., Stuart, G., Sudhof, T. C., Tonnesen, J., Trevino, M., Uthman, B. M., Venter, J., Verkhratsky, A., Weiss, C., Wiesel, T. N., Yaksi, E., Yizhar, O., Young, L. J., Young, P., Zawia, N. H., Zugaza, J. L., Hasan, M. T. 2017; 96 (4): 730–35

  Abstract

  Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge" initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities.

  View details for PubMedID 29144972

• Synaptic Neurexin Complexes: A Molecular Code for the Logic of Neural Circuits CELL Sudhof, T. C. 2017; 171 (4): 745–69

  Abstract

  Synapses are specialized junctions between neurons in brain that transmit and compute information, thereby connecting neurons into millions of overlapping and interdigitated neural circuits. Here, we posit that the establishment, properties, and dynamics of synapses are governed by a molecular logic that is controlled by diverse trans-synaptic signaling molecules. Neurexins, expressed in thousands of alternatively spliced isoforms, are central components of this dynamic code. Presynaptic neurexins regulate synapse properties via differential binding to multifarious postsynaptic ligands, such as neuroligins, cerebellin/GluD complexes, and latrophilins, thereby shaping the input/output relations of their resident neural circuits. Mutations in genes encoding neurexins and their ligands are associated with diverse neuropsychiatric disorders, especially schizophrenia, autism, and Tourette syndrome. Thus, neurexins nucleate an overall trans-synaptic signaling network that controls synapse properties, which thereby determines the precise responses of synapses to spike patterns in a neuron and circuit and which is vulnerable to impairments in neuropsychiatric disorders.

  View details for PubMedID 29100073

• Molecular Neuroscience in the 21st Century: A Personal Perspective NEURON Sudhof, T. C. 2017; 96 (3): 536–41

  Abstract

  Neuroscience is inherently interdisciplinary in its quest to explain the brain. Like all biological structures, the brain operates at multiple levels, from nano-scale molecules to meter-scale systems. Here, I argue that understanding the nano-scale organization of the brain is not only helpful for insight into its function, but is a requisite for such insight. I propose that one impediment to a better understanding of the brain is that most of its molecular processes are incompletely understood, and suggest a number of key questions that require our attention so that progress can be achieved in neuroscience beyond a description of the activity of neural circuits.

  View details for PubMedID 29096071

• Postsynaptic adhesion GPCR latrophilin-2 mediates target recognition in entorhinal-hippocampal synapse assembly JOURNAL OF CELL BIOLOGY Anderson, G. R., Maxeiner, S., Sando, R., Tsetsenis, T., Malenka, R. C., Sudhof, T. C. 2017; 216 (11): 3831–46

  Abstract

  Synapse assembly likely requires postsynaptic target recognition by incoming presynaptic afferents. Using newly generated conditional knock-in and knockout mice, we show in this study that latrophilin-2 (Lphn2), a cell-adhesion G protein-coupled receptor and presumptive α-latrotoxin receptor, controls the numbers of a specific subset of synapses in CA1-region hippocampal neurons, suggesting that Lphn2 acts as a synaptic target-recognition molecule. In cultured hippocampal neurons, Lphn2 maintained synapse numbers via a postsynaptic instead of a presynaptic mechanism, which was surprising given its presumptive role as an α-latrotoxin receptor. In CA1-region neurons in vivo, Lphn2 was specifically targeted to dendritic spines in the stratum lacunosum-moleculare, which form synapses with presynaptic entorhinal cortex afferents. In this study, postsynaptic deletion of Lphn2 selectively decreased spine numbers and impaired synaptic inputs from entorhinal but not Schaffer-collateral afferents. Behaviorally, loss of Lphn2 from the CA1 region increased spatial memory retention but decreased learning of sequential spatial memory tasks. Thus, Lphn2 appears to control synapse numbers in the entorhinal cortex/CA1 region circuit by acting as a domain-specific postsynaptic target-recognition molecule.

  View details for PubMedID 28972101

• Cerebellins are differentially expressed in selective subsets of neurons throughout the brain JOURNAL OF COMPARATIVE NEUROLOGY Seigneur, E., Sudhof, T. C. 2017; 525 (15): 3286–3311

  Abstract

  Cerebellins are secreted hexameric proteins that form tripartite complexes with the presynaptic cell-adhesion molecules neurexins or 'deleted-in-colorectal-cancer', and the postsynaptic glutamate-receptor-related proteins GluD1 and GluD2. These tripartite complexes are thought to regulate synapses. However, cerebellins are expressed in multiple isoforms whose relative distributions and overall functions are not understood. Three of the four cerebellins, Cbln1, Cbln2, and Cbln4, autonomously assemble into homohexamers, whereas the Cbln3 requires Cbln1 for assembly and secretion. Here, we show that Cbln1, Cbln2, and Cbln4 are abundantly expressed in nearly all brain regions, but exhibit strikingly different expression patterns and developmental dynamics. Using newly generated knockin reporter mice for Cbln2 and Cbln4, we find that Cbln2 and Cbln4 are not universally expressed in all neurons, but only in specific subsets of neurons. For example, Cbln2 and Cbln4 are broadly expressed in largely non-overlapping subpopulations of excitatory cortical neurons, but only sparse expression was observed in excitatory hippocampal neurons of the CA1- or CA3-region. Similarly, Cbln2 and Cbln4 are selectively expressed, respectively, in inhibitory interneurons and excitatory mitral projection neurons of the main olfactory bulb; here, these two classes of neurons form dendrodendritic reciprocal synapses with each other. A few brain regions, such as the nucleus of the lateral olfactory tract, exhibit astoundingly high Cbln2 expression levels. Viewed together, our data show that cerebellins are abundantly expressed in relatively small subsets of neurons, suggesting specific roles restricted to subsets of synapses.

  View details for PubMedID 28714144

• Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C(2)A domain in asynchronous neurotransmitter release PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Voleti, R., Tomchick, D. R., Sudhof, T. C., Rizo, J. 2017; 114 (40): E8518–E8527

  Abstract

  Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of l-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.

  View details for PubMedID 28923929

• Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+ channels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Luo, F., Liu, X., Sudhof, T. C., Acuna, C. 2017; 114 (38): E8081–E8090

  Abstract

  Fast neurotransmitter release from ribbon synapses via Ca2+-triggered exocytosis requires tight coupling of L-type Ca2+ channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca2+ channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca2+ buffer EGTA. These findings suggest that RBPs tether L-type Ca2+ channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

  View details for PubMedID 28874522

• IGF1-Dependent Synaptic Plasticity of Mitral Cells in Olfactory Memory during Social Learning NEURON Liu, Z., Chen, Z., Shang, C., Yan, F., Shi, Y., Zhang, J., Qu, B., Han, H., Wang, Y., Li, D., Udhof, T. S., Cao, P. 2017; 95 (1): 106-+

  Abstract

  During social transmission of food preference (STFP), mice form long-term memory of food odors presented by a social partner. How does the brain associate a social context with odor signals to promote memory encoding? Here we show that odor exposure during STFP, but not unconditioned odor exposure, induces glomerulus-specific long-term potentiation (LTP) of synaptic strength selectively at the GABAergic component of dendrodendritic synapses of granule and mitral cells in the olfactory bulb. Conditional deletion of synaptotagmin-10, the Ca2+ sensor for IGF1 secretion from mitral cells, or deletion of IGF1 receptor in the olfactory bulb prevented the socially relevant GABAergic LTP and impaired memory formation after STFP. Conversely, the addition of IGF1 to acute olfactory bulb slices elicited the GABAergic LTP in mitral cells by enhancing postsynaptic GABA receptor responses. Thus, our data reveal a synaptic substrate for a socially conditioned long-term memory that operates at the level of the initial processing of sensory information.

  View details for PubMedID 28683263

• Generation of pure GABAergic neurons by transcription factor programming. Nature methods Yang, N., Chanda, S., Marro, S., Ng, Y., Janas, J. A., Haag, D., Ang, C. E., Tang, Y., Flores, Q., Mall, M., Wapinski, O., Li, M., Ahlenius, H., Rubenstein, J. L., Chang, H. Y., Buylla, A. A., Südhof, T. C., Wernig, M. 2017; 14 (6): 621-628

  Abstract

  Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

  View details for DOI 10.1038/nmeth.4291

  View details for PubMedID 28504679

• Synaptotagmin-7-Mediated Asynchronous Release Boosts High-Fidelity Synchronous Transmission at a Central Synapse NEURON Luo, F., Sudhof, T. C. 2017; 94 (4): 826-?

  Abstract

  Synchronous release triggered by Ca(2+) binding to synaptotagmin-1, -2, or -9 is thought to drive fast synaptic transmission, whereas asynchronous release induced by Ca(2+) binding to synaptotagmin-7 is thought to produce delayed synaptic signaling, enabling prolonged synaptic computations. However, it is unknown whether synaptotagmin-7-dependent asynchronous release performs a physiological function at fast synapses lacking a prolonged signaling mode, such as the calyx of Held synapse. Here, we show at the calyx synapse that synaptotagmin-7-dependent asynchronous release indeed does not produce a prolonged synaptic signal after a stimulus train and does not contribute to short-term plasticity, but induces a steady-state, asynchronous postsynaptic current during stimulus trains. This steady-state postsynaptic current does not increase overall synaptic transmission but instead sustains reliable generation of postsynaptic spikes that are precisely time locked to presynaptic spikes. Thus, asynchronous release surprisingly functions, at least at some synapses, to sustain high-fidelity neurotransmission driven by synchronous release during high-frequency stimulus trains.

  View details for DOI 10.1016/j.neuron.2017.04.020

  View details for PubMedID 28521135

• Conditional Deletion of All Neurexins Defines Diversity of Essential Synaptic Organizer Functions for Neurexins NEURON Chen, L. Y., Jiang, M., Zhang, B., Gokce, O., Sudhof, T. C. 2017; 94 (3): 611-?

  Abstract

  Neurexins are recognized as key organizers of synapses that are essential for normal brain function. However, it is unclear whether neurexins are fundamental building blocks of all synapses with similar overall functions or context-dependent specifiers of synapse properties. To address this question, we produced triple cKO (conditional knockout) mice that allow ablating all neurexin expression in mice. Using neuron-specific manipulations combined with immunocytochemistry, paired recordings, and two-photon Ca(2+) imaging, we analyzed excitatory synapses formed by climbing fibers on Purkinje cells in cerebellum and inhibitory synapses formed by parvalbumin- or somatostatin-positive interneurons on pyramidal layer 5 neurons in the medial prefrontal cortex. After pan-neurexin deletions, we observed in these synapses severe but dramatically different synaptic phenotypes that ranged from major impairments in their distribution and function (climbing-fiber synapses) to large decreases in synapse numbers (parvalbumin-positive synapses) and severe alterations in action potential-induced presynaptic Ca(2+) transients (somatostatin-positive synapses). Thus, neurexins function primarily as context-dependent specifiers of synapses.

  View details for DOI 10.1016/j.neuron.2017.04.011

  View details for PubMedID 28472659

• Anatomical and Behavioral Investigation of C1ql3 in the Mouse Suprachiasmatic Nucleus. Journal of biological rhythms Chew, K. S., Fernandez, D. C., Hattar, S., Südhof, T. C., Martinelli, D. C. 2017: 748730417704766-?

  Abstract

  Many biochemical, physiological, and behavioral processes such as glucose metabolism, body temperature, and sleep-wake cycles show regular daily rhythms. These circadian rhythms are adjusted to the environmental light-dark cycle by a central pacemaker located in the suprachiasmatic nucleus (SCN) in order for the processes to occur at appropriate times of day. Here, we investigated the expression and function of a synaptic organizing protein, C1QL3, in the SCN. We found that C1ql3 is robustly expressed in the SCN. C1ql3 knockout mice have a reduced density of excitatory synapses in the SCN. In addition, these mice exhibited less consolidated activity to the active portions of the day and period lengthening following a 15-minute phase-delaying light pulse. These data identify C1QL3 as a signaling molecule that is highly expressed in SCN neurons, where it contributes to the formation and/or maintenance of glutamatergic synapses and plays a role in circadian behaviors, which may include circadian aftereffects.

  View details for DOI 10.1177/0748730417704766

  View details for PubMedID 28553739

• Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP NATURE Wu, D., Bacaj, T., Morishita, W., Goswami, D., Arendt, K. L., Xu, W., Chen, L., Malenka, R. C., Sudhof, T. C. 2017; 544 (7650): 316-?

  Abstract

  Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca(2+) influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca(2+) induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca(2+)-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca(2+)-dependent presynaptic vesicle exocytosis, also blocked Ca(2+)-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca(2+)-sensors for Ca(2+)-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.

  View details for DOI 10.1038/nature21720

  View details for PubMedID 28355182

• Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates NATURE Mall, M., Kareta, M. S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S. D., Ge, X., Drake, S., Ang, C. E., Walker, B. M., Vierbuchen, T., Fuentes, D. R., Brennecke, P., Nitta, K. R., Jolma, A., Steinmetz, L. M., Taipale, J., Sudhof, T. C., Wernig, M. 2017; 544 (7649): 245-?

  Abstract

  Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar 'many-but-one' lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.

  View details for DOI 10.1038/nature21722

  View details for PubMedID 28379941

• ELKS1 localizes the synaptic vesicle priming protein bMunc 13-2 to a specific subset of active zones JOURNAL OF CELL BIOLOGY Kawabe, H., Mitkovski, M., Kaeser, P. S., Hirrlinger, J., Opazo, F., Nestvogel, D., Kalla, S., Fejtova, A., Verrier, S. E., Bungers, S. R., Cooper, B. H., Varoqueaux, F., Wang, Y., Nehring, R. B., Gundelfinger, E. D., Rosenmund, C., Rizzoli, S. O., Sudhof, T. C., Rhee, J., Brose, N. 2017; 216 (4): 1143-1161

  Abstract

  Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s-Munc13-1 and bMunc13-2-mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.

  View details for DOI 10.1083/jcb.201606086

  View details for PubMedID 28264913

• Structural and Functional Studies of Latrophilin-Family Adhesion G-Protein Coupled Receptors Leon, K., Lu, Y., Nazarko, O., Sando, R., Salzman, G., Sudhof, T., Arac, D. FEDERATION AMER SOC EXP BIOL. 2017

  View details for Web of Science ID 000405986501710

• Conditional ablation of neuroligin-1 in CA1 pyramidal neurons blocks LTP by a cell-autonomous NMDA receptor-independent mechanism MOLECULAR PSYCHIATRY Jiang, M., Polepalli, J., Chen, L. Y., Zhang, B., Sudhof, T. C., Malenka, R. C. 2017; 22 (3): 375-383

  View details for DOI 10.1038/mp.2016.80

  View details for Web of Science ID 000394537100007

• Carbonic anhydrase-related protein CA10 is an evolutionarily conserved pan-neurexin ligand. Proceedings of the National Academy of Sciences of the United States of America Sterky, F. H., Trotter, J. H., Lee, S., Recktenwald, C. V., Du, X., Zhou, B., Zhou, P., Schwenk, J., Fakler, B., Südhof, T. C. 2017; 114 (7): E1253-E1262

  Abstract

  Establishment, specification, and validation of synaptic connections are thought to be mediated by interactions between pre- and postsynaptic cell-adhesion molecules. Arguably, the best-characterized transsynaptic interactions are formed by presynaptic neurexins, which bind to diverse postsynaptic ligands. In a proteomic screen of neurexin-1 (Nrxn1) complexes immunoisolated from mouse brain, we identified carbonic anhydrase-related proteins CA10 and CA11, two homologous, secreted glycoproteins of unknown function that are predominantly expressed in brain. We found that CA10 directly binds in a cis configuration to a conserved membrane-proximal, extracellular sequence of α- and β-neurexins. The CA10-neurexin complex is stable and stoichiometric, and results in formation of intermolecular disulfide bonds between conserved cysteine residues in neurexins and CA10. CA10 promotes surface expression of α- and β-neurexins, suggesting that CA10 may form a complex with neurexins in the secretory pathway that facilitates surface transport of neurexins. Moreover, we observed that the Nrxn1 gene expresses from an internal 3' promoter a third isoform, Nrxn1γ, that lacks all Nrxn1 extracellular domains except for the membrane-proximal sequences and that also tightly binds to CA10. Our data expand the understanding of neurexin-based transsynaptic interaction networks by providing further insight into the interactions nucleated by neurexins at the synapse.

  View details for DOI 10.1073/pnas.1621321114

  View details for PubMedID 28154140

• Towards Understanding the Molecular Mechanism of Synchronous Neurotransmitter Release Zhou, Q., Sudhof, T., Brunger, A. CELL PRESS. 2017: 77A–78A

  View details for DOI 10.1016/j.bpj.2016.11.465

  View details for Web of Science ID 000402328000399

• Modulation of excitation on parvalbumin interneurons by neuroligin-3 regulates the hippocampal network NATURE NEUROSCIENCE Polepalli, J. S., Wu, H., Goswami, D., Halpern, C. H., Sudhof, T. C., Malenka, R. C. 2017; 20 (2): 219-229

  Abstract

  Hippocampal network activity is generated by a complex interplay between excitatory pyramidal cells and inhibitory interneurons. Although much is known about the molecular properties of excitatory synapses on pyramidal cells, comparatively little is known about excitatory synapses on interneurons. Here we show that conditional deletion of the postsynaptic cell adhesion molecule neuroligin-3 in parvalbumin interneurons causes a decrease in NMDA-receptor-mediated postsynaptic currents and an increase in presynaptic glutamate release probability by selectively impairing the inhibition of glutamate release by presynaptic Group III metabotropic glutamate receptors. As a result, the neuroligin-3 deletion altered network activity by reducing gamma oscillations and sharp wave ripples, changes associated with a decrease in extinction of contextual fear memories. These results demonstrate that neuroligin-3 specifies the properties of excitatory synapses on parvalbumin-containing interneurons by a retrograde trans-synaptic mechanism and suggest a molecular pathway whereby neuroligin-3 mutations contribute to neuropsychiatric disorders.

  View details for DOI 10.1038/nn.4471

  View details for PubMedID 28067903

• Presynaptic Neuronal Pentraxin Receptor Organizes Excitatory and Inhibitory Synapses. journal of neuroscience Lee, S., Wei, M., Zhang, C., Maxeiner, S., Pak, C., Calado Botelho, S., Trotter, J., Sterky, F. H., Südhof, T. C. 2017; 37 (5): 1062-1080

  Abstract

  Three neuronal pentraxins are expressed in brain, the membrane-bound "neuronal pentraxin receptor" (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1β-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses.Neuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.

  View details for DOI 10.1523/JNEUROSCI.2768-16.2016

  View details for PubMedID 27986928

• ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aß Secretion. Cell Huang, Y. A., Zhou, B., Wernig, M., Südhof, T. C. 2017; 168 (3): 427-441 e21

  Abstract

  Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis.

  View details for DOI 10.1016/j.cell.2016.12.044

  View details for PubMedID 28111074

• Cell Biology and Pathophysiology of a-Synuclein. Cold Spring Harbor perspectives in medicine Burré, J., Sharma, M., Südhof, T. C. 2017

  Abstract

  α-Synuclein is an abundant neuronal protein that is highly enriched in presynaptic nerve terminals. Genetics and neuropathology studies link α-synuclein to Parkinson's disease (PD) and other neurodegenerative disorders. Accumulation of misfolded oligomers and larger aggregates of α-synuclein defines multiple neurodegenerative diseases called synucleinopathies, but the mechanisms by which α-synuclein acts in neurodegeneration are unknown. Moreover, the normal cellular function of α-synuclein remains debated. In this perspective, we review the structural characteristics of α-synuclein, its developmental expression pattern, its cellular and subcellular localization, and its function in neurons. We also discuss recent progress on secretion of α-synuclein, which may contribute to its interneuronal spread in a prion-like fashion, and describe the neurotoxic effects of α-synuclein that are thought to be responsible for its role in neurodegeneration.

  View details for DOI 10.1101/cshperspect.a024091

  View details for PubMedID 28108534

• Unique versus Redundant Functions of Neuroligin Genes in Shaping Excitatory and Inhibitory Synapse Properties. The Journal of neuroscience : the official journal of the Society for Neuroscience Chanda, S. n., Hale, W. D., Zhang, B. n., Wernig, M. n., Südhof, T. C. 2017; 37 (29): 6816–36

  Abstract

  Neuroligins are evolutionarily conserved postsynaptic cell adhesion molecules that interact with presynaptic neurexins. Neurons express multiple neuroligin isoforms that are targeted to specific synapses, but their synaptic functions and mechanistic redundancy are not completely understood. Overexpression or RNAi-mediated knockdown of neuroligins, respectively, causes a dramatic increase or decrease in synapse density, whereas genetic deletions of neuroligins impair synapse function with only minor effects on synapse numbers, raising fundamental questions about the overall physiological role of neuroligins. Here, we have systematically analyzed the effects of conditional genetic deletions of all major neuroligin isoforms (i.e., NL1, NL2, and NL3), either individually or in combinations, in cultured mouse hippocampal and cortical neurons. We found that conditional genetic deletions of neuroligins caused no change or only a small change in synapses numbers, but strongly impaired synapse function. This impairment was isoform specific, suggesting that neuroligins are not functionally redundant. Sparse neuroligin deletions produced phenotypes comparable to those of global deletions, indicating that neuroligins function in a cell-autonomous manner. Mechanistically, neuroligin deletions decreased the synaptic levels of neurotransmitter receptors and had no effect on presynaptic release probabilities. Overexpression of neuroligin-1 in control or neuroligin-deficient neurons increased synaptic transmission and synapse density but not spine numbers, suggesting that these effects reflect a gain-of-function mechanism; whereas overexpression of neuroligin-3, which, like neuroligin-1 is also targeted to excitatory synapses, had no comparable effect. Our data demonstrate that neuroligins are required for the physiological organization of neurotransmitter receptors in postsynaptic specializations and suggest that they do not play a major role in synapse formation.SIGNIFICANCE STATEMENT Human neuroligin genes have been associated with autism, but the cellular functions of different neuroligins and their molecular mechanisms remain incompletely understood. Here, we performed comparative analyses in cultured mouse neurons of all major neuroligin isoforms, either individually or in combinations, using conditional knockouts. We found that neuroligin deletions did not affect synapse numbers but differentially impaired excitatory or inhibitory synaptic functions in an isoform-specific manner. These impairments were due, at least in part, to a decrease in synaptic distribution of neurotransmitter receptors upon deletion of neuroligins. Conversely, the overexpression of neuroligin-1 increased synapse numbers but not spine numbers. Our results suggest that various neuroligin isoforms perform unique postsynaptic functions in organizing synapses but are not essential for synapse formation or maintenance.

  View details for PubMedID 28607166

• The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis. Nature Zhou, Q. n., Zhou, P. n., Wang, A. L., Wu, D. n., Zhao, M. n., Südhof, T. C., Brunger, A. T. 2017; 548 (7668): 420–25

  Abstract

  Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca(2+) binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

  View details for PubMedID 28813412

• C-terminal domain of mammalian complexin-1 localizes to highly curved membranes. Proceedings of the National Academy of Sciences of the United States of America Gong, J., Lai, Y., Li, X., Wang, M., Leitz, J., Hu, Y., Zhang, Y., Choi, U. B., Cipriano, D., Pfuetzner, R. A., Südhof, T. C., Yang, X., Brunger, A. T., Diao, J. 2016

  Abstract

  In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca(2+)-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca(2+)-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

  View details for PubMedID 27821736

• Expression of C1ql3 in Discrete Neuronal Populations Controls Efferent Synapse Numbers and Diverse Behaviors. Neuron Martinelli, D. C., Chew, K. S., Rohlmann, A., Lum, M. Y., Ressl, S., Hattar, S., Brunger, A. T., Missler, M., Südhof, T. C. 2016; 91 (5): 1034-1051

  Abstract

  C1ql3 is a secreted neuronal protein that binds to BAI3, an adhesion-class GPCR. C1ql3 is homologous to other gC1q-domain proteins that control synapse numbers, but a role for C1ql3 in regulating synapse density has not been demonstrated. We show in cultured neurons that C1ql3 expression is activity dependent and supports excitatory synapse density. Using newly generated conditional and constitutive C1ql3 knockout mice, we found that C1ql3-deficient mice exhibited fewer excitatory synapses and diverse behavioral abnormalities, including marked impairments in fear memories. Using circuit-tracing tools and conditional ablation of C1ql3 targeted to specific brain regions, we demonstrate that C1ql3-expressing neurons in the basolateral amygdala project to the medial prefrontal cortex, that these efferents contribute to fear memory behavior, and that C1ql3 is required for formation and/or maintenance of these synapses. Our results suggest that C1ql3 is a signaling protein essential for subsets of synaptic projections and the behaviors controlled by these projections.

  View details for DOI 10.1016/j.neuron.2016.07.002

  View details for PubMedID 27478018

• Neuroligins Are Selectively Essential for NMDAR Signaling in Cerebellar Stellate Interneurons JOURNAL OF NEUROSCIENCE Zhang, B., Sudhof, T. C. 2016; 36 (35): 9070-9083

  Abstract

  Neuroligins are postsynaptic cell-adhesion molecules that contribute to synapse specification. However, many other postsynaptic cell-adhesion molecules are known and the relative contributions of neuroligins versus other such molecules in different types of synapses and neurons remains largely unknown. Here, we have studied the role of neuroligins in cerebellar stellate interneurons that participate in a well defined circuit that converges on Purkinje cells as the major output neurons of cerebellar cortex. By crossing triple conditional knock-out (cKO) mice targeting all three major neuroligins [neuroligin-1 to neuroligin-3 (NL123)] with parvalbumin-Cre (PV-Cre) transgenic mice, we deleted neuroligins from inhibitory cerebellar interneurons and Purkinje cells, allowing us to study the effects of neuroligin deletions on cerebellar stellate cell synapses by electrophysiology in acute slices. PV-Cre/NL123 cKO mice did not exhibit gross alterations of cerebellar structure or cerebellar interneuron morphology. Strikingly, electrophysiological recordings in stellate cells from these PV-Cre/NL123 cKO mice revealed a large decrease in NMDAR-mediated excitatory synaptic responses, which, in stellate cells, are largely extrasynaptic, without a change in AMPA-receptor-mediated responses. Parallel analyses in PV-Cre/NL1 mice that are single NL1 cKO mice uncovered the same phenotype, demonstrating that NL1 is responsible for recruiting extrasynaptic NMDARs. Moreover, we observed only a modest impairment in inhibitory synaptic responses in stellate cells lacking NL123 despite a nearly complete suppression of inhibitory synaptic transmission in Purkinje cells by the same genetic manipulation. Our results suggest that, unlike other types of neurons investigated, neuroligins are selectively essential in cerebellar stellate interneurons for enabling the function of extrasynaptic NMDARs.Neuroligins are postsynaptic cell-adhesion molecules genetically linked to autism. However, the contributions of neuroligins to interneuron functions remain largely unknown. Here, we analyzed the role of neuroligins in cerebellar stellate interneurons. We deleted neuroligin-1, neuroligin-2, and neuroligin-3, the major cerebellar neuroligin isoforms, from stellate cells in triple NL123 conditional knock-out mice and analyzed synaptic responses by acute slice electrophysiology. We find that neuroligins are selectively essential for extrasynaptic NMDAR-mediated signaling, but dispensable for both AMPAR-mediated and inhibitory synaptic transmission. Our results reveal a critical and selective role for neuroligins in the regulation of NMDAR responses in cerebellar stellate interneurons.

  View details for DOI 10.1523/JNEUROSCI.1356-16.2016

  View details for PubMedID 27581450

• Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons. Proceedings of the National Academy of Sciences of the United States of America Földy, C., Darmanis, S., Aoto, J., Malenka, R. C., Quake, S. R., Südhof, T. C. 2016; 113 (35): E5222-31

  Abstract

  In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known. Here, we devised a comprehensive list of genes involved in cell adhesion, and used single-cell RNA sequencing (RNAseq) to analyze their expression in electrophysiologically defined interneurons and projection neurons. We compared the cell type-specific expression of these genes with that of genes involved in transmembrane ion conductances (i.e., channels), exocytosis, and rho/rac signaling, which regulates the actin cytoskeleton. Using these data, we identified two independent, developmentally regulated networks of interacting genes encoding molecules involved in cell adhesion, exocytosis, and signal transduction. Our approach provides a framework for a presumed cell adhesion and signaling code in neurons, enables correlating electrophysiological with molecular properties of neurons, and suggests avenues toward understanding synaptic specificity.

  View details for DOI 10.1073/pnas.1610155113

  View details for PubMedID 27531958

• How to Make an Active Zone: Unexpected Universal Functional Redundancy between RIMs and RIM-BPs. Neuron Acuna, C., Liu, X., Südhof, T. C. 2016; 91 (4): 792-807

  Abstract

  RIMs and RIM-binding proteins (RBPs) are evolutionary conserved multidomain proteins of presynaptic active zones that are known to recruit Ca(2+) channels; in addition, RIMs perform well-recognized functions in tethering and priming synaptic vesicles for exocytosis. However, deletions of RIMs or RBPs in mice cause only partial impairments in various active zone functions and have no effect on active zone structure, as visualized by electron micrographs, suggesting that their contribution to active zone functions is limited. Here, we show in synapses of the calyx of Held in vivo and hippocampal neurons in culture that combined, but not individual, deletions of RIMs and RBPs eliminate tethering and priming of synaptic vesicles, deplete presynaptic Ca(2+) channels, and ablate active zone complexes, as analyzed by electron microscopy of chemically fixed synapses. Thus, RBPs perform unexpectedly broad roles at the active zone that together with those of RIMs are essential for all active zone functions.

  View details for DOI 10.1016/j.neuron.2016.07.042

  View details for PubMedID 27537484

• Truth in Science Publishing: A Personal Perspective PLOS BIOLOGY Sudhof, T. C. 2016; 14 (8)

  Abstract

  Scientists, public servants, and patient advocates alike increasingly question the validity of published scientific results, endangering the public's acceptance of science. Here, I argue that emerging flaws in the integrity of the peer review system are largely responsible. Distortions in peer review are driven by economic forces and enabled by a lack of accountability of journals, editors, and authors. One approach to restoring trust in the validity of published results may be to establish basic rules that render peer review more transparent, such as publishing the reviews (a practice already embraced by some journals) and monitoring not only the track records of authors but also of editors and journals.

  View details for DOI 10.1371/journal.pbio.1002547

  View details for PubMedID 27564858

• Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq CELL REPORTS Gokce, O., Stanley, G. M., Treutlein, B., Neff, N. F., Camp, J. G., Malenka, R. C., Rothwell, P. E., Fuccillo, M. V., Sudhof, T. C., Quake, S. R. 2016; 16 (4): 1126-1137

  Abstract

  The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

  View details for DOI 10.1016/j.celrep.2016.06.059

  View details for PubMedID 27425622

• FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ahlenius, H., Chanda, S., Webb, A. E., Yousif, I., Karmazin, J., Prusiner, S. B., Brunet, A., Suedhof, T. C., Wernig, M. 2016; 113 (30): 8514-8519

  Abstract

  We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.

  View details for DOI 10.1073/pnas.1607079113

  View details for PubMedID 27402759

• Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction PLOS ONE Sclip, A., Bacaj, T., Giam, L. R., Sudhof, T. C. 2016; 11 (6)

  Abstract

  Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges.

  View details for DOI 10.1371/journal.pone.0158295

  View details for PubMedID 27348751

• Conditional ablation of neuroligin-1 in CA1 pyramidal neurons blocks LTP by a cell-autonomous NMDA receptor-independent mechanism. Molecular psychiatry Jiang, M., Polepalli, J., Chen, L. Y., Zhang, B., Südhof, T. C., Malenka, R. C. 2016

  Abstract

  Neuroligins are postsynaptic cell-adhesion molecules implicated in autism and other neuropsychiatric disorders. Despite extensive work, the role of neuroligins in synapse function and plasticity, especially N-methyl-d-aspartate (NMDA) receptor (NMDAR)-dependent long-term potentiation (LTP), remains unclear. To establish which synaptic functions unequivocally require neuroligins, we analyzed single and triple conditional knockout (cKO) mice for all three major neuroligin isoforms (NL1-NL3). We inactivated neuroligins by stereotactic viral expression of Cre-recombinase in hippocampal CA1 region pyramidal neurons at postnatal day 0 (P0) or day 21 (P21) and measured synaptic function, synaptic plasticity and spine numbers in acute hippocampal slices 2-3 weeks later. Surprisingly, we find that ablation of neuroligins in newborn or juvenile mice only modestly impaired basal synaptic function in hippocampus and caused no alteration in postsynaptic spine numbers. However, triple cKO of NL1-NL3 or single cKO of NL1 impaired NMDAR-mediated excitatory postsynaptic currents and abolished NMDAR-dependent LTP. Strikingly, the NL1 cKO also abolished LTP elicited by activation of L-type Ca(2+)-channels during blockade of NMDARs. These findings demonstrate that neuroligins are generally not essential for synapse formation in CA1 pyramidal neurons but shape synaptic properties and that NL1 specifically is required for LTP induced by postsynaptic Ca(2+)-elevations, a function which may contribute to the pathophysiological role of neuroligins in brain disorders.Molecular Psychiatry advance online publication, 24 May 2016; doi:10.1038/mp.2016.80.

  View details for DOI 10.1038/mp.2016.80

  View details for PubMedID 27217145

• How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release EMBO JOURNAL Maxeiner, S., Luo, F., Tan, A., Schmitz, F., Sudhof, T. C. 2016; 35 (10): 1098-1114

  Abstract

  Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon-specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca(2+)-buffer EGTA, suggesting that synaptic ribbons mediate nano-domain coupling of Ca(2+) channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano-domains that position release-ready synaptic vesicles adjacent to Ca(2+) channels.

  View details for DOI 10.15252/embj.201592701

  View details for PubMedID 26929012

• Autism-associated SHANK3 haploinsufficiency causes I-h channelopathy in human neurons SCIENCE Yi, F., Danko, T., Botelho, S. C., Patzke, C., Pak, C., Wernig, M., Sudhof, T. C. 2016; 352 (6286): 672-?

  Abstract

  Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired Ih channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) forming Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.

  View details for DOI 10.1126/science.aaf2669

  View details for PubMedID 26966193

• Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation JOURNAL OF EXPERIMENTAL MEDICINE Patzke, C., Acuna, C., Giam, L. R., Wernig, M., Suedhof, T. C. 2016; 213 (4): 499-515

  Abstract

  Hundreds ofL1CAMgene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditionalL1CAMloss-of-function mutation and produced precisely matching control andL1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion ofL1CAMdramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20-50% and ∼20-30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that theL1CAMmutation selectively decreased activity-dependent Na(+)-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations ofL1CAMmutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.

  View details for Web of Science ID 000373394100005

  View details for PubMedCentralID PMC4821644

• Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation. journal of experimental medicine Patzke, C., Acuna, C., Giam, L. R., Wernig, M., Südhof, T. C. 2016; 213 (4): 499-515

  Abstract

  Hundreds ofL1CAMgene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditionalL1CAMloss-of-function mutation and produced precisely matching control andL1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion ofL1CAMdramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20-50% and ∼20-30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that theL1CAMmutation selectively decreased activity-dependent Na(+)-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations ofL1CAMmutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.

  View details for DOI 10.1084/jem.20150951

  View details for PubMedID 27001749

  View details for PubMedCentralID PMC4821644

• Cellular Taxonomy of the Mouse Striatum as Revealed by Single Cell RNA Sequencing Stanley, G., Gokce, O., Treutlein, B., Sudhof, T. C., Quake, S. CELL PRESS. 2016: 321A

  View details for DOI 10.1016/j.bpj.2015.11.1723

  View details for Web of Science ID 000375142200043

• Pathogenic mechanism of an autism-associated neuroligin mutation involves altered AMPA-receptor trafficking. Molecular psychiatry Chanda, S., Aoto, J., Lee, S., Wernig, M., Südhof, T. C. 2016; 21 (2): 169-177

  Abstract

  Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Although the general synaptic role of neuroligins is undisputed, their specific functions at a synapse remain unclear, even controversial. Moreover, many neuroligin gene mutations were associated with autism, but the pathophysiological relevance of these mutations is often unknown, and their mechanisms of action uninvestigated. Here, we examine the synaptic effects of an autism-associated neuroligin-4 substitution (called R704C), which mutates a cytoplasmic arginine residue that is conserved in all neuroligins. We show that the R704C mutation, when introduced into neuroligin-3, enhances the interaction between neuroligin-3 and AMPA receptors, increases AMPA-receptor internalization and decreases postsynaptic AMPA-receptor levels. When introduced into neuroligin-4, conversely, the R704C mutation unexpectedly elevated AMPA-receptor-mediated synaptic responses. These results suggest a general functional link between neuroligins and AMPA receptors, indicate that both neuroligin-3 and -4 act at excitatory synapses but perform surprisingly distinct functions, and demonstrate that the R704C mutation significantly impairs the normal function of neuroligin-4, thereby validating its pathogenicity.Molecular Psychiatry advance online publication, 17 March 2015; doi:10.1038/mp.2015.20.

  View details for DOI 10.1038/mp.2015.20

  View details for PubMedID 25778475

• The conditional KO approach: Cre/Lox technology in human neurons. Rare diseases (Austin, Tex.) Patzke, C., Südhof, T. C. 2016; 4 (1)

  Abstract

  The use of human pluripotent stem cells to model human diseases has become a new standard in biomedical sciences. To this end, patient-derived somatic cells are studied in vitro to mimic human pathological conditions. Here, we describe an alternative experimental strategy, the 'conditional KO approach', which allows engineering disease-relevant mutations in pluripotent stem cells from healthy donors. In combination with the Cre/Lox technology, this strategy enables us to study the molecular causes of human diseases independent of the genetic background or of genetic alterations induced by clonal selection. As a proof-of-principle, we generated pluripotent stem cells with conditional loss-of-function mutations in the human STXBP1 gene that encodes Munc18-1. Using neurons derived from these cells, we show that heterozygous disruption of STXBP1 produces a specific and selective impairment in synaptic transmission that may account for the severe neurological disease caused by such mutations in human patients.

  View details for DOI 10.1080/21675511.2015.1131884

  View details for PubMedID 27141410

• The conditional KO approach: Cre/Lox technology in human RARE DISEASES Patzke, C., Suedhof, T. C. 2016; 4 (1)

  View details for DOI 10.1080/21675511.2015.1131884

  View details for Web of Science ID 000399083900003

• REPRODUCIBILITY Experimental mismatch in neural circuits NATURE Suedhof, T. C. 2015; 528 (7582): 338–39

  View details for PubMedID 26649825

• Harness the power of endogenous neural stem cells by biomaterials to treat spinal cord injury SCIENCE CHINA-LIFE SCIENCES De Filippis, L., Suedhof, T. C., Pang, Z. P. 2015; 58 (11): 1167–68

  View details for DOI 10.1007/s11427-015-4943-z

  View details for Web of Science ID 000365130000018

  View details for PubMedID 26481248

• Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles. PLoS biology Bacaj, T., Wu, D., Burré, J., Malenka, R. C., Liu, X., Südhof, T. C. 2015; 13 (10)

  Abstract

  In forebrain neurons, Ca2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca2+-independent, mutations in Ca2+-binding sequences of synaptotagmin-1 or synaptotagmin-7-which are contained in flexible top-loop sequences of their C2 domains-blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca2+ triggering of different phases of release.

  View details for DOI 10.1371/journal.pbio.1002267

  View details for PubMedID 26437117

• Evolutionary conservation of complexins: from choanoflagellates to mice EMBO REPORTS Yang, X., Pei, J., Kaeser-Woo, Y., Bacaj, T., Grishin, N. V., Suedhof, T. C. 2015; 16 (10): 1308–17

  Abstract

  Complexins are synaptic SNARE complex-binding proteins that cooperate with synaptotagmins in activating Ca(2+)-stimulated, synaptotagmin-dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin-independent synaptic vesicle exocytosis. Here, we show that complexin sequences are conserved in some non-metazoan unicellular organisms and in all metazoans, suggesting that complexins are a universal feature of metazoans that predate metazoan evolution. We show that complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse complexins in activating Ca(2+)-triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of complexins is likely conserved throughout metazoan evolution.

  View details for PubMedID 26338476

• Synaptotagmin-1 and-7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles PLOS BIOLOGY Bacaj, T., Wu, D., Burre, J., Malenka, R. C., Liu, X., Suedhof, T. C. 2015; 13 (10)

  Abstract

  In forebrain neurons, Ca2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca2+-independent, mutations in Ca2+-binding sequences of synaptotagmin-1 or synaptotagmin-7-which are contained in flexible top-loop sequences of their C2 domains-blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca2+ triggering of different phases of release.

  View details for DOI 10.1371/journal.pbio.1002267

  View details for Web of Science ID 000364457500003

• RIM-BPs Mediate Tight Coupling of Action Potentials to Ca(2+)-Triggered Neurotransmitter Release. Neuron Acuna, C., Liu, X., Gonzalez, A., Südhof, T. C. 2015; 87 (6): 1234-1247

  Abstract

  Ultrafast neurotransmitter release requires tight colocalization of voltage-gated Ca(2+) channels with primed, release-ready synaptic vesicles at the presynaptic active zone. RIM-binding proteins (RIM-BPs) are multidomain active zone proteins that bind to RIMs and to Ca(2+) channels. In Drosophila, deletion of RIM-BPs dramatically reduces neurotransmitter release, but little is known about RIM-BP function in mammalian synapses. Here, we generated double conditional knockout mice for RIM-BP1 and RIM-BP2, and analyzed RIM-BP-deficient synapses in cultured hippocampal neurons and the calyx of Held. Surprisingly, we find that in murine synapses, RIM-BPs are not essential for neurotransmitter release as such, but are selectively required for high-fidelity coupling of action potential-induced Ca(2+) influx to Ca(2+)-stimulated synaptic vesicle exocytosis. Deletion of RIM-BPs decelerated action-potential-triggered neurotransmitter release and rendered it unreliable, thereby impairing the fidelity of synaptic transmission. Thus, RIM-BPs ensure optimal organization of the machinery for fast release in mammalian synapses without being a central component of the machinery itself.

  View details for DOI 10.1016/j.neuron.2015.08.027

  View details for PubMedID 26402606

• Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1. Cell stem cell Pak, C., Danko, T., Zhang, Y., Aoto, J., Anderson, G., Maxeiner, S., Yi, F., Wernig, M., Südhof, T. C. 2015; 17 (3): 316-328

  Abstract

  Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

  View details for DOI 10.1016/j.stem.2015.07.017

  View details for PubMedID 26279266

  View details for PubMedCentralID PMC4560990

• Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis NATURE Zhou, Q., Lai, Y., Bacaj, T., Zhao, M., Lyubimov, A. Y., Uervirojnangkoorn, M., Zeldin, O. B., Brewster, A. S., Sauter, N. K., Cohen, A. E., Soltis, S. M., Alonso-Mori, R., Chollet, M., Lemke, H. T., Pfuetzner, R. A., Choi, U. B., Weis, W. I., Diao, J., Suedhof, T. C., Brunger, A. T. 2015; 525 (7567): 62-?

  Abstract

  Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

  View details for DOI 10.1038/nature14975

  View details for Web of Science ID 000360594100025

• Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis. Nature Zhou, Q., Lai, Y., Bacaj, T., Zhao, M., Lyubimov, A. Y., Uervirojnangkoorn, M., Zeldin, O. B., Brewster, A. S., Sauter, N. K., Cohen, A. E., Soltis, S. M., Alonso-Mori, R., Chollet, M., Lemke, H. T., Pfuetzner, R. A., Choi, U. B., Weis, W. I., Diao, J., Südhof, T. C., Brunger, A. T. 2015; 525 (7567): 62-67

  Abstract

  Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

  View details for DOI 10.1038/nature14975

  View details for PubMedID 26280336

• Propagation of prions causing synucleinopathies in cultured cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Woerman, A. L., Stoehr, J., Aoyagi, A., Rampersaud, R., Krejciova, Z., Watts, J. C., Ohyama, T., Patel, S., Widjaja, K., Oehler, A., Sanders, D. W., Diamond, M. I., Seeley, W. W., Middleton, L. T., Gentleman, S. M., Mordes, D. A., Suedhof, T. C., Giles, K., Prusiner, S. B. 2015; 112 (35): E4949-E4958

  Abstract

  Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

  View details for DOI 10.1073/pnas.1513426112

  View details for Web of Science ID 000360383200018

  View details for PubMedCentralID PMC4568231

• Structural Basis of Latrophilin-FLRT-UNC5 Interaction in Cell Adhesion. Structure Lu, Y. C., Nazarko, O. V., Sando, R., Salzman, G. S., Südhof, T. C., Araç, D. 2015; 23 (9): 1678-1691

  Abstract

  Fibronectin leucine-rich repeat transmembrane proteins (FLRTs) are cell-adhesion molecules with emerging functions in cortical development and synapse formation. Their extracellular regions interact with latrophilins (LPHNs) to mediate synapse development, and with Uncoordinated-5 (UNC5)/netrin receptors to control the migration of neurons in the developing cortex. Here, we present the crystal structures of FLRT3 in isolation and in complex with LPHN3. The LPHN3/FLRT3 structure reveals that LPHN3 binds to FLRT3 at a site distinct from UNC5. Structure-based mutations specifically disrupt LPHN3/FLRT3 binding, but do not disturb their interactions with other proteins or their cell-membrane localization. Thus, they can be used as molecular tools to dissect the functions of FLRTs and LPHNs in vivo. Our results suggest that UNC5 and LPHN3 can simultaneously bind to FLRT3, forming a trimeric complex, and that FLRT3 may form transsynaptic complexes with both LPHN3 and UNC5. These findings provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  View details for DOI 10.1016/j.str.2015.06.024

  View details for PubMedID 26235030

• Propagation of prions causing synucleinopathies in cultured cells. Proceedings of the National Academy of Sciences of the United States of America Woerman, A. L., Stöhr, J., Aoyagi, A., Rampersaud, R., Krejciova, Z., Watts, J. C., Ohyama, T., Patel, S., Widjaja, K., Oehler, A., Sanders, D. W., Diamond, M. I., Seeley, W. W., Middleton, L. T., Gentleman, S. M., Mordes, D. A., Südhof, T. C., Giles, K., Prusiner, S. B. 2015; 112 (35): E4949-58

  Abstract

  Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

  View details for DOI 10.1073/pnas.1513426112

  View details for PubMedID 26286986

• Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1 CELL STEM CELL Pak, C., Danko, T., Zhang, Y., Aoto, J., Anderson, G., Maxeiner, S., Yi, F., Wernig, M., Suedhof, T. C. 2015; 17 (3): 316-328

  Abstract

  Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

  View details for DOI 10.1016/j.stem.2015.07.017

  View details for Web of Science ID 000364011000010

  View details for PubMedCentralID PMC4560990

• Analysis of conditional heterozygous STXBP1 mutations in human neurons JOURNAL OF CLINICAL INVESTIGATION Patzke, C., Han, Y., Covy, J., Yi, F., Maxeiner, S., Wernig, M., Suedhof, T. C. 2015; 125 (9): 3560-3571

  Abstract

  Heterozygous mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes Munc18-1, a core component of the presynaptic membrane-fusion machinery, cause infantile early epileptic encephalopathy (Ohtahara syndrome), but it is unclear how a partial loss of Munc18-1 produces this severe clinical presentation. Here, we generated human ES cells designed to conditionally express heterozygous and homozygous STXBP1 loss-of-function mutations and studied isogenic WT and STXBP1-mutant human neurons derived from these conditionally mutant ES cells. We demonstrated that heterozygous STXBP1 mutations lower the levels of Munc18-1 protein and its binding partner, the t-SNARE-protein Syntaxin-1, by approximately 30% and decrease spontaneous and evoked neurotransmitter release by nearly 50%. Thus, our results confirm that using engineered human embryonic stem (ES) cells is a viable approach to studying disease-associated mutations in human neurons on a controlled genetic background, demonstrate that partial STXBP1 loss of function robustly impairs neurotransmitter release in human neurons, and suggest that heterozygous STXBP1 mutations cause early epileptic encephalopathy specifically through a presynaptic impairment.

  View details for DOI 10.1172/JCI78612

  View details for Web of Science ID 000362303600030

  View details for PubMedCentralID PMC4588304

• Structural Basis of Latrophilin-FLRT-UNC5 Interaction in Cell Adhesion STRUCTURE Lu, Y. C., Nazarko, O. V., Sando, R., Salzman, G. S., Suedhof, T. C., Arac, D. 2015; 23 (9): 1678-1691

  Abstract

  Fibronectin leucine-rich repeat transmembrane proteins (FLRTs) are cell-adhesion molecules with emerging functions in cortical development and synapse formation. Their extracellular regions interact with latrophilins (LPHNs) to mediate synapse development, and with Uncoordinated-5 (UNC5)/netrin receptors to control the migration of neurons in the developing cortex. Here, we present the crystal structures of FLRT3 in isolation and in complex with LPHN3. The LPHN3/FLRT3 structure reveals that LPHN3 binds to FLRT3 at a site distinct from UNC5. Structure-based mutations specifically disrupt LPHN3/FLRT3 binding, but do not disturb their interactions with other proteins or their cell-membrane localization. Thus, they can be used as molecular tools to dissect the functions of FLRTs and LPHNs in vivo. Our results suggest that UNC5 and LPHN3 can simultaneously bind to FLRT3, forming a trimeric complex, and that FLRT3 may form transsynaptic complexes with both LPHN3 and UNC5. These findings provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  View details for DOI 10.1016/j.str.2015.06.024

  View details for Web of Science ID 000361113000013

• Neuroligins Sculpt Cerebellar Purkinje-Cell Circuits by Differential Control of Distinct Classes of Synapses. Neuron Zhang, B., Chen, L. Y., Liu, X., Maxeiner, S., Lee, S., Gokce, O., Südhof, T. C. 2015; 87 (4): 781-796

  Abstract

  Neuroligins are postsynaptic cell-adhesion molecules that bind presynaptic neurexins and are genetically linked to autism. Neuroligins are proposed to organize synaptogenesis and/or synaptic transmission, but no systematic analysis of neuroligins in a defined circuit is available. Here, we show that conditional deletion of all neuroligins in cerebellar Purkinje cells caused loss of distal climbing-fiber synapses and weakened climbing-fiber but not parallel-fiber synapses, consistent with alternative use of neuroligins and cerebellins as neurexin ligands for the excitatory climbing-fiber versus parallel-fiber synapses. Moreover, deletion of neuroligins increased the size of inhibitory basket/stellate-cell synapses but simultaneously severely impaired their function. Multiple neuroligin isoforms differentially contributed to climbing-fiber and basket/stellate-cell synapse functions, such that inhibitory synapse-specific neuroligin-2 was unexpectedly essential for maintaining normal climbing-fiber synapse numbers. Using systematic analyses of all neuroligins in a defined neural circuit, our data thus show that neuroligins differentially contribute to various Purkinje-cell synapses in the cerebellum in vivo.

  View details for DOI 10.1016/j.neuron.2015.07.020

  View details for PubMedID 26291161

• Ubiquitin-Synaptobrevin Fusion Protein Causes Degeneration of Presynaptic Motor Terminals in Mice JOURNAL OF NEUROSCIENCE Liu, Y., Li, H., Sugiura, Y., Han, W., Gallardo, G., Khvotchev, M., Zhang, Y., Kavalali, E. T., Suedhof, T. C., Lin, W. 2015; 35 (33): 11514–31

  Abstract

  Protein aggregates containing ubiquitin (Ub) are commonly observed in neurodegenerative disorders, implicating the involvement of the ubiquitin proteasome system (UPS) in their pathogenesis. Here, we aimed to generate a mouse model for monitoring UPS function using a green fluorescent protein (GFP)-based substrate that carries a "noncleavable" N-terminal ubiquitin moiety (Ub(G76V)). We engineered transgenic mice expressing a fusion protein, consisting of the following: (1) Ub(G76V), GFP, and a synaptic vesicle protein synaptobrevin-2 (Ub(G76V)-GFP-Syb2); (2) GFP-Syb2; or (3) Ub(G76V)-GFP-Syntaxin1, all under the control of a neuron-specific Thy-1 promoter. As expected, Ub(G76V)-GFP-Syb2, GFP-Syb2, and Ub(G76V)-GFP-Sytaxin1 were highly expressed in neurons, such as motoneurons and motor nerve terminals of the neuromuscular junction (NMJ). Surprisingly, Ub(G76V)-GFP-Syb2 mice developed progressive adult-onset degeneration of motor nerve terminals, whereas GFP-Syb2 and Ub(G76V)-GFP-Syntaxin1 mice were normal. The degeneration of nerve terminals in Ub(G76V)-GFP-Syb2 mice was preceded by a progressive impairment of synaptic transmission at the NMJs. Biochemical analyses demonstrated that Ub(G76V)-GFP-Syb2 interacted with SNAP-25 and Syntaxin1, the SNARE partners of synaptobrevin. Ultrastructural analyses revealed a marked reduction in synaptic vesicle density, accompanying an accumulation of tubulovesicular structures at presynaptic nerve terminals. These morphological defects were largely restricted to motor nerve terminals, as the ultrastructure of motoneuron somata appeared to be normal at the stages when synaptic nerve terminals degenerated. Furthermore, synaptic vesicle endocytosis and membrane trafficking were impaired in Ub(G76V)-GFP-Syb2 mice. These findings indicate that Ub(G76V)-GFP-Syb2 may compete with endogenous synaptobrevin, acting as a gain-of-function mutation that impedes SNARE function, resulting in the depletion of synaptic vesicles and degeneration of the nerve terminals.Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.

  View details for PubMedID 26290230

• Neuroligins Sculpt Cerebellar Purkinje-Cell Circuits by Differential Control of Distinct Classes of Synapses NEURON Zhang, B., Chen, L. Y., Liu, X., Maxeiner, S., Lee, S., Gokce, O., Suedhof, T. C. 2015; 87 (4): 781-796

  Abstract

  Neuroligins are postsynaptic cell-adhesion molecules that bind presynaptic neurexins and are genetically linked to autism. Neuroligins are proposed to organize synaptogenesis and/or synaptic transmission, but no systematic analysis of neuroligins in a defined circuit is available. Here, we show that conditional deletion of all neuroligins in cerebellar Purkinje cells caused loss of distal climbing-fiber synapses and weakened climbing-fiber but not parallel-fiber synapses, consistent with alternative use of neuroligins and cerebellins as neurexin ligands for the excitatory climbing-fiber versus parallel-fiber synapses. Moreover, deletion of neuroligins increased the size of inhibitory basket/stellate-cell synapses but simultaneously severely impaired their function. Multiple neuroligin isoforms differentially contributed to climbing-fiber and basket/stellate-cell synapse functions, such that inhibitory synapse-specific neuroligin-2 was unexpectedly essential for maintaining normal climbing-fiber synapse numbers. Using systematic analyses of all neuroligins in a defined neural circuit, our data thus show that neuroligins differentially contribute to various Purkinje-cell synapses in the cerebellum in vivo.

  View details for DOI 10.1016/j.neuron.2015.07.020

  View details for Web of Science ID 000361145600011

  View details for PubMedCentralID PMC4545494

• Synaptotagmin-7 phosphorylation mediates GLP-1-dependent potentiation of insulin secretion from beta-cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, B., Wei, S., Petersen, N., Ali, Y., Wang, X., Bacaj, T., Rorsman, P., Hong, W., Suedhof, T. C., Han, W. 2015; 112 (32): 9996–10001

  Abstract

  Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca(2+)-triggered exocytosis by direct phosphorylation of a synaptotagmin.

  View details for PubMedID 26216970

• Synaptotagmin-7 Is Essential for Ca2+-Triggered Delayed Asynchronous Release But Not for Ca2+-Dependent Vesicle Priming in Retinal Ribbon Synapses JOURNAL OF NEUROSCIENCE Luo, F., Bacaj, T., Suedhof, T. C. 2015; 35 (31): 11024–33

  Abstract

  Most synapses release neurotransmitters in two phases: (1) a fast synchronous phase lasting a few milliseconds; and (2) a delayed "asynchronous" phase lasting hundreds of milliseconds. Ca(2+) triggers fast synchronous neurotransmitter release by binding to synaptotagmin-1, synaptotagmin-2, or synaptotagmin-9, but how Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Recent results suggested that consistent with the Ca(2+)-sensor function of synaptotagmin-7 in neuroendocrine exocytosis, synaptotagmin-7 also functions as a Ca(2+) sensor for synaptic vesicle exocytosis but operates during delayed asynchronous release. Puzzlingly, a subsequent study postulated that synaptotagmin-7 is not a Ca(2+) sensor for release but mediates Ca(2+)-dependent vesicle repriming after intense stimulation. To address these issues, we here analyzed synaptic transmission at rod bipolar neuron-AII amacrine cell synapses in acute mouse retina slices as a model system. Using paired recordings, we show that knock-out of synaptotagmin-7 selectively impairs delayed asynchronous release but not fast synchronous release. Delayed asynchronous release was blocked in wild-type synapses by intracellular addition of high concentrations of the slow Ca(2+)-chelator EGTA, but EGTA had no effect in synaptotagmin-7 knock-out neurons because delayed asynchronous release was already impaired. Moreover, direct measurements of vesicle repriming failed to uncover an effect of the synaptotagmin-7 knock-out on vesicle repriming. Our data demonstrate that synaptotagmin-7 is selectively essential for Ca(2+)-dependent delayed asynchronous release in retinal rod bipolar cell synapses, that its function can be blocked by simply introducing a slow Ca(2+) buffer into the cells, and that synaptotagmin-7 is not required for normal vesicle repriming.How Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Synaptotagmin-7 has been implicated recently as Ca(2+) sensor in mediating delayed asynchronous release, or vesicle repriming, in cultured neurons. To test the precise function of synaptotagmin-7 in a physiologically important synapse in situ, we have used pair recordings to study the synaptic transmission between retinal rod bipolar cells and AII amacrine cells. Our data demonstrate that the knock-out of synaptotagmin-7 selectively impaired delayed asynchronous release but not synchronous release. In contrast, the readily releasable vesicles after depletion recover normally in knock-out mice. Therefore, our findings extend our knowledge of synaptotagmins as Ca(2+) sensors in vesicle fusion and support the idea that synapses are governed universally by different synaptotagmin Ca(2+) sensors mediating distinct release.

  View details for PubMedID 26245964

• ß-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling. Cell Anderson, G. R., Aoto, J., Tabuchi, K., Földy, C., Covy, J., Yee, A. X., Wu, D., Lee, S., Chen, L., Malenka, R. C., Südhof, T. C. 2015; 162 (3): 593-606

  Abstract

  α- and β-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although β-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of β-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The β-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of β-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of β-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic β-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for β-neurexins in the endocannabinoid-dependent regulation of neural circuits.

  View details for DOI 10.1016/j.cell.2015.06.056

  View details for PubMedID 26213384

• beta-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling CELL Anderson, G. R., Aoto, J., Tabuchi, K., Foeldy, C., Covy, J., Yee, A. X., Wu, D., Lee, S., Chen, L., Malenka, R. C., Suedhof, T. C. 2015; 162 (3): 593-606

  Abstract

  α- and β-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although β-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of β-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The β-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of β-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of β-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic β-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for β-neurexins in the endocannabinoid-dependent regulation of neural circuits.

  View details for DOI 10.1016/j.cell.2015.06.056

  View details for Web of Science ID 000358801800018

• Single-Cell mRNA Profiling Reveals Cell-Type-Specific Expression of Neurexin Isoforms. Neuron Fuccillo, M. V., Földy, C., Gökce, Ö., Rothwell, P. E., Sun, G. L., Malenka, R. C., Südhof, T. C. 2015; 87 (2): 326-340

  Abstract

  Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell-type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell-type-specific expression patterns of multiple neurexins at the single-cell level and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity.

  View details for DOI 10.1016/j.neuron.2015.06.028

  View details for PubMedID 26182417

• Distinct circuit-dependent functions of presynaptic neurexin-3 at GABAergic and glutamatergic synapses. Nature neuroscience Aoto, J., Földy, C., Ilcus, S. M., Tabuchi, K., Südhof, T. C. 2015; 18 (7): 997-1007

  Abstract

  α- and β-neurexins are presynaptic cell-adhesion molecules whose general importance for synaptic transmission is well documented. The specific functions of neurexins, however, remain largely unknown because no conditional neurexin knockouts are available and targeting all α- and β-neurexins produced by a particular gene is challenging. Using newly generated constitutive and conditional knockout mice that target all neurexin-3α and neurexin-3β isoforms, we found that neurexin-3 was differentially required for distinct synaptic functions in different brain regions. Specifically, we found that, in cultured neurons and acute slices of the hippocampus, extracellular sequences of presynaptic neurexin-3 mediated trans-synaptic regulation of postsynaptic AMPA receptors. In cultured neurons and acute slices of the olfactory bulb, however, intracellular sequences of presynaptic neurexin-3 were selectively required for GABA release. Thus, our data indicate that neurexin-3 performs distinct essential pre- or postsynaptic functions in different brain regions by distinct mechanisms.

  View details for DOI 10.1038/nn.4037

  View details for PubMedID 26030848

• Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution. Nature structural & molecular biology Brewer, K. D., Bacaj, T., Cavalli, A., Camilloni, C., Swarbrick, J. D., Liu, J., Zhou, A., Zhou, P., Barlow, N., Xu, J., Seven, A. B., Prinslow, E. A., Voleti, R., Häussinger, D., Bonvin, A. M., Tomchick, D. R., Vendruscolo, M., Graham, B., Südhof, T. C., Rizo, J. 2015; 22 (7): 555-564

  Abstract

  Rapid neurotransmitter release depends on the Ca(2+) sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.

  View details for DOI 10.1038/nsmb.3035

  View details for PubMedID 26030874

• Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution NATURE STRUCTURAL & MOLECULAR BIOLOGY Brewer, K. D., Bacaj, T., Cavalli, A., Camilloni, C., Swarbrick, J. D., Liu, J., Zhou, A., Zhou, P., Barlow, N., Xu, J., Seven, A. B., Prinslow, E. A., Voleti, R., Haeussinger, D., Bonvin, A. M., Tomchick, D. R., Vendruscolo, M., Graham, B., Suedhof, T. C., Rizo, J. 2015; 22 (7): 555-?

  Abstract

  Rapid neurotransmitter release depends on the Ca(2+) sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.

  View details for DOI 10.1038/nsmb.3035

  View details for Web of Science ID 000357614900007

  View details for PubMedID 26030874

  View details for PubMedCentralID PMC4496268

• Distinct circuit-dependent functions of presynaptic neurexin-3 at GABAergic and glutamatergic synapses NATURE NEUROSCIENCE Aoto, J., Foeldy, C., Ilcus, S. M., Tabuchi, K., Suedhof, T. C. 2015; 18 (7): 997-?

  Abstract

  α- and β-neurexins are presynaptic cell-adhesion molecules whose general importance for synaptic transmission is well documented. The specific functions of neurexins, however, remain largely unknown because no conditional neurexin knockouts are available and targeting all α- and β-neurexins produced by a particular gene is challenging. Using newly generated constitutive and conditional knockout mice that target all neurexin-3α and neurexin-3β isoforms, we found that neurexin-3 was differentially required for distinct synaptic functions in different brain regions. Specifically, we found that, in cultured neurons and acute slices of the hippocampus, extracellular sequences of presynaptic neurexin-3 mediated trans-synaptic regulation of postsynaptic AMPA receptors. In cultured neurons and acute slices of the olfactory bulb, however, intracellular sequences of presynaptic neurexin-3 were selectively required for GABA release. Thus, our data indicate that neurexin-3 performs distinct essential pre- or postsynaptic functions in different brain regions by distinct mechanisms.

  View details for DOI 10.1038/nn.4037

  View details for Web of Science ID 000356866200015

  View details for PubMedCentralID PMC4482778

• Synaptic Function of Rab11Fip5: Selective Requirement for Hippocampal Long-Term Depression JOURNAL OF NEUROSCIENCE Bacaj, T., Ahmad, M., Jurado, S., Malenka, R. C., Suedhof, T. C. 2015; 35 (19): 7460-7474

  Abstract

  Postsynaptic AMPA-type glutamate receptors (AMPARs) are among the major determinants of synaptic strength and can be trafficked into and out of synapses. Neuronal activity regulates AMPAR trafficking during synaptic plasticity to induce long-term changes in synaptic strength, including long-term potentiation (LTP) and long-term depression (LTD). Rab family GTPases regulate most membrane trafficking in eukaryotic cells; particularly, Rab11 and its effectors are implicated in mediating postsynaptic AMPAR insertion during LTP. To explore the synaptic function of Rab11Fip5, a neuronal Rab11 effector and a candidate autism-spectrum disorder gene, we performed shRNA-mediated knock-down and genetic knock-out (KO) studies. Surprisingly, we observed robust shRNA-induced synaptic phenotypes that were rescued by a Rab11Fip5 cDNA but that were nevertheless not observed in conditional KO neurons. Both in cultured neurons and acute slices, KO of Rab11Fip5 had no significant effect on basic parameters of synaptic transmission, indicating that Rab11Fip5 is not required for fundamental synaptic operations, such as neurotransmitter release or postsynaptic AMPAR insertion. KO of Rab11Fip5 did, however, abolish hippocampal LTD as measured both in acute slices or using a chemical LTD protocol in cultured neurons but did not affect hippocampal LTP. The Rab11Fip5 KO mice performed normally in several behavioral tasks, including fear conditioning, but showed enhanced contextual fear extinction. These are the first findings to suggest a requirement for Rab11Fip5, and presumably Rab11, during LTD.

  View details for DOI 10.1523/JNEUROSCI.1581-14.2015

  View details for PubMedID 25972173

• Retinoic Acid and LTP Recruit Postsynaptic AMPA Receptors Using Distinct SNARE-Dependent Mechanisms NEURON Arendt, K. L., Zhang, Y., Jurado, S., Malenka, R. C., Suedhof, T. C., Chen, L. 2015; 86 (2): 442-456

  Abstract

  Retinoic acid (RA)-dependent homeostatic plasticity and NMDA receptor-dependent long-term potentiation (LTP), a form of Hebbian plasticity, both enhance synaptic strength by increasing the abundance of postsynaptic AMPA receptors (AMPARs). However, it is unclear whether the molecular mechanisms mediating AMPAR trafficking during homeostatic and Hebbian plasticity differ, and it is unknown how RA signaling impacts Hebbian plasticity. Here, we show that RA increases postsynaptic AMPAR abundance using an activity-dependent mechanism that requires a unique SNARE (soluble NSF-attachment protein receptor)-dependent fusion machinery different from that mediating LTP. Specifically, RA-induced AMPAR trafficking did not involve complexin, which activates SNARE complexes containing syntaxin-1 or -3, but not complexes containing syntaxin-4, whereas LTP required complexin. Moreover, RA-induced AMPAR trafficking utilized the Q-SNARE syntaxin-4, whereas LTP utilized syntaxin-3; both additionally required the Q-SNARE SNAP-47 and the R-SNARE synatobrevin-2. Finally, acute RA treatment blocked subsequent LTP expression, probably by increasing AMPAR trafficking. Thus, RA-induced homeostatic plasticity involves a novel, activity-dependent postsynaptic AMPAR-trafficking pathway mediated by a unique SNARE-dependent fusion machinery.

  View details for DOI 10.1016/j.neuron.2015.03.009

  View details for PubMedID 25843403

• Structures of C1q-like Proteins Reveal Unique Features among the C1q/TNF Superfamily STRUCTURE Ressl, S., Vu, B. K., Vivona, S., Martinelli, D. C., Suedhof, T. C., Brunger, A. T. 2015; 23 (4): 688-699

  Abstract

  C1q-like (C1QL) -1, -2, and -3 proteins are encoded by homologous genes that are highly expressed in brain. C1QLs bind to brain-specific angiogenesis inhibitor 3 (BAI3), an adhesion-type G-protein coupled receptor that may regulate dendritic morphology by organizing actin filaments. To begin to understand the function of C1QLs, we determined high-resolution crystal structures of the globular C1q-domains of C1QL1, C1QL2, and C1QL3. Each structure is a trimer, with each protomer forming a jelly-roll fold consisting of 10 β strands. Moreover, C1QL trimers may assemble into higher-order oligomers similar to adiponectin and contain four Ca(2+)-binding sites along the trimeric symmetry axis, as well as additional surface Ca(2+)-binding sites. Mutation of Ca(2+)-coordinating residues along the trimeric symmetry axis lowered the Ca(2+)-binding affinity and protein stability. Our results reveal unique structural features of C1QLs among C1q/TNF superfamily proteins that may be associated with their specific brain functions.

  View details for DOI 10.1016/j.str.2015.01.019

  View details for PubMedID 25752542

• Definition of a Molecular Pathway Mediating alpha-Synuclein Neurotoxicity JOURNAL OF NEUROSCIENCE Burre, J., Sharma, M., Suedhof, T. C. 2015; 35 (13): 5221-5232

  Abstract

  α-Synuclein physiologically chaperones SNARE-complex assembly at the synapse but pathologically misfolds into neurotoxic aggregates that are characteristic for neurodegenerative disorders, such as Parkinson's disease, and that may spread from one neuron to the next throughout the brain during Parkinson's disease pathogenesis. In normal nerve terminals, α-synuclein is present in an equilibrium between a cytosolic form that is natively unfolded and monomeric and a membrane-bound form that is composed of an α-helical multimeric species that chaperones SNARE-complex assembly. Although the neurotoxicity of α-synuclein is well established, the relationship between the native conformations of α-synuclein and its pathological aggregation remain incompletely understood; most importantly, it is unclear whether α-synuclein aggregation originates from its monomeric cytosolic or oligomeric membrane-bound form. Here, we address this question by introducing into α-synuclein point mutations that block membrane binding and by then assessing the effect of blocking membrane binding on α-synuclein aggregation and neurotoxicity. We show that membrane binding inhibits α-synuclein aggregation; conversely, blocking membrane binding enhances α-synuclein aggregation. Stereotactic viral expression of wild-type and mutant α-synuclein in the substantia nigra of mice demonstrated that blocking α-synuclein membrane binding significantly enhanced its neurotoxicity in vivo. Our data delineate a folding pathway for α-synuclein that ranges from a physiological multimeric, α-helical, and membrane-bound species that acts as a SNARE-complex chaperone over a monomeric, natively unfolded form to an amyloid-like aggregate that is neurotoxic in vivo.

  View details for DOI 10.1523/JNEUROSCI.4650-14.2015

  View details for Web of Science ID 000352208200014

  View details for PubMedCentralID PMC4380997

• Definition of a molecular pathway mediating a-synuclein neurotoxicity. journal of neuroscience Burré, J., Sharma, M., Südhof, T. C. 2015; 35 (13): 5221-5232

  Abstract

  α-Synuclein physiologically chaperones SNARE-complex assembly at the synapse but pathologically misfolds into neurotoxic aggregates that are characteristic for neurodegenerative disorders, such as Parkinson's disease, and that may spread from one neuron to the next throughout the brain during Parkinson's disease pathogenesis. In normal nerve terminals, α-synuclein is present in an equilibrium between a cytosolic form that is natively unfolded and monomeric and a membrane-bound form that is composed of an α-helical multimeric species that chaperones SNARE-complex assembly. Although the neurotoxicity of α-synuclein is well established, the relationship between the native conformations of α-synuclein and its pathological aggregation remain incompletely understood; most importantly, it is unclear whether α-synuclein aggregation originates from its monomeric cytosolic or oligomeric membrane-bound form. Here, we address this question by introducing into α-synuclein point mutations that block membrane binding and by then assessing the effect of blocking membrane binding on α-synuclein aggregation and neurotoxicity. We show that membrane binding inhibits α-synuclein aggregation; conversely, blocking membrane binding enhances α-synuclein aggregation. Stereotactic viral expression of wild-type and mutant α-synuclein in the substantia nigra of mice demonstrated that blocking α-synuclein membrane binding significantly enhanced its neurotoxicity in vivo. Our data delineate a folding pathway for α-synuclein that ranges from a physiological multimeric, α-helical, and membrane-bound species that acts as a SNARE-complex chaperone over a monomeric, natively unfolded form to an amyloid-like aggregate that is neurotoxic in vivo.

  View details for DOI 10.1523/JNEUROSCI.4650-14.2015

  View details for PubMedID 25834048

• Building bridges between neuroscientific evidence and policy. The Lancet. Neurology Quaglio, G., McGuinness, M., Rubig, P., Nutt, D., Karapiperis, T., Pandolfo, M., Sudhof, T. C. 2015; 14 (3): 242–45

  View details for PubMedID 25728434

• Building bridges between neuroscientific evidence and policy LANCET NEUROLOGY Quaglio, G., McGuinness, M., Ruebig, P., Nutt, D., Karapiperis, T., Pandolfo, M., Suedhof, T. 2015; 14 (3): 243-245

  View details for DOI 10.1016/S1474-4422(15)70014-1

  View details for Web of Science ID 000349594000007

• La machinerie moleculaire de secretion des neurotransmetteurs. Biologie aujourd'hui Sudhof, T. C. 2015; 209 (1): 3–33

  View details for DOI 10.1051/jbio/2015012

  View details for PubMedID 26115711

• RIM1 and RIM2 redundantly determine Ca2+ channel density and readily releasable pool size at a large hindbrain synapse JOURNAL OF NEUROPHYSIOLOGY Han, Y., Babai, N., Kaeser, P., Suedhof, T. C., Schneggenburger, R. 2015; 113 (1): 255–63

  Abstract

  The localization and density of voltage-gated Ca(2+) channels at active zones are essential for the amount and kinetics of transmitter release at synapses. RIM proteins are scaffolding proteins at the active zone that bind to several other presynaptic proteins, including voltage-gated Ca(2+) channel α-subunits. The long isoforms of RIM proteins, which contain NH2-terminal Rab3- and Munc13-interacting domains, as well as a central PDZ domain and two COOH-terminal C2 domains, are encoded by two genes, Rim1 and Rim2. Here, we used the ideal accessibility of the large calyx of Held synapse for direct presynaptic electrophysiology to investigate whether the two Rim genes have redundant, or separate, functions in determining the presynaptic Ca(2+) channel density, and the size of a readily releasable vesicle pool (RRP). Quantitative PCR showed that cochlear nucleus neurons, which include calyx of Held generating neurons, express both RIM1 and RIM2. Conditional genetic inactivation of RIM2 at the calyx of Held led to a subtle reduction in presynaptic Ca(2+) current density, whereas deletion of RIM1 was ineffective. The release efficiency of brief presynaptic Ca(2+) "tail" currents and the RRP were unaffected in conditional single RIM1 and RIM2 knockout (KO) mice, whereas both parameters were strongly reduced in RIM1/2 double KO mice. Thus, despite a somewhat more decisive role for RIM2 in determining presynaptic Ca(2+) channel density, RIM1 and RIM2 can overall replace each other's presynaptic functions at a large relay synapse in the hindbrain, the calyx of Held.

  View details for PubMedID 25343783

• The Molecular Machinery of Neurotransmitter Release (Nobel Lecture) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Suedhof, T. C. 2014; 53 (47): 12696–717

  Abstract

  The most important property of synaptic transmission is its speed, which is crucial for the overall workings of the brain. In his Nobel Lecture, T. C. Südhof explains how the synaptic vesicle and the plasma membrane undergo rapid fusion during neurotransmitter release and how this process is spatially organized, such that opening of Ca(2+) -channels allows rapid translation of the entering Ca(2+) signal into a fusion event.

  View details for PubMedID 25339369

• Direct Visualization of Trans-Synaptic Neurexin-Neuroligin Interactions during Synapse Formation JOURNAL OF NEUROSCIENCE Tsetsenis, T., Boucard, A. A., Arac, D., Brunger, A. T., Suedhof, T. C. 2014; 34 (45): 15083-15096

  Abstract

  Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1β and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1β to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1β and neuroligin-1 if and after neurexin-1β bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1β and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1β forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.

  View details for DOI 10.1523/JNEUROSCI.0348-14.2014

  View details for Web of Science ID 000345220300025

  View details for PubMedCentralID PMC4220035

• Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation. journal of neuroscience Tsetsenis, T., Boucard, A. A., Araç, D., Brunger, A. T., Südhof, T. C. 2014; 34 (45): 15083-15096

  Abstract

  Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1β and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1β to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1β and neuroligin-1 if and after neurexin-1β bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1β and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1β forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.

  View details for DOI 10.1523/JNEUROSCI.0348-14.2014

  View details for PubMedID 25378172

  View details for PubMedCentralID PMC4220035

• The Morphological and Molecular Nature of Synaptic Vesicle Priming at Presynaptic Active Zones NEURON Imig, C., Min, S., Krinner, S., Arancillo, M., Rosenmund, C., Suedhof, T. C., Rhee, J., Brose, N., Cooper, B. H. 2014; 84 (2): 416-431

  Abstract

  Synaptic vesicle docking, priming, and fusion at active zones are orchestrated by a complex molecular machinery. We employed hippocampal organotypic slice cultures from mice lacking key presynaptic proteins, cryofixation, and three-dimensional electron tomography to study the mechanism of synaptic vesicle docking in the same experimental setting, with high precision, and in a near-native state. We dissected previously indistinguishable, sequential steps in synaptic vesicle active zone recruitment (tethering) and membrane attachment (docking) and found that vesicle docking requires Munc13/CAPS family priming proteins and all three neuronal SNAREs, but not Synaptotagmin-1 or Complexins. Our data indicate that membrane-attached vesicles comprise the readily releasable pool of fusion-competent vesicles and that synaptic vesicle docking, priming, and trans-SNARE complex assembly are the respective morphological, functional, and molecular manifestations of the same process, which operates downstream of vesicle tethering by active zone components.

  View details for DOI 10.1016/j.neuron.2014.10.009

  View details for Web of Science ID 000344167900020

• The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron Imig, C., Min, S. W., Krinner, S., Arancillo, M., Rosenmund, C., Südhof, T. C., Rhee, J., Brose, N., Cooper, B. H. 2014; 84 (2): 416-31

  Abstract

  Synaptic vesicle docking, priming, and fusion at active zones are orchestrated by a complex molecular machinery. We employed hippocampal organotypic slice cultures from mice lacking key presynaptic proteins, cryofixation, and three-dimensional electron tomography to study the mechanism of synaptic vesicle docking in the same experimental setting, with high precision, and in a near-native state. We dissected previously indistinguishable, sequential steps in synaptic vesicle active zone recruitment (tethering) and membrane attachment (docking) and found that vesicle docking requires Munc13/CAPS family priming proteins and all three neuronal SNAREs, but not Synaptotagmin-1 or Complexins. Our data indicate that membrane-attached vesicles comprise the readily releasable pool of fusion-competent vesicles and that synaptic vesicle docking, priming, and trans-SNARE complex assembly are the respective morphological, functional, and molecular manifestations of the same process, which operates downstream of vesicle tethering by active zone components.

  View details for DOI 10.1016/j.neuron.2014.10.009

  View details for PubMedID 25374362

• alpha-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Burre, J., Sharma, M., Suedhof, T. C. 2014; 111 (40): E4274-E4283

  Abstract

  Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.

  View details for DOI 10.1073/pnas.1416598111

  View details for Web of Science ID 000342633900017

  View details for PubMedCentralID PMC4210039

• a-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proceedings of the National Academy of Sciences of the United States of America Burré, J., Sharma, M., Südhof, T. C. 2014; 111 (40): E4274-83

  Abstract

  Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.

  View details for DOI 10.1073/pnas.1416598111

  View details for PubMedID 25246573

• The Active Zone Protein Family ELKS Supports Ca2+ Influx at Nerve Terminals of Inhibitory Hippocampal Neurons JOURNAL OF NEUROSCIENCE Liu, C., Bickford, L. S., Held, R. G., Nyitrai, H., Suedhof, T. C., Kaeser, P. S. 2014; 34 (37): 12289–303

  Abstract

  In a presynaptic nerve terminal, synaptic vesicle exocytosis is restricted to specialized sites called active zones. At these sites, neurotransmitter release is determined by the number of releasable vesicles and their probability of release. Proteins at the active zone set these parameters by controlling the presynaptic Ca(2+) signal, and through docking and priming of synaptic vesicles. Vertebrate ELKS proteins are enriched at presynaptic active zones, but their functions are not well understood. ELKS proteins are produced by two genes in vertebrates, and each gene contributes ∼50% to total brain ELKS. We generated knock-out mice for ELKS1 and found that its constitutive removal causes lethality. To bypass lethality, and to circumvent redundancy between ELKS1 and ELKS2 in synaptic transmission, we used a conditional genetic approach to remove both genes in cultured hippocampal neurons after synapses are established. Simultaneous removal of ELKS1 and ELKS2 resulted in a 50% decrease of neurotransmitter release at inhibitory synapses, paralleled by a reduction in release probability. Removal of ELKS did not affect synapse numbers or their electron microscopic appearance. Using Ca(2+) imaging, we found that loss of ELKS caused a 30% reduction in single action potential-triggered Ca(2+) influx in inhibitory nerve terminals, consistent with the deficits in synaptic transmission and release probability. Unlike deletion of the active zone proteins RIM, RIM-BP, or bruchpilot, ELKS removal did not lead to a measurable reduction in presynaptic Ca(2+) channel levels. Our results reveal that ELKS is required for normal Ca(2+) influx at nerve terminals of inhibitory hippocampal neurons.

  View details for PubMedID 25209271

• Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem cell reports Chanda, S., Ang, C. E., Davila, J., Pak, C., Mall, M., Lee, Q. Y., Ahlenius, H., Jung, S. W., Südhof, T. C., Wernig, M. 2014; 3 (2): 282-296

  Abstract

  Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

  View details for DOI 10.1016/j.stemcr.2014.05.020

  View details for PubMedID 25254342

  View details for PubMedCentralID PMC4176533

• Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1 STEM CELL REPORTS Chanda, S., Ang, C. E., Davila, J., Pak, C., Mall, M., Lee, Q. Y., Ahlenius, H., Jung, S. W., Suedhof, T. C., Wernig, M. 2014; 3 (2): 282-296

  Abstract

  Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

  View details for DOI 10.1016/j.stemcr.2014.05.020

  View details for Web of Science ID 000340882400008

  View details for PubMedCentralID PMC4176533

• Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors. Cell Rothwell, P. E., Fuccillo, M. V., Maxeiner, S., Hayton, S. J., Gokce, O., Lim, B. K., Fowler, S. C., Malenka, R. C., Südhof, T. C. 2014; 158 (1): 198-212

  Abstract

  In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology. PAPERFLICK:

  View details for DOI 10.1016/j.cell.2014.04.045

  View details for PubMedID 24995986

• Synaptic function of nicastrin in hippocampal neurons PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, S. H., Sharma, M., Suedhof, T. C., Shen, J. 2014; 111 (24): 8973-8978

  Abstract

  Synaptic dysfunction is widely thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Presenilins, the major gene products involved in familial AD, are essential for short- and long-term synaptic plasticity in mature neurons as well as for the survival of cortical neurons during aging. Presenilin and nicastrin are both indispensable components of the γ-secretase complex, but it remains unknown whether presenilin regulates synaptic function in a γ-secretase-dependent or γ-secretase-independent manner and whether nicastrin plays similar roles in central synapses. In the current study, we address these questions using an electrophysiological approach to analyze nicastrin conditional knockout (cKO) mice in the hippocampal Schaffer collateral pathway. In these mice, we found that, even at 2 mo of age, deletion of nicastrin in excitatory neurons of the postnatal forebrain using Cre recombinase expressed under the control of the αCaMKII promoter led to deficits in presynaptic short-term plasticity including paired-pulse facilitation and frequency facilitation. Depletion of Ca(2+) in the endoplasmic reticulum mimics and occludes the presynaptic facilitation deficits in nicastrin cKO mice, suggesting that disrupted intracellular Ca(2+) homeostasis underlies the presynaptic deficits. In addition, NMDA receptor-mediated responses and long-term potentiation induced by theta-burst stimulation were decreased in nicastrin cKO mice at 3 mo but not at 2 mo of age. Together, these findings show that, similar to presenilins, nicastrin plays essential roles in the regulation of short- and long-term synaptic plasticity, highlighting the importance of γ-secretase in the function of mature synapses.

  View details for DOI 10.1073/pnas.1408554111

  View details for PubMedID 24889619

• Mi(c)rosecond Dissection of Neurotransmitter Release: SNARE-Complex Assembly Dictates Speed and Ca2+ Sensitivity NEURON Acuna, C., Guo, Q., Burre, J., Sharma, M., Sun, J., Suedhof, T. C. 2014; 82 (5): 1088-1100

  Abstract

  SNARE-complex assembly mediates synaptic vesicle fusion during neurotransmitter release and requires that the target-SNARE protein syntaxin-1 switches from a closed to an open conformation. Although many SNARE proteins are available per vesicle, only one to three SNARE complexes are minimally needed for a fusion reaction. Here, we use high-resolution measurements of synaptic transmission in the calyx-of-Held synapse from mutant mice in which syntaxin-1 is rendered constitutively open and SNARE-complex assembly is enhanced to examine the relation between SNARE-complex assembly and neurotransmitter release. We show that enhancing SNARE-complex assembly dramatically increases the speed of evoked release, potentiates the Ca(2+)-affinity of release, and accelerates fusion-pore expansion during individual vesicle fusion events. Our data indicate that the number of assembled SNARE complexes per vesicle during fusion determines the presynaptic release probability and fusion kinetics and suggest a mechanism whereby proteins (Munc13 or RIM) may control presynaptic plasticity by regulating SNARE-complex assembly.

  View details for DOI 10.1016/j.neuron.2014.04.020

  View details for Web of Science ID 000337359800015

• Microsecond Dissection of Neurotransmitter Release: SNARE-Complex Assembly Dictates Speed and Ca(2+) Sensitivity. Neuron Acuna, C., Guo, Q., Burré, J., Sharma, M., Sun, J., Südhof, T. C. 2014; 82 (5): 1088-1100

  Abstract

  SNARE-complex assembly mediates synaptic vesicle fusion during neurotransmitter release and requires that the target-SNARE protein syntaxin-1 switches from a closed to an open conformation. Although many SNARE proteins are available per vesicle, only one to three SNARE complexes are minimally needed for a fusion reaction. Here, we use high-resolution measurements of synaptic transmission in the calyx-of-Held synapse from mutant mice in which syntaxin-1 is rendered constitutively open and SNARE-complex assembly is enhanced to examine the relation between SNARE-complex assembly and neurotransmitter release. We show that enhancing SNARE-complex assembly dramatically increases the speed of evoked release, potentiates the Ca(2+)-affinity of release, and accelerates fusion-pore expansion during individual vesicle fusion events. Our data indicate that the number of assembled SNARE complexes per vesicle during fusion determines the presynaptic release probability and fusion kinetics and suggest a mechanism whereby proteins (Munc13 or RIM) may control presynaptic plasticity by regulating SNARE-complex assembly.

  View details for DOI 10.1016/j.neuron.2014.04.020

  View details for PubMedID 24908488

• Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing. Proceedings of the National Academy of Sciences of the United States of America Treutlein, B., Gokce, O., Quake, S. R., Südhof, T. C. 2014; 111 (13): E1291-9

  Abstract

  Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

  View details for DOI 10.1073/pnas.1403244111

  View details for PubMedID 24639501

• Calsyntenins function as synaptogenic adhesion molecules in concert with neurexins. Cell reports Um, J. W., Pramanik, G., Ko, J. S., Song, M., Lee, D., Kim, H., Park, K., Südhof, T. C., Tabuchi, K., Ko, J. 2014; 6 (6): 1096-1109

  Abstract

  Multiple synaptic adhesion molecules govern synapse formation. Here, we propose calsyntenin-3/alcadein-β as a synapse organizer that specifically induces presynaptic differentiation in heterologous synapse-formation assays. Calsyntenin-3 (CST-3) is highly expressed during various postnatal periods of mouse brain development. The simultaneous knockdown of all three CSTs, but not CST-3 alone, decreases inhibitory, but not excitatory, synapse densities in cultured hippocampal neurons. Moreover, the knockdown of CSTs specifically reduces inhibitory synaptic transmission in vitro and in vivo. Remarkably, the loss of CSTs induces a concomitant decrease in neuron soma size in a non-cell-autonomous manner. Furthermore, α-neurexins (α-Nrxs) are components of a CST-3 complex involved in CST-3-mediated presynaptic differentiation. However, CST-3 does not directly bind to Nrxs. Viewed together, these data suggest that the three CSTs redundantly regulate inhibitory synapse formation, inhibitory synapse function, and neuron development in concert with Nrxs.

  View details for DOI 10.1016/j.celrep.2014.02.010

  View details for PubMedID 24613359

• Structure and ca(2+)-binding properties of the tandem c2 domains of e-syt2. Structure Xu, J., Bacaj, T., Zhou, A., Tomchick, D. R., Südhof, T. C., Rizo, J. 2014; 22 (2): 269-280

  Abstract

  Contacts between the endoplasmic reticulum and the plasma membrane involve extended synaptotagmins (E-Syts) in mammals or tricalbins in yeast, proteins with multiple C2 domains. One of the tandem C2 domains of E-Syt2 is predicted to bind Ca(2+), but no Ca(2+)-dependent function has been attributed to this protein. We have determined the crystal structures of the tandem C2 domains of E-Syt2 in the absence and presence of Ca(2+) and analyzed their Ca(2+)-binding properties by nuclear magnetic resonance spectroscopy. Our data reveal an unexpected V-shaped structure with a rigid orientation between the two C2 domains that is not substantially altered by Ca(2+). The E-Syt2 C2A domain binds up to four Ca(2+) ions, whereas the C2B domain does not bind Ca(2+). These results suggest that E-Syt2 performs an as yet unidentified Ca(2+)-dependent function through its C2A domain and uncover fundamental differences between the properties of the tandem C2 domains of E-Syts and synaptotagmins.

  View details for DOI 10.1016/j.str.2013.11.011

  View details for PubMedID 24373768

• Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing. journal of biological chemistry Boucard, A. A., Maxeiner, S., Südhof, T. C. 2014; 289 (1): 387-402

  Abstract

  Latrophilin-1, -2, and -3 are adhesion-type G protein-coupled receptors that are auxiliary α-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, we here identify teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. We show that teneurin binds to latrophilins with nanomolar affinity and that this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas our findings suggest a heterophilic interaction mechanism. Thus, we tested whether mammalian teneurins also are homophilic cell-adhesion molecules, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, we find that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance.

  View details for DOI 10.1074/jbc.M113.504779

  View details for PubMedID 24273166

• Deconstructing complexin function in activating and clamping Ca2+-triggered exocytosis by comparing knockout and knockdown phenotypes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, X., Cao, P., Suedhof, T. C. 2013; 110 (51): 20777–82

  Abstract

  Complexin, a presynaptic protein that avidly binds to assembled SNARE complexes, is widely acknowledged to activate Ca(2+)-triggered exocytosis. In addition, studies of invertebrate complexin mutants and of mouse neurons with a double knockdown (DKD) of complexin-1 and -2 suggested that complexin maintains the readily releasable pool (RRP) of vesicles and clamps spontaneous exocytosis. In contrast, studies of mouse neurons with a double knockout (DKO) of complexin-1 and -2, largely carried out in hippocampal autapses, did not detect changes in the RRP size or in spontaneous exocytosis. To clarify complexin function, we here directly compared in two different preparations, cultured cortical and olfactory bulb neurons, the phenotypes of complexin DKD and DKO neurons. We find that complexin-deficient DKD and DKO neurons invariably exhibit a ~50% decrease in vesicle priming. Moreover, the DKD consistently increased spontaneous exocytosis, but the DKO did so in cortical but not olfactory bulb neurons. Furthermore, the complexin DKD but not the complexin DKO caused a compensatory increase in complexin-3 and -4 mRNA levels; overexpression of complexin-3 but not complexin-1 increased spontaneous exocytosis. Complexin-3 but not complexin-1 contains a C-terminal lipid anchor attaching it to the plasma membrane; addition of a similar lipid anchor to complexin-1 converted complexin-1 from a clamp into an activator of spontaneous exocytosis. Viewed together, our data suggest that complexin generally functions in priming and Ca(2+) triggering of exocytosis, and additionally contributes to the control of spontaneous exocytosis dependent on the developmental history of a neuron and on the subcellular localization of the complexin.

  View details for PubMedID 24297916

• Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release. Neuron Bacaj, T., Wu, D., Yang, X., Morishita, W., Zhou, P., Xu, W., Malenka, R. C., Südhof, T. C. 2013; 80 (4): 947-959

  Abstract

  In forebrain neurons, knockout of synaptotagmin-1 blocks fast Ca(2+)-triggered synchronous neurotransmitter release but enables manifestation of slow Ca(2+)-triggered asynchronous release. Here, we show using single-cell PCR that individual hippocampal neurons abundantly coexpress two Ca(2+)-binding synaptotagmin isoforms, synaptotagmin-1 and synaptotagmin-7. In synaptotagmin-1-deficient synapses of excitatory and inhibitory neurons, loss of function of synaptotagmin-7 suppressed asynchronous release. This phenotype was rescued by wild-type but not mutant synaptotagmin-7 lacking functional Ca(2+)-binding sites. Even in synaptotagmin-1-containing neurons, synaptotagmin-7 ablation partly impaired asynchronous release induced by extended high-frequency stimulus trains. Synaptotagmins bind Ca(2+) via two C2 domains, the C2A and C2B domains. Surprisingly, synaptotagmin-7 function selectively required its C2A domain Ca(2+)-binding sites, whereas synaptotagmin-1 function required its C2B domain Ca(2+)-binding sites. Our data show that nearly all Ca(2+)-triggered release at a synapse is due to synaptotagmins, with synaptotagmin-7 mediating a slower form of Ca(2+)-triggered release that is normally occluded by faster synaptotagmin-1-induced release but becomes manifest upon synaptotagmin-1 deletion.

  View details for DOI 10.1016/j.neuron.2013.10.026

  View details for PubMedID 24267651

• Neurotransmitter Release: The Last Millisecond in the Life of a Synaptic Vesicle NEURON Suedhof, T. C. 2013; 80 (3): 675–90

  Abstract

  During an action potential, Ca(2+) entering a presynaptic terminal triggers synaptic vesicle exocytosis and neurotransmitter release in less than a millisecond. How does Ca(2+) stimulate release so rapidly and precisely? Work over the last decades revealed that Ca(2+) binding to synaptotagmin triggers release by stimulating synaptotagmin binding to a core fusion machinery composed of SNARE and SM proteins that mediates membrane fusion during exocytosis. Complexin adaptor proteins assist synaptotagmin by activating and clamping this core fusion machinery. Synaptic vesicles containing synaptotagmin are positioned at the active zone, the site of vesicle fusion, by a protein complex containing RIM proteins. RIM proteins activate docking and priming of synaptic vesicles and simultaneously recruit Ca(2+) channels to active zones, thereby connecting in a single complex primed synaptic vesicles to Ca(2+) channels. This architecture allows direct flow of Ca(2+) ions from Ca(2+) channels to synaptotagmin, which then triggers fusion, thus mediating tight millisecond coupling of an action potential to neurotransmitter release.

  View details for PubMedID 24183019

• Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons CELL Wapinski, O. L., Vierbuchen, T., Qu, K., Lee, Q. Y., Chanda, S., Fuentes, D. R., Giresi, P. G., Ng, Y. H., Marro, S., Neff, N. F., Drechsel, D., Martynoga, B., Castro, D. S., Webb, A. E., Suedhof, T. C., Brunet, A., Guillemot, F., Chang, H. Y., Wernig, M. 2013; 155 (3): 621-635

  Abstract

  Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

  View details for DOI 10.1016/j.cell.2013.09.028

  View details for PubMedID 24243019

• Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability JOURNAL OF NEUROSCIENCE Arancillo, M., Min, S., Gerber, S., Muenster-Wandowski, A., Wu, Y., Herman, M., Trimbuch, T., Rah, J., Ahnert-Hilger, G., Riedel, D., Suedhof, T. C., Rosenmund, C. 2013; 33 (42): 16698-16714

  Abstract

  Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.

  View details for DOI 10.1523/JNEUROSCI.0187-13.2013

  View details for Web of Science ID 000325809800026

• Titration of syntaxin1 in Mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability. The Journal of neuroscience : the official journal of the Society for Neuroscience Arancillo, M., Min, S. W., Gerber, S., Münster-Wandowski, A., Wu, Y. J., Herman, M., Trimbuch, T., Rah, J. C., Ahnert-Hilger, G., Riedel, D., Südhof, T. C., Rosenmund, C. 2013; 33 (42): 16698-714

  Abstract

  Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.

  View details for DOI 10.1523/JNEUROSCI.0187-13.2013

  View details for PubMedID 24133272

• Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release. Neuron Zhou, P., Bacaj, T., Yang, X., Pang, Z. P., Südhof, T. C. 2013; 80 (2): 470-483

  Abstract

  Synaptic vesicle fusion during neurotransmitter release is mediated by assembly of SNARE- and SM-protein complexes composed of syntaxin-1, SNAP-25, synaptobrevin-2/VAMP2, and Munc18-1. Current models suggest that SNARE-complex assembly catalyzes membrane fusion by pulling the transmembrane regions (TMRs) of SNARE proteins together, thus allowing their TMRs to form a fusion pore. These models are consistent with the requirement for TMRs in viral fusion proteins. However, the role of the SNARE TMRs in synaptic vesicle fusion has not yet been tested physiologically. Here, we examined whether synaptic SNAREs require TMRs for catalysis of synaptic vesicle fusion, which was monitored electrophysiologically at millisecond time resolution. Surprisingly, we find that both lipid-anchored syntaxin-1 and lipid-anchored synaptobrevin-2 lacking TMRs efficiently promoted spontaneous and Ca(2+)-triggered membrane fusion. Our data suggest that SNARE proteins function during fusion primarily as force generators, consistent with the notion that forcing lipid membranes close together suffices to induce membrane fusion.

  View details for DOI 10.1016/j.neuron.2013.09.010

  View details for PubMedID 24120845

• Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation. Proceedings of the National Academy of Sciences of the United States of America Chanda, S., Marro, S., Wernig, M., Südhof, T. C. 2013; 110 (41): 16622-16627

  Abstract

  Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling.

  View details for DOI 10.1073/pnas.1316240110

  View details for PubMedID 24046374

  View details for PubMedCentralID PMC3799342

• Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chanda, S., Marro, S., Wernig, M., Suedhof, T. C. 2013; 110 (41): 16622-16627

  Abstract

  Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling.

  View details for DOI 10.1073/pnas.1316240110

  View details for Web of Science ID 000325395600075

  View details for PubMedCentralID PMC3799342

• A molecular machine for neurotransmitter release: synaptotagmin and beyond NATURE MEDICINE Suedhof, T. C. 2013; 19 (10): 1227–31

  View details for PubMedID 24100992

• Membrane-Tethered Monomeric Neurexin LNS-Domain Triggers Synapse Formation. journal of neuroscience Gokce, O., Südhof, T. C. 2013; 33 (36): 14617-14628

  Abstract

  Neurexins are presynaptic cell-adhesion molecules that bind to postsynaptic cell-adhesion molecules such as neuroligins and leucine-rich repeat transmembrane proteins (LRRTMs). When neuroligins or LRRTMs are expressed in a nonneuronal cell, cocultured neurons avidly form heterologous synapses onto that cell. Here we show that knockdown of all neurexins in cultured hippocampal mouse neurons did not impair synapse formation between neurons, but blocked heterologous synapse formation induced by neuroligin-1 or LRRTM2. Rescue experiments demonstrated that all neurexins tested restored heterologous synapse formation in neurexin-deficient neurons. Neurexin-deficient neurons exhibited a decrease in the levels of the PDZ-domain protein CASK (a calcium/calmodulin-activated serine/threonine kinase), which binds to neurexins, and mutation of the PDZ-domain binding sequence of neurexin-3β blocked its transport to the neuronal surface and impaired heterologous synapse formation. However, replacement of the C-terminal neurexin sequence with an unrelated PDZ-domain binding sequence that does not bind to CASK fully restored surface transport and heterologous synapse formation in neurexin-deficient neurons, suggesting that no particular PDZ-domain protein is essential for neurexin surface transport or heterologous synapse formation. Further mutagenesis revealed, moreover, that the entire neurexin cytoplasmic tail was dispensable for heterologous synapse formation in neurexin-deficient neurons, as long as the neurexin protein was transported to the neuronal cell surface. Furthermore, the single LNS-domain (for laminin/neurexin/sex hormone-binding globulin-domain) of neurexin-1β or neurexin-3β, when tethered to the presynaptic plasma membrane by a glycosylinositolphosphate anchor, was sufficient for rescuing heterologous synapse formation in neurexin-deficient neurons. Our data suggest that neurexins mediate heterologous synapse formation via an extracellular interaction with presynaptic and postsynaptic ligands without the need for signal transduction by the neurexin cytoplasmic tail.

  View details for DOI 10.1523/JNEUROSCI.1232-13.2013

  View details for PubMedID 24005312

• Leucine-Rich Repeat Transmembrane Proteins Are Essential for Maintenance of Long-Term Potentiation NEURON Soler-Llavina, G. J., Arstikaitis, P., Morishita, W., Ahmad, M., Suedhof, T. C., Malenka, R. C. 2013; 79 (3): 439-446

  Abstract

  Leucine-rich repeat transmembrane proteins (LRRTMs) are synaptic cell adhesion molecules that trigger excitatory synapse assembly in cultured neurons and influence synaptic function in vivo, but their role in synaptic plasticity is unknown. shRNA-mediated knockdown (KD) of LRRTM1 and LRRTM2 in vivo in CA1 pyramidal neurons of newborn mice blocked long-term potentiation (LTP) in acute hippocampal slices. Molecular replacement experiments revealed that the LRRTM2 extracellular domain is sufficient for LTP, probably because it mediates binding to neurexins (Nrxs). Examination of surface expression of endogenous AMPA receptors (AMPARs) in cultured neurons suggests that LRRTMs maintain newly delivered AMPARs at synapses after LTP induction. LRRTMs are also required for LTP of mature synapses on adult CA1 pyramidal neurons, indicating that the block of LTP in neonatal synapses by LRRTM1 and LRRTM2 KD is not due to impairment of synapse maturation.

  View details for DOI 10.1016/j.neuron.2013.06.007

  View details for PubMedID 23931994

• Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ. Proceedings of the National Academy of Sciences of the United States of America Pertsinidis, A., Mukherjee, K., Sharma, M., Pang, Z. P., Park, S. R., Zhang, Y., Brunger, A. T., Südhof, T. C., Chu, S. 2013; 110 (30): E2812-20

  Abstract

  Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

  View details for DOI 10.1073/pnas.1310654110

  View details for PubMedID 23821748

• Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pertsinidis, A., Mukherjee, K., Sharma, M., Pang, Z. P., Park, S. R., Zhang, Y., Brunger, A. T., Suedhof, T. C., Chu, S. 2013; 110 (30): E2812-E2820

  Abstract

  Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

  View details for DOI 10.1073/pnas.1310654110

  View details for Web of Science ID 000322112300011

  View details for PubMedID 23821748

• Presynaptic Neurexin-3 Alternative Splicing trans-Synaptically Controls Postsynaptic AMPA Receptor Trafficking CELL Aoto, J., Martinelli, D. C., Malenka, R. C., Tabuchi, K., Suedhof, T. C. 2013; 154 (1): 75-88

  Abstract

  Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.

  View details for DOI 10.1016/j.cell.2013.05.060

  View details for PubMedID 23827676

• Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts JOURNAL OF NEUROSCIENCE METHODS Davila, J., Chanda, S., Ang, C. E., Suedhof, T. C., Wernig, M. 2013; 216 (2): 104-109

  Abstract

  We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O₂). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O₂ tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O₂ conditions compared to 50% under high O₂ growth conditions, confirming the beneficial aspect of reprogramming under low O₂. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3β and Smad-mediated pathways. Our results showed that lowering the O₂ tension at the initiation of reprogramming is a simple and efficient strategy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

  View details for DOI 10.1016/j.jneumeth.2013.03.020

  View details for Web of Science ID 000321168200004

  View details for PubMedCentralID PMC4009399

• Properties of native brain alpha-synuclein NATURE Burre, J., Vivona, S., Diao, J., Sharma, M., Brunger, A. T., Suedhof, T. C. 2013; 498 (7453): E4-E6

  View details for DOI 10.1038/nature12125

  View details for Web of Science ID 000320283400001

• Synaptotagmin-12 Phosphorylation by cAMP-Dependent Protein Kinase Is Essential for Hippocampal Mossy Fiber LTP JOURNAL OF NEUROSCIENCE Kaeser-Woo, Y. J., Younts, T. J., Yang, X., Zhou, P., Wu, D., Castillo, P. E., Suedhof, T. C. 2013; 33 (23): 9769-9780

  Abstract

  Synaptotagmin-12 (Syt12) is an abundant synaptic vesicle protein that-different from other synaptic vesicle-associated synaptotagmins-does not bind Ca(2+). Syt12 is phosphorylated by cAMP-dependent protein kinase-A at serine-97 in an activity-dependent manner, suggesting a function for Syt12 in cAMP-dependent synaptic plasticity. To test this hypothesis, we here generated (1) Syt12 knock-out mice and (2) Syt12 knockin mice carrying a single amino-acid substitution [the serine-97-to-alanine- (S97A)-substitution]. Both Syt12 knock-out mice and Syt12 S97A-knockin mice were viable and fertile, and exhibited no measurable change in basal synaptic strength or short-term plasticity as analyzed in cultured cortical neurons or in acute hippocampal slices. However, both Syt12 knock-out and Syt12 S97A-knockin mice displayed a major impairment in cAMP-dependent mossy-fiber long-term potentiation (LTP) in the CA3 region of the hippocampus. This impairment was observed using different experimental configurations for inducing and monitoring mossy-fiber LTP. Moreover, although the Syt12 knock-out had no effect on the short-term potentiation of synaptic transmission induced by the adenylate-cyclase activator forskolin in cultured cortical neurons and in the CA1 region of the hippocampus, both the Syt12 knock-out and the Syt12 S97A-knockin impaired the long-term increase in mossy-fiber synaptic transmission induced by forskolin. Thus, Syt12 is essential for cAMP-dependent presynaptic LTP at mossy-fiber synapses, and a single amino-acid substitution that blocks the cAMP-dependent phosphorylation of Syt12 is sufficient to impair the function of Syt12 in mossy-fiber LTP, suggesting that cAMP-dependent phosphorylation of Syt12 on serine-97 contributes to the induction of mossy-fiber LTP.

  View details for DOI 10.1523/JNEUROSCI.5814-12.2013

  View details for PubMedID 23739973

• Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron Zhang, Y., Pak, C., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W., Yang, N., Danko, T., Chen, L., Wernig, M., Südhof, T. C. 2013; 78 (5): 785-798

  Abstract

  Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.

  View details for DOI 10.1016/j.neuron.2013.05.029

  View details for PubMedID 23764284

• Autism-associated neuroligin-3 mutations commonly disrupt tonic endocannabinoid signaling. Neuron Földy, C., Malenka, R. C., Südhof, T. C. 2013; 78 (3): 498-509

  Abstract

  Neuroligins are postsynaptic cell-adhesion molecules that interact with presynaptic neurexins. Rare mutations in neuroligins and neurexins predispose to autism, including a neuroligin-3 amino acid substitution (R451C) and a neuroligin-3 deletion. Previous analyses showed that neuroligin-3 R451C-knockin mice exhibit robust synaptic phenotypes but failed to uncover major changes in neuroligin-3 knockout mice, questioning the notion that a common synaptic mechanism mediates autism pathogenesis in patients with these mutations. Here, we used paired recordings in mice carrying these mutations to measure synaptic transmission at GABAergic synapses formed by hippocampal parvalbumin- and cholecystokinin-expressing basket cells onto pyramidal neurons. We demonstrate that in addition to unique gain-of-function effects produced by the neuroligin-3 R451C-knockin but not the neuroligin-3 knockout mutation, both mutations dramatically impaired tonic but not phasic endocannabinoid signaling. Our data thus suggest that neuroligin-3 is specifically required for tonic endocannabinoid signaling, raising the possibility that alterations in endocannabinoid signaling may contribute to autism pathophysiology.

  View details for DOI 10.1016/j.neuron.2013.02.036

  View details for PubMedID 23583622

• Native alpha-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2 ELIFE Diao, J., Burre, J., Vivona, S., Cipriano, D. J., Sharma, M., Kyoung, M., Suedhof, T. C., Brunger, A. T. 2013; 2

  Abstract

  α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca(2+)-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a 'buffer' of synaptic vesicles, without affecting neurotransmitter release itself. DOI:http://dx.doi.org/10.7554/eLife.00592.001.

  View details for DOI 10.7554/eLife.00592

  View details for Web of Science ID 000328614700004

  View details for PubMedCentralID PMC3639508

• A neural circuit for memory specificity and generalization. Science Xu, W., Südhof, T. C. 2013; 339 (6125): 1290-1295

  Abstract

  Increased fear memory generalization is associated with posttraumatic stress disorder, but the circuit mechanisms that regulate memory specificity remain unclear. Here, we define a neural circuit-composed of the medial prefrontal cortex, the nucleus reuniens (NR), and the hippocampus-that controls fear memory generalization. Inactivation of prefrontal inputs into the NR or direct silencing of NR projections enhanced fear memory generalization, whereas constitutive activation of NR neurons decreased memory generalization. Direct optogenetic activation of phasic and tonic action-potential firing of NR neurons during memory acquisition enhanced or reduced memory generalization, respectively. We propose that the NR determines the specificity and generalization of memory attributes for a particular context by processing information from the medial prefrontal cortex en route to the hippocampus.

  View details for DOI 10.1126/science.1229534

  View details for PubMedID 23493706

• LTP Requires a Unique Postsynaptic SNARE Fusion Machinery NEURON Jurado, S., Goswami, D., Zhang, Y., Minano Molina, A. J., Suedhof, T. C., Malenka, R. C. 2013; 77 (3): 542-558

  Abstract

  Membrane fusion during exocytosis is mediated by assemblies of SNARE (soluble NSF-attachment protein receptor) and SM (Sec1/Munc18-like) proteins. The SNARE/SM proteins involved in vesicle fusion during neurotransmitter release are well understood, whereas little is known about the protein machinery that mediates activity-dependent AMPA receptor (AMPAR) exocytosis during long-term potentiation (LTP). Using direct measurements of LTP in acute hippocampal slices and an in vitro LTP model of stimulated AMPAR exocytosis, we demonstrate that the Q-SNARE proteins syntaxin-3 and SNAP-47 are required for regulated AMPAR exocytosis during LTP but not for constitutive basal AMPAR exocytosis. In contrast, the R-SNARE protein synaptobrevin-2/VAMP2 contributes to both regulated and constitutive AMPAR exocytosis. Both the central complexin-binding and the N-terminal Munc18-binding sites of syntaxin-3 are essential for its postsynaptic role in LTP. Thus, postsynaptic exocytosis of AMPARs during LTP is mediated by a unique fusion machinery that is distinct from that used during presynaptic neurotransmitter release.

  View details for DOI 10.1016/j.neuron.2012.11.029

  View details for PubMedID 23395379

• Complexin Activates Exocytosis of Distinct Secretory Vesicles Controlled by Different Synaptotagmins JOURNAL OF NEUROSCIENCE Cao, P., Yang, X., Suedhof, T. C. 2013; 33 (4): 1714-?

  Abstract

  Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.

  View details for DOI 10.1523/JNEUROSCI.4087-12.2013

  View details for PubMedID 23345244

• Syntaxin-1 N-peptide and H-abc-domain perform distinct essential functions in synaptic vesicle fusion EMBO JOURNAL Zhou, P., Pang, Z. P., Yang, X., Zhang, Y., Rosenmund, C., Bacaj, T., Suedhof, T. C. 2013; 32 (1): 159-171

  Abstract

  Among SNARE proteins mediating synaptic vesicle fusion, syntaxin-1 uniquely includes an N-terminal peptide ('N-peptide') that binds to Munc18-1, and a large, conserved H(abc)-domain that also binds to Munc18-1. Previous in vitro studies suggested that the syntaxin-1 N-peptide is functionally important, whereas the syntaxin-1 H(abc)-domain is not, but limited information is available about the in vivo functions of these syntaxin-1 domains. Using rescue experiments in cultured syntaxin-deficient neurons, we now show that the N-peptide and the H(abc)-domain of syntaxin-1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N-peptide is essential for vesicle fusion as such, whereas the H(abc)-domain regulates this fusion, in part by forming the closed syntaxin-1 conformation. Moreover, we observed that deletion of the H(abc)-domain but not deletion of the N-peptide caused a loss of Munc18-1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the H(abc)-domain stabilizes Munc18-1. Thus, the N-terminal syntaxin-1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE-protein complexes.

  View details for DOI 10.1038/emboj.2012.307

  View details for PubMedID 23188083

• MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, K., Kim, Y., Lee, S., Qiang, Y., Lee, D., Lee, H. W., Kim, H., Je, H. S., Suedhof, T. C., Ko, J. 2013; 110 (1): 336-341

  Abstract

  The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2-dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.

  View details for DOI 10.1073/pnas.1219987110

  View details for Web of Science ID 000313630300073

  View details for PubMedCentralID PMC3538197

• Native a-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. eLife Diao, J., Burré, J., Vivona, S., Cipriano, D. J., Sharma, M., Kyoung, M., Südhof, T. C., Brunger, A. T. 2013; 2

  Abstract

  α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca(2+)-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a 'buffer' of synaptic vesicles, without affecting neurotransmitter release itself. DOI:http://dx.doi.org/10.7554/eLife.00592.001.

  View details for DOI 10.7554/eLife.00592

  View details for PubMedID 23638301

  View details for PubMedCentralID PMC3639508

• Local use of geographic information systems to improve data utilisation and health services: mapping caesarean section coverage in rural Rwanda TROPICAL MEDICINE & INTERNATIONAL HEALTH Sudhof, L., Amoroso, C., Barebwanuwe, P., Munyaneza, F., Karamaga, A., Zambotti, G., Drobac, P., Hirschhorn, L. R. 2013; 18 (1): 18-26

  Abstract

  To show the utility of combining routinely collected data with geographic location using a Geographic Information System (GIS) in order to facilitate a data-driven approach to identifying potential gaps in access to emergency obstetric care within a rural Rwandan health district.Total expected births in 2009 at sub-district levels were estimated using community health worker collected population data. Clinical data were extracted from birth registries at eight health centres (HCs) and the district hospital (DH). C-section rates as a proportion of total expected births were mapped by cell. Peri-partum foetal mortality rates per facility-based births, as well as the rate of uterine rupture as an indication for C-section, were compared between areas of low and high C-section rates.The lowest C-section rates were found in the more remote part of the hospital catchment area. The sector with significantly lower C-section rates had significantly higher facility-based peri-partum foetal mortality and incidence of uterine rupture than the sector with the highest C-section rates (P < 0.034).This simple approach for geographic monitoring and evaluation leveraging existing health service and GIS data facilitated evidence-based decision making and represents a feasible approach to further strengthen local data-driven decisions for resource allocation and quality improvement.

  View details for DOI 10.1111/tmi.12016

  View details for Web of Science ID 000312739700004

  View details for PubMedID 23279379

• Candidate autism gene screen identifies critical role for cell-adhesion molecule CASPR2 in dendritic arborization and spine development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, G. R., Galfin, T., Xu, W., Aoto, J., Malenka, R. C., Suedhof, T. C. 2012; 109 (44): 18120-18125

  Abstract

  Mutations in the contactin-associated protein 2 (CNTNAP2) gene encoding CASPR2, a neurexin-related cell-adhesion molecule, predispose to autism, but the function of CASPR2 in neural circuit assembly remains largely unknown. In a knockdown survey of autism candidate genes, we found that CASPR2 is required for normal development of neural networks. RNAi-mediated knockdown of CASPR2 produced a cell-autonomous decrease in dendritic arborization and spine development in pyramidal neurons, leading to a global decline in excitatory and inhibitory synapse numbers and a decrease in synaptic transmission without a detectable change in the properties of these synapses. Our data suggest that in addition to the previously described role of CASPR2 in mature neurons, where CASPR2 organizes nodal microdomains of myelinated axons, CASPR2 performs an earlier organizational function in developing neurons that is essential for neural circuit assembly and operates coincident with the time of autism spectrum disorder (ASD) pathogenesis.

  View details for DOI 10.1073/pnas.1216398109

  View details for PubMedID 23074245

• Systematic mutagenesis of a-synuclein reveals distinct sequence requirements for physiological and pathological activities. journal of neuroscience Burré, J., Sharma, M., Südhof, T. C. 2012; 32 (43): 15227-15242

  Abstract

  -Synuclein is an abundant presynaptic protein that binds to phospholipids and synaptic vesicles. Physiologically, ?-synuclein functions as a SNARE-protein chaperone that promotes SNARE-complex assembly for neurotransmitter release. Pathologically, ?-synuclein mutations and ?-synuclein overexpression cause Parkinson's disease, and aggregates of ?-synuclein are found as Lewy bodies in multiple neurodegenerative disorders ("synucleinopathies"). The relation of the physiological functions to the pathological effects of ?-synuclein remains unclear. As an initial avenue of addressing this question, we here systematically examined the effect of ?-synuclein mutations on its physiological and pathological activities. We generated 26 ?-synuclein mutants spanning the entire molecule, and analyzed them compared with wild-type ?-synuclein in seven assays that range from biochemical studies with purified ?-synuclein, to analyses of ?-synuclein expression in cultured neurons, to examinations of the effects of virally expressed ?-synuclein introduced into the mouse substantia nigra by stereotactic injections. We found that both the N-terminal and C-terminal sequences of ?-synuclein were required for its physiological function as SNARE-complex chaperone, but that these sequences were not essential for its neuropathological effects. In contrast, point mutations in the central region of ?-synuclein, referred to as nonamyloid ? component (residues 61-95), as well as point mutations linked to Parkinson's disease (A30P, E46K, and A53T) increased the neurotoxicity of ?-synuclein but did not affect its physiological function in SNARE-complex assembly. Thus, our data show that the physiological function of ?-synuclein, although protective of neurodegeneration in some contexts, is fundamentally distinct from its neuropathological effects, thereby dissociating the two activities of ?-synuclein.

  View details for DOI 10.1523/JNEUROSCI.3545-12.2012

  View details for PubMedID 23100443

  View details for PubMedCentralID PMC3506191

• Proteasome Inhibition Alleviates SNARE-Dependent Neurodegeneration SCIENCE TRANSLATIONAL MEDICINE Sharma, M., Burre, J., Suedhof, T. C. 2012; 4 (147)

  Abstract

  Activation of the proteasomal degradation of misfolded proteins has been proposed as a therapeutic strategy for treating neurodegenerative diseases, but it is unclear whether proteasome dysfunction contributes to neurodegeneration. We tested the role of proteasome activity in neurodegeneration developed by mice lacking cysteine string protein-α (CSPα). Unexpectedly, we found that proteasome inhibitors alleviated neurodegeneration in CSPα-deficient mice, reversing impairment of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-complex assembly and extending life span. We tested whether dysfunctional SNARE-complex assembly could contribute to neurodegeneration in Alzheimer's and Parkinson's disease by analyzing postmortem brain tissue from these patients; we found reduced SNARE-complex assembly in the brain tissue samples. Our results suggest that proteasomal activation may not always be beneficial for alleviating neurodegeneration and that blocking the proteasome may represent a potential therapeutic avenue for treating some forms of neurodegenerative disease.

  View details for DOI 10.1126/scitranslmed.3004028

  View details for PubMedID 22896677

• Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, S., Shi, Y., Yang, G., Li, Y., Yu, L., Shin, O., Bacaj, T., Suedhof, T. C., Yu, J., Berggren, P. 2012; 109 (30): 12183-12188

  Abstract

  Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.

  View details for DOI 10.1073/pnas.1115070109

  View details for Web of Science ID 000306992700063

  View details for PubMedID 22778403

  View details for PubMedCentralID PMC3409763

• RIM genes differentially contribute to organizing presynaptic release sites PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaeser, P. S., Deng, L., Fan, M., Suedhof, T. C. 2012; 109 (29): 11830-11835

  Abstract

  Tight coupling of Ca(2+) channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca(2+) channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca(2+)-responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca(2+) influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca(2+) channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca(2+) responsiveness and synchronization of release. RIM proteins may act on Ca(2+) channels in two modes: They tether Ca(2+) channels to active zones, and they directly modulate Ca(2+)-channel inactivation. The first mechanism is essential for localizing presynaptic Ca(2+) influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C(2)B domain by itself significantly decreased Ca(2+)-channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca(2+)-channel tethers and not as Ca(2+)-channel modulators. Different RIM proteins compensate for each other in recruiting Ca(2+) channels to active zones, but contribute independently and incrementally to vesicle priming.

  View details for DOI 10.1073/pnas.1209318109

  View details for PubMedID 22753485

• The Presynaptic Active Zone NEURON Suedhof, T. C. 2012; 75 (1): 11-25

  Abstract

  Neurotransmitters are released by synaptic vesicle exocytosis at the active zone of a presynaptic nerve terminal. In this review, I discuss the molecular composition and function of the active zone. Active zones are composed of an evolutionarily conserved protein complex containing as core constituents RIM, Munc13, RIM-BP, α-liprin, and ELKS proteins. This complex docks and primes synaptic vesicles for exocytosis, recruits Ca(2+) channels to the site of exocytosis, and positions the active zone exactly opposite to postsynaptic specializations via transsynaptic cell-adhesion molecules. Moreover, this complex mediates short- and long-term plasticity in response to bursts of action potentials, thus critically contributing to the computational power of a synapse.

  View details for DOI 10.1016/j.neuron.2012.06.012

  View details for PubMedID 22794257

• Neurotransmitter Release at the Thalamocortical Synapse Instructs Barrel Formation But Not Axon Patterning in the Somatosensory Cortex JOURNAL OF NEUROSCIENCE Narboux-Neme, N., Evrard, A., Ferezou, I., Erzurumlu, R. S., Kaeser, P. S., Laine, J., Rossier, J., Ropert, N., Suedhof, T. C., Gaspar, P. 2012; 32 (18): 6183-6196

  Abstract

  To assess the impact of synaptic neurotransmitter release on neural circuit development, we analyzed barrel cortex formation after thalamic or cortical ablation of RIM1 and RIM2 proteins, which control synaptic vesicle fusion. Thalamus-specific deletion of RIMs reduced neurotransmission efficacy by 67%. A barrelless phenotype was found with a dissociation of effects on the presynaptic and postsynaptic cellular elements of the barrel. Presynaptically, thalamocortical axons formed a normal whisker map, whereas postsynaptically the cytoarchitecture of layer IV neurons was altered as spiny stellate neurons were evenly distributed and their dendritic trees were symmetric. Strikingly, cortex-specific deletion of the RIM genes did not modify barrel development. Adult mice with thalamic-specific RIM deletion showed a lack of activity-triggered immediate early gene expression and altered sensory-related behaviors. Thus, efficient synaptic release is required at thalamocortical but not at corticocortical synapses for building the whisker to barrel map and for efficient sensory function.

  View details for DOI 10.1523/JNEUROSCI.0343-12.2012

  View details for Web of Science ID 000303598900014

  View details for PubMedID 22553025

  View details for PubMedCentralID PMC3526954

• Synapses and Alzheimer's disease. Cold Spring Harbor perspectives in biology Sheng, M., Sabatini, B. L., Südhof, T. C. 2012; 4 (5)

  Abstract

  Alzheimer's disease (AD) is a major cause of dementia in the elderly. Pathologically, AD is characterized by the accumulation of insoluble aggregates of Aβ-peptides that are proteolytic cleavage products of the amyloid-β precursor protein ("plaques") and by insoluble filaments composed of hyperphosphorylated tau protein ("tangles"). Familial forms of AD often display increased production of Aβ peptides and/or altered activity of presenilins, the catalytic subunits of γ-secretase that produce Aβ peptides. Although the pathogenesis of AD remains unclear, recent studies have highlighted two major themes that are likely important. First, oligomeric Aβ species have strong detrimental effects on synapse function and structure, particularly on the postsynaptic side. Second, decreased presenilin function impairs synaptic transmission and promotes neurodegeneration. The mechanisms underlying these processes are beginning to be elucidated, and, although their relevance to AD remains debated, understanding these processes will likely allow new therapeutic avenues to AD.

  View details for DOI 10.1101/cshperspect.a005777

  View details for PubMedID 22491782

  View details for PubMedCentralID PMC3331702

• Synaptic cell adhesion. Cold Spring Harbor perspectives in biology Missler, M., Südhof, T. C., Biederer, T. 2012; 4 (4)

  Abstract

  Chemical synapses are asymmetric intercellular junctions that mediate synaptic transmission. Synaptic junctions are organized by trans-synaptic cell adhesion molecules bridging the synaptic cleft. Synaptic cell adhesion molecules not only connect pre- and postsynaptic compartments, but also mediate trans-synaptic recognition and signaling processes that are essential for the establishment, specification, and plasticity of synapses. A growing number of synaptic cell adhesion molecules that include neurexins and neuroligins, Ig-domain proteins such as SynCAMs, receptor phosphotyrosine kinases and phosphatases, and several leucine-rich repeat proteins have been identified. These synaptic cell adhesion molecules use characteristic extracellular domains to perform complementary roles in organizing synaptic junctions that are only now being revealed. The importance of synaptic cell adhesion molecules for brain function is highlighted by recent findings implicating several such molecules, notably neurexins and neuroligins, in schizophrenia and autism.

  View details for DOI 10.1101/cshperspect.a005694

  View details for PubMedID 22278667

  View details for PubMedCentralID PMC3312681

• A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis EMBO JOURNAL Arac, D., Boucard, A. A., Bolliger, M. F., Nguyen, J., Soltis, S. M., Suedhof, T. C., Brunger, A. T. 2012; 31 (6): 1364-1378

  Abstract

  The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40-residue GPS motif represents an integral part of a much larger ∼320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.

  View details for DOI 10.1038/emboj.2012.26

  View details for Web of Science ID 000302131600005

  View details for PubMedID 22333914

  View details for PubMedCentralID PMC3321182

• High Affinity Neurexin Binding to Cell Adhesion G-protein-coupled Receptor CIRL1/Latrophilin-1 Produces an Intercellular Adhesion Complex JOURNAL OF BIOLOGICAL CHEMISTRY Boucard, A. A., Ko, J., Suedhof, T. C. 2012; 287 (12): 9399-9413

  Abstract

  The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

  View details for DOI 10.1074/jbc.M111.318659

  View details for PubMedID 22262843

• Distinct Neuronal Coding Schemes in Memory Revealed by Selective Erasure of Fast Synchronous Synaptic Transmission NEURON Xu, W., Morishita, W., Buckmaster, P. S., Pang, Z. P., Malenka, R. C., Suedhof, T. C. 2012; 73 (5): 990-1001

  Abstract

  Neurons encode information by firing spikes in isolation or bursts and propagate information by spike-triggered neurotransmitter release that initiates synaptic transmission. Isolated spikes trigger neurotransmitter release unreliably but with high temporal precision. In contrast, bursts of spikes trigger neurotransmission reliably (i.e., boost transmission fidelity), but the resulting synaptic responses are temporally imprecise. However, the relative physiological importance of different spike-firing modes remains unclear. Here, we show that knockdown of synaptotagmin-1, the major Ca(2+) sensor for neurotransmitter release, abrogated neurotransmission evoked by isolated spikes but only delayed, without abolishing, neurotransmission evoked by bursts of spikes. Nevertheless, knockdown of synaptotagmin-1 in the hippocampal CA1 region did not impede acquisition of recent contextual fear memories, although it did impair the precision of such memories. In contrast, knockdown of synaptotagmin-1 in the prefrontal cortex impaired all remote fear memories. These results indicate that different brain circuits and types of memory employ distinct spike-coding schemes to encode and transmit information.

  View details for DOI 10.1016/j.neuron.2011.12.036

  View details for PubMedID 22405208

• Region-specific deletions of RIM1 reproduce a subset of global RIM1a-/- phenotypes GENES BRAIN AND BEHAVIOR Haws, M. E., Kaeser, P. S., Jarvis, D. L., SUEDHOF, T. C., Powell, C. M. 2012; 11 (2): 201-213

  Abstract

  The presynaptic protein RIM1α mediates multiple forms of presynaptic plasticity at both excitatory and inhibitory synapses. Previous studies of mice lacking RIM1α (RIM1α(-/-) throughout the brain showed that deletion of RIM1α results in multiple behavioral abnormalities. In an effort to begin to delineate the brain regions in which RIM1 deletion mediates these abnormal behaviors, we used conditional (floxed) RIM1 knockout mice (fRIM1). By crossing these fRIM1 mice to previously characterized transgenic cre lines, we aimed to delete RIM1 selectively in the dentate gyrus (DG), using a specific preproopiomelanocortin promoter driving cre recombinase (POMC-cre) line , and in pyramidal neurons of the CA3 region of hippocampus, using the kainate receptor subunit 1 promoter driving cre recombinase (KA-cre). Neither of these cre driver lines was uniquely selective to the targeted regions. In spite of this, we were able to reproduce a subset of the global RIM1α(-/-) behavioral abnormalities, thereby narrowing the brain regions in which loss of RIM1 is sufficient to produce these behavioral differences. Most interestingly, hypersensitivity to the pyschotomimetic MK-801 was shown in mice lacking RIM1 selectively in the DG, arcuate nucleus of the hypothalamus and select cerebellar neurons, implicating novel brain regions and neuronal subtypes in this behavior.

  View details for DOI 10.1111/j.1601-183X.2011.00755.x

  View details for Web of Science ID 000299634400009

  View details for PubMedID 22103334

  View details for PubMedCentralID PMC3268893

• C-Terminal Complexin Sequence Is Selectively Required for Clamping and Priming But Not for Ca2+ Triggering of Synaptic Exocytosis JOURNAL OF NEUROSCIENCE Kaeser-Woo, Y. J., Yang, X., Suedhof, T. C. 2012; 32 (8): 2877-2885

  Abstract

  Complexins are small soluble proteins that bind to assembling SNARE complexes during synaptic vesicle exocytosis, which in turn mediates neurotransmitter release. Complexins are required for clamping of spontaneous "mini " release and for the priming and synaptotagmin-dependent Ca(2+) triggering of evoked release. Mammalian genomes encode four complexins that are composed of an N-terminal unstructured sequence that activates synaptic exocytosis, an accessory α-helix that clamps exocytosis, an essential central α-helix that binds to assembling SNARE complexes and is required for all of its functions, and a long, apparently unstructured C-terminal sequence whose function remains unclear. Here, we used cultured mouse neurons to show that the C-terminal sequence of complexin-1 is not required for its synaptotagmin-activating function but is essential for its priming and clamping functions. Wild-type complexin-3 did not clamp exocytosis but nevertheless fully primed and activated exocytosis. Strikingly, exchanging the complexin-1 C terminus for the complexin-3 C terminus abrogated clamping, whereas exchanging the complexin-3 C terminus for the complexin-1 C terminus enabled clamping. Analysis of point mutations in the complexin-1 C terminus identified two single amino-acid substitutions that impaired clamping without altering the activation function of complexin-1. Examination of release induced by stimulus trains revealed that clamping-deficient C-terminal complexin mutants produced a modest relative increase in delayed release. Overall, our results show that the relatively large C-terminal complexin-1 sequence acts in priming and clamping synaptic exocytosis and demonstrate that the clamping function is not conserved in complexin-3, presumably because of its distinct C-terminal sequences.

  View details for DOI 10.1523/JNEUROSCI.3360-11.2012

  View details for PubMedID 22357870

• CSPa knockout causes neurodegeneration by impairing SNAP-25 function. EMBO journal Sharma, M., Burré, J., Bronk, P., Zhang, Y., Xu, W., Südhof, T. C. 2012; 31 (4): 829-841

  Abstract

  At a synapse, the synaptic vesicle protein cysteine-string protein-α (CSPα) functions as a co-chaperone for the SNARE protein SNAP-25. Knockout (KO) of CSPα causes fulminant neurodegeneration that is rescued by α-synuclein overexpression. The CSPα KO decreases SNAP-25 levels and impairs SNARE-complex assembly; only the latter but not the former is reversed by α-synuclein. Thus, the question arises whether the CSPα KO phenotype is due to decreased SNAP-25 function that then causes neurodegeneration, or due to the dysfunction of multiple as-yet uncharacterized CSPα targets. Here, we demonstrate that decreasing SNAP-25 levels in CSPα KO mice by either KO or knockdown of SNAP-25 aggravated their phenotype. Conversely, increasing SNAP-25 levels by overexpression rescued their phenotype. Inactive SNAP-25 mutants were unable to rescue, showing that the rescue was specific. Under all conditions, the neurodegenerative phenotype precisely correlated with SNARE-complex assembly, indicating that impaired SNARE-complex assembly due to decreased SNAP-25 levels is the ultimate correlate of neurodegeneration. Our findings suggest that the neurodegeneration in CSPα KO mice is primarily produced by defective SNAP-25 function, which causes neurodegeneration by impairing SNARE-complex assembly.

  View details for DOI 10.1038/emboj.2011.467

  View details for PubMedID 22187053

  View details for PubMedCentralID PMC3280561

• Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lujan, E., Chanda, S., Ahlenius, H., Suedhof, T. C., Wernig, M. 2012; 109 (7): 2527-2532

  Abstract

  We recently showed that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as "induced neuronal" (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. We hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, we infected mouse embryonic fibroblasts derived from Sox2-EGFP mice with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP(+) colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, we found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When we added the Pou and Homeobox domain-containing transcription factor Brn2 to Sox2 and FoxG1, we were able to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. Our data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord.

  View details for DOI 10.1073/pnas.1121003109

  View details for PubMedID 22308465

• Postsynaptic Complexin Controls AMPA Receptor Exocytosis during LTP NEURON Ahmad, M., Polepalli, J. S., Goswami, D., Yang, X., Kaeser-Woo, Y. J., Suedhof, T. C., Malenka, R. C. 2012; 73 (2): 260-267

  Abstract

  Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.

  View details for DOI 10.1016/j.neuron.2011.11.020

  View details for PubMedID 22284181

• The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged? Annual review of cell and developmental biology Rizo, J., Südhof, T. C. 2012; 28: 279-308

  Abstract

  Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

  View details for DOI 10.1146/annurev-cellbio-101011-155818

  View details for PubMedID 23057743

• Calcium control of neurotransmitter release. Cold Spring Harbor perspectives in biology Südhof, T. C. 2012; 4 (1)

  Abstract

  Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.

  View details for DOI 10.1101/cshperspect.a011353

  View details for PubMedID 22068972

• Comprehensive qPCR profiling of gene expression in single neuronal cells NATURE PROTOCOLS Citri, A., Pang, Z. P., Suedhof, T. C., Wernig, M., Malenka, R. C. 2012; 7 (1): 118-127

  Abstract

  A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe uses Fluidigm Biomark dynamic arrays for high-throughput expression profiling from single neuronal cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment, which can be completed within 2-3 d. The protocol enables simple and cost-effective profiling of several hundred transcripts from a single cell, and it could have numerous utilities.

  View details for DOI 10.1038/nprot.2011.430

  View details for PubMedID 22193304

• Induced Neuronal Cells: How to Make and Define a Neuron CELL STEM CELL Yang, N., Ng, Y. H., Pang, Z. P., Suedhof, T. C., Wernig, M. 2011; 9 (6): 517-525

  Abstract

  Cellular plasticity is a major focus of investigation in developmental biology. The recent discovery that induced neuronal (iN) cells can be generated from mouse and human fibroblasts by expression of defined transcription factors suggested that cell fate plasticity is much wider than previously anticipated. In this review, we summarize the most recent developments in this nascent field and suggest criteria to help define and categorize iN cells that take into account the complexity of neuronal identity.

  View details for DOI 10.1016/j.stem.2011.11.015

  View details for PubMedID 22136927

• Synaptic Vesicle Exocytosis COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Suedhof, T. C., Rizo, J. 2011; 3 (12)

  Abstract

  Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for "soluble NSF-attachment protein receptor") and SM (for "Sec1/Munc18-like") proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for "N-ethylmaleimide sensitive factor"). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the "active zone") that includes Munc13 and RIM proteins as central components.

  View details for DOI 10.1101/cshperspect.a005637

  View details for PubMedID 22026965

• Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons CELL STEM CELL Marro, S., Pang, Z. P., Yang, N., Tsai, M., Qu, K., Chang, H. Y., Suedhof, T. C., Wernig, M. 2011; 9 (4): 374-382

  Abstract

  Several recent studies have showed that mouse and human fibroblasts can be directly reprogrammed into induced neuronal (iN) cells, bypassing a pluripotent intermediate state. However, fibroblasts represent heterogeneous mesenchymal progenitor cells that potentially contain neural crest lineages, and the cell of origin remained undefined. This raises the fundamental question of whether lineage reprogramming is possible between cell types derived from different germ layers. Here, we demonstrate that terminally differentiated hepatocytes can be directly converted into functional iN cells. Importantly, single-cell and genome-wide expression analyses showed that fibroblast- and hepatocyte-derived iN cells not only induced a neuronal transcriptional program, but also silenced their donor transcriptome. The remaining donor signature decreased over time and could not support functional hepatocyte properties. Thus, the reprogramming factors lead to a binary lineage switch decision rather than an induction of hybrid phenotypes, but iN cells retain a small but detectable epigenetic memory of their donor cells.

  View details for DOI 10.1016/j.stem.2011.09.002

  View details for PubMedID 21962918

• The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Soler-Llavina, G. J., Fuccillo, M. V., Ko, J., Suedhof, T. C., Malenka, R. C. 2011; 108 (40): 16502-16509

  Abstract

  Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.

  View details for DOI 10.1073/pnas.1114028108

  View details for PubMedID 21953696

• Rab3B protein is required for long-term depression of hippocampal inhibitory synapses and for normal reversal learning PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tsetsenis, T., Younts, T. J., Chiu, C. Q., Kaeser, P. S., Castillo, P. E., Suedhof, T. C. 2011; 108 (34): 14300-14305

  Abstract

  Rab3B, similar to other Rab3 isoforms, is a synaptic vesicle protein that interacts with the Rab3-interacting molecule (RIM) isoforms RIM1α and RIM2α as effector proteins in a GTP-dependent manner. Previous studies showed that at excitatory synapses, Rab3A and RIM1α are essential for presynaptically expressed long-term potentiation (LTP), whereas at inhibitory synapses RIM1α is required for endocannabinoid-dependent long-term depression (referred to as "i-LTD"). However, it remained unknown whether i-LTD also involves a Rab3 isoform and whether i-LTD, similar to other forms of long-term plasticity, is important for learning and memory. Here we show that Rab3B is highly enriched in inhibitory synapses in the CA1 region of the hippocampus. Using electrophysiological recordings in acute slices, we demonstrate that knockout (KO) of Rab3B does not alter the strength or short-term plasticity of excitatory or inhibitory synapses but does impair i-LTD significantly without changing classical NMDA receptor-dependent LTP. Behaviorally, we found that Rab3B KO mice exhibit no detectable changes in all basic parameters tested, including the initial phase of learning and memory. However, Rab3B KO mice did display a selective enhancement in reversal learning, as measured using Morris water-maze and fear-conditioning assays. Our data support the notion that presynaptic forms of long-term plasticity at excitatory and inhibitory synapses generally are mediated by a common Rab3/RIM-dependent pathway, with various types of synapses using distinct Rab3 isoforms. Moreover, our results suggest that i-LTD contributes to learning and memory, presumably by stabilizing circuits established in previous learning processes.

  View details for DOI 10.1073/pnas.1112237108

  View details for PubMedID 21844341

• Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Etherton, M., Foeldy, C., Sharma, M., Tabuchi, K., Liu, X., Shamloo, M., Malenka, R. C., Suedhof, T. C. 2011; 108 (33): 13764-13769

  Abstract

  Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3(R451C) knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3(R451C) mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3(R451C) mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3(R451C) mutation caused an ∼1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3(R451C) mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3(R451C) mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

  View details for DOI 10.1073/pnas.1111093108

  View details for PubMedID 21808020

• Induction of human neuronal cells by defined transcription factors NATURE Pang, Z. P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D. R., Yang, T. Q., Citri, A., Sebastiano, V., Marro, S., Suedhof, T. C., Wernig, M. 2011; 476 (7359): 220-U122

  Abstract

  Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

  View details for DOI 10.1038/nature10202

  View details for PubMedID 21617644

• Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons JOURNAL OF CELL BIOLOGY Ko, J., Soler-Llavina, G. J., Fuccillo, M. V., Malenka, R. C., Suedhof, T. C. 2011; 194 (2): 323-334

  Abstract

  Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. In this paper, we show that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an ∼40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. Our results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway.

  View details for DOI 10.1083/jcb.201101072

  View details for PubMedID 21788371

• An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus EMBO JOURNAL Etherton, M. R., Tabuchi, K., Sharma, M., Ko, J., Suedhof, T. C. 2011; 30 (14): 2908-2919

  Abstract

  Neuroligins are evolutionarily conserved postsynaptic cell-adhesion molecules that function, at least in part, by forming trans-synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino-acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin-4. We introduced the R704C mutation into mouse neuroligin-3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin-3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor-mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor-mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin-3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.

  View details for DOI 10.1038/emboj.2011.182

  View details for PubMedID 21642956

• Doc2 Supports Spontaneous Synaptic Transmission by a Ca2+-Independent Mechanism NEURON Pang, Z. P., Bacaj, T., Yang, X., Zhou, P., Xu, W., Suedhof, T. C. 2011; 70 (2): 244-251

  Abstract

  Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2 family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2 knockdown in wild-type synapses is fully reversed by mutant Doc2B lacking Ca(2+)-binding sites. Thus, our data suggest that Doc2s are modulators of spontaneous synaptic transmission that act by a Ca(2+)-independent mechanism.

  View details for DOI 10.1016/j.neuron.2011.03.011

  View details for PubMedID 21521611

• Activity-Dependent IGF-1 Exocytosis Is Controlled by the Ca2+-Sensor Synaptotagmin-10 CELL Cao, P., Maximov, A., Suedhof, T. C. 2011; 145 (2): 300-311

  Abstract

  Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca(2+)-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca(2+)-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 colocalized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca(2+)-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca(2+)-dependent exocytosis that is spatially and temporally distinct from Ca(2+)-dependent synaptic vesicle exocytosis controlled by Syt1. Our findings thereby reveal that two different synaptotagmins can regulate functionally distinct Ca(2+)-dependent membrane fusion reactions in the same neuron.

  View details for DOI 10.1016/j.cell.2011.03.034

  View details for PubMedID 21496647

• Cerebellins meet neurexins (Commentary on Matsuda & Yuzaki) EUROPEAN JOURNAL OF NEUROSCIENCE Martinelli, D. C., Suedhof, T. C. 2011; 33 (8): 1445-1446

  View details for DOI 10.1111/j.1460-9568.2011.07665.x

  View details for PubMedID 21496123

• The cell-adhesion G protein-coupled receptor BAI3 is a high-affinity receptor for C1q-like proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bolliger, M. F., Martinelli, D. C., Suedhof, T. C. 2011; 108 (6): 2534-2539

  Abstract

  C1q-like genes (C1ql1-C1ql4) encode small, secreted proteins that are expressed in differential patterns in the brain but whose receptors and functions remain unknown. BAI3 protein, in contrast, is a member of the cell-adhesion class of G protein-coupled receptors that are expressed at high levels in the brain but whose ligands have thus far escaped identification. Using a biochemical approach, we show that all four C1ql proteins bind to the extracellular thrombospondin-repeat domain of BAI3 with high affinity, and that this binding is mediated by the globular C1q domains of the C1ql proteins. Moreover, we demonstrate that addition of submicromolar concentrations of C1ql proteins to cultured neurons causes a significant decrease in synapse density, and that this decrease was prevented by simultaneous addition of the thrombospondin-repeat fragment of BAI3, which binds to C1ql proteins. Our data suggest that C1ql proteins are secreted signaling molecules that bind to BAI3 and act, at least in part, to regulate synapse formation and/or maintenance.

  View details for DOI 10.1073/pnas.1019577108

  View details for PubMedID 21262840

• Delayed onset of hyperglycaemia in a mouse model with impaired glucagon secretion demonstrates that dysregulated glucagon secretion promotes hyperglycaemia and type 2 diabetes DIABETOLOGIA Gustavsson, N., SEAH, T., Lao, Y., Radda, G. K., SUEDHOF, T. C., Han, W. 2011; 54 (2): 415-422

  Abstract

  Type 2 diabetes is caused by relative deficiency of insulin secretion and is associated with dysregulation of glucagon secretion during the late stage of diabetes development. Like insulin secretion from beta cells, glucagon secretion is dependent on calcium signals and a calcium sensing protein, synaptotagmin-7. In this study, we tested the relative contribution of dysregulated glucagon secretion and reduced insulin release in the development of hyperglycaemia and type 2 diabetes by using synaptotagmin-7 knockout (KO) mice, which exhibit glucose intolerance, reduced insulin secretion and nearly abolished Ca(2+)-stimulated glucagon secretion.We fed the synaptotagmin-7 KO and control mice with a high-fat diet (HFD) for 14 weeks, and compared their body weight, glucose levels, glucose and insulin tolerance, and insulin and glucagon secretion.On the HFD, synaptotagmin-7 KO mice showed progressive impairment of glucose tolerance and insulin secretion, along with continued maintenance of a low glucagon level. The control mice were less affected in terms of glucose intolerance, and showed enhanced insulin secretion with a concurrent increase in glucagon levels. Unexpectedly, after 14 weeks of HFD feeding, only the control mice displayed resting hyperglycaemia, whereas in synaptotagmin-7 KO mice defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion.Our data uncover a previously overlooked role of dysregulated glucagon secretion in promoting hyperglycaemia and the ensuing diabetes, and strongly suggest maintenance of adequate regulation of glucagon secretion as an important therapeutic target in addition to the preservation of beta cell function and mass in the prevention and treatment of diabetes.

  View details for DOI 10.1007/s00125-010-1950-2

  View details for Web of Science ID 000286001800027

  View details for PubMedID 20978738

• RIM Proteins Activate Vesicle Priming by Reversing Autoinhibitory Homodimerization of Munc13 NEURON Deng, L., Kaeser, P. S., Xu, W., Suedhof, T. C. 2011; 69 (2): 317-331

  Abstract

  At a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active zone scaffolding molecules that--among others--mediate vesicle priming and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn²+ finger domain of RIMs binds to the C₂A domain of the priming factor Munc13, which forms a homodimer in the absence of RIM but a heterodimer with it. Here, we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought but by directly activating Munc13. Specifically, we found that the isolated Zn²+ finger domain of RIMs autonomously promoted vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.

  View details for DOI 10.1016/j.neuron.2011.01.005

  View details for PubMedID 21262469

• RIM Determines Ca2+ Channel Density and Vesicle Docking at the Presynaptic Active Zone NEURON Han, Y., Kaeser, P. S., Suedhof, T. C., Schneggenburger, R. 2011; 69 (2): 304-316

  Abstract

  At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca²+ channels close to docked vesicles. The mechanisms that enrich Ca²+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knockout approach at a presynaptically accessible central nervous system synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca²+ channel density, revealing a role of RIM proteins in Ca²+ channel targeting. Removal of RIMs also reduced the readily releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca²+ channel-vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release, enabling a high presynaptic Ca²+ channel density and vesicle docking at the active zone.

  View details for DOI 10.1016/j.neuron.2010.12.014

  View details for Web of Science ID 000286792900013

  View details for PubMedID 21262468

  View details for PubMedCentralID PMC3259453

• RIM Proteins Tether Ca2+ Channels to Presynaptic Active Zones via a Direct PDZ-Domain Interaction CELL Kaeser, P. S., Deng, L., Wang, Y., Dulubova, I., Liu, X., Rizo, J., Suedhof, T. C. 2011; 144 (2): 282-295

  Abstract

  At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.

  View details for DOI 10.1016/j.cell.2010.12.029

  View details for PubMedID 21241895

• CSP alpha promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity NATURE CELL BIOLOGY Sharma, M., Burre, J., Suedhof, T. C. 2011; 13 (1): 30-U74

  Abstract

  A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα-Hsc70-SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.

  View details for DOI 10.1038/ncb2131

  View details for PubMedID 21151134

• Activity-dependent synapse validation by neurexin ligands, LRRTMs and neuroligins Ko, J., Sudhof, T. C. ELSEVIER IRELAND LTD. 2011: E17–E18

  View details for DOI 10.1016/j.neures.2011.07.071

  View details for Web of Science ID 000308218100068

• Testing the SNARE/SM protein model of membrane fusion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bacaj, T., Pang, Z. P., Suedhof, T. C. 2010; 107 (52): 22365-22366

  View details for DOI 10.1073/pnas.1017268108

  View details for PubMedID 21169506

• Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory alpha Helix NEURON Yang, X., Kaeser-Woo, Y. J., Pang, Z. P., Xu, W., Suedhof, T. C. 2010; 68 (5): 907-920

  Abstract

  Complexin activates and clamps neurotransmitter release; impairing complexin function decreases synchronous, but increases spontaneous and asynchronous synaptic vesicle exocytosis. Here, we show that complexin-different from the Ca(2+) sensor synaptotagmin-1-activates synchronous exocytosis by promoting synaptic vesicle priming, but clamps spontaneous and asynchronous exocytosis-similar to synaptotagmin-1-by blocking a secondary Ca(2+) sensor. Activation and clamping functions of complexin depend on distinct, autonomously acting sequences, namely its N-terminal region and accessory α helix, respectively. Mutations designed to test whether the accessory α helix of complexin clamps exocytosis by inserting into SNARE-complexes support this hypothesis, suggesting that the accessory α helix blocks completion of trans-SNARE-complex assembly until Ca(2+) binding to synaptotagmin relieves this block. Moreover, a juxtamembranous mutation in the SNARE-protein synaptobrevin-2, which presumably impairs force transfer from nascent trans-SNARE complexes onto fusing membranes, also unclamps spontaneous fusion by disinhibiting a secondary Ca(2+) sensor. Thus, complexin performs mechanistically distinct activation and clamping functions that operate in conjunction with synaptotagmin-1 by controlling trans-SNARE-complex assembly.

  View details for DOI 10.1016/j.neuron.2010.11.001

  View details for PubMedID 21145004

• SynCAM 1 Adhesion Dynamically Regulates Synapse Number and Impacts Plasticity and Learning NEURON Robbins, E. M., Krupp, A. J., de Arce, K. P., Ghosh, A. K., Fogel, A. I., Boucard, A., Suedhof, T. C., Stein, V., Biederer, T. 2010; 68 (5): 894-906

  Abstract

  Synaptogenesis is required for wiring neuronal circuits in the developing brain and continues to remodel adult networks. However, the molecules organizing synapse development and maintenance in vivo remain incompletely understood. We now demonstrate that the immunoglobulin adhesion molecule SynCAM 1 dynamically alters synapse number and plasticity. Overexpression of SynCAM 1 in transgenic mice promotes excitatory synapse number, while loss of SynCAM 1 results in fewer excitatory synapses. By turning off SynCAM 1 overexpression in transgenic brains, we show that it maintains the newly induced synapses. SynCAM 1 also functions at mature synapses to alter their plasticity by regulating long-term depression. Consistent with these effects on neuronal connectivity, SynCAM 1 expression affects spatial learning, with knock-out mice learning better. The reciprocal effects of increased SynCAM 1 expression and loss reveal that this adhesion molecule contributes to the regulation of synapse number and plasticity, and impacts how neuronal networks undergo activity-dependent changes.

  View details for DOI 10.1016/j.neuron.2010.11.003

  View details for Web of Science ID 000285664900011

  View details for PubMedID 21145003

  View details for PubMedCentralID PMC3026433

• Inactivation of presenilins causes pre-synaptic impairment prior to post-synaptic dysfunction JOURNAL OF NEUROCHEMISTRY Zhang, D., Zhang, C., Ho, A., Kirkwood, A., Suedhof, T. C., Shen, J. 2010; 115 (5): 1215-1221

  Abstract

  J. Neurochem. (2010) 115, 1215-1221. ABSTRACT: Synaptic dysfunction is widely thought to be a pathogenic precursor to neurodegeneration in Alzheimer's disease (AD), and the extent of synaptic loss provides the best correlate for the severity of dementia in AD patients. Presenilins 1 and 2 are the major causative genes of early-onset familial AD. Conditional inactivation of presenilins in the adult cerebral cortex results in synaptic dysfunction and memory impairment, followed by age-dependent neurodegeneration. To characterize further the consequence of presenilin inactivation in the synapse, we evaluated the temporal development of pre-synaptic and post-synaptic deficits in the Schaeffer-collateral pathway of presenilin conditional double knockout (PS cDKO) mice prior to onset of neurodegeneration. Following presenilin inactivation at 4 weeks, synaptic facilitation and probability of neurotransmitter release are impaired in PS cDKO mice at 5 weeks of age, whereas post-synaptic NMDA receptor (NMDAR)-mediated responses are normal at 5 weeks but impaired at 6 weeks of age. Long-term potentiation induced by theta burst stimulation is also reduced in PS cDKO mice at 6 weeks of age. These results show that loss of presenilins results in pre-synaptic deficits in short-term plasticity and probability of neurotransmitter release prior to post-synaptic NMDAR dysfunction, raising the possibility that presenilins may regulate post-synaptic NMDAR function in part via a trans-synaptic mechanism.

  View details for DOI 10.1111/j.1471-4159.2010.07011.x

  View details for Web of Science ID 000283951300013

  View details for PubMedID 20854432

  View details for PubMedCentralID PMC2972413

• Neuronal Calcium Sensor Synaptotagmin-9 Is Not Involved in the Regulation of Glucose Homeostasis or Insulin Secretion PLOS ONE Gustavsson, N., Wang, X., Wang, Y., Seah, T., Xu, J., Radda, G. K., Suedhof, T. C., Han, W. 2010; 5 (11)

  Abstract

  Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7.In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice.Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic β-cells.

  View details for DOI 10.1371/journal.pone.0015414

  View details for Web of Science ID 000284035900029

  View details for PubMedID 21085706

  View details for PubMedCentralID PMC2976867

• Push-and-pull regulation of the fusion pore by synaptotagmin-7 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segovia, M., Ales, E., Angeles Montes, M., Bonifas, I., Jemal, I., Lindau, M., Maximov, A., Suedhof, T. C., de Toledo, G. A. 2010; 107 (44): 19032-19037

  Abstract

  In chromaffin cells, Ca(2+) binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca(2+) and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca(2+) binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca(2+) binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca(2+) binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

  View details for DOI 10.1073/pnas.1014070107

  View details for Web of Science ID 000283749000054

  View details for PubMedID 20956309

  View details for PubMedCentralID PMC2973888

• Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells TRAFFIC Schonn, J., van Weering, J. R., Mohrmann, R., Schlueter, O. M., Suedhof, T. C., de Wit, H., Verhage, M., Sorensen, J. B. 2010; 11 (11): 1415-1428

  Abstract

  The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD(-/-) ) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense-core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD(-/-) cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short-term (4-6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.

  View details for DOI 10.1111/j.1600-0854.2010.01107.x

  View details for Web of Science ID 000282571500004

  View details for PubMedID 20716109

• Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons JOURNAL OF BIOLOGICAL CHEMISTRY Pang, Z. P., Xu, W., Cao, P., Suedhof, T. C. 2010; 285 (44): 33930-33939

  Abstract

  Calmodulin (CaM) is a ubiquitous Ca(2+) sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca(2+) binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca(2+) binding to an unidentified secondary Ca(2+) sensor. To test whether CaM could be the enigmatic Ca(2+) sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca(2+) binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca(2+)/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.

  View details for DOI 10.1074/jbc.M110.150151

  View details for PubMedID 20729199

• QnAs with Thomas C. Südhof. Interview by Prashant Nair. Proceedings of the National Academy of Sciences of the United States of America Südhof, T. C. 2010; 107 (42): 17863-?

  View details for DOI 10.1073/pnas.1012628107

  View details for PubMedID 20855597

• Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, H., Wang, B., Wang, Z., Guo, Q., Tabuchi, K., Hammer, R. E., Suedhof, T. C., Zheng, H. 2010; 107 (40): 17362-17367

  Abstract

  Amyloidogenic processing of the amyloid precursor protein (APP) generates a large secreted ectodomain fragment (APPsβ), β-amyloid (Aβ) peptides, and an APP intracellular domain (AICD). Whereas Aβ is viewed as critical for Alzheimer's disease pathogenesis, the role of other APP processing products remains enigmatic. Of interest, the AICD has been implicated in transcriptional regulation, and N-terminal cleavage of APPsβ has been suggested to produce an active fragment that may mediate axonal pruning and neuronal cell death. We previously reported that mice deficient in APP and APP-like protein 2 (APLP2) exhibit early postnatal lethality and neuromuscular synapse defects, whereas mice with neuronal conditional deletion of APP and APLP2 are viable. Using transcriptional profiling, we now identify transthyretin (TTR) and Klotho as APP/APLP2-dependent genes whose expression is decreased in loss-of-function states but increased in gain-of-function states. Significantly, by creating an APP knockin allele that expresses only APPsβ protein, we demonstrate that APPsβ is not normally cleaved in vivo and is fully capable of mediating the APP-dependent regulation of TTR and Klotho gene expression. Despite being an active regulator of gene expression, APPsβ did not rescue the lethality and neuromuscular synapse defects of APP and APLP2 double-KO animals. Our studies identify TTR and Klotho as physiological targets of APP that are regulated by soluble APPsβ independent of developmental APP functions. This unexpected APP-mediated signaling pathway may play an important role in maintaining TTR and Klotho levels and their respective functions in Aβ sequestration and aging.

  View details for DOI 10.1073/pnas.1012568107

  View details for Web of Science ID 000282512000057

  View details for PubMedID 20855613

  View details for PubMedCentralID PMC2951422

• Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis JOURNAL OF BIOLOGICAL CHEMISTRY Li, H., Wang, Z., Wang, B., Guo, Q., Dolios, G., Tabuchi, K., Hammer, R. E., Suedhof, T. C., Wang, R., Zheng, H. 2010; 285 (40): 30598-30605

  Abstract

  Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.

  View details for DOI 10.1074/jbc.P110.137729

  View details for Web of Science ID 000282135500028

  View details for PubMedID 20693289

  View details for PubMedCentralID PMC2945554

• An interview with the Kavli prize winners. Interview by Claudia Wiedemann. Nature reviews. Neuroscience Rothman, J., Scheller, R., Südhof, T. 2010; 11 (10): 669-673

  View details for PubMedID 21080540

• alpha-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro SCIENCE Burre, J., Sharma, M., Tsetsenis, T., Buchman, V., Etherton, M. R., Suedhof, T. C. 2010; 329 (5999): 1663-1667

  Abstract

  Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we show that maintenance of continuous presynaptic SNARE-complex assembly required a nonclassical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) and promoted SNARE-complex assembly. Moreover, triple-knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and died prematurely. Thus, synucleins may function to sustain normal SNARE-complex assembly in a presynaptic terminal during aging.

  View details for DOI 10.1126/science.1195227

  View details for PubMedID 20798282

• Structural and Mutational Analysis of Functional Differentiation between Synaptotagmins-1 and-7 PLOS ONE Xue, M., Craig, T. K., Shin, O., Li, L., Brautigam, C. A., Tomchick, D. R., Suedhof, T. C., Rosenmund, C., Rizo, J. 2010; 5 (9)

  Abstract

  Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 A resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.

  View details for DOI 10.1371/journal.pone.0012544

  View details for Web of Science ID 000281480900025

  View details for PubMedID 20824061

  View details for PubMedCentralID PMC2932738

• Cell biology of Ca2+-triggered exocytosis CURRENT OPINION IN CELL BIOLOGY Pang, Z. P., Suedhof, T. C. 2010; 22 (4): 496-505

  Abstract

  Ca(2+) triggers many forms of exocytosis in different types of eukaryotic cells, for example synaptic vesicle exocytosis in neurons, granule exocytosis in mast cells, and hormone exocytosis in endocrine cells. Work over the past two decades has shown that synaptotagmins function as the primary Ca(2+)-sensors for most of these forms of exocytosis, and that synaptotagmins act via Ca(2+)-dependent interactions with both the fusing phospholipid membranes and the membrane fusion machinery. However, some forms of Ca(2+)-induced exocytosis may utilize other, as yet unidentified Ca(2+)-sensors, for example, slow synaptic exocytosis mediating asynchronous neurotransmitter release. In the following overview, we will discuss the synaptotagmin-based mechanism of Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells, and its potential extension to other types of Ca(2+)-stimulated exocytosis for which no synaptotagmin Ca(2+)-sensor has been identified.

  View details for DOI 10.1016/j.ceb.2010.05.001

  View details for PubMedID 20561775

• Neurexins Physically and Functionally Interact with GABA(A) Receptors NEURON Zhang, C., Atasoy, D., Arac, D., Yang, X., Fucillo, M. V., Robison, A. J., Ko, J., Brunger, A. T., Sudhof, T. C. 2010; 66 (3): 403-416

  Abstract

  Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in nonneuronal cells, neurexins induce formation of postsynaptic specializations in cocultured neurons, suggesting that neurexins are synaptogenic. However, we find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at submicromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABA(A) receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABA(A) receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABA(A) receptors, which may contribute to regulate the excitatory/inhibitory balance in brain.

  View details for DOI 10.1016/j.neuron.2010.04.008

  View details for PubMedID 20471353

• Climate Change and the Integrity of Science SCIENCE Gleick, P. H., ADAMS, R. M., Amasino, R. M., ANDERS, E., ANDERSON, D. J., Anderson, W. W., Anselin, L. E., Arroyo, M. K., Asfaw, B., Ayala, F. J., Bax, A., Bebbington, A. J., Bell, G., Bennett, M. V., Bennetzen, J. L., Berenbaum, M. R., Berlin, O. B., BJORKMAN, P. J., Blackburn, E., Blamont, J. E., Botchan, M. R., Boyer, J. S., Boyle, E. A., Branton, D., Briggs, S. P., Briggs, W. R., BRILL, W. J., Britten, R. J., Broecker, W. S., Brown, J. H., Brown, P. O., Brunger, A. T., Cairns, J., Canfield, D. E., Carpenter, S. R., Carrington, J. C., Cashmore, A. R., CASTILLA, J. C., Cazenave, A., Chapin, F. S., CIECHANOVER, A. J., Clapham, D. E., CLARK, W. C., Clayton, R. N., Coe, M. D., Conwell, E. M., Cowling, E. B., Cowling, R. M., Cox, C. S., Croteau, R. B., Crothers, D. M., Crutzen, P. J., Daily, G. C., Dalrymple, G. B., Dangl, J. L., Darst, S. A., Davies, D. R., Davis, M. B., De Camilli, P. V., Dean, C., DeFries, R. S., Deisenhofer, J., Delmer, D. P., DeLong, E. F., DeRosier, D. J., Diener, T. O., Dirzo, R., Dixon, J. E., Donoghue, M. J., Doolittle, R. F., Dunne, T., Ehrlich, P. R., Eisenstadt, S. N., Eisner, T., Emanuel, K. A., Englander, S. W., Ernst, W. G., Falkowski, P. G., Feher, G., FEREJOHN, J. A., Fersht, A., Fischer, E. H., Fischer, R., Flannery, K. V., Frank, J., Frey, P. A., Fridovich, I., FRIEDEN, C., Futuyma, D. J., Gardner, W. R., GARRETT, C. J., Gilbert, W., Goldberg, R. B., GOODENOUGH, W. H., Goodman, C. S., Goodman, M., Greengard, P., Hake, S., HAMMEL, G., Hanson, S., HARRISON, S. C., Hart, S. R., Hartl, D. L., Haselkorn, R., Hawkes, K., Hayes, J. M., Hille, B., Hoekfelt, T., House, J. S., HOUT, M., Hunten, D. M., Izquierdo, I. A., Jagendorf, A. T., Janzen, D. H., Jeanloz, R., Jencks, C. S., Jury, W. A., Kaback, H. R., KAILATH, T., Kay, P., Kay, S. A., Kennedy, D., Kerr, A., Kessler, R. C., Khush, G. S., Kieffer, S. W., Kirch, P. V., Kirk, K., Kivelson, M. G., Klinman, J. P., Klug, A., Knopoff, L., Kornberg, H., Kutzbach, J. E., Lagarias, J. C., Lambeck, K., Landy, A., Langmuir, C. H., Larkins, B. A., Le Pichon, X. T., Lenski, R. E., Leopold, E. B., Levin, S. A., Levitt, M., Likens, G. E., Lippincott-Schwartz, J., Lorand, L., Lovejoy, C. O., Lynch, M., Mabogunje, A. L., Malone, T. F., Manabe, S., Marcus, J., Massey, D. S., McWilliams, J. C., Medina, E., Melosh, H. J., Meltzer, D. J., Michener, C. D., Miles, E. L., Mooney, H. A., Moore, P. B., Morel, F. M., Mosley-Thompson, E. S., Moss, B., Munk, W. H., Myers, N., Nair, G. B., Nathans, J., Nester, E. W., Nicoll, R. A., Novick, R. P., O'Connell, J. F., Olsen, P. E., Opdyke, N. D., Oster, G. F., Ostrom, E., Pace, N. R., Paine, R. T., Palmiter, R. D., Pedlosky, J., Petsko, G. A., Pettengill, G. H., Philander, S. G., Piperno, D. R., Pollard, T. D., Price, P. B., REICHARD, P. A., Reskin, B. F., Ricklefs, R. E., Rivest, R. L., Roberts, J. D., Romney, A. K., Rossmann, M. G., Russell, D. W., Rutter, W. J., Sabloff, J. A., SAGDEEV, R. Z., Sahlins, M. D., Salmond, A., Sanes, J. R., Schekman, R., Schellnhuber, J., Schindler, D. W., Schmitt, J., Schneider, S. H., Schramm, V. L., SEDEROFF, R. R., Shatz, C. J., Sherman, F., Sidman, R. L., Sieh, K., Simons, E. L., Singer, B. H., Singer, M. F., Skyrms, B., Sleep, N. H., Smith, B. D., Snyder, S. H., Sokal, R. R., Spencer, C. S., Steitz, T. A., Strier, K. B., SUEDHOF, T. C., Taylor, S. S., Terborgh, J., Thomas, D. H., Thompson, L. G., Jian, R. T., Turner, M. G., Uyeda, S., Valentine, J. W., Valentine, J. S., Van Etten, J. L., van Holde, K. E., Vaughan, M., Verba, S., von Hippel, P. H., Wake, D. B., Walker, A., Walker, J. E., Watson, E. B., Watson, P. J., Weigel, D., Wessler, S. R., West-Eberhard, M. J., White, T. D., Wilson, W. J., Wolfenden, R. V., Wood, J. A., Woodwell, G. M., Wright, H. E., Wu, C., Wunsch, C., Zoback, M. L. 2010; 328 (5979): 689-690

  View details for Web of Science ID 000277357100011

  View details for PubMedID 20448167

• Evolution of CASK into a Mg2+-Sensitive Kinase SCIENCE SIGNALING Mukherjee, K., Sharma, M., Jahn, R., Wahl, M. C., Suedhof, T. C. 2010; 3 (119)

  Abstract

  All known protein kinases, except CASK [calcium/calmodulin (CaM)-activated serine-threonine kinase], require magnesium ions (Mg(2+)) to stimulate the transfer of a phosphate from adenosine 5'-triphosphate (ATP) to a protein substrate. The CaMK (calcium/calmodulin-dependent kinase) domain of CASK shows activity in the absence of Mg(2+); indeed, it is inhibited by divalent ions including Mg(2+). Here, we converted the Mg(2+)-inhibited wild-type CASK kinase (CASK(WT)) into a Mg(2+)-stimulated kinase (CASK(4M)) by substituting four residues within the ATP-binding pocket. Crystal structures of CASK(4M) with and without bound nucleotide and Mn(2+), together with kinetic analyses, demonstrated that Mg(2+) accelerates catalysis of CASK(4M) by stabilizing the transition state, enhancing the leaving group properties of adenosine 5'-diphosphate, and indirectly shifting the position of the gamma-phosphate of ATP. Phylogenetic analysis revealed that the four residues conferring Mg(2+)-mediated stimulation were substituted from CASK during early animal evolution, converting a primordial, Mg(2+)-coordinating form of CASK into a Mg(2+)-inhibited kinase. This emergence of Mg(2+) sensitivity (inhibition by Mg(2+)) conferred regulation of CASK activity by divalent cations, in parallel with the evolution of the animal nervous systems.

  View details for DOI 10.1126/scisignal.2000800

  View details for PubMedID 20424264

• RIM1 alpha and Interacting Proteins Involved in Presynaptic Plasticity Mediate Prepulse Inhibition and Additional Behaviors Linked to Schizophrenia JOURNAL OF NEUROSCIENCE Blundell, J., Kaeser, P. S., Sudhof, T. C., Powell, C. M. 2010; 30 (15): 5326-5333

  Abstract

  Several presynaptic proteins involved in neurotransmitter release in the CNS have been implicated in schizophrenia in human clinical genetic studies, in postmortem studies, and in studies of putative animal models of schizophrenia. The presynaptic protein RIM1alpha mediates presynaptic plasticity and cognitive function. We now demonstrate that mice deficient in RIM1alpha exhibit abnormalities in multiple schizophrenia-relevant behavioral tasks including prepulse inhibition, response to psychotomimetic drugs, and social interaction. These schizophrenia-relevant behavioral findings are relatively selective to RIM1alpha-deficient mice, as mice bearing mutations in the RIM1alpha binding partners Rab3A or synaptotagmin 1 only show decreased prepulse inhibition. In addition to RIM1alpha's involvement in multiple behavioral abnormalities, these data suggest that alterations in presynaptic forms of short-term plasticity are linked to alterations in prepulse inhibition, a measure of sensorimotor gating.

  View details for DOI 10.1523/JNEUROSCI.0328-10.2010

  View details for Web of Science ID 000276685100020

  View details for PubMedID 20392954

  View details for PubMedCentralID PMC2860606

• Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mukherjee, K., Yang, X., Gerber, S. H., Kwon, H., Ho, A., Castillo, P. E., Liu, X., Suedhof, T. C. 2010; 107 (14): 6504-6509

  Abstract

  Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

  View details for DOI 10.1073/pnas.1002307107

  View details for PubMedID 20332206

• Calmodulin Controls Synaptic Strength via Presynaptic Activation of Calmodulin Kinase II JOURNAL OF NEUROSCIENCE Pang, Z. P., Cao, P., Xu, W., Suedhof, T. C. 2010; 30 (11): 4132-4142

  Abstract

  Calmodulin regulates multifarious cellular processes via a panoply of target interactions. However, the central role, multiple isoforms, and complex target interactions of calmodulin make it difficult to examine its precise functions. Here, we analyzed calmodulin function in neurons using lentivirally delivered short-hairpin RNAs that suppressed expression of all calmodulin isoforms by approximately 70%. Calmodulin knockdown did not significantly alter neuronal survival or synapse formation but depressed spontaneous neuronal network activity. Strikingly, calmodulin knockdown decreased the presynaptic release probability almost twofold, without altering the presynaptic readily-releasable vesicle pool or postsynaptic neurotransmitter reception. In calmodulin knockdown neurons, presynaptic release was restored to wild-type levels by expression of constitutively active calmodulin-dependent kinase-IIalpha (CaMKIIalpha); in contrast, in control neurons, expression of constitutively active CaMKIIalpha had no effect on presynaptic release. Viewed together, these data suggest that calmodulin performs a major function in boosting synaptic strength via direct activation of presynaptic calmodulin-dependent kinase II.

  View details for DOI 10.1523/JNEUROSCI.3129-09.2010

  View details for PubMedID 20237283

• Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis NATURE STRUCTURAL & MOLECULAR BIOLOGY Shin, O., Lu, J., Rhee, J., Tomchick, D. R., Pang, Z. P., Wojcik, S. M., Camacho-Perez, M., Brose, N., Machius, M., Rizo, J., Rosenmund, C., Suedhof, T. C. 2010; 17 (3): 280-U42

  Abstract

  Munc13 is a multidomain protein present in presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here we use biophysical, biochemical and electrophysiological approaches to show that the central C(2)B domain of Munc13 functions as a Ca(2+) regulator of short-term synaptic plasticity. The crystal structure of the C(2)B domain revealed an unusual Ca(2+)-binding site with an amphipathic alpha-helix. This configuration confers onto the C(2)B domain unique Ca(2+)-dependent phospholipid-binding properties that favor phosphatidylinositolphosphates. A mutation that inactivated Ca(2+)-dependent phospholipid binding to the C(2)B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress release evoked by action-potential trains. In contrast, a mutation that increased Ca(2+)-dependent phosphatidylinositolbisphosphate binding to the C(2)B domain enhanced release evoked by isolated action potentials and by action-potential trains. Our data suggest that, during repeated action potentials, Ca(2+) and phosphatidylinositolphosphate binding to the Munc13 C(2)B domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

  View details for DOI 10.1038/nsmb.1758

  View details for Web of Science ID 000275182700006

  View details for PubMedID 20154707

  View details for PubMedCentralID PMC2916016

• Direct conversion of fibroblasts to functional neurons by defined factors NATURE Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Suedhof, T. C., Wernig, M. 2010; 463 (7284): 1035-U50

  Abstract

  Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

  View details for DOI 10.1038/nature08797

  View details for PubMedID 20107439

• Neuroligin-1 Deletion Results in Impaired Spatial Memory and Increased Repetitive Behavior JOURNAL OF NEUROSCIENCE Blundell, J., Blaiss, C. A., Etherton, M. R., Espinosa, F., Tabuchi, K., Walz, C., Bolliger, M. F., Suedhof, T. C., Powell, C. M. 2010; 30 (6): 2115-2129

  Abstract

  Neuroligins (NLs) are a family of neural cell-adhesion molecules that are involved in excitatory/inhibitory synapse specification. Multiple members of the NL family (including NL1) and their binding partners have been linked to cases of human autism and mental retardation. We have now characterized NL1-deficient mice in autism- and mental retardation-relevant behavioral tasks. NL1 knock-out (KO) mice display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation. In addition, NL1 KO mice exhibit a dramatic increase in repetitive, stereotyped grooming behavior, a potential autism-relevant abnormality. This repetitive grooming abnormality in NL1 KO mice is associated with a reduced NMDA/AMPA ratio at corticostriatal synapses. Interestingly, we further demonstrate that the increased repetitive grooming phenotype can be rescued in adult mice by administration of the NMDA receptor partial coagonist d-cycloserine. Broadly, these data are consistent with a role of synaptic cell-adhesion molecules in general, and NL1 in particular, in autism and implicate reduced excitatory synaptic transmission as a potential mechanism and treatment target for repetitive behavioral abnormalities.

  View details for DOI 10.1523/JNEUROSCI.4517-09.2010

  View details for Web of Science ID 000274398200013

  View details for PubMedID 20147539

  View details for PubMedCentralID PMC2824441

• beta-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC PLOS PATHOGENS Barbu, E. M., Ganesh, V. K., Gurusiddappa, S., Mackenzie, R. C., Foster, T. J., Sudhof, T. C., Hoeoek, M. 2010; 6 (1)

  Abstract

  Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in beta-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the beta-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing beta-neurexin on their surface. Taken together, our experimental results demonstrate that beta-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N(2)N(3) domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed.

  View details for DOI 10.1371/journal.ppat.1000726

  View details for Web of Science ID 000274227100017

  View details for PubMedID 20090838

  View details for PubMedCentralID PMC2800189

• LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation NEURON Ko, J., Fuccillo, M. V., Malenka, R. C., Suedhof, T. C. 2009; 64 (6): 791-798

  Abstract

  Recently, leucine-rich repeat transmembrane proteins (LRRTMs) were found to be synaptic cell-adhesion molecules that, when expressed in nonneuronal cells, induce presynaptic differentiation in contacting axons. We now demonstrate that LRRTM2 induces only excitatory synapses, and that it also acts to induce synapses in transfected neurons similarly to neuroligin-1. Using affinity chromatography, we identified alpha- and beta-neurexins as LRRTM2 ligands, again rendering LRRTM2 similar to neuroligin-1. However, whereas neuroligins bind neurexins containing or lacking an insert in splice site #4, LRRTM2 only binds neurexins lacking an insert in splice site #4. Binding of neurexins to LRRTM2 can produce cell-adhesion junctions, consistent with a trans-interaction regulated by neurexin alternative splicing, and recombinant neurexin-1beta blocks LRRTM2's ability to promote presynaptic differentiation. Thus, our data suggest that two unrelated postsynaptic cell-adhesion molecules, LRRTMs and neuroligins, unexpectedly bind to neurexins as the same presynaptic receptor, but that their binding is subject to distinct regulatory mechanisms.

  View details for DOI 10.1016/j.neuron.2009.12.012

  View details for PubMedID 20064387

• Neuroligin-2 Deletion Selectively Decreases Inhibitory Synaptic Transmission Originating from Fast-Spiking but Not from Somatostatin-Positive Interneurons JOURNAL OF NEUROSCIENCE Gibson, J. R., Huber, K. M., Suedhof, T. C. 2009; 29 (44): 13883-13897

  Abstract

  Neuroligins are cell adhesion molecules involved in synapse formation and/or function. Neurons express four neuroligins (NL1-NL4), of which NL1 is specific to excitatory and NL2 to inhibitory synapses. Excitatory and inhibitory synapses include numerous subtypes. However, it is unknown whether NL1 performs similar functions in all excitatory and NL2 in all inhibitory synapses, or whether they regulate the formation and/or function of specific subsets of synapses. To address this central question, we performed paired recordings in primary somatosensory cortex of mice lacking NL1 or NL2. Using this system, we examined neocortical microcircuits formed by reciprocal synapses between excitatory neurons and two subtypes of inhibitory interneurons, namely, fast-spiking and somatostatin-positive interneurons. We find that the NL1 deletion had little effect on inhibitory synapses, whereas the NL2 deletion decreased (40-50%) the unitary (cell-to-cell) IPSC amplitude evoked from single fast-spiking interneurons. Strikingly, the NL2 deletion had no effect on IPSC amplitude evoked from single somatostatin-positive inhibitory interneurons. Moreover, the frequency of unitary synaptic connections between individual fast-spiking and somatostatin-positive interneurons and excitatory neurons was unchanged. The decrease in unitary IPSC amplitude originating from fast-spiking interneurons in NL2-deficient mice was due to a multiplicative and uniform downscaling of the amplitude distribution, which in turn was mediated by a decrease in both synaptic quantal amplitude and quantal content, the latter inferred from an increase in the coefficient of variation. Thus, NL2 is not necessary for establishing unitary inhibitory synaptic connections but is selectively required for "scaling up" unitary connections originating from a subset of interneurons.

  View details for DOI 10.1523/JNEUROSCI.2457-09.2009

  View details for Web of Science ID 000271471400015

  View details for PubMedID 19889999

  View details for PubMedCentralID PMC2814361

• ELKS2 alpha/CAST Deletion Selectively Increases Neurotransmitter Release at Inhibitory Synapses NEURON Kaeser, P. S., Deng, L., Chavez, A. E., Liu, X., Castillo, P. E., Suedhof, T. C. 2009; 64 (2): 227-239

  Abstract

  The presynaptic active zone is composed of a protein network that contains ELKS2alpha (a.k.a. CAST) as a central component. Here we demonstrate that in mice, deletion of ELKS2alpha caused a large increase in inhibitory, but not excitatory, neurotransmitter release, and potentiated the size, but not the properties, of the readily-releasable pool of vesicles at inhibitory synapses. Quantitative electron microscopy revealed that the ELKS2alpha deletion did not change the number of docked vesicles or other ultrastructural parameters of synapses, except for a small decrease in synaptic vesicle numbers. The ELKS2alpha deletion did, however, alter the excitatory/inhibitory balance and exploratory behaviors, possibly as a result of the increased synaptic inhibition. Thus, as opposed to previous studies indicating that ELKS2alpha is essential for mediating neurotransmitter release, our results suggest that ELKS2alpha normally restricts release and limits the size of the readily-releasable pool of synaptic vesicles at the active zone of inhibitory synapses.

  View details for DOI 10.1016/j.neuron.2009.09.019

  View details for PubMedID 19874790

• Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation EMBO JOURNAL Ko, J., Zhang, C., Arac, D., Boucard, A. A., Brunger, A. T., Suedhof, T. C. 2009; 28 (20): 3244-3255

  Abstract

  Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic alpha- and beta-neurexins. To test this hypothesis, we examined the functional effects of neuroligin-1 mutations that impair only alpha-neurexin binding, block both alpha- and beta-neurexin binding, or abolish neuroligin-1 dimerization. Abolishing alpha-neurexin binding abrogated neuroligin-induced generation of neuronal synapses onto transfected non-neuronal cells in the so-called artificial synapse-formation assay, even though beta-neurexin binding was retained. Thus, in this assay, neuroligin-1 induces apparent synapse formation by binding to presynaptic alpha-neurexins. In transfected neurons, however, neither alpha- nor beta-neurexin binding was essential for the ability of postsynaptic neuroligin-1 to dramatically increase synapse density, suggesting a neurexin-independent mechanism of synapse formation. Moreover, neuroligin-1 dimerization was not required for either the non-neuronal or the neuronal synapse-formation assay. Nevertheless, both alpha-neurexin binding and neuroligin-1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin-1 in transfected neurons. Thus, neuroligin-1 performs diverse synaptic functions by mechanisms that include as essential components of alpha-neurexin binding and neuroligin dimerization, but extend beyond these activities.

  View details for DOI 10.1038/emboj.2009.249

  View details for PubMedID 19730411

• Mouse neurexin-1 alpha deletion causes correlated electrophysiological and behavioral changes consistent with cognitive impairments PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Etherton, M. R., Blaiss, C. A., Powell, C. M., Suedhof, T. C. 2009; 106 (42): 17998-18003

  Abstract

  Deletions in the neurexin-1alpha gene were identified in large-scale unbiased screens for copy-number variations in patients with autism or schizophrenia. To explore the underlying biology, we studied the electrophysiological and behavioral phenotype of mice lacking neurexin-1alpha. Hippocampal slice physiology uncovered a defect in excitatory synaptic strength in neurexin-1alpha deficient mice, as revealed by a decrease in miniature excitatory postsynaptic current (EPSC) frequency and in the input-output relation of evoked postsynaptic potentials. This defect was specific for excitatory synaptic transmission, because no change in inhibitory synaptic transmission was observed in the hippocampus. Behavioral studies revealed that, compared with littermate control mice, neurexin-1alpha deficient mice displayed a decrease in prepulse inhibition, an increase in grooming behaviors, an impairment in nest-building activity, and an improvement in motor learning. However, neurexin-1alpha deficient mice did not exhibit any obvious changes in social behaviors or in spatial learning. Together, these data indicate that the neurexin-1alpha deficiency induces a discrete neural phenotype whose extent correlates, at least in part, with impairments observed in human patients.

  View details for DOI 10.1073/pnas.0910297106

  View details for PubMedID 19822762

• Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C-2 domains mediate neurotransmitter release PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shin, O., Xu, J., Rizo, J., Suedhof, T. C. 2009; 106 (38): 16469-16474

  Abstract

  Neurotransmitter release is triggered by cooperative Ca2+-binding to the Ca2+-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca2+ with similar properties and affinities. However, Ca2+ binding to the C2A domain is not required for release, whereas Ca2+ binding to the C2B domain is essential for release. We now demonstrate that despite its expendability, Ca2+-binding to the C2A domain significantly contributes to the overall triggering of neurotransmitter release, and determines its Ca2+ cooperativity. Biochemically, Ca2+ induces more tight binding of the isolated C2A domain than of the isolated C2B domain to standard liposomes composed of phosphatidylcholine and phosphatidylserine. However, here we show that surprisingly, the opposite holds true when the double C2A/B-domain fragment of synaptotagmin-1 is used instead of isolated C2 domains, and when liposomes containing a physiological lipid composition are used. Under these conditions, Ca2+ binding to the C2B domain but not the C2A domain becomes the primary determinant of phospholipid binding. Thus, the unique requirement for Ca2+ binding to the C2B domain for synaptotagmin-1 in Ca2+-triggered neurotransmitter release may be accounted for, at least in part, by the unusual phospholipid-binding properties of its double C2A/B-domain fragment.

  View details for DOI 10.1073/pnas.0908798106

  View details for Web of Science ID 000270071600084

  View details for PubMedID 19805322

  View details for PubMedCentralID PMC2752550

• A Neuroligin-4 Missense Mutation Associated with Autism Impairs Neuroligin-4 Folding and Endoplasmic Reticulum Export JOURNAL OF NEUROSCIENCE Zhang, C., Milunsky, J. M., Newton, S., Ko, J., Zhao, G., Maher, T. A., Tager-Flusberg, H., Bolliger, M. F., Carter, A. S., Boucard, A. A., Powell, C. M., Suedhof, T. C. 2009; 29 (35): 10843-10854

  Abstract

  Neuroligins (NLs) are postsynaptic cell-adhesion molecules essential for normal synapse function. Mutations in neuroligin-4 (NL4) (gene symbol: NLGN4) have been reported in some patients with autism spectrum disorder (ASD) and other neurodevelopmental impairments. However, the low frequency of NL4 mutations and the limited information about the affected patients and the functional consequences of their mutations cast doubt on the causal role of NL4 mutations in these disorders. Here, we describe two brothers with classical ASD who carry a single amino-acid substitution in NL4 (R87W). This substitution was absent from the brothers' asymptomatic parents, suggesting that it arose in the maternal germ line. R87 is conserved in all NL isoforms, and the R87W substitution is not observed in control individuals. At the protein level, the R87W substitution impaired glycosylation processing of NL4 expressed in HEK293 and COS cells, destabilized NL4, caused NL4 retention in the endoplasmic reticulum in non-neuronal cells and neurons, and blocked NL4 transport to the cell surface. As a result, the R87W substitution inactivated the synapse-formation activity of NL4 and abolished the functional effect of NL4 on synapse strength. Viewed together, these observations suggest that a point mutation in NL4 can cause ASD by a loss-of-function mechanism.

  View details for DOI 10.1523/JNEUROSCI.1248-09.2009

  View details for PubMedID 19726642

• Presenilins are essential for regulating neurotransmitter release NATURE Zhang, C., Wu, B., Beglopoulos, V., Wines-Samuelson, M., Zhang, D., Dragatsis, I., Suedhof, T. C., Shen, J. 2009; 460 (7255): 632-U100

  Abstract

  Mutations in the presenilin genes are the main cause of familial Alzheimer's disease. Loss of presenilin activity and/or accumulation of amyloid-beta peptides have been proposed to mediate the pathogenesis of Alzheimer's disease by impairing synaptic function. However, the precise site and nature of the synaptic dysfunction remain unknown. Here we use a genetic approach to inactivate presenilins conditionally in either presynaptic (CA3) or postsynaptic (CA1) neurons of the hippocampal Schaeffer-collateral pathway. We show that long-term potentiation induced by theta-burst stimulation is decreased after presynaptic but not postsynaptic deletion of presenilins. Moreover, we found that presynaptic but not postsynaptic inactivation of presenilins alters short-term plasticity and synaptic facilitation. The probability of evoked glutamate release, measured with the open-channel NMDA (N-methyl-D-aspartate) receptor antagonist MK-801, is reduced by presynaptic inactivation of presenilins. Notably, depletion of endoplasmic reticulum Ca(2+) stores by thapsigargin, or blockade of Ca(2+) release from these stores by ryanodine receptor inhibitors, mimics and occludes the effects of presynaptic presenilin inactivation. Collectively, these results indicate a selective role for presenilins in the activity-dependent regulation of neurotransmitter release and long-term potentiation induction by modulation of intracellular Ca(2+) release in presynaptic terminals, and further suggest that presynaptic dysfunction might be an early pathogenic event leading to dementia and neurodegeneration in Alzheimer's disease.

  View details for DOI 10.1038/nature08177

  View details for Web of Science ID 000268454300051

  View details for PubMedID 19641596

  View details for PubMedCentralID PMC2744588

• alpha-Latrotoxin Stimulates a Novel Pathway of Ca2+-Dependent Synaptic Exocytosis Independent of the Classical Synaptic Fusion Machinery JOURNAL OF NEUROSCIENCE Deak, F., Liu, X., Khvotchev, M., Li, G., Kavalali, E. T., Sugita, S., Sudhof, T. C. 2009; 29 (27): 8639-8648

  Abstract

  Alpha-latrotoxin induces neurotransmitter release by stimulating synaptic vesicle exocytosis via two mechanisms: (1) A Ca(2+)-dependent mechanism with neurexins as receptors, in which alpha-latrotoxin acts like a Ca(2+) ionophore, and (2) a Ca(2+)-independent mechanism with CIRL/latrophilins as receptors, in which alpha-latrotoxin directly stimulates the transmitter release machinery. Here, we show that the Ca(2+)-independent release mechanism by alpha-latrotoxin requires the synaptic SNARE-proteins synaptobrevin/VAMP and SNAP-25, and, at least partly, the synaptic active-zone protein Munc13-1. In contrast, the Ca(2+)-dependent release mechanism induced by alpha-latrotoxin does not require any of these components of the classical synaptic release machinery. Nevertheless, this type of exocytotic neurotransmitter release appears to fully operate at synapses, and to stimulate exocytosis of the same synaptic vesicles that participate in physiological action potential-triggered release. Thus, synapses contain two parallel and independent pathways of Ca(2+)-triggered exocytosis, a classical, physiological pathway that operates at the active zone, and a novel reserve pathway that is recruited only when Ca(2+) floods the synaptic terminal.

  View details for DOI 10.1523/JNEUROSCI.0898-09.2009

  View details for Web of Science ID 000267818400004

  View details for PubMedID 19587270

  View details for PubMedCentralID PMC2739239

• Conditional Forebrain Inactivation of Nicastrin Causes Progressive Memory Impairment and Age-Related Neurodegeneration JOURNAL OF NEUROSCIENCE Tabuchi, K., Chen, G., Suedhof, T. C., Shen, J. 2009; 29 (22): 7290-7301

  Abstract

  Loss of presenilin function in adult mouse brains causes memory loss and age-related neurodegeneration. Since presenilin possesses gamma-secretase-dependent and -independent activities, it remains unknown which activity is required for presenilin-dependent memory formation and neuronal survival. To address this question, we generated postnatal forebrain-specific nicastrin conditional knock-out (cKO) mice, in which nicastrin, a subunit of gamma-secretase, is inactivated selectively in mature excitatory neurons of the cerebral cortex. nicastrin cKO mice display progressive impairment in learning and memory and exhibit age-dependent cortical neuronal loss, accompanied by astrocytosis, microgliosis, and hyperphosphorylation of the microtubule-associated protein Tau. The neurodegeneration observed in nicastrin cKO mice likely occurs via apoptosis, as evidenced by increased numbers of apoptotic neurons. These findings demonstrate an essential role of nicastrin in the execution of learning and memory and the maintenance of neuronal survival in the brain and suggest that presenilin functions in memory and neuronal survival via its role as a gamma-secretase subunit.

  View details for DOI 10.1523/JNEUROSCI.1320-09.2009

  View details for PubMedID 19494151

• Common circuit defect of excitatory-inhibitory balance in mouse models of autism JOURNAL OF NEURODEVELOPMENTAL DISORDERS Gogolla, N., LeBlanc, J. J., Quast, K. B., Sudhof, T. C., Fagiolini, M., Hensch, T. K. 2009; 1 (2): 172-181

  Abstract

  One unifying explanation for the complexity of Autism Spectrum Disorders (ASD) may lie in the disruption of excitatory/inhibitory (E/I) circuit balance during critical periods of development. We examined whether Parvalbumin (PV)-positive inhibitory neurons, which normally drive experience-dependent circuit refinement (Hensch Nat Rev Neurosci 6:877-888, 1), are disrupted across heterogeneous ASD mouse models. We performed a meta-analysis of PV expression in previously published ASD mouse models and analyzed two additional models, reflecting an embryonic chemical insult (prenatal valproate, VPA) or single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C). PV-cells were reduced in the neocortex across multiple ASD mouse models. In striking contrast to controls, both VPA and NL-3 mouse models exhibited an asymmetric PV-cell reduction across hemispheres in parietal and occipital cortices (but not the underlying area CA1). ASD mouse models may share a PV-circuit disruption, providing new insight into circuit development and potential prevention by treatment of autism.The online version of this article (doi:10.1007/s11689-009-9023-x) contains supplementary material, which is available to authorized users.

  View details for DOI 10.1007/s11689-009-9023-x

  View details for Web of Science ID 000274848700007

  View details for PubMedID 20664807

  View details for PubMedCentralID PMC2906812

• Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release NATURE NEUROSCIENCE Xu, J., Pang, Z. P., Shin, O., Suedhof, T. C. 2009; 12 (6): 759-U111

  Abstract

  Spontaneous 'mini' release occurs at all synapses, but its nature remains enigmatic. We found that >95% of spontaneous release in murine cortical neurons was induced by Ca2+-binding to synaptotagmin-1 (Syt1), the Ca2+ sensor for fast synchronous neurotransmitter release. Thus, spontaneous and evoked release used the same Ca2+-dependent release mechanism. As a consequence, Syt1 mutations that altered its Ca2+ affinity altered spontaneous and evoked release correspondingly. Paradoxically, Syt1 deletions (as opposed to point mutations) massively increased spontaneous release. This increased spontaneous release remained Ca2+ dependent but was activated at lower Ca2+ concentrations and with a lower Ca2+ cooperativity than synaptotagmin-driven spontaneous release. Thus, in addition to serving as a Ca2+ sensor for spontaneous and evoked release, Syt1 clamped a second, more sensitive Ca2+ sensor for spontaneous release that resembles the Ca2+ sensor for evoked asynchronous release. These data suggest that Syt1 controls both evoked and spontaneous release at a synapse as a simultaneous Ca2+-dependent activator and clamp of exocytosis.

  View details for DOI 10.1038/nn.2320

  View details for PubMedID 19412166

• Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas JOURNAL OF PHYSIOLOGY-LONDON Gustavsson, N., Wei, S., Hoang, D. N., Lao, Y., Zhang, Q., Radda, G. K., Rorsman, P., Sudhof, T. C., Han, W. 2009; 587 (6): 1169-1178

  Abstract

  Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

  View details for DOI 10.1113/jphysiol.2008.168005

  View details for Web of Science ID 000264188400005

  View details for PubMedID 19171650

  View details for PubMedCentralID PMC2674989

• Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming JOURNAL OF CELL BIOLOGY Deak, F., Xu, Y., Chang, W., Dulubova, I., Khvotchev, M., Liu, X., Suedhof, T. C., Rizo, J. 2009; 184 (5): 751-764

  Abstract

  Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca(2+)-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1-SNARE-complexin macromolecular assembly that is poised for Ca(2+) triggering of fusion.

  View details for DOI 10.1083/jcb.200812026

  View details for Web of Science ID 000264016700012

  View details for PubMedID 19255244

  View details for PubMedCentralID PMC2686405

• Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 GENES BRAIN AND BEHAVIOR Blundell, J., Tabuchi, K., Bolliger, M. F., Blaiss, C. A., Brose, N., Liu, X., Sudhof, T. C., Powell, C. M. 2009; 8 (1): 114-126

  Abstract

  Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autism-associated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2-deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2-deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety-like behavior, a decrease in pain sensitivity and a slight decrease in motor co-ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.

  View details for DOI 10.1111/j.1601-183X.2008.00455.x

  View details for Web of Science ID 000262888000015

  View details for PubMedID 19016888

• SV2 Renders Primed Synaptic Vesicles Competent for Ca2+-Induced Exocytosis JOURNAL OF NEUROSCIENCE Chang, W., Suedhof, T. C. 2009; 29 (4): 883-897

  Abstract

  Synaptic vesicle protein 2 (SV2), one of the first synaptic vesicle proteins identified, is characterized by multiple transmembrane regions that exhibit homology to sugar transporters, and by a highly glycosylated intravesicular sequence. Deletion of SV2 causes postnatal lethality in mice, primarily because of fulminant epilepsy. At the cellular level, deletion of SV2 impairs neurotransmitter release, but its function is unknown, and even the exact point at which release is affected in SV2-deleted synapses remains unclear. Using electrophysiological approaches, we now examine at what step in exocytosis the deletion of SV2 impairs release. Our data demonstrate that deletion of SV2 produces a decrease in evoked synaptic responses without causing changes in mini frequency, mini amplitude, the readily releasable pool of vesicles, or the apparent Ca(2+) sensitivity of vesicle fusion. These findings indicate that a previously unidentified step may couple priming of synaptic vesicles to Ca(2+) triggering of fusion, and that SV2 acts in this step to render primed synaptic vesicles fully Ca(2+) responsive. To investigate the structural requirements for this function of SV2, we used rescue experiments. We demonstrate that conserved charged residues within the transmembrane regions and the intravesicular glycosylation of SV2 are required for its normal folding and trafficking. In contrast, the conserved putative synaptotagmin-binding sequence of SV2 is fully dispensable. Viewed together, these observations suggest that SV2 functions in a maturation step of primed vesicles that converts the vesicles into a Ca(2+)- and synaptotagmin-responsive state.

  View details for DOI 10.1523/JNEUROSCI.4521-08.2009

  View details for Web of Science ID 000262859000001

  View details for PubMedID 19176798

  View details for PubMedCentralID PMC2693337

• Membrane Fusion: Grappling with SNARE and SM Proteins SCIENCE Sudhof, T. C., Rothman, J. E. 2009; 323 (5913): 474-477

  Abstract

  The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.

  View details for DOI 10.1126/science.1161748

  View details for PubMedID 19164740

• Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion SCIENCE Maximov, A., Tang, J., Yang, X., Pang, Z. P., Sudhof, T. C. 2009; 323 (5913): 516-521

  Abstract

  Trans-SNAP receptor (SNARE, where SNAP is defined as soluble NSF attachment protein, and NSF is defined as N-ethylmaleimide-sensitive factor) complexes catalyze synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast calcium ion-evoked fusion. The dual function of complexin required SNARE binding and also involved distinct amino-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/vesicle-associated membrane protein (VAMP) phenocopied the complexin loss-of-function state without impairing complexin binding to SNARE complexes. Thus, complexin probably activates and clamps the force transfer from assembled trans-SNARE complexes onto fusing membranes.

  View details for DOI 10.1126/science.1166505

  View details for Web of Science ID 000262587900046

  View details for PubMedID 19164751

  View details for PubMedCentralID PMC3235366

• Analysis of neuroligin-3 knock-in mice relevant to autism spectrum disorders Tabuchi, K., Etherton, M., Shigemoto, R., Sudhof, T. ELSEVIER IRELAND LTD. 2009: S257

  View details for DOI 10.1016/j.neures.2009.09.1464

  View details for Web of Science ID 000272421101828

• Deletion of Mint Proteins Decreases Amyloid Production in Transgenic Mouse Models of Alzheimer's Disease JOURNAL OF NEUROSCIENCE Ho, A., Liu, X., Sudhof, T. C. 2008; 28 (53): 14392-14400

  Abstract

  Mints/X11s are neuronal adaptor proteins that bind to amyloid-beta precursor protein (APP). Previous studies suggested that Mint/X11 proteins influence APP cleavage and affect production of pathogenic amyloid-beta (Abeta) peptides in Alzheimer's disease; however, the biological significance of Mint/X11 binding to APP and their possible role in Abeta production remain unclear. Here, we crossed conditional and constitutive Mint1, Mint2, and Mint3 knock-out mice with transgenic mouse models of Alzheimer's disease overproducing human Abeta peptides. We show that deletion of all three individual Mint proteins delays the age-dependent production of amyloid plaque numbers and Abeta40 and Abeta42 levels with loss of Mint2 having the largest effect. Acute conditional deletion of all three Mints in cultured neurons suppresses the accumulation of APP C-terminal fragments and the secretion of ectodomain APP by decreasing beta-cleavage but does not impair subsequent gamma-cleavage. These results suggest that the three Mint/X11 proteins regulate Abeta production by a novel mechanism that may have implications for therapeutic approaches to altering APP cleavage in Alzheimer's disease.

  View details for DOI 10.1523/JNEUROSCI.2481-08.2008

  View details for Web of Science ID 000262137800008

  View details for PubMedID 19118172

• RIM1 alpha and RIM1 beta Are Synthesized from Distinct Promoters of the RIM1 Gene to Mediate Differential But Overlapping Synaptic Functions JOURNAL OF NEUROSCIENCE Kaeser, P. S., Kwon, H., Chiu, C. Q., Deng, L., Castillo, P. E., Suedhof, T. C. 2008; 28 (50): 13435-13447

  Abstract

  At a synapse, presynaptic terminals form a specialized area of the plasma membrane called the active zone that mediates neurotransmitter release. RIM1alpha is a multidomain protein that constitutes a central component of the active zone by binding to other active zone proteins such as Munc13 s, alpha-liprins, and ELKS, and to synaptic vesicle proteins such as Rab3 and synaptotagmin-1. In mice, knockout of RIM1alpha significantly impairs synaptic vesicle priming and presynaptic long-term plasticity, but is not lethal. We now find that the RIM1 gene encodes a second, previously unknown RIM1 isoform called RIM1beta that is upregulated in RIM1alpha knock-out mice. RIM1beta is identical to RIM1alpha except for the N terminus where RIM1beta lacks the N-terminal Rab3-binding sequence of RIM1alpha. Using newly generated knock-out mice lacking both RIM1alpha and RIM1beta, we demonstrate that different from the deletion of only RIM1alpha, deletion of both RIM1alpha and RIM1beta severely impairs mouse survival. Electrophysiological analyses show that the RIM1alphabeta deletion abolishes long-term presynaptic plasticity, as does RIM1alpha deletion alone. In contrast, the impairment in synaptic strength and short-term synaptic plasticity that is caused by the RIM1alpha deletion is aggravated by the deletion of both RIM1alpha and RIM1beta. Thus, our data indicate that the RIM1 gene encodes two different isoforms that perform overlapping but distinct functions in neurotransmitter release.

  View details for DOI 10.1523/JNEUROSCI.3235-08.2008

  View details for Web of Science ID 000261601800009

  View details for PubMedID 19074017

  View details for PubMedCentralID PMC2701653

• Understanding Synapses: Past, Present, and Future NEURON Suedhof, T. C., Malenka, R. C. 2008; 60 (3): 469-476

  Abstract

  Classical physiological work by Katz, Eccles, and others revealed the central importance of synapses in brain function, and characterized the mechanisms involved in synaptic transmission. Building on this work, major advances in the past two decades have elucidated how synapses work molecularly. In the present perspective, we provide a short description of our personal view of these advances, suggest a series of important future questions about synapses, and discuss ideas about how best to achieve further progress in the field.

  View details for DOI 10.1016/j.neuron.2008.10.011

  View details for PubMedID 18995821

• Neuroligins and neurexins link synaptic function to cognitive disease NATURE Sudhof, T. C. 2008; 455 (7215): 903-911

  Abstract

  The brain processes information by transmitting signals at synapses, which connect neurons into vast networks of communicating cells. In these networks, synapses not only transmit signals but also transform and refine them. Neurexins and neuroligins are synaptic cell-adhesion molecules that connect presynaptic and postsynaptic neurons at synapses, mediate signalling across the synapse, and shape the properties of neural networks by specifying synaptic functions. In humans, alterations in genes encoding neurexins or neuroligins have recently been implicated in autism and other cognitive diseases, linking synaptic cell adhesion to cognition and its disorders.

  View details for DOI 10.1038/nature07456

  View details for PubMedID 18923512

• RiM1 alpha phosphorylation at serine-413 by protein kinase A is not required for presynaptic long-term plasticity or learning PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaeser, P. S., Kwon, H., Blundell, J., Chevaleyre, V., Morishita, W., Malenka, R. C., Powell, C. M., Castillo, P. E., Sudhof, T. C. 2008; 105 (38): 14680-14685

  Abstract

  Activation of presynaptic cAMP-dependent protein kinase A (PKA) triggers presynaptic long-term plasticity in synapses such as cerebellar parallel fiber and hippocampal mossy fiber synapses. RIM1alpha, a large multidomain protein that forms a scaffold at the presynaptic active zone, is essential for presynaptic long-term plasticity in these synapses and is phosphorylated by PKA at serine-413. Previous studies suggested that phosphorylation of RIM1alpha at serine-413 is required for presynaptic long-term potentiation in parallel fiber synapses formed in vitro by cultured cerebellar neurons and that this type of presynaptic long-term potentiation is mediated by binding of 14-3-3 proteins to phosphorylated serine-413. To test the role of serine-413 phosphorylation in vivo, we have now produced knockin mice in which serine-413 is mutated to alanine. Surprisingly, we find that in these mutant mice, three different forms of presynaptic PKA-dependent long-term plasticity are normal. Furthermore, we observed that in contrast to RIM1alpha KO mice, RIM1 knockin mice containing the serine-413 substitution exhibit normal learning capabilities. The lack of an effect of the serine-413 mutation of RIM1alpha is not due to compensation by RIM2alpha because mice carrying both the serine-413 substitution and a RIM2alpha deletion still exhibited normal long-term presynaptic plasticity. Thus, phosphorylation of serine-413 of RIM1alpha is not essential for PKA-dependent long-term presynaptic plasticity in vivo, suggesting that PKA operates by a different mechanism despite the dependence of long-term presynaptic plasticity on RIM1alpha.

  View details for DOI 10.1073/pnas.0806679105

  View details for Web of Science ID 000259592400081

  View details for PubMedID 18799741

  View details for PubMedCentralID PMC2567150

• Conformational switch of syntaxin-1 controls synaptic vesicle fusion SCIENCE Gerber, S. H., Rah, J., Min, S., Liu, X., de Wit, H., Dulubova, I., Meyer, A. C., Rizo, J., Arancillo, M., Hammer, R. E., Verhage, M., Rosenmund, C., Sudhof, T. C. 2008; 321 (5895): 1507-1510

  Abstract

  During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.

  View details for DOI 10.1126/science.1163174

  View details for Web of Science ID 000259121800046

  View details for PubMedID 18703708

• Cysteine string protein-alpha is essential for the high calcium sensitivity of exocytosis in a vertebrate synapse EUROPEAN JOURNAL OF NEUROSCIENCE Ruiz, R., Casanas, J. J., Sudhof, T. C., Tabares, L. 2008; 27 (12): 3118-3131

  Abstract

  Cysteine string protein (CSPalpha) is a synaptic vesicle protein present in most central and peripheral nervous system synapses. Previous studies demonstrated that the deletion of CSPalpha results in postnatal sensorial and motor impairment and premature lethality. To understand the participation of CSPalpha in neural function in vertebrates, we have studied the properties of synaptic transmission of motor terminals in wild-type and CSPalpha knockout mice. Our results demonstrate that, in the absence of CSPalpha, fast Ca2+-triggered release was not affected at postnatal day (P)14 but was dramatically reduced at P18 and P30 without a change in release kinetics. Although mutant terminals also exhibited a reduction in functional vesicle pool size by P30, further analysis showed that neurotransmission could be 'rescued' by high extracellular [Ca2+] or by the presence of a phorbol ester, suggesting that an impairment in the fusion machinery, or in vesicle recycling, was not the primary cause of the dysfunction of this synapse. The specific shift to the right of the Ca2+ dependence of synchronous release, and the lineal dependence of secretion on extracellular [Ca2+] in mutant terminals after P18, suggests that CSPalpha is indispensable for a normal Ca2+ sensitivity of exocytosis in vertebrate mature synapses.

  View details for DOI 10.1111/j.1460-9568.2008.06301.x

  View details for Web of Science ID 000256853800004

  View details for PubMedID 18598257

• Unusually rapid evolution of Neuroligin-4 in mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bolliger, M. F., Pei, J., Maxeiner, S., Boucard, A. A., Grishin, N. V., Sudhof, T. C. 2008; 105 (17): 6421-6426

  Abstract

  Neuroligins (NLs) are postsynaptic cell-adhesion molecules that are implicated in humans in autism spectrum disorders because the genes encoding NL3 and NL4 are mutated in rare cases of familial autism. NLs are highly conserved evolutionarily, except that no NL4 was detected in the currently available mouse genome sequence assemblies. We now demonstrate that mice express a distant NL4 variant that rapidly evolved from other mammalian NL4 genes and that exhibits sequence variations even between different mouse strains. Despite its divergence, mouse NL4 binds neurexins and is transported into dendritic spines, suggesting that the core properties of NLs are retained in this divergent NL isoform. The selectively rapid evolution of NL4 in mice suggests that its function in the brain is under less stringent control than that of other NLs, shedding light on why its mutation in autism spectrum disorder patients is not lethal, but instead leads to a discrete developmental brain disorder.

  View details for DOI 10.1073/pnas.0801383105

  View details for Web of Science ID 000255534100038

  View details for PubMedID 18434543

• CASK functions as a Mg2+-independent neurexin kinase CELL Mukherjee, K., Sharma, M., Urlaub, H., Bourenkov, G. P., Jahn, R., Suedhof, T. C., Wahl, M. C. 2008; 133 (2): 328-339

  Abstract

  CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.

  View details for DOI 10.1016/j.cell.2008.02.036

  View details for Web of Science ID 000255052000021

  View details for PubMedID 18423203

• Synaptotagmin-1 and-7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schonn, J., Maximov, A., Lao, Y., Suedhof, T. C., Sorensen, J. B. 2008; 105 (10): 3998-4003

  Abstract

  Synaptotagmin-1, the canonical isoform of the synaptotagmin family, is a Ca(2+) sensor for fast synchronous neurotransmitter release in forebrain neurons and chromaffin cells. Even though deletion of synaptotagmin-1 abolishes fast exocytosis in chromaffin cells, it reduces overall secretion by only 20% because of the persistence of slow exocytosis. Therefore, another Ca(2+) sensor dominates release in these cells. Synaptotagmin-7 has a higher Ca(2+) affinity and slower binding kinetics than synaptotagmin-1, matching the proposed properties for the second, slower Ca(2+) sensor. Here, we examined Ca(2+)-triggered exocytosis in chromaffin cells from KO mice lacking synaptotagmin-7, and from knockin mice containing normal levels of a mutant synaptotagmin-7 whose C(2)B domain does not bind Ca(2+). In both types of mutant chromaffin cells, Ca(2+)-triggered exocytosis was decreased dramatically. Moreover, in chromaffin cells lacking both synaptotagmin-1 and -7, only a very slow release component, accounting for approximately 30% of WT exocytosis, persisted. These data establish synaptotagmin-7 as a major Ca(2+) sensor for exocytosis in chromaffin cells, which, together with synaptotagmin-1, mediates almost all of the Ca(2+) triggering of exocytosis in these cells, a surprising result, considering the lack of a role of synaptotagmin-7 in synaptic vesicle exocytosis.

  View details for DOI 10.1073/pnas.0712373105

  View details for Web of Science ID 000253930600061

  View details for PubMedID 18308932

• Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maximov, A., Lao, Y., Li, H., Chen, X., Rizo, J., Sorensen, J. B., Suedhof, T. C. 2008; 105 (10): 3986-3991

  Abstract

  Synaptotagmin-7 is a candidate Ca(2+) sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca(2+) sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C(2)B domains that exhibit Ca(2+)-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca(2+) sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca(2+)-binding properties. Here, we examine how synaptotagmin-7 binds Ca(2+) and test whether this Ca(2+) binding regulates Ca(2+)-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca(2+)-binding mode similar to that of the synaptotagmin-1 C(2)A domain, suggesting that the synaptotagmin-1 and -7 C(2) domains generally employ comparable Ca(2+)-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca(2+) binding either to both C(2) domains of synaptotagmin-7 or only to its C(2)B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca(2+) binding to both C(2) domains caused an approximately 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca(2+) binding to only the C(2)B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca(2+) binding to the synaptotagmin-7 C(2) domains is physiologically important for stabilizing synaptotagmin-7, but that Ca(2+) binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca(2+)-dependent synaptic exocytosis.

  View details for DOI 10.1073/pnas.0712372105

  View details for Web of Science ID 000253930600059

  View details for PubMedID 18308933

• Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gustavsson, N., Lao, Y., Maximov, A., Chuang, J., Kostromina, E., Repa, J. J., Li, C., Radda, G. K., Suedhof, T. C., Han, W. 2008; 105 (10): 3992-3997

  Abstract

  Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.

  View details for DOI 10.1073/pnas.0711700105

  View details for Web of Science ID 000253930600060

  View details for PubMedID 18308938

• A molecular pathway of neurodegeneration linking alpha-synuclein to ApoE and A beta peptides NATURE NEUROSCIENCE Gallardo, G., Schlueter, O. M., Suedhof, T. C. 2008; 11 (3): 301-308

  Abstract

  Pathogenic aggregates of alpha-synuclein are thought to contribute to the development of Parkinson's disease. Inclusion bodies containing alpha-synuclein are present in Parkinson's disease and other neurodegenerative diseases, including Alzheimer's disease. Moreover, alpha-synuclein mutations are found in cases of familial Parkinson's disease, and transgenic overexpression of alpha-synuclein causes neurodegeneration in mice. The molecular mechanisms involved, however, remain incompletely understood. Here we show that, in transgenic mice, alpha-synuclein induced neurodegeneration involves activation of the ubiquitin/proteasome system, a massive increase in apolipoprotein E (ApoE) levels and accumulation of insoluble mouse Abeta. ApoE was not protective, but was injurious, as deletion of ApoE delayed the neurodegeneration caused by alpha-synuclein and suppressed the accumulation of Abeta. Our data reveal a molecular link between central pathogenic mechanisms implicated in Parkinson's disease and Alzheimer's disease and suggest that intracellular alpha-synuclein is pathogenic, at least in part, by activation of extracellular signaling pathways involving ApoE.

  View details for DOI 10.1038/nn2058

  View details for Web of Science ID 000253548300013

  View details for PubMedID 18297066

• Structures of neuroligin-1 and the Neuroligin-l/Neurexin-1 beta complex reveal specificprotein-protein and protein-Ca2+ interactions NEURON Arac, D., Boucard, A. A., Ozkan, E., Strop, P., Newell, E., Sudhof, T. C., Brunger, A. T. 2007; 56 (6): 992-1003

  Abstract

  Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1 beta. Neuroligin-1 forms a constitutive dimer, and two neurexin-1 beta monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1 beta complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca2+. Alternatively spliced sites in neurexin-1 beta and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1 beta, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1 beta complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  View details for DOI 10.1016/j.neuron.2007.12.002

  View details for PubMedID 18093522

• A dual-Ca2+-sensor model for neurotransmitter release in a central synapse NATURE Sun, J., Pang, Z. P., Qin, D., Fahim, A. T., Adachi, R., Suedhof, T. C. 2007; 450 (7170): 676-U4

  Abstract

  Ca2+-triggered synchronous neurotransmitter release is well described, but asynchronous release-in fact, its very existence-remains enigmatic. Here we report a quantitative description of asynchronous neurotransmitter release in calyx-of-Held synapses. We show that deletion of synaptotagmin 2 (Syt2) in mice selectively abolishes synchronous release, allowing us to study pure asynchronous release in isolation. Using photolysis experiments of caged Ca2+, we demonstrate that asynchronous release displays a Ca2+ cooperativity of approximately 2 with a Ca2+ affinity of approximately 44 microM, in contrast to synchronous release, which exhibits a Ca2+ cooperativity of approximately 5 with a Ca2+ affinity of approximately 38 muM. Our results reveal that release triggered in wild-type synapses at low Ca2+ concentrations is physiologically asynchronous, and that asynchronous release completely empties the readily releasable pool of vesicles during sustained elevations of Ca2+. We propose a dual-Ca2+-sensor model of release that quantitatively describes the contributions of synchronous and asynchronous release under conditions of different presynaptic Ca2+ dynamics.

  View details for DOI 10.1038/nature06308

  View details for Web of Science ID 000251209700046

  View details for PubMedID 18046404

• Dual modes of Munc18-1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 n terminus JOURNAL OF NEUROSCIENCE Khvotchev, M., Dulubova, I., Sun, J., Dai, H., Rizo, J., Suedhof, T. C. 2007; 27 (45): 12147-12155

  Abstract

  The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal syntaxin/Munc18 interaction is essential. Introduction of an N-terminal syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.

  View details for DOI 10.1523/JNEUROSCI.3655-07.2007

  View details for Web of Science ID 000250758600006

  View details for PubMedID 17989281

• Neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice SCIENCE Tabuchi, K., Blundell, J., Etherton, M. R., Hammer, R. E., Liu, X., Powell, C. M., Suedhof, T. C. 2007; 318 (5847): 71-76

  Abstract

  Autism spectrum disorders (ASDs) are characterized by impairments in social behaviors that are sometimes coupled to specialized cognitive abilities. A small percentage of ASD patients carry mutations in genes encoding neuroligins, which are postsynaptic cell-adhesion molecules. We introduced one of these mutations into mice: the Arg451-->Cys451 (R451C) substitution in neuroligin-3. R451C mutant mice showed impaired social interactions but enhanced spatial learning abilities. Unexpectedly, these behavioral changes were accompanied by an increase in inhibitory synaptic transmission with no apparent effect on excitatory synapses. Deletion of neuroligin-3, in contrast, did not cause such changes, indicating that the R451C substitution represents a gain-of-function mutation. These data suggest that increased inhibitory synaptic transmission may contribute to human ASDs and that the R451C knockin mice may be a useful model for studying autism-related behaviors.

  View details for DOI 10.1126/science.1146221

  View details for Web of Science ID 000249915400041

  View details for PubMedID 17823315

• Membrane fusion as a team effort PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Suedhof, T. C. 2007; 104 (34): 13541-13542

  View details for Web of Science ID 000249064700004

  View details for PubMedID 17699625

• Integrative genomic and functional analyses reveal neuronal subtype differentiation bias in human embryonic stem cell lines PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, H., Xu, J., Pang, Z. P., Ge, W., Kim, K. J., Blanchi, B., Chen, C., Suedhof, T. C., Sun, Y. E. 2007; 104 (34): 13821-13826

  Abstract

  The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, because hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows stepwise generation from hESCs of cultures composed of approximately 70-80% human neurons that exhibit spontaneous synaptic network activity. Comparison of neurons derived from the well characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are preprogrammed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs.

  View details for DOI 10.1073/pnas.0706199104

  View details for Web of Science ID 000249064700054

  View details for PubMedID 17693548

• Crystal structure of the RIM1 alpha C2B domain at 1.7 angstrom resolution BIOCHEMISTRY Guan, R., Dai, H., Tomchick, D. R., Dulubova, I., Machius, M., Suedhof, T. C., Rizo, J. 2007; 46 (31): 8988-8998

  Abstract

  RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1alpha C2B domain. We also find that the isolated RIM1alpha C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1alpha C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1alpha C2B domain forms weak dimers in solution. The crystal structure of the RIM1alpha C2B domain dimer at 1.7 A resolution reveals that it forms a beta-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the beta-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.

  View details for DOI 10.1021/bi700698a

  View details for Web of Science ID 000248439000009

  View details for PubMedID 17630786

• Differential effects of SNAP-25 deletion on Ca2+-dependent and Ca2+-independent neurotransmission JOURNAL OF NEUROPHYSIOLOGY Bronk, P., Deak, F., Wilson, M. C., Liu, X., Sudhof, T. C., Kavalali, E. T. 2007; 98 (2): 794-806

  Abstract

  At the synapse, SNAP-25, along with syntaxin/HPC-1 and synaptobrevin/VAMP, forms SNARE N-ethylmaleimide-sensitive factor [soluble (NSF) attachment protein receptor] complexes that are thought to catalyze membrane fusion. Results from neuronal cultures of synaptobrevin-2 knockout (KO) mice showed that loss of synaptobrevin has a more severe effect on calcium-evoked release than on spontaneous release or on release evoked by hypertonicity. In this study, we recorded neurotransmitter release from neuronal cultures of SNAP-25 KO mice to determine whether they share this property. In neurons lacking SNAP-25, as those deficient in synaptobrevin-2, we found that approximately 10-12% of calcium-independent excitatory and inhibitory neurotransmitter release persisted. However, in contrast to synaptobrevin-2 knockouts, this remaining readily releasable pool in SNAP-25-deficient synapses was virtually insensitive to calcium-dependent-evoked stimulation. Although field stimulation reliably evoked neurotransmitter release in synaptobrevin-2 KO neurons, responses were rare in neurons lacking SNAP-25, and unlike synaptobrevin-2-deficient synapses, SNAP-25-deficient synapses did not exhibit facilitation of release during high-frequency stimulation. This severe loss of evoked exocytosis was matched by a reduction, but not a complete loss, of endocytosis during evoked stimulation. Moreover, synaptic vesicle turnover probed by FM-dye uptake and release during hypertonic stimulation was relatively unaffected by the absence of SNAP-25. This last difference indicates that in contrast to synaptobrevin, SNAP-25 does not directly function in endocytosis. Together, these results suggest that SNAP-25 has a more significant role in calcium-secretion coupling than synaptobrevin-2.

  View details for DOI 10.1152/jn.00226.2007

  View details for Web of Science ID 000248601100021

  View details for PubMedID 17553942

• Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2 NEURON Chubykin, A. A., Atasoy, D., Etherton, M. R., Brose, N., Kavalali, E. T., Gibson, J. R., Suedhof, T. C. 2007; 54 (6): 919-931

  Abstract

  Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.

  View details for DOI 10.1016/j.neuron.2007.05.029

  View details for Web of Science ID 000247645600011

  View details for PubMedID 17582332

• Endocannabinoid-mediated long-term plasticity requires cAMP/PKA signaling and RIM1 alpha NEURON Chevaleyre, V., Heifets, B. D., Kaeser, P. S., Sudhof, T. C., Purpura, D. P., Castillo, P. E. 2007; 54 (5): 801-812

  Abstract

  Endocannabinoids (eCBs) have emerged as key activity-dependent signals that, by activating presynaptic cannabinoid receptors (i.e., CB1) coupled to G(i/o) protein, can mediate short-term and long-term synaptic depression (LTD). While the presynaptic mechanisms underlying eCB-dependent short-term depression have been identified, the molecular events linking CB1 receptors to LTD are unknown. Here we show in the hippocampus that long-term, but not short-term, eCB-dependent depression of inhibitory transmission requires presynaptic cAMP/PKA signaling. We further identify the active zone protein RIM1alpha as a key mediator of both CB1 receptor effects on the release machinery and eCB-dependent LTD in the hippocampus. Moreover, we show that eCB-dependent LTD in the amygdala and hippocampus shares major mechanistic features. These findings reveal the signaling pathway by which CB1 receptors mediate long-term effects of eCBs in two crucial brain structures. Furthermore, our results highlight a conserved mechanism of presynaptic plasticity in the brain.

  View details for DOI 10.1016/j.neuron.2007.05.020

  View details for Web of Science ID 000247329900012

  View details for PubMedID 17553427

• Synaptotagmin-1,-2, and-9: Ca2+ sensors for fast release that specify distinct presynaptic properties in subsets of neurons NEURON Xu, J., Mashimo, T., Sudhof, T. C. 2007; 54 (4): 567-581

  Abstract

  Synaptotagmin-1 and -2 are known Ca(2+) sensors for fast synchronous neurotransmitter release, but the potential Ca(2+)-sensor functions of other synaptotagmins in release remain uncharacterized. We now show that besides synaptotagmin-1 and -2, only synaptotagmin-9 (also called synaptotagmin-5) mediates fast Ca(2+) triggering of release. Release induced by the three different synaptotagmin Ca(2+) sensors exhibits distinct kinetics and apparent Ca(2+) sensitivities, suggesting that the synaptotagmin isoform expressed by a neuron determines the release properties of its synapses. Conditional knockout mice producing GFP-tagged synaptotagmin-9 revealed that synaptotagmin-9 is primarily expressed in the limbic system and striatum. Acute deletion of synaptotagmin-9 in striatal neurons severely impaired fast synchronous release without changing the size of the readily-releasable vesicle pool. These data show that in mammalian brain, only synaptotagmin-1, -2, and -9 function as Ca(2+) sensors for fast release, and that these synaptotagmins are differentially expressed to confer distinct release properties onto synapses formed by defined subsets of neurons.

  View details for DOI 10.1016/j.neuron.2007.05.004

  View details for Web of Science ID 000246970800012

  View details for PubMedID 17521570

• Deletion of alpha-neurexins does not cause a major impairment of axonal pathfinding or synapse formation JOURNAL OF COMPARATIVE NEUROLOGY Dudanova, I., Tabuchi, K., Rohlmann, A., Suedhof, T. C., Missler, M. 2007; 502 (2): 261-274

  Abstract

  Alpha-neurexins are synaptic cell-surface molecules that are required for Ca(2+)-triggered exocytosis. Mice lacking all three alpha-neurexins show drastically reduced neurotransmitter release at excitatory and inhibitory synapses and die early postnatally. Although previous histological analysis of newborn alpha-neurexin triple mutants revealed only a moderate reduction in the density of type II synapses in the brainstem, cell culture studies proposed that neurexins are prominently involved in synapse formation. To assess the contribution of alpha-neurexins to the formation and structural properties of synapses in vivo, we performed a detailed morphological analysis of the brains from surviving adult double knockout mice lacking two of the three alpha-neurexins. Despite their impaired neurotransmission, we did not observe any gross anatomical defects or changes in the distribution of synaptic proteins in adult mutants. Only mild structural alterations were found: a approximately 20% reduction of neuropil area in many brain regions, resulting predominantly from shortened distal dendritic branches and fewer spines, as demonstrated by Golgi impregnation of pyramidal neurons. Quantitative electron microscopy revealed ultrastructurally normal type I and II terminals and a approximately 30% decrease in the density of type II synapses in the neocortex. To exclude errors in pathfinding, we investigated axonal projections in the olfactory bulb of newborn knockouts and did not observe any changes. Therefore, alpha-neurexins are not essential for the formation of the vast majority of synapses in vivo but rather regulate the function of these synapses.

  View details for DOI 10.1002/cne.21305

  View details for Web of Science ID 000245688600007

  View details for PubMedID 17347997

• Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation JOURNAL OF NEUROSCIENCE METHODS Maximov, A., Pang, Z. P., Tervo, D. G., Sudhof, T. C. 2007; 161 (1): 75-87

  Abstract

  Various techniques have been applied for the functional analysis of synaptic transmission in cultured neurons. Here, we describe a method of studying synaptic transmission in neurons cultured at high-density from different brain regions such as the cortex, striatum and spinal cord. We use postsynaptic whole-cell recordings to monitor synaptic currents triggered by presynaptic action potentials that are induced by brief stimulations with a nearby extracellular bipolar electrode. Pharmacologically isolated excitatory or inhibitory postsynaptic currents can be reliably induced, with amplitudes, synaptic charge transfers, and short-term plasticity properties that are reproducible from culture to culture. We show that the size and kinetics of pharmacologically isolated inhibitory postsynaptic currents triggered by single action potentials or stimulus trains depend on the Ca2+ concentration, temperature and stimulation frequency. This method can be applied to study synaptic transmission in wildtype neurons infected with lentiviruses encoding various components of presynaptic release machinery, or in neurons from genetically modified mice, for example neurons carrying floxed genes in which gene expression can be acutely ablated by expression of Cre recombinase. The preparation described in this paper should be useful for analysis of synaptic transmission in inter-neuronal synapses formed by different types of neurons.

  View details for DOI 10.1016/j.jneumeth.2006.10.009

  View details for Web of Science ID 000245490900010

  View details for PubMedID 17118459

• E-Syts, a family of membranous Ca2+-sensor proteins with multiple C-2 domains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Min, S., Chang, W., Sudhof, T. C. 2007; 104 (10): 3823-3828

  Abstract

  C(2) domains are autonomously folded protein modules that generally act as Ca(2+)- and phospholipid-binding domains and/or as protein-protein interaction domains. We now report the primary structures and biochemical properties of a family of evolutionarily conserved mammalian proteins, referred to as E-Syts, for extended synaptotagmin-like proteins. E-Syts contain an N-terminal transmembrane region, a central juxtamembranous domain that is conserved from yeast to human, and five (E-Syt1) or three (E-Syt2 and E-Syt3) C-terminal C(2) domains. Only the first E-Syt C(2) domain, the C(2)A domain, includes the complete sequence motif that is required for Ca(2+) binding in C(2) domains. Recombinant protein fragments of E-Syt2 that include the first C(2) domain are capable of Ca(2+)-dependent phospholipid binding at micromolar concentrations of free Ca(2+), suggesting that E-Syts bind Ca(2+) through their first C(2) domain in a phospholipid complex. E-Syts are ubiquitously expressed, but enriched in brain. Expression of myc-tagged E-Syt proteins in transfected cells demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3. Structure/function studies showed that the plasma-membrane localization of E-Syt2 and E-Syt3 was directed by their C-terminal C(2)C domains. This result reveals an unexpected mechanism by which the C(2)C domains of E-Syt2 and E-Syt3 functions as a targeting motif that localizes these proteins into the plasma membrane independent of their transmembrane region. Viewed together, our findings suggest that E-Syts function as Ca(2+)-regulated intrinsic membrane proteins with multiple C(2) domains, expanding the repertoire of such proteins to a fourth class beyond synaptotagmins, ferlins, and MCTPs (multiple C(2) domain and transmembrane region proteins).

  View details for DOI 10.1073/pnas.0611725104

  View details for Web of Science ID 000244972400030

  View details for PubMedID 17360437

• cAMP oscillations and retinal activity are permissive for ephrin signaling during the establishment of the retinotopic map NATURE NEUROSCIENCE Nicol, X., Voyatzis, S., Muzerelle, A., Narboux-Neme, N., Sudhof, T. C., Miles, R., Gaspar, P. 2007; 10 (3): 340-347

  Abstract

  Spontaneous activity generated in the retina is necessary to establish a precise retinotopic map, but the underlying mechanisms are poorly understood. We demonstrate here that neural activity controls ephrin-A-mediated responses. In the mouse retinotectal system, we show that spontaneous activity of the retinal ganglion cells (RGCs) is needed, independently of synaptic transmission, for the ordering of the retinotopic map and the elimination of exuberant retinal axons. Activity blockade suppressed the repellent action of ephrin-A on RGC growth cones by cyclic AMP (cAMP)-dependent pathways. Unexpectedly, the ephrin-A5-induced retraction required cAMP oscillations rather than sustained increases in intracellular cAMP concentrations. Periodic photo-induced release of caged cAMP in growth cones rescued the response to ephrin-A5 when activity was blocked. These results provide a direct molecular link between spontaneous neural activity and axon guidance mechanisms during the refinement of neural maps.

  View details for DOI 10.1038/nn1842

  View details for Web of Science ID 000244480300016

  View details for PubMedID 17259982

• Munc18-1 binds directly to the neuronal SNARE complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Sudhof, T. C., Rizo, J. 2007; 104 (8): 2697-2702

  Abstract

  Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.

  View details for DOI 10.1073/pnas.0611318104

  View details for Web of Science ID 000244511200026

  View details for PubMedID 17301226

• Deletion of CASK in mice is lethal and impairs synaptic function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atasoy, D., Schoch, S., Ho, A., Nadasy, K. A., Liu, X., Zhang, W., Mukherjee, K., Nosyreva, E. D., Fernandez-Chacon, R., Missler, M., Kavalali, E. T., Suedhof, T. C. 2007; 104 (7): 2525-2530

  Abstract

  CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca2+/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK "knockdown" mice in which CASK expression was suppressed by approximately 70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca2+/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca2+-triggered presynaptic release, or postsynaptic receptor functions.

  View details for DOI 10.1073/pnas.0611003104

  View details for Web of Science ID 000244438500086

  View details for PubMedID 17287346

• Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release JOURNAL OF CELL BIOLOGY Maximov, A., Shin, O., Liu, X., Sudhof, T. C. 2007; 176 (1): 113-124

  Abstract

  Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

  View details for DOI 10.1083/jcb.200607021

  View details for Web of Science ID 000243581600012

  View details for PubMedID 17190793

• Synaptotagmin-2 is essential for survival and contributes to Ca2+ triggering of neurotransmitter release in central and neuromuscular synapses JOURNAL OF NEUROSCIENCE Pang, Z. P., Melicoff, E., Padgett, D., Liu, Y., Teich, A. F., Dickey, B. F., Lin, W., Adachi, R., Sudhof, T. C. 2006; 26 (52): 13493-13504

  Abstract

  Biochemical and genetic data suggest that synaptotagmin-2 functions as a Ca2+ sensor for fast neurotransmitter release in caudal brain regions, but animals and/or synapses lacking synaptotagmin-2 have not been examined. We have now generated mice in which the 5' end of the synaptotagmin-2 gene was replaced by lacZ. Using beta-galactosidase as a marker, we show that, consistent with previous studies, synaptotagmin-2 is widely expressed in spinal cord, brainstem, and cerebellum, but is additionally present in selected forebrain neurons, including most striatal neurons and some hypothalamic, cortical, and hippocampal neurons. Synaptotagmin-2-deficient mice were indistinguishable from wild-type littermates at birth, but subsequently developed severe motor dysfunction, and perished at approximately 3 weeks of age. Electrophysiological studies in cultured striatal neurons revealed that the synaptotagmin-2 deletion slowed the kinetics of evoked neurotransmitter release without altering the total amount of release. In contrast, synaptotagmin-2-deficient neuromuscular junctions (NMJs) suffered from a large reduction in evoked release and changes in short-term synaptic plasticity. Furthermore, in mutant NMJs, the frequency of spontaneous miniature release events was increased both at rest and during stimulus trains. Viewed together, our results demonstrate that the synaptotagmin-2 deficiency causes a lethal impairment in synaptic transmission in selected synapses. This impairment, however, is less severe than that produced in forebrain neurons by deletion of synaptotagmin-1, presumably because at least in NMJs, synaptotagmin-1 is coexpressed with synaptotagmin-2, and both together mediate fast Ca2+-triggered release. Thus, synaptotagmin-2 is an essential synaptotagmin isoform that functions in concert with other synaptotagmins in the Ca2+ triggering of neurotransmitter release.

  View details for DOI 10.1523/JNEUROSCI.3519-06.2006

  View details for Web of Science ID 000243277800010

  View details for PubMedID 17192432

• Redundant functions of RIM1 alpha and RIM2 alpha in Ca2+-triggered neurotransmitter release EMBO JOURNAL Schoch, S., Mittelstaedt, T., Kaeser, P. S., Padgett, D., Feldmann, N., Chevaleyre, V., Castillo, P. E., Hammer, R. E., Han, W., Schmitz, F., Lin, W., Sudhof, T. C. 2006; 25 (24): 5852-5863

  Abstract

  Alpha-RIMs (RIM1alpha and RIM2alpha) are multidomain active zone proteins of presynaptic terminals. Alpha-RIMs bind to Rab3 on synaptic vesicles and to Munc13 on the active zone via their N-terminal region, and interact with other synaptic proteins via their central and C-terminal regions. Although RIM1alpha has been well characterized, nothing is known about the function of RIM2alpha. We now show that RIM1alpha and RIM2alpha are expressed in overlapping but distinct patterns throughout the brain. To examine and compare their functions, we generated knockout mice lacking RIM2alpha, and crossed them with previously produced RIM1alpha knockout mice. We found that deletion of either RIM1alpha or RIM2alpha is not lethal, but ablation of both alpha-RIMs causes postnatal death. This lethality is not due to a loss of synapse structure or a developmental change, but to a defect in neurotransmitter release. Synapses without alpha-RIMs still contain active zones and release neurotransmitters, but are unable to mediate normal Ca(2+)-triggered release. Our data thus demonstrate that alpha-RIMs are not essential for synapse formation or synaptic exocytosis, but are required for normal Ca(2+)-triggering of exocytosis.

  View details for DOI 10.1038/sj.emboj.7601425

  View details for Web of Science ID 000242891100021

  View details for PubMedID 17124501

• Genetic analysis of Mint/X11 proteins: Essential presynaptic functions of a neuronal adaptor protein family JOURNAL OF NEUROSCIENCE Ho, A., Morishita, W., Atasoy, D., Liu, X., Tabuchi, K., Hammer, R. E., Malenka, R. C., Sudhof, T. C. 2006; 26 (50): 13089-13101

  Abstract

  Mints/X11s are adaptor proteins composed of three isoforms: neuron-specific Mints 1 and 2, and the ubiquitously expressed Mint 3. We have now analyzed constitutive and conditional knock-out mice for all three Mints/X11s. We found that approximately 80% of mice lacking both neuron-specific Mint isoforms (Mints 1 and 2) die at birth, whereas mice lacking any other combination of Mint isoforms survive normally. The approximately 20% surviving Mint 1/2 double knock-out mice exhibit a decrease in weight and deficits in motor behaviors. Hippocampal slice electrophysiology uncovered a decline in spontaneous neurotransmitter release, lowered synaptic strength, and enhanced paired-pulse facilitation in Mint-deficient mice, suggesting a decreased presynaptic release probability. Acute ablation of Mint expression in cultured neurons from conditional Mint 1/2/3 triple knock-in mice also revealed a decline in spontaneous release, confirming that deletion of Mints impair presynaptic function. Quantitation of synaptic proteins showed that acute deletion of Mints caused a selective increase in Munc18-1 and Fe65 proteins, and overexpression of Munc18-1 in wild-type neurons also produced a decrease in spontaneous release, suggesting that the interaction of Mints with Munc18-1 may contribute to the presynaptic phenotype observed in Mint-deficient mice. Our studies thus indicate that Mints are important regulators of presynaptic neurotransmitter release that are essential for mouse survival.

  View details for DOI 10.1523/JNEUROSCI.2855-06.2006

  View details for Web of Science ID 000242996200024

  View details for PubMedID 17167098

• Differential expression of active zone proteins in neuromuscular junctions suggests functional diversification EUROPEAN JOURNAL OF NEUROSCIENCE Juranek, J., Mukherjee, K., Rickmann, M., Martens, H., Calka, J., Suedhof, T. C., Jahn, R. 2006; 24 (11): 3043-3052

  Abstract

  Nerve terminals of the central nervous system (CNS) contain specialized release sites for synaptic vesicles, referred to as active zones. They are characterized by electron-dense structures that are tightly associated with the presynaptic plasma membrane and organize vesicle docking and priming sites. Recently, major protein constituents of active zones have been identified, including the proteins Piccolo, Bassoon, RIM, Munc13, ERCs/ELKs/CASTs and liprins. While it is becoming apparent that each of these proteins is essential for synaptic function in the CNS, it is not known to what extent these proteins are involved in synaptic function of the peripheral nervous system. Somatic neuromuscular junctions contain morphologically and functionally defined active zones with similarities to CNS synapses. In contrast, sympathetic neuromuscular varicosities lack active zone-like morphological specializations. Using immunocytochemistry at the light and electron microscopic level we have now performed a systematic investigation of all five major classes of active zone proteins in peripheral neuromuscular junctions. Our results show that somatic neuromuscular endplates contain a full complement of all active zone proteins. In contrast, varicosities of the vas deferens contain a subset of active zone proteins including Bassoon and ELKS2, with the other four components being absent. We conclude that Bassoon and ELKS2 perform independent and specialized functions in synaptic transmission of autonomic synapses.

  View details for DOI 10.1111/j.1460-9568.2006.05183.x

  View details for Web of Science ID 000243361700008

  View details for PubMedID 17156365

• A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis JOURNAL OF NEUROSCIENCE Pang, Z. P., Shin, O., Meyer, A. C., Rosenmund, C., Suedhof, T. C. 2006; 26 (48): 12556-12565

  Abstract

  Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

  View details for DOI 10.1523/JNEUROSCI.3804-06.2006

  View details for Web of Science ID 000242387900019

  View details for PubMedID 17135417

• Synaptic vesicles: An organelle comes of age CELL Sudhof, T. C. 2006; 127 (4): 671-673

  Abstract

  Synaptic vesicles mediate the release of neurotransmitters at nerve terminals. In this issue of Cell, Takamori et al. (2006) analyze the lipid and protein components of synaptic vesicles, providing the most comprehensive description of synaptic vesicles to date.

  View details for DOI 10.1016/j.cell.2006.10.033

  View details for Web of Science ID 000242330600013

  View details for PubMedID 17110326

• Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for beta-neurexins BIOCHEMISTRY Comoletti, D., Flynn, R. E., Boucard, A. A., Demeler, B., Schirf, V., Shi, J., Jennings, L. L., Newlin, H. R., Sudhof, T. C., Taylor, P. 2006; 45 (42): 12816-12827

  Abstract

  Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.

  View details for DOI 10.1021/bi0614131

  View details for Web of Science ID 000241325700021

  View details for PubMedID 17042500

• A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis CELL Tang, J., Maximov, A., Shin, O., Dai, H., Rizo, J., Sudhof, T. C. 2006; 126 (6): 1175-1187

  Abstract

  Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.

  View details for DOI 10.1016/j.cell.2006.08.030

  View details for Web of Science ID 000240897600022

  View details for PubMedID 16990140

• Neuroligins determine synapse maturation and function NEURON Varoqueaux, F., Aramuni, G., Rawson, R. L., Mohrmann, R., Missler, M., Gottmann, K., Zhang, W., Suedhof, T. C., Brose, N. 2006; 51 (6): 741-754

  Abstract

  Synaptogenesis, the generation and maturation of functional synapses between nerve cells, is an essential step in the development of neuronal networks in the brain. It is thought to be triggered by members of the neuroligin family of postsynaptic cell adhesion proteins, which may form transsynaptic contacts with presynaptic alpha- and beta-neurexins and have been implicated in the etiology of autism. We show that deletion mutant mice lacking neuroligin expression die shortly after birth due to respiratory failure. This respiratory failure is a consequence of reduced GABAergic/glycinergic and glutamatergic synaptic transmission and network activity in brainstem centers that control respiration. However, the density of synaptic contacts is not altered in neuroligin-deficient brains and cultured neurons. Our data show that neuroligins are required for proper synapse maturation and brain function, but not for the initial formation of synaptic contacts.

  View details for DOI 10.1016/j.neuron.2006.09.003

  View details for Web of Science ID 000240997900013

  View details for PubMedID 16982420

• Crystal structure of the second LNS/LG domain from neurexin 1 alpha - Ca2+ binding and the effects of alternative splicing JOURNAL OF BIOLOGICAL CHEMISTRY Sheckler, L. R., Henry, L., Sugita, S., Sudhof, T. C., Rudenko, G. 2006; 281 (32): 22896-22905

  Abstract

  Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca2+. The crystal structure of n1alpha_LNS#2 (the second LNS/LG domain of bovine neurexin 1alpha) reveals large structural differences compared with n1alpha_LNS#6 (or n1beta_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca2+-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca2+-binding site in n1alpha_LNS#2 has low affinity (Kd approximately 400 microm). Ca2+ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca2+-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.

  View details for DOI 10.1074/jbc.M603464200

  View details for Web of Science ID 000239542600052

  View details for PubMedID 16772286

• Structural basis for a Munc13-1 homodimer to Munc13-1/RIM heterodimer switch PLOS BIOLOGY Lu, J., Machius, M., Dulubova, I., Dai, H., Sudhof, T. C., Tomchick, D. R., Rizo, J. 2006; 4 (7): 1159-1172

  Abstract

  C(2) domains are well characterized as Ca(2+)/phospholipid-binding modules, but little is known about how they mediate protein-protein interactions. In neurons, a Munc13-1 C(2)A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13-1 C(2)A domain homodimerizes, and that homodimerization competes with Munc13-1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13-1 C(2)A-domain homodimer and the Munc13-1 C(2)A-domain/RIM ZF heterodimer at 1.44 A and 1.78 A resolution, respectively. The C(2)A domain adopts a beta-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded beta-barrel. In contrast, heterodimerization involves the bottom tip of the C(2)A-domain beta-sandwich and a C-terminal alpha-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13-1 homodimer-Munc13-1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C(2) domains as protein-protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes.

  View details for DOI 10.1371/journal.pbio.0040192

  View details for Web of Science ID 000238974300009

  View details for PubMedID 16732694

• Structural determinants of synaptobrevin 2 function in synaptic vesicle fusion JOURNAL OF NEUROSCIENCE Deak, F., Shin, O. H., Kavalali, E. T., Sudhof, T. C. 2006; 26 (25): 6668-6676

  Abstract

  Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca2+-evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca2+-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.

  View details for DOI 10.1523/JNEUROSCI.5272-05.2006

  View details for Web of Science ID 000238473600004

  View details for PubMedID 16793874

• Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion EMBO JOURNAL Deak, F., Shin, O., Tang, J., Hanson, P., Ubach, J., Jahn, R., Rizo, J., Kavalali, E. T., Sudhof, T. C. 2006; 25 (12): 2856-2866

  Abstract

  Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.

  View details for DOI 10.1038/sj.embjol.7601165

  View details for Web of Science ID 000238709800018

  View details for PubMedID 16763567

• Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C-2 domains of synaptotagmin 1 JOURNAL OF BIOLOGICAL CHEMISTRY Li, L., Shin, O., Rhee, J., Arac, D., Rah, J., Rizo, J., Sudhof, T., Rosenmund, C. 2006; 281 (23): 15845-15852

  Abstract

  Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.

  View details for DOI 10.1074/jbc.M600888200

  View details for Web of Science ID 000237996000033

  View details for PubMedID 16595652

• Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+-triggered neurotransmitter release EMBO JOURNAL Pang, Z. P., Sun, J., Rizo, J., Maximov, A., Sudhof, T. C. 2006; 25 (10): 2039-2050

  Abstract

  Synaptotagmin 2 resembles synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling 'release slots' for synaptic vesicles at the active zone.

  View details for DOI 10.1038/sj.emboj.7601103

  View details for Web of Science ID 000237590000001

  View details for PubMedID 16642042

• Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids NATURE STRUCTURAL & MOLECULAR BIOLOGY Arac, D., Chen, X. C., Khant, H. A., Ubach, J., Ludtke, S. J., Kikkawa, M., Johnson, A. E., Chiu, W., Sudhof, T. C., Rizo, J. 2006; 13 (3): 209-217

  Abstract

  Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

  View details for DOI 10.1038/nsmb1056

  View details for Web of Science ID 000235776900011

  View details for PubMedID 16491093

• Synapsins regulate use-dependent synaptic plasticity in the calyx of Held by a Ca2+/calmodulin-dependent pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, J. Y., Bronk, P., Liu, X. R., Han, W. P., Sudhof, T. C. 2006; 103 (8): 2880-2885

  Abstract

  Synapsins are abundant synaptic-vesicle phosphoproteins that are known to regulate neurotransmitter release but whose precise function has been difficult to pinpoint. Here, we use knockout mice to analyze the role of synapsins 1 and 2 in the calyx of Held synapse, allowing precise measurements of neurotransmitter release. We find that deletion of synapsins did not induce significant changes in spontaneous release or release evoked by isolated action potentials (APs) and did not alter the size of the readily releasable vesicle pool (RRP), the kinetics of RRP depletion, or the rate of recovery of the RRP after depletion. Deletion of synapsins, however, did increase use-dependent synaptic depression induced by a high-frequency stimulus train (> or = 50 Hz). The increased depression was due to a decrease in the fraction of the RRP, whose release was evoked by APs late in the stimulus train. The effect of synapsin deletions was occluded by intracellular application of the Ca2+-chelator EGTA or of a calmodulin inhibitor. Our results show that synapsins boost the release probability during high-frequency stimulation and suggest that this effect involves Ca2+/calmodulin-dependent phosphorylation of synapsins.

  View details for Web of Science ID 000235554900072

  View details for PubMedID 16481620

• CSP alpha-deficiency causes massive and rapid photoreceptor degeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schmitz, F., Tabares, L., Khimich, D., Strenzke, N., de la Villa-Polo, P., Castellano-Munoz, M., Bulankina, A., Moser, T., Fernandez-Chacon, R., Sudhof, T. C. 2006; 103 (8): 2926-2931

  Abstract

  Cysteine string protein (CSP) alpha is an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40-type cochaperones. Previous studies showed that deletion of CSPalpha in mice leads to massive lethal neurodegeneration but did not clarify how the neurodegeneration affects specific subpopulations of neurons. Here, we analyzed the effects of the CSPalpha deficiency on tonically active ribbon synapses of the retina and the inner ear. We show that CSPalpha-deficient photoreceptor terminals undergo dramatic and rapidly progressive neurodegeneration that starts before eye opening and initially does not affect other retinal synapses. These changes are associated with progressive blindness. In contrast, ribbon synapses of auditory hair cells did not exhibit presynaptic impairments in CSPalpha-deficient mice. Hair cells, but not photoreceptor cells or central neurons, express CSPbeta, thereby accounting for the lack of a hair-cell phenotype in CSPalpha knockout mice. Our data demonstrate that tonically active ribbon synapses in retina are particularly sensitive to the deletion of CSPalpha and that expression of at least one CSP isoform is essential to protect such tonically active synapses from neurodegeneration.

  View details for DOI 10.1073/pnas.0510060103

  View details for Web of Science ID 000235554900080

  View details for PubMedID 16477021

• Rab3 superprimes synaptic vesicles for release: Implications for short-term synaptic plasticity JOURNAL OF NEUROSCIENCE Schluter, O. M., Basu, J., Sudhof, T. C., Rosenmund, C. 2006; 26 (4): 1239-1246

  Abstract

  Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. The four closely related small GTP-binding proteins Rab3A, Rab3B, Rab3C, and Rab3D are believed to be important for these steps. In mice, the complete absence of all Rab3s leads to perinatal lethality accompanied by a 30% reduction of probability of Ca2+-triggered synaptic release. This study examines the role of Rab3 during Ca2+-triggered release in more detail and identifies its impact on short-term plasticity. Using patch-clamp electrophysiology of autaptic neuronal cultures from Rab3-deficient mouse hippocampus, we show that excitatory Rab3-deficient neurons display unique time- and frequency-dependent short-term plasticity characteristics in response to spike trains. Analysis of vesicle release and repriming kinetics as well as Ca2+ sensitivity of release indicate that Rab3 acts on a subset of primed, fusion competent vesicles. They lower the amount of Ca2+ required for action potential-triggered release, which leads to a boosting of release probability, but their action also introduces a significant delay in the supply of these modified vesicles. As a result, Rab3-induced modifications to primed vesicles causes a transient increase in the transduction efficacy of synaptic action potential trains and optimizes the encoding of synaptic information at an intermediate spike frequency range.

  View details for DOI 10.1523/JNEUROSCI.3553-05.2006

  View details for Web of Science ID 000234896200022

  View details for PubMedID 16436611

• Different effects on fast exocytosis induced by synaptotagmin 1 and 2 isoforms and abundance but not by phosphorylation JOURNAL OF NEUROSCIENCE Nagy, G., Kim, J. H., Pang, Z. P., Matti, U., Rettig, J., Sudhof, T. C., Sorensen, J. B. 2006; 26 (2): 632-643

  Abstract

  Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+ sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells expressing Syt1. Biochemical experiments showed that Syt2 has a slightly lower Ca2+ affinity for phospholipid binding than Syt1 because of a difference in the C2A domain. These data constitute evidence for the function of Syt1 and Syt2 as alternative, but not identical, calcium-sensors for RRP fusion. By overexpression of Syt1 mutated in the shared PKC/calcium/calmodulin-dependent kinase phosphorylation site, we show that phorbol esters act independently and upstream of Syt1 to regulate the size of the releasable pools. We conclude that exocytosis from mouse chromaffin cells can be modified by the differential expression of Syt isoforms and by Syt abundance but not by phosphorylation of Syt1.

  View details for DOI 10.1523/JNEUROSCI.2589-05.2006

  View details for Web of Science ID 000234556200032

  View details for PubMedID 16407561

• Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rhee, J. S., Li, L. Y., Shin, O. H., Rah, J. C., Rizo, J., Sudhof, T. C., Rosenmund, C. 2005; 102 (51): 18664-18669

  Abstract

  Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.

  View details for DOI 10.1073/pnas.0509153102

  View details for Web of Science ID 000234174300075

  View details for PubMedID 16352718

• RIM function in short- and long-term synaptic plasticity BioScience 2005 Conference Kaeser, P. S., Sudhof, T. C. PORTLAND PRESS LTD. 2005: 1345–1349

  Abstract

  RIM1alpha (Rab3-interacting molecule 1alpha) is a large multidomain protein that is localized to presynaptic active zones [Wang, Okamoto, Schmitz, Hofmann and Südhof (1997) Nature (London) 388, 593-598] and is the founding member of the RIM protein family that also includes RIM2alpha, 2beta, 2gamma, 3gamma and 4gamma [Wang and Südhof (2003) Genomics 81, 126-137]. In presynaptic nerve termini, RIM1alpha interacts with a series of presynaptic proteins, including the synaptic vesicle GTPase Rab3 and the active zone proteins Munc13, liprins and ELKS (a protein rich in glutamate, leucine, lysine and serine). Mouse KOs (knockouts) revealed that, in different types of synapses, RIM1alpha is essential for different forms of synaptic plasticity. In CA1-region Schaffer-collateral excitatory synapses and in GABAergic synapses (where GABA is gamma-aminobutyric acid), RIM1alpha is required for maintaining normal neurotransmitter release and short-term synaptic plasticity. In contrast, in excitatory CA3-region mossy fibre synapses and cerebellar parallel fibre synapses, RIM1alpha is necessary for presynaptic long-term, but not short-term, synaptic plasticity. In these synapses, the function of RIM1alpha in presynaptic long-term plasticity depends, at least in part, on phosphorylation of RIM1alpha at a single site, suggesting that RIM1alpha constitutes a 'phosphoswitch' that determines synaptic strength. However, in spite of the progress in understanding RIM1alpha function, the mechanisms by which RIM1alpha acts remain unknown. For example, how does phosphorylation regulate RIM1alpha, what is the relationship of the function of RIM1alpha in basic release to synaptic plasticity and what is the physiological significance of different forms of RIM-dependent plasticity? Moreover, the roles of other RIM isoforms are unclear. Addressing these important questions will contribute to our view of how neurotransmitter release is regulated at the presynaptic active zone.

  View details for Web of Science ID 000233930800030

  View details for PubMedID 16246115

• Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release NEURON Maximov, A., Sudhof, T. C. 2005; 48 (4): 547-554

  Abstract

  Ca(2+) triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release. Synaptotagmin 1 functions as a Ca(2+) sensor for synchronous release, but its role in asynchronous release remains unclear. We now show that in cultured cortical neurons stimulated at low frequency (or Hz), deletion of synaptotagmin 1 also alters only synchronous, not asynchronous, release during the stimulus train, but dramatically enhances "delayed asynchronous release" following the stimulus train. Thus synaptotagmin 1 functions as an autonomous Ca(2+) sensor independent of asynchronous release during isolated action potentials and action potential trains, but restricts asynchronous release induced by residual Ca(2+) after action potential trains. We propose that synaptotagmin 1 occupies release "slots" at the active zone, possibly in a Ca(2+)-independent complex with SNARE proteins that are freed when action potential-induced Ca(2+) influx activates synaptotagmin 1.

  View details for DOI 10.1016/j.neuron.2005.09.006

  View details for Web of Science ID 000233677300009

  View details for PubMedID 16301172

• alpha-synuclein cooperates with CSP alpha in preventing neurodegeneration CELL Chandra, S., Gallardo, G., Fernandez-Chacon, R., Schluter, O. M., Sudhof, T. C. 2005; 123 (3): 383-396

  Abstract

  Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.

  View details for DOI 10.1016/j.cell.2005.09.028

  View details for Web of Science ID 000233264300008

  View details for PubMedID 16269331

• A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alpha- and beta-neurexins NEURON Boucard, A. A., Chubykin, A. A., Comoletti, D., Taylor, P., Sudhof, T. C. 2005; 48 (2): 229-236

  Abstract

  Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic beta-neurexins, but not with presynaptic alpha-neurexins. Unexpectedly, we now find that neuroligins also bind alpha-neurexins and that alpha- and beta-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines alpha-neurexin binding but leaves beta-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only beta-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both alpha- and beta-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to alpha- and beta-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.

  View details for DOI 10.1016/j.neuron.2005.08.026

  View details for Web of Science ID 000232838700013

  View details for PubMedID 16242404

• Crystal structure of the RIM2 C(2)A-domain at 1.4 angstrom resolution BIOCHEMISTRY Dai, H., Tomchick, D. R., Garcia, J., Sudhof, T. C., Machius, M., Rizo, J. 2005; 44 (41): 13533-13542

  Abstract

  RIMs are large proteins that contain two C2-domains and are localized at presynaptic active zones, where neurotransmitters are released. RIMs play key roles in synaptic vesicle priming and regulation of presynaptic plasticity. A mutation in the RIM1 C2A-domain has been implicated in autosomal dominant cone-rod dystrophy (CORD7). The RIM C2A-domain does not contain the full complement of aspartate residues that commonly mediate Ca2+ binding at the top loops of C2-domains, and has been reported to interact with SNAP-25 and synaptotagmin 1, two proteins from the Ca2+-dependent membrane fusion machinery. Here we have used NMR spectroscopy and X-ray crystallography to analyze the structure and biochemical properties of the RIM2 C2A-domain, which is closely related to the RIM1 C2A-domain. We find that the RIM2 C2A-domain does not bind Ca2+. Moreover, little binding of the RIM2 C2A-domain to SNAP-25 and to the C2-domains of synaptotagmin 1 was detected by NMR experiments, suggesting that as yet unidentified interactions of the RIM C2A-domain mediate its function. The crystal structure of the RIM2 C2A-domain using data to 1.4 A resolution reveals a beta-sandwich that resembles those observed for other C2-domains, but exhibits a unique dipolar distribution of electrostatic charges whereby one edge of the beta-sandwich is highly positive and the other edge is highly negative. The location of the mutation site implicated in CORD7 at the bottom of the domain and the pattern of sequence conservation suggest that, in contrast to most C2-domains, the RIM C2A-domains may function through Ca2+-independent interactions involving their bottom face.

  View details for DOI 10.1021/bi0513608

  View details for Web of Science ID 000232632100011

  View details for PubMedID 16216076

• Solution structure of the RIM1 alpha PDZ domain in complex with an ELKS1 b C-terminal peptide JOURNAL OF MOLECULAR BIOLOGY Lu, J., Li, H. M., Wang, Y., Sudhof, T. C., Rizo, J. 2005; 352 (2): 455-466

  Abstract

  PDZ domains are widespread protein modules that commonly recognize C-terminal sequences of target proteins and help to organize macromolecular signaling complexes. These sequences usually bind in an extended conformation to relatively shallow grooves formed between a beta-strand and an alpha-helix in the corresponding PDZ domains. Because of this binding mode, many PDZ domains recognize primarily the C-terminal and the antepenultimate side-chains of the target protein, which commonly conform to motifs that have been categorized into different classes. However, an increasing number of PDZ domains have been found to exhibit unusual specificities. These include the PDZ domain of RIMs, which are large multidomain proteins that regulate neurotransmitter release and help to organize presynaptic active zones. The RIM PDZ domain binds to the C-terminal sequence of ELKS with a unique specificity that involves each of the four ELKS C-terminal residues. To elucidate the structural basis for this specificity, we have determined the 3D structure in solution of an RIM/ELKS C-terminal peptide complex using NMR spectroscopy. The structure shows that the RIM PDZ domain contains an unusually deep and narrow peptide-binding groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. This groove is formed, in part, by a set of side-chains that is conserved selectively in RIM PDZ domains and that hence determines, at least in part, their unique specificity.

  View details for DOI 10.1016/j.jmb.2005.07.047

  View details for Web of Science ID 000231921700018

  View details for PubMedID 16095618

• A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity? EMBO JOURNAL Dulubova, I., Lou, X. L., Lu, J., Huryeva, I., Alam, A., Schneggenburger, R., Sudhof, T. C., Rizo, J. 2005; 24 (16): 2839-2850

  Abstract

  alpha-RIMs and Munc13s are active zone proteins that control priming of synaptic vesicles to a readily releasable state, and interact with each other via their N-terminal sequences. The alpha-RIM N-terminal sequence also binds to Rab3s (small synaptic vesicle GTPases), an interaction that regulates presynaptic plasticity. We now demonstrate that alpha-RIMs contain adjacent but separate Munc13- and Rab3-binding sites, allowing formation of a tripartite Rab3/RIM/Munc13 complex. Munc13 binding is mediated by the alpha-RIM zinc-finger domain. Elucidation of the three-dimensional structure of this domain by NMR spectroscopy facilitated the design of a mutation that abolishes alpha-RIM/Munc13 binding. Selective disruption of this interaction in the calyx of Held synapse decreased the size of the readily releasable vesicle pool. Our data suggest that the ternary Rab3/RIM/Munc13 interaction approximates synaptic vesicles to the priming machinery, providing a substrate for presynaptic plasticity. The modular architecture of alpha-RIMs, with nested binding sites for Rab3 and other targets, may be a general feature of Rab effectors that share homology with the alpha-RIM N-terminal sequence.

  View details for DOI 10.1038/sj.emboj.7600753

  View details for Web of Science ID 000231789300001

  View details for PubMedID 16052212

• Nicastrin functions as a gamma-secretase-substrate receptor CELL Shah, S., Lee, S. F., Tabuchi, K., Hao, Y. H., Yu, C., LaPlant, Q., Ball, H., Dann, C. E., Sudhof, T., Yu, G. 2005; 122 (3): 435-447

  Abstract

  gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.

  View details for DOI 10.1016/j.cell.2005.05.022

  View details for Web of Science ID 000231254400014

  View details for PubMedID 16096062

• Genetic evidence for a protein-kinase-A-mediated presynaptic component in NMDA-receptor-dependent forms of long-term synaptic potentiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huang, Y. Y., Zakharenko, S. S., Schoch, S., Kaeser, P. S., Janz, R., Sudhof, T. C., Siegelbaum, S. A., Kandel, E. R. 2005; 102 (26): 9365-9370

  Abstract

  The synaptic vesicle protein Rab3A is a small GTP-binding protein that interacts with rabphilin and RIM1alpha, two presynaptic substrates of protein kinase A (PKA). Mice lacking RIM1alpha and Rab3A have a defect in PKA-dependent and NMDA receptor (NMDAR)-independent presynaptic long-term potentiation (LTP) at hippocampal mossy-fiber and cerebellar parallel-fiber synapses. In contrast, the NMDAR-dependent and PKA-independent early phase of LTP at hippocampal CA3-CA1 synapses does not require these presynaptic proteins. Here, we ask whether Rab3A and RIM1alpha participate in forms of LTP that require both PKA and NMDAR activation. We find that Rab3A is necessary for corticoamygdala LTP and late-phase LTP at CA3-CA1 synapses, two forms of LTP that require NMDAR and PKA activation. The latter form of LTP also requires RIM1alpha. These results provide genetic evidence that presynaptic proteins are required in LTP induced through the postsynaptic activation of NMDARs. Thus Rab3A and its effectors are general modules for four distinct types of PKA-dependent LTP in the brain.

  View details for DOI 10.1073/pnas.0503777102

  View details for Web of Science ID 000230191400050

  View details for PubMedID 15967982

• v-SNAREs control exocytosis of vesicles from priming to fusion EMBO JOURNAL Borisovska, M., Zhao, Y., Tsytsyura, Y., Glyvuk, N., Takamori, S., Matti, U., Rettig, J., Sudhof, T., Bruns, D. 2005; 24 (12): 2114-2126

  Abstract

  SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

  View details for DOI 10.1038/sj.emboj.7600696

  View details for Web of Science ID 000230511000007

  View details for PubMedID 15920476

• Dissection of synapse induction by neuroligins - Effect of a neuroligin mutation associated with autism JOURNAL OF BIOLOGICAL CHEMISTRY Chubykin, A. A., Liu, X. R., Comoletti, D., Tsigelny, I., Taylor, P., Sudhof, T. C. 2005; 280 (23): 22365-22374

  Abstract

  To study synapse formation by neuroligins, we co-cultured hippocampal neurons with COS cells expressing wild type and mutant neuroligins. The large size of COS cells makes it possible to test the effect of neuroligins presented over an extended surface area. We found that a uniform lawn of wild type neuroligins displayed on the cell surface triggers the formation of hundreds of uniformly sized, individual synaptic contacts that are labeled with neurexin antibodies. Electron microscopy revealed that these artificial synapses contain a presynaptic active zone with docked vesicles and often feature a postsynaptic density. Neuroligins 1, 2, and 3 were active in this assay. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta, a presumptive presynaptic binding partner for postsynaptic neuroligins, and blocked synapse formation. An analysis of mutant neuroligins with an amino acid substitution that corresponds to a mutation described in patients with an autistic syndrome confirmed previous reports that these mutant neuroligins have a compromised capacity to be transported to the cell surface. Nevertheless, the small percentage of mutant neuroligins that reached the cell surface still induced synapse formation. Viewed together, our data suggest that neuroligins generally promote artificial synapse formation in a manner that is associated with beta-neurexin binding and results in morphologically well differentiated synapses and that a neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synapse formation activity.

  View details for Web of Science ID 000229557900082

  View details for PubMedID 15797875

• Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and SNARE complex formation JOURNAL OF NEUROCHEMISTRY Toonen, R. F., de Vries, K. J., Zalm, R., Sudhof, T. C., Verhage, M. 2005; 93 (6): 1393-1400

  Abstract

  Munc18-1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18-1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18-1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18-1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18-1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18-1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18-1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18-1 knockout mice indicates that Munc18-1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself.

  View details for DOI 10.1111/j.1471-4159.2005.03128.x

  View details for Web of Science ID 000229425800004

  View details for PubMedID 15935055

• Extracellular domains of alpha-neurexins participate in regulating synaptic transmission by selectively affecting N- and P/Q-type Ca2+ channels JOURNAL OF NEUROSCIENCE Zhang, W. Q., Rohlmann, A., Sargsyan, V., Aramuni, G., Hammer, R. E., Sudhof, T. C., Missler, M. 2005; 25 (17): 4330-4342

  Abstract

  Neurexins constitute a large family of highly variable cell-surface molecules that may function in synaptic transmission and/or synapse formation. Each of the three known neurexin genes encodes two major neurexin variants, alpha- and beta-neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three alpha-neurexins in mice recently demonstrated their essential role at synapses. In multiple alpha-neurexin knock-outs, neurotransmitter release from excitatory and inhibitory synapses was severely reduced, primarily probably because voltage-dependent Ca2+ channels were impaired. It remained unclear, however, which neurexin variants actually influence exocytosis and Ca2+ channels, which domain of neurexins is required for this function, and which Ca2+-channel subtypes are regulated. Here, we show by electrophysiological recordings that transgenic neurexin 1alpha rescues the release and Ca2+-current phenotypes, whereas transgenic neurexin 1beta has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1alpha rescued the knock-out phenotype independent of the alpha-neurexin gene deleted, these data are consistent with a redundant function among different alpha-neurexins. In both knock-out and transgenically rescued mice, alpha-neurexins selectively affected the component of neurotransmitter release that depended on activation of N- and P/Q-type Ca2+ channels, but left L-type Ca2+ channels unscathed. Our findings indicate that alpha-neurexins represent organizer molecules in neurotransmission that regulate N- and P/Q-type Ca2+ channels, constituting an essential role at synapses that critically involves the extracellular domains of neurexins.

  View details for DOI 10.1523/JNEUROSCI.0497-05.2005

  View details for Web of Science ID 000228702900014

  View details for PubMedID 15858059

• CAPS in search of a lost function NEURON Sudhof, T. C. 2005; 46 (1): 2-4

  Abstract

  Ca2+-dependent activator protein for secretion (CAPS) is an evolutionarily conserved secretory protein that was previously thought to mediate Ca2+-triggered fusion of dense-core vesicles. In an elegant study of CAPS1-deficient mice, Speidel et al. (this issue of Neuron) now show that CAPS function may have been misunderstood. CAPS appears to act upstream of fusion in the biogenesis or maintenance of mature secretory vesicles, raising the possibility of a completely new type of function for an essential component of the secretory machinery.

  View details for DOI 10.1016/j.neuron.2005.03.017

  View details for Web of Science ID 000228228600002

  View details for PubMedID 15820687

• Are neuronal SNARE proteins Ca2+ sensors? JOURNAL OF MOLECULAR BIOLOGY Chen, X. C., Tang, J., Sudhof, T. C., Rizo, J. 2005; 347 (1): 145-158

  Abstract

  The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.

  View details for DOI 10.1016/j.jmb.2005.01.024

  View details for Web of Science ID 000227505200012

  View details for PubMedID 15733924

• Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitin-proteasome system and alpha-synuclein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fornai, F., Schluter, O. M., Lenzi, P., Gesi, M., Ruffoli, R., Ferrucci, M., Lazzeri, G., Busceti, C. L., Pontarelli, F., Battaglia, G., Pellegrini, A., Nicoletti, F., Ruggieri, S., Paparelli, A., Sudhof, T. C. 2005; 102 (9): 3413-3418

  Abstract

  In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking alpha-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an alpha-synuclein-dependent manner.

  View details for DOI 10.1073/pnas.0409713102

  View details for Web of Science ID 000227423700045

  View details for PubMedID 15716361

• C-terminal ECFP fusion impairs synaptotagmin 1 function - Crowding out synaptotagmin 1 JOURNAL OF BIOLOGICAL CHEMISTRY Han, W. P., Rhee, J. S., Maximov, A., Lin, W. C., Hammer, R. E., Rosenmund, C., Sudhof, T. C. 2005; 280 (6): 5089-5100

  Abstract

  To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.

  View details for DOI 10.1074/jbc.M408757200

  View details for Web of Science ID 000227096600131

  View details for PubMedID 15561725

• Evolutionarily conserved multiple C-2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties JOURNAL OF BIOLOGICAL CHEMISTRY Shin, O. H., Han, W. P., Wang, Y., Sudhof, T. C. 2005; 280 (2): 1641-1651

  Abstract

  C2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C2 domains. In both classes of proteins, C2 domains usually regulate the respective protein's function by forming Ca(2+)-dependent or Ca(2+)-independent phospholipid complexes. We now describe MCTPs (multiple C2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C2 domain proteins with unusual Ca(2+)-dependent properties. MCTPs are composed of a variable N-terminal sequence, three C2 domains, two transmembrane regions, and a short C-terminal sequence. The invertebrate organisms Caenorhabditis elegans and Drosophila melanogaster express a single MCTP gene, whereas vertebrates express two MCTP genes (MCTP1 and MCTP2) whose primary transcripts are extensively alternatively spliced. Most of the MCTP sequences, in particular the C2 domains, are highly conserved. All MCTP C2 domains except for the second C2 domain of MCTP2 include a perfect Ca2+/phospholipid-binding consensus sequence. To determine whether the C2 domains of MCTPs actually function as Ca2+/phospholipid-binding modules, we analyzed their Ca2+ and phospholipid binding properties. Surprisingly, we found that none of the three MCTP1 C2 domains interacted with negatively charged or neutral phospholipids in the presence or absence of Ca2+. However, Ca2+ titrations monitored via intrinsic tryptophan fluorescence revealed that all three C2 domains bound Ca2+ in the absence of phospholipids with a high apparent affinity (EC50 of approximately 1.3-2.3 microM). Our data thus reveal that MCTPs are evolutionarily conserved C2 domain proteins that are unusual in that the C2 domains are anchored in the membrane by two closely spaced transmembrane regions and represent Ca(2+)-binding but not phospholipid-binding modules.

  View details for DOI 10.1074/jbc.M407305200

  View details for Web of Science ID 000226195200095

  View details for PubMedID 15528213

• Selective capability of SynCAM and neuroligin for functional synapse assembly JOURNAL OF NEUROSCIENCE Sara, Y., Biederer, T., Atasoy, D., Chubykin, A., Mozhayeva, M. G., Sudhof, T. C., Kavalali, E. T. 2005; 25 (1): 260-270

  Abstract

  Synaptic cell adhesion is central for synapse formation and function. Recently, the synaptic cell adhesion molecules neuroligin 1 (NL1) and SynCAM were shown to induce presynaptic differentiation in cocultured neurons when expressed in a non-neuronal cell. However, it is uncertain how similar the resulting artificial synapses are to regular synapses. Are these molecules isofunctional, or do all neuronal cell adhesion molecules nonspecifically activate synapse formation? To address these questions, we analyzed the properties of artificial synapses induced by NL1 and SynCAM, compared the actions of these molecules with those of other neuronal cell adhesion molecules, and examined the functional effects of NL1 and SynCAM overexpression in neurons. We found that only NL1 and SynCAM specifically induced presynaptic differentiation in cocultured neurons. The induced nerve terminals were capable of both spontaneous and evoked neurotransmitter release, suggesting that a full secretory apparatus was assembled. By all measures, SynCAM- and NL1-induced artificial synapses were identical. Overexpression in neurons demonstrated that only SynCAM, but not NL1, increased synaptic function in immature developing excitatory neurons after 8 d in vitro. Tests of chimeric molecules revealed that the dominant-positive effect of SynCAM on synaptic function in developing neurons was mediated by its intracellular cytoplasmic tail. Interestingly, morphological analysis of neurons overexpressing SynCAM or NL1 showed the opposite of the predictions from electrophysiological results. In this case, only NL1 increased the synapse number, suggesting a role for NL1 in morphological synapse induction. These results suggest that both NL1 and SynCAM act similarly and specifically in artificial synapse induction but that this process does not reflect a shared physiological function of these molecules.

  View details for DOI 10.1523/JNEUROSCI.3165-04.2005

  View details for Web of Science ID 000226130200032

  View details for PubMedID 15634790

• Multiple roles for the active zone protein RIM1 alpha in late stages of neurotransmitter release NEURON Calakos, N., Schoch, S., Sudhof, T. C., Malenka, R. C. 2004; 42 (6): 889-896

  Abstract

  The active zone protein RIM1alpha interacts with multiple active zone and synaptic vesicle proteins and is implicated in short- and long-term synaptic plasticity, but it is unclear how RIM1alpha's biochemical interactions translate into physiological functions. To address this question, we analyzed synaptic transmission in autaptic neurons cultured from RIM1alpha-/- mice. Deletion of RIM1alpha causes a large reduction in the readily releasable pool of vesicles, alters short-term plasticity, and changes the properties of evoked asynchronous release. Lack of RIM1alpha, however, had no effect on synapse formation, spontaneous release, overall Ca2+ sensitivity of release, or synaptic vesicle recycling. These results suggest that RIM1alpha modulates sequential steps in synaptic vesicle exocytosis through serial protein-protein interactions and that this modulation is the basis for RIM1alpha's role in synaptic plasticity.

  View details for PubMedID 15207234

• Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine-induced parkinsonism in mice NEUROSCIENCE Schluter, O. M., Fornai, F., Alessandri, M. G., Takamori, S., Geppert, M., Jahn, R., Sudhof, T. C. 2003; 118 (4): 985-1002

  Abstract

  In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

  View details for DOI 10.1016/S0306-4522(03)00036-8

  View details for Web of Science ID 000182926900011

  View details for PubMedID 12732244

• Section 1: Insulin release: Some molecular requisites - Molecular determinants of regulated exocytosis 2nd Servier-IGIS Symposium Gerber, S. H., Sudhof, T. C. AMER DIABETES ASSOC. 2002: S3–S11

  View details for Web of Science ID 000173599900002

• RIM1 alpha is required for presynaptic long-term potentiation NATURE Castillo, P. E., Schoch, S., Schmitz, F., Sudhof, T. C., Malenka, R. C. 2002; 415 (6869): 327-330

  Abstract

  Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.

  View details for PubMedID 11797010

• Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Song, J. Y., Ichtchenko, K., Sudhof, T. C., Brose, N. 1999; 96 (3): 1100-1105

  Abstract

  At the synapse, presynaptic membranes specialized for vesicular traffic are linked to postsynaptic membranes specialized for signal transduction. The mechanisms that connect pre- and postsynaptic membranes into synaptic junctions are unknown. Neuroligins and beta-neurexins are neuronal cell-surface proteins that bind to each other and form asymmetric intercellular junctions. To test whether the neuroligin/beta-neurexin junction is related to synapses, we generated and characterized monoclonal antibodies to neuroligin 1. With these antibodies, we show that neuroligin 1 is synaptic. The neuronal localization, subcellular distribution, and developmental expression of neuroligin 1 are similar to those of the postsynaptic marker proteins PSD-95 and NMDA-R1 receptor. Quantitative immunogold electron microscopy demonstrated that neuroligin 1 is clustered in synaptic clefts and postsynaptic densities. Double immunofluorescence labeling revealed that neuroligin 1 colocalizes with glutamatergic but not gamma-aminobutyric acid (GABA)ergic synapses. Thus neuroligin 1 is a synaptic cell-adhesion molecule that is enriched in postsynaptic densities where it may recruit receptors, channels, and signal-transduction molecules to synaptic sites of cell adhesion. In addition, the neuroligin/beta-neurexin junction may be involved in the specification of excitatory synapses.

  View details for Web of Science ID 000078484100055

  View details for PubMedID 9927700

• Neurexophilin binding to alpha-neurexins - A single LNS domain functions as an independently folding ligand-binding unit JOURNAL OF BIOLOGICAL CHEMISTRY Missler, M., Hammer, R. E., Sudhof, T. C. 1998; 273 (52): 34716-34723

  Abstract

  alpha-Neurexins (Ialpha, IIalpha, and IIIalpha) are receptor-like proteins expressed in hundreds of isoforms on the neuronal cell surface. The extracellular domains of alpha-neurexins are composed of six LNS repeats, named after homologous sequences in the Laminin A G domain, Neurexins, and Sex hormone-binding globulin, with three interspersed epidermal growth factor-like domains. Purification of neurexin Ialpha revealed that it is tightly complexed to a secreted glycoprotein called neurexophilin 1. Neurexophilin 1 is a member of a family of at least four genes and resembles a neuropeptide, suggesting a function as an endogenous ligand for alpha-neurexins. We have now used recombinant proteins and knockout mice to investigate which isoforms and domains of different neurexins and neurexophilins interact with each other. We show that neurexophilins 1 and 3 but not 4 (neurexophilin 2 is not expressed in rodents) bind to a single individual LNS domain, the second overall LNS domain in all three alpha-neurexins. Although this domain is alternatively spliced, all splice variants bind, suggesting that alternative splicing does not regulate binding. Using homologous recombination to disrupt the neurexophilin 1 gene, we generated mutant mice that do not express detectable neurexophilin 1 mRNA. Mice lacking neurexophilin 1 are viable with no obvious morbidity or mortality. However, homozygous mutant mice exhibit male sterility, probably because homologous recombination resulted in the co-insertion into the neurexophilin gene of herpes simplex virus thymidine kinase, which is known to cause male sterility. In the neurexophilin 1 knockout mice, neurexin Ialpha is complexed with neurexophilin 3 but not neurexophilin 4, suggesting that neurexophilin 1 is redundant with neurexophilin 3 and that neurexophilins 1 and 3 but not 4 bind to neurexins. This hypothesis was confirmed using expression experiments. Our data reveal that the six LNS and three epidermal growth factor domains of neurexins are independently folding ligand-binding domains that may interact with distinct targets. The results support the notion that neurexophilins represent a family of extracellular signaling molecules that interact with multiple receptors including all three alpha-neurexins.

  View details for Web of Science ID 000077719700012

  View details for PubMedID 9856994

• alpha-latrotoxin receptor CIRL/latrophilin 1 (CL1) defines an unusual family of ubiquitous G-protein-linked receptors - G-protein coupling not required for triggering exocytosis JOURNAL OF BIOLOGICAL CHEMISTRY Sugita, S., Ichtchenko, K., Khvotchev, M., Sudhof, T. C. 1998; 273 (49): 32715-32724

  Abstract

  alpha-Latrotoxin, a potent excitatory neurotoxin, binds to two receptors: a G-protein-coupled receptor called CIRL/latrophilin 1 (CL1) and a cell-surface protein called neurexin Ialpha. We now show that CL1 belongs to a family of closely related receptors called CL1, CL2, and CL3. CLs exhibit an unusual multidomain structure with similar alternative splicing and large extra- and intracellular sequences. CLs share domains with other G-protein-coupled receptors, lectins, and olfactomedins/myocilin. In addition, CLs contain a novel, widespread cysteine-rich domain that may direct endoproteolytic processing of CLs during transport to the cell surface. Although the mRNAs for CLs are enriched in brain, CLs are ubiquitously expressed in all tissues. To examine how binding of alpha-latrotoxin to CL1 triggers exocytosis, we used PC12 cells transfected with human growth hormone. Ca2+-dependent secretion of human growth hormone from transfected PC12 cells was triggered by KCl depolarization or alpha-latrotoxin and was inhibited by tetanus toxin and by phenylarsine oxide, a phosphoinositide kinase inhibitor. When CL1 was transfected into PC12 cells, their response to alpha-latrotoxin was sensitized dramatically. A similar sensitization to alpha-latrotoxin was observed with different splice variants of CL1, whereas CL2 and CL3 were inactive in this assay. A truncated form of CL1 that contains only a single transmembrane region and presumably is unable to mediate G-protein-signaling was as active as wild type CL1 in alpha-latrotoxin-triggered exocytosis. Our data show that CL1, CL2, and CL3 perform a general and ubiquitous function as G-protein-coupled receptors in cellular signaling. In addition, CL1 serves a specialized role as an alpha-latrotoxin receptor that does not require G-protein-signaling for triggering exocytosis. This suggests that as an alpha-latrotoxin receptor, CL1 recruits alpha-latrotoxin to target membranes without participating in exocytosis directly.

  View details for Web of Science ID 000077329100053

  View details for PubMedID 9830014

• alpha-latrotoxin action probed with recombinant toxin: receptors recruit alpha-latrotoxin but do not transduce an exocytotic signal EMBO JOURNAL Ichtchenko, K., Khvotchev, M., Kiyatkin, N., Simpson, L., Sugita, S., Sudhof, T. C. 1998; 17 (21): 6188-6199

  Abstract

  alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. We have now produced recombinant alpha-latrotoxin (LtxWT) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.

  View details for Web of Science ID 000076984900009

  View details for PubMedID 9799228

• Mechanism of action of rab3A in mossy fiber LTP NEURON Lonart, G., Janz, R., Johnson, K. M., Sudhof, T. C. 1998; 21 (5): 1141-1150

  Abstract

  In mossy fiber synapses of the hippocampal CA3 region, LTP is induced by cAMP and requires the synaptic vesicle protein rab3A. In contrast, CA1-region synapses do not exhibit this type of LTP. We now show that cAMP enhances glutamate release from CA3 but not CA1 synaptosomes by (1) increasing the readily releasable pool as tested by hypertonic sucrose; (2) potentiating release evoked by KCl depolarization, which opens voltage-gated Ca2+ channels; and (3) by enhancing Ca2+ action on the secretory apparatus as monitored by the Ca2+-ionophore ionomycin. In rab3A-deficient synaptosomes, forskolin still enhances KCl- and sucrose-induced glutamate release but not ionomycin-induced release. Our results show that cAMP has multiple actions in mossy fiber synapses, of which only the direct activation of the secretory apparatus requires rab3A and functions in mfLTP.

  View details for Web of Science ID 000077386100024

  View details for PubMedID 9856469

• Mechanics of membrane fusion NATURE STRUCTURAL BIOLOGY Rizo, J., Sudhof, T. C. 1998; 5 (10): 839-842

  View details for Web of Science ID 000076303800002

  View details for PubMedID 9783736

• A role for cAMP in long-term depression at hippocampal mossy fiber synapses NEURON Tzounopoulos, T., Janz, R., Sudhof, T. C., Nicoll, R. A., Malenka, R. C. 1998; 21 (4): 837-845

  Abstract

  Mossy fiber synapses on hippocampal CA3 pyramidal cells, in addition to expressing an NMDA receptor-independent form of long-term potentiation (LTP), have recently been shown to express a novel presynaptic form of long-term depression (LTD). We have studied the mechanisms underlying mossy fiber LTD and present evidence that it is triggered, at least in part, by a metabotropic glutamate receptor-mediated decrease in adenylyl cyclase activity, which leads to a decrease in the activity of the cAMP-dependent protein kinase (PKA) and a reversal of the presynaptic processes responsible for mossy fiber LTP. The bidirectional control of synaptic strength at mossy fiber synapses by activity therefore appears to be due to modulation of the cAMP-PKA signaling pathway in mossy fiber boutons.

  View details for Web of Science ID 000076697300026

  View details for PubMedID 9808469

• Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A CELL Fernandez, I., Ubach, J., Dulubova, I., Zhang, X. Y., Sudhof, T. C., Rizo, J. 1998; 94 (6): 841-849

  Abstract

  Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.

  View details for Web of Science ID 000076021200016

  View details for PubMedID 9753330

• Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release NEURON Betz, A., Ashery, U., Rickmann, M., Augustin, I., Neher, E., Sudhof, T. C., Rettig, J., Brose, N. 1998; 21 (1): 123-136

  Abstract

  Munc13-1, a mammalian homolog of C. elegans unc-13p, is thought to be involved in the regulation of synaptic transmission. We now demonstrate that Munc13-1 is a presynaptic high-affinity phorbol ester and diacylglycerol receptor with ligand affinities similar to those of protein kinase C. Munc13-1 associates with the plasma membrane in response to phorbol ester binding and acts as a phorbol ester-dependent enhancer of transmitter release when overexpressed presynaptically in the Xenopus neuromuscular junction. These observations establish Munc13-1 as a novel presynaptic target of the diacylglycerol second messenger pathway that acts in parallel with protein kinase C to regulate neurotransmitter secretion.

  View details for Web of Science ID 000075061900012

  View details for PubMedID 9697857

• Neurexophilins form a conserved family of neuropeptide-like glycoproteins JOURNAL OF NEUROSCIENCE Missler, M., Sudhof, T. C. 1998; 18 (10): 3630-3638

  Abstract

  Neurexophilin was discovered as a neuronal glycoprotein that is copurified with neurexin Ialpha during affinity chromatography on immobilized alpha-latrotoxin (Petrenko et al., 1996). We have now investigated how neurexophilin interacts with neurexins, whether it is post-translationally processed by site-specific cleavage similar to neuropeptides, and whether related neuropeptide-like proteins are expressed in brain. Our data show that mammalian brains contain four genes for neurexophilins the products of which share a common structure composed of five domains: an N-terminal signal peptide, a variable N-terminal domain, a highly conserved central domain that is N-glycosylated, a short linker region, and a conserved C-terminal domain that is cysteine-rich. When expressed in pheochromocytoma (PC12) cells with a replication-deficient adenovirus, neurexophilin 1 was rapidly N-glycosylated and then slowly processed to a smaller mature form, probably by endoproteolytic cleavage. Similar expression experiments in other neuron-like cells and in fibroblastic cells revealed that N-glycosylation of neurexophilin 1 occurred in all cell types tested, whereas proteolytic processing was observed only in neuron-like cells. Finally, only recombinant neurexin Ialpha and IIIalpha but not neurexin Ibeta interacted with neurexophilin 1 and were preferentially bound to the processed mature form of neurexophilin. Together our data demonstrate that neurexophilins form a family of related glycoproteins that are proteolytically processed after synthesis and bind to alpha-neurexins. The structure and characteristics of neurexophilins indicate that they function as neuropeptides that may signal via alpha-neurexins.

  View details for Web of Science ID 000073484300015

  View details for PubMedID 9570794

• Neurexin I alpha is a major alpha-latratoxin receptor that cooperates in alpha-latrotoxin action JOURNAL OF BIOLOGICAL CHEMISTRY Geppert, M., Khvotchev, M., Krasnoperov, V., Goda, Y., Missler, M., Hammer, R. E., Ichtchenko, K., Petrenko, A. G., Sudhof, T. C. 1998; 273 (3): 1705-1710

  Abstract

  alpha-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description of two distinct cell surface proteins that bind alpha-latrotoxin with nanomolar affinities; Neurexin I alpha binds alpha-latrotoxin in a Ca(2+)-dependent manner, and CIRL/latrophilin binds in a Ca(2+)-independent manner. We have now generated and analyzed mice that lack neurexin I alpha to test its importance in alpha-latrotoxin action. alpha-Latrotoxin binding to brain membranes from mutant mice was decreased by almost 50% compared with wild type membranes; the decrease was almost entirely due to a loss of Ca(2+)-dependent alpha-latrotoxin binding sites. In cultured hippocampal neurons, alpha-latrotoxin was still capable of activating neurotransmission in the absence of neurexin I alpha. Direct measurements of [3H]glutamate release from synaptosomes, however, showed a major decrease in the amount of release triggered by alpha-latrotoxin in the presence of Ca2+. Thus neurexin I alpha is not essential for alpha-latrotoxin action but contributes to alpha-latrotoxin action when Ca2+ is present. Viewed as a whole, our results show that mice contain two distinct types of alpha-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of alpha-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin I alpha pathway contributes to the Ca(2+)-dependent action of alpha-latrotoxin.

  View details for Web of Science ID 000071411500063

  View details for PubMedID 9430716

• Postsynaptic membrane fusion and long-term potentiation SCIENCE Lledo, P. M., Zhang, X. Y., Sudhof, T. C., Malenka, R. C., Nicoll, R. A. 1998; 279 (5349): 399-403

  Abstract

  The possibility that membrane fusion events in the postsynaptic cell may be required for the change in synaptic strength resulting from long-term potentiation (LTP) was examined. Introducing substances into the postsynaptic cell that block membrane fusion at a number of different steps reduced LTP. Introducing SNAP, a protein that promotes membrane fusion, into cells enhanced synaptic transmission, and this enhancement was significantly less when generated in synapses that expressed LTP. Thus, postsynaptic fusion events, which could be involved either in retrograde signaling or in regulating postsynaptic receptor function or both, contribute to LTP.

  View details for Web of Science ID 000071570800051

  View details for PubMedID 9430593

• Region-specific phosphorylation of rabphilin in mossy fiber nerve terminals of the hippocampus JOURNAL OF NEUROSCIENCE Lonart, G., Sudhof, T. C. 1998; 18 (2): 634-640

  Abstract

  In mossy fiber synapses of the CA3 region of the hippocampus, long-term potentiation (LTP) is induced presynaptically by activation of cAMP-dependent protein kinase A (PKA). Rab3A is a synaptic vesicle protein that regulates vesicle fusion and is essential for mossy fiber LTP. Rab3A probably acts via two effector proteins, rabphilin and RIM, of which rabphilin is an in vitro substrate for PKA. To test if rabphilin is phosphorylated in nerve terminals and if its PKA-dependent phosphorylation correlates with the PKA-dependent induction of LTP in mossy fiber terminals, we have studied the phosphorylation of rabphilin in synaptosomes isolated from the CA1 and CA3 regions of the hippocampus. Rabphilin was phosphorylated in both CA1 and CA3 synaptosomes. However, when we treated the CA1 and CA3 synaptosomes with forskolin (an agent that enhances PKA activity) or induced Ca2+ influx into synaptosomes with high K+, rabphilin phosphorylation was increased selectively in mossy fiber CA3 synaptosomes, but not in CA1 synaptosomes. In contrast, the phosphorylation of synapsin, studied as a control for the specificity of the region-specific phosphorylation of rabphilin, was augmented similarly by both treatments in CA1 and CA3 synaptosomes. These results reveal that the phosphorylation states of two synaptic substrates for PKA and CaM KII, rabphilin and synapsin, are regulated differentially in a region-specific manner, an unexpected finding because rabphilin and synapsin are similarly present in CA1 and CA3 synaptosomes and are colocalized on the same synaptic vesicles. The region-specific phosphorylation of rabphilin agrees well with the restricted induction of LTP by presynaptic PKA activation in mossy fiber, but not CA1, nerve terminals.

  View details for Web of Science ID 000071414200006

  View details for PubMedID 9425005

• Neurexins: three genes and 1001 products TRENDS IN GENETICS Missler, M., Sudhof, T. C. 1998; 14 (1): 20-26

  Abstract

  The human brain has approximately 10(12) neurons, three orders of magnitude more than there are basepairs in the human genome. Each neuron is connected to other neurons by thousands of synapses, creating a dense network of communicating neurons. Cell-recognition events between neurons at, and outside of synapses, are likely to guide the development and maintenance of the complex network formed by neurons. However, little is known about which proteins are important for neuronal cell recognition. Neurexins, a family of polymorphic cell-surface proteins, might mediate some of these cell recognition events. Thousands of neurexin isoforms are generated from three genes by usage of alternative promoters and alternative splicing. These isoforms are displayed on the neuronal cell surface, with different classes of neurons expressing distinct combinations of isoforms. Neurexins probably have a multitude of ligands, some of which interact only with subsets of neurexin isoforms. This review describes the properties of the neurexin protein family and their potential roles in neuronal cell adhesion and intercellular signaling.

  View details for Web of Science ID 000071421400008

  View details for PubMedID 9448462

• Metal binding motifs in cholinesterases and neuroligins - Structural comparison 6th International Meeting on Cholinesterases and Related Proteins (Cholinesterases 98) Tsigelny, I., Matsumura, T., Sudhof, T., Taylor, P. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1998: 407–412

  View details for Web of Science ID 000082926200111

• Rab3A is essential for mossy fibre long-term potentiation in the hippocampus NATURE Castillo, P. E., Janz, R., Sudhof, T. C., Tzounopoulos, T., Malenka, R. C., Nicoll, R. A. 1997; 388 (6642): 590-593

  Abstract

  Repetitive activation of excitatory synapses in the central nervous system results in a long-lasting increase in synaptic transmission called long-term potentiation (LTP). It is generally believed that this synaptic plasticity may underlie certain forms of learning and memory. LTP at most synapses involves the activation of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor, but LTP at hippocampal mossy fibre synapses is independent of NMDA receptors and has a component that is induced and expressed presynaptically. It appears to be triggered by a rise in presynaptic Ca2+, and requires the activation of protein kinase A, which leads to an increased release of glutamate. A great deal is known about the biochemical steps involved in the vesicular release of transmitter, but none of these steps has been directly implicated in long-term synaptic plasticity. Here we show that, although a variety of short-term plasticities are normal, LTP at mossy fibre synapses is abolished in mice lacking the synaptic vesicle protein Rab3A.

  View details for Web of Science ID A1997XP72200051

  View details for PubMedID 9252190

• Calcium regulation of neurotransmitter release: reliably unreliable? CURRENT OPINION IN CELL BIOLOGY Goda, Y., Sudhof, T. C. 1997; 9 (4): 513-518

  Abstract

  Recent studies of central synaptic transmission reveal that neurotransmitter release is more unreliable than was previously thought. Nerve stimulation does not always elicit transmitter release, and when release events occur vesicle fusion with the presynaptic membrane is limited to at most a single quantum.

  View details for Web of Science ID A1997XM84400008

  View details for PubMedID 9261057

• The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion NATURE Geppert, M., Goda, Y., Stevens, C. F., Sudhof, T. C. 1997; 387 (6635): 810-814

  Abstract

  The Rab family of low-molecular-mass GTP-binding proteins are thought to guide membrane fusion between a transport vesicle and the target membrane, and to determine the specificity of docking. The docking and fusion of vesicles is, however, a complex multistep reaction, and the precise point at which Rab proteins act in these sequential processes is unknown. In brain, the Rab protein Rab3A is specific to synaptic vesicles, whose exocytosis can be monitored with submillisecond resolution by following synaptic transmission. We have now determined the precise point at which Rab3A acts in the sequence of synaptic vesicle docking and fusion by using electrophysiological analysis of neurotransmitter release in Rab3A-deficient mice. Unexpectedly, the size of the readily releasable pool of vesicles is normal, whereas Ca2+-triggered fusion is altered in the absence of Rab3A in that a more-than-usual number of exocytic events occur within a brief time after arrival of the nerve impulse.

  View details for Web of Science ID A1997XF14400052

  View details for PubMedID 9194562

• The evolutionary pressure to inactivate - A subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding JOURNAL OF BIOLOGICAL CHEMISTRY VONPOSER, C., Ichtchenko, K., Shao, X. G., Rizo, J., Sudhof, T. C. 1997; 272 (22): 14314-14319

  Abstract

  Synaptotagmin I is a Ca2+-binding protein of synaptic vesicles that serves as a Ca2+ sensor for neurotransmitter release and was the first member found of a large family of trafficking proteins. We have now identified a novel synaptotagmin, synaptotagmin XI, that is highly expressed in brain and at lower levels in other tissues. Like other synaptotagmins, synaptotagmin XI has a single transmembrane region and two cytoplasmic C2-domains but is most closely related to synaptotagmin IV with which it forms a new subclass of synaptotagmins. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids as a function of Ca2+ and contains a Ca2+-binding site, the C2-motif, that binds at least two Ca2+ ions via five aspartate residues and is conserved in most C2-domains (Shao, X., Davletov, B., Sutton, B., Südhof, T. C., Rizo, J. R. (1996) Science 273, 248-253). In the C2A-domains of synaptotagmins IV and XI, however, one of the five Ca2+-binding aspartates in the C2-motif is substituted for a serine, suggesting that these C2-domains do not bind Ca2+. To test this, we produced recombinant C2A-domains from synaptotagmins IV and XI with either wild type serine or mutant aspartate in the C2-motif. Circular dichroism showed that Ca2+ stabilizes both mutant but not wild type C2-domains against temperature-induced denaturation, indicating that the mutations restore Ca2+-binding to the wild type C2-domains. Furthermore, wild type C2A-domains of synaptotagmins IV and XI exhibited no Ca2+-dependent phospholipid binding, whereas mutant C2A-domains bound phospholipids as a function of Ca2+ similarly to wild type synaptotagmin I. These experiments suggest that a class of synaptotagmins was selected during evolution in which the Ca2+-binding site of the C2A-domain was inactivated by a single point mutation. Thus, synaptotagmins must have Ca2+-independent functions as well as Ca2+-dependent functions that are selectively maintained in distinct members of this gene family.

  View details for Web of Science ID A1997XB49200053

  View details for PubMedID 9162066

• Structure and evolution of neurexophilin JOURNAL OF NEUROSCIENCE Petrenko, A. G., Ullrich, B., Missler, M., Krasnoperov, V., Rosahl, T. W., Sudhof, T. C. 1996; 16 (14): 4360-4369

  Abstract

  Using affinity chromatography on immobilized alpha-latrotoxin, we have purified a novel 29 kDa protein, neurexophilin, in a complex with neurexin l alpha. Cloning revealed that rat and bovine neurexophilins are composed of N-terminal signal peptides, nonconserved N-terminal domains (20% identity over 80 residues), and highly homologous C-terminal sequences (85% identity over 169 residues). Analysis of genomic clones from mice identified two distinct neurexophilin genes, one of which is more homologous to rat neurexophilin and the other to bovine neurexophilin. The first neurexophilin gene is expressed abundantly in adult rat and mouse brain, whereas no mRNA corresponding to the second gene was detected in rodents despite its abundant expression in bovine brain, suggesting that rodents and cattle primarily express distinct neurexophilin genes. RNA blots and in situ hybridizations revealed that neurexophilin is expressed in adult rat brain at high levels only in a scattered subpopulation of neurons that probably represent inhibitory interneurons; by contrast, neurexins are expressed in all neurons. Neurexophilin contains a signal sequence and is N-glycosylated at multiple sites, suggesting that it is secreted and binds to the extracellular domain of neurexin l alpha. This hypothesis was confirmed by binding recombinant neurexophilin to the extracellular domains of neurexin l alpha. Together our data suggest that neurexophilin constitutes a secreted glycoprotein that is synthesized in a subclass of neurons and may be a ligand for neurexins.

  View details for Web of Science ID A1996UW87900007

  View details for PubMedID 8699246

• Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C - Its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin JOURNAL OF BIOLOGICAL CHEMISTRY Fujita, Y., Sasaki, T., Fukui, K., Kotani, H., Kimura, T., Hata, Y., Sudhof, T. C., Scheller, R. H., Takai, Y. 1996; 271 (13): 7265-7268

  Abstract

  Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca2+-dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser306 and Ser313. The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane.

  View details for Web of Science ID A1996UC77400006

  View details for PubMedID 8631738

• Structures, alternative splicing, and neurexin binding of multiple neuroligins JOURNAL OF BIOLOGICAL CHEMISTRY Ichtchenko, K., Nguyen, T., Sudhof, T. C. 1996; 271 (5): 2676-2682

  Abstract

  Neuroligin 1 is a neuronal cell surface protein that binds to a subset of neurexins, polymorphic cell surface proteins that are also localized on neurons (Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C., and Südhof, T. C. (1995) Cell 81, 435-443). We now describe two novel neuroligins called neuroligins 2 and 3 that are similar in structure and sequence to neuroligin 1. All neuroligins contain an N-terminal hydrophobic sequence with the characteristics of a cleaved signal peptide followed by a large esterase homology domain, a highly conserved single transmembrane region, and a short cytoplasmic domain. The three neuroligins are alternatively spliced at the same position and are expressed at high levels only in brain. Binding studies demonstrate that all three neuroligins bind to beta-neurexins both as native brain proteins and as recombinant proteins. Tight binding of the three neuroligins to beta-neurexins is observed only for beta-neurexins lacking an insert in splice site 4. Thus, neuroligins constitute a multigene family of brain-specific proteins with distinct isoforms that may have overlapping functions in mediating recognition processes between neurons.

  View details for Web of Science ID A1996TT48800054

  View details for PubMedID 8576240

• LONG-TERM POTENTIATION IN MICE LACKING SYNAPSINS Neuropharmacology Symposium on Presynaptic Mechanisms of Neurotransmission SPILLANE, D. M., Rosahl, T. W., Sudhof, T. C., Malenka, R. C. PERGAMON-ELSEVIER SCIENCE LTD. 1995: 1573–79

  Abstract

  Synapsin I and synapsin II are widely expressed synaptic vesicle phosphoproteins that have been proposed to play an important role in synaptic transmission and synaptic plasticity. To gain further insight into the functional significance of the phosphorylation sites on the synapsins, we have examined a number of synaptic processes thought to be mediated by protein kinases in knockout mice lacking both forms of synapsin (Rosahl et al., 1995). Long-term potentiation (LTP) at both the mossy fiber (MF)-CA3 pyramidal cell synapse and the Schaffer collateral-CA1 pyramidal cell synapse appears normal in hippocampal slices prepared from mice lacking synapsins. Moreover, the effects on synaptic transmission of forskolin at MF synapses and H-7 at synapses on CA1 cells are also normal in the mutant mice. These results indicate that the synapsins are not necessary for: (1) the induction or expression of two different forms of LTP in the hippocampus, (2) the enhancement in transmitter release elicited by activation of the cAMP-dependent protein kinase (PKA) and (3) the depression of synaptic transmission caused by H-7. Although disappointing, these results are important in that they exclude the most abundant family of synaptic phosphoproteins as an essential component of long-term synaptic plasticity.

  View details for Web of Science ID A1995TD88400024

  View details for PubMedID 8606805

• ESSENTIAL FUNCTIONS OF SYNAPSIN-I AND SYNAPSIN-II IN SYNAPTIC VESICLE REGULATION NATURE Rosahl, T. W., Spillane, D., Missler, M., Herz, J., Selig, D. K., Wolff, J. R., Hammer, R. E., Malenka, R. C., Sudhof, T. C. 1995; 375 (6531): 488-493

  Abstract

  Synaptic vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.

  View details for Web of Science ID A1995RC18800048

  View details for PubMedID 7777057

• NEUROLIGIN-1 - A SPLICE SITE-SPECIFIC LIGAND FOR BETA-NEUREXINS CELL Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C., Sudhof, T. C. 1995; 81 (3): 435-443

  Abstract

  Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.

  View details for Web of Science ID A1995QW89400015

  View details for PubMedID 7736595

• MAPPING OF SYNAPSIN-II (SYN2) GENES TO HUMAN-CHROMOSOME-3P AND MOUSE CHROMOSOME-6 BAND-F CYTOGENETICS AND CELL GENETICS Li, X., Rosahl, T. W., Sudhof, T. C., FRANCKE, U. 1995; 71 (3): 301-305

  Abstract

  Synapsins are neuron-specific phosphoproteins of small synaptic vesicles encoded by two different genes. While the gene for synapsin I (SYN1) is on the X chromosome, we have now assigned the human and mouse synapsin II (SYN2) genes to autosomes. By using PCR primers derived from rat synapsin II cDNA sequences we were able to amplify homologous sequences of the 3'-untranslated regions and to localize the human SYN2 gene to 3p and the mouse Syn2 gene to mouse chromosome 6 by single strand conformation analysis of PCR products from panels of somatic hybrid cell lines. The mouse gene was further mapped by FISH to chromosome 6 band F in a region of known conserved synteny with human 3p. Genotyping of a M. musculus x M. spretus backcross panel placed Syn2 close to a cluster of previously mapped loci on chromosome 6 in an interval between interleukin 5 receptor alpha (Il5ra) and hematopoietic cell phosphatase 1C (Hcph). Both physical and genetic mapping data indicate that Syn2 is near two mutant loci defined by neuromuscular disorders, opisthotonus (opt) and deaf waddler (dfw).

  View details for Web of Science ID A1995TD38500023

  View details for PubMedID 7587399

• A systematic approach to studying synaptic function in vertebrates Cold Spring Harbor Symposia on Quantitative Biology - Protein Kinesis: The Dynamics of Protein Trafficking and Stability Janz, R., Sudhof, T. C. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1995: 309–314

  View details for Web of Science ID A1995VA12500034

  View details for PubMedID 8824404

• SYNAPTOTAGMIN-I - A MAJOR CA2+ SENSOR FOR TRANSMITTER RELEASE AT A CENTRAL SYNAPSE CELL Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., Sudhof, T. C. 1994; 79 (4): 717-727

  Abstract

  Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca(2+)-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or alpha-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca2+ triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca(2+)-independent release. We propose that synaptotagmin I is the major low affinity Ca2+ sensor mediating Ca2+ regulation of synchronous neurotransmitter release in hippocampal neurons.

  View details for Web of Science ID A1994PT48100018

  View details for PubMedID 7954835

• SHORT-TERM SYNAPTIC PLASTICITY IS ALTERED IN MICE LACKING SYNAPSIN-I CELL Rosahl, T. W., Geppert, M., Spillane, D., Herz, J., Hammer, R. E., Malenka, R. C., Sudhof, T. C. 1993; 75 (4): 661-670

  Abstract

  Synapsin I, the major phosphoprotein of synaptic vesicles, is thought to play a central role in neurotransmitter release. Here we introduce a null mutation into the murine synapsin I gene by homologous recombination. Mice with no detectable synapsin I manifest no apparent changes in well-being or gross nervous system function. Thus, synapsin I is not essential for neurotransmitter release. Electrophysiology reveals that mice lacking synapsin I exhibit a selective increase in paired pulse facilitation, with no major alterations in other synaptic parameters such as long-term potentiation. In addition to potential redundant functions shared with other proteins, synapsin I in normal mice may function to limit increases in neurotransmitter release elicited by residual Ca2+ after an initial stimulus.

  View details for Web of Science ID A1993MH74900012

  View details for PubMedID 7902212

• MOLECULAR APPROACHES TO SYNAPTIC VESICLE EXOCYTOSIS 8TH INTERNATIONAL CHOLINERGIC SYMP Sudhof, T. C., Petrenko, A. G., Whittaker, V. P., Jahn, R. ELSEVIER SCIENCE PUBL B V. 1993: 235–240

  View details for Web of Science ID A1993BZ03G00030

• MOLECULAR APPROACHES TO SYNAPTIC VESICLE EXOCYTOSIS PROGRESS IN BRAIN RESEARCH Sudhof, T. C., Petrenko, A. G., Whittaker, V. P., Jahn, R. 1993; 98: 235-240

  View details for Web of Science ID A1993MA26900030

  View details for PubMedID 8248512

• SYNAPTOTAGMIN - A CALCIUM SENSOR ON THE SYNAPTIC VESICLE SURFACE SCIENCE Brose, N., Petrenko, A. G., Sudhof, T. C., Jahn, R. 1992; 256 (5059): 1021-1025

  Abstract

  Neurons release neurotransmitters by calcium-dependent exocytosis of synaptic vesicles. However, the molecular steps transducing the calcium signal into membrane fusion are still an enigma. It is reported here that synaptotagmin, a highly conserved synaptic vesicle protein, binds calcium at physiological concentrations in a complex with negatively charged phospholipids. This binding is specific for calcium and involves the cytoplasmic domain of synaptotagmin. Calcium binding is dependent on the intact oligomeric structure of synaptotagmin (it is abolished by proteolytic cleavage at a single site). These results suggest that synaptotagmin acts as a cooperative calcium receptor in exocytosis.

  View details for Web of Science ID A1992HU22400039

  View details for PubMedID 1589771

• STRUCTURAL AND FUNCTIONAL CONSERVATION OF SYNAPTOTAGMIN (P65) IN DROSOPHILA AND HUMANS JOURNAL OF BIOLOGICAL CHEMISTRY Perin, M. S., Johnston, P. A., Ozcelik, T., Jahn, R., FRANCKE, U., Sudhof, T. C. 1991; 266 (1): 615-622

  Abstract

  Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C. Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution. The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species). The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent. The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bound phosphatidylserine similar to rat synaptotagmin. They also hemagglutinated trypsinized erythrocytes at nanomolar concentrations. Hemagglutination was inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contained only a single copy of the two internal repeats. Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone. The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding. The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B.

  View details for Web of Science ID A1991EQ33900089

  View details for PubMedID 1840599

• DOMAIN-STRUCTURE OF SYNAPTOTAGMIN (P65) JOURNAL OF BIOLOGICAL CHEMISTRY Perin, M. S., Brose, N., Jahn, R., Sudhof, T. C. 1991; 266 (1): 623-629

  Abstract

  Synaptotagmin (p65) is an abundant and evolutionarily conserved protein of synaptic vesicles that contains two copies of an internal repeat homologous to the regulatory region of protein kinase C. In the current study, we have investigated the biochemical properties of synaptotagmin, demonstrating that it contains five protein domains: an intravesicular amino-terminal domain that is glycosylated but lacks a cleavable signal sequence; a single transmembrane region; a sequence separating the transmembrane region from the two repeats homologous to protein kinase C; the two protein kinase C-homologous repeats; and a conserved carboxyl-terminal sequence following the two repeats homologous to protein kinase C. Sucrose density gradient centrifugations and gel electrophoresis indicate that synaptotagmin monomers associate into dimers and are part of a larger molecular weight complex. A sequence predicted to form an amphipathic alpha-helix that may cause the stable dimerization of synaptotagmin is found in its third domain between the transmembrane region and the protein kinase C-homologous repeats. Synaptotagmin contains a single hypersensitive proteolytic site that is located immediately amino-terminal to the amphipathic alpha-helix, suggesting that synaptotagmin contains a particularly exposed region as the peptide backbone emerges from the dimer. Finally, subcellular fractionation and antibody bead purification demonstrate that synaptotagmin co-purifies with synaptophysin and other synaptic vesicle markers in brain. However, in the adrenal medulla, synaptotagmin was found in both synaptophysin-containing microvesicles and in chromaffin granules that are devoid of synaptophysin, suggesting a shared role for synaptotagmin in the exocytosis of small synaptic vesicles and large dense core catecholaminergic vesicles.

  View details for Web of Science ID A1991EQ33900090

  View details for PubMedID 1985919

• STRUCTURES AND CHROMOSOMAL LOCALIZATIONS OF 2 HUMAN GENES ENCODING SYNAPTOBREVIN-1 AND SYNAPTOBREVIN-2 JOURNAL OF BIOLOGICAL CHEMISTRY ARCHER, B. T., Ozcelik, T., Jahn, R., FRANCKE, U., Sudhof, T. C. 1990; 265 (28): 17267-17273

  Abstract

  Synaptobrevins 1 and 2 are small integral membrane proteins specific for synaptic vesicles in neurons. Two cosmid clones containing the human genes encoding synaptobrevins 1 and 2 (gene symbols SYB1 and SYB2, respectively) were isolated and characterized. The coding regions of the synaptobrevin genes are highly homologous to each other and are interrupted at identical positions by introns of different size and sequence. Each gene is organized into five exons whose boundaries correspond to those of the protein domains. Exon I contains part of the initiator methionine codon whereas exon II encodes the variable and immunogenic amino-terminal domain of the synaptobrevins. The third exon comprises the highly conserved central domain of the synaptobrevins, exon IV encodes most of the transmembrane region, and exon V contains the last residues of the transmembrane region and the small intravesicular carboxyl terminus. Comparisons of the synaptobrevin sequences in five species from Drosophila with man indicate a selective conservation of sequences adjacent to the synaptic vesicle surface, suggesting a function at the membrane-cystosol interface. The chromosomal localizations of the human and mouse SYB1 and SYB2 genes were determined using hybrid cell lines. SYB1 was localized to the short arm of human chromosome 12 and to mouse chromosome 6 whereas SYB2 was found on the distal portion of the short arm of human chromosome 17 and on mouse chromosome 11. A PstI restriction fragment length polymorphism was identified at the SYB2 locus.

  View details for Web of Science ID A1990EA85700086

  View details for PubMedID 1976629

• SYNAPTOPHYSIN - STRUCTURE OF THE HUMAN GENE AND ASSIGNMENT TO THE X-CHROMOSOME IN MAN AND MOUSE AMERICAN JOURNAL OF HUMAN GENETICS Ozcelik, T., Lafreniere, R. G., ARCHER, B. T., Johnston, P. A., WILLARD, H. F., FRANCKE, U., Sudhof, T. C. 1990; 47 (3): 551-561

  Abstract

  Synaptophysin is an integral membrane protein of small synaptic vesicles in brain and endocrine cells. We have determined the structure and organization of the human synaptophysin gene and have established the chromosome localizations in man and mouse. Analysis of a cosmid clone containing the human synaptophysin gene (SYP) revealed seven exons distributed over approximately 20 kb, when compared with the previously published cDNA sequence. The exon-intron boundaries have been identified and do not correlate with functional domains. One intron interrupts the 3' untranslated region. Chromosomal localization of the human and murine genes for synaptophysin established the human SYP locus on the X chromosome in subbands Xp11.22-p11.23 and the mouse synaptophysin gene locus (Syp) on the X chromosome in region A-D. In addition, an Eco0109 RFLP has been identified and used in genetic mapping of the human SYP locus and supports the order TIMP-SYP-DXS14 within a span of approximately 4-7 centimorgans.

  View details for Web of Science ID A1990DX22300020

  View details for PubMedID 1975480