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  • Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching. Cell Xu, C., Li, Z., Lyu, C., Hu, Y., McLaughlin, C. N., Wong, K. K., Xie, Q., Luginbuhl, D. J., Li, H., Udeshi, N. D., Svinkina, T., Mani, D. R., Han, S., Li, T., Li, Y., Guajardo, R., Ting, A. Y., Carr, S. A., Li, J., Luo, L. 2024

    Abstract

    In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

    View details for DOI 10.1016/j.cell.2024.06.022

    View details for PubMedID 38996528

  • Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons SCIENCE TRANSLATIONAL MEDICINE Qian, J., Guan, X., Xie, B., Xu, C., Niu, J., Tang, X., Li, C. H., Colecraft, H. M., Jaenisch, R., Liu, X. 2023; 15 (679): eadd4666

    Abstract

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 (MECP2) on the X chromosome in young females. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target single-guide RNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation-edited MECP2 locus by dCpf1-CTCF (a catalytically dead Cpf1 fused with CCCTC-binding factor) with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.

    View details for DOI 10.1126/scitranslmed.add4666

    View details for Web of Science ID 000932397900001

    View details for PubMedID 36652535

  • In situ cell-type-specific cell-surface proteomic profiling in mice. Neuron Shuster, S. A., Li, J., Chon, U., Sinantha-Hu, M. C., Luginbuhl, D. J., Udeshi, N. D., Carey, D. K., Takeo, Y. H., Xie, Q., Xu, C., Mani, D. R., Han, S., Ting, A. Y., Carr, S. A., Luo, L. 2022

    Abstract

    Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

    View details for DOI 10.1016/j.neuron.2022.09.025

    View details for PubMedID 36220098

  • In Situ Cell-Surface Proteomics: Method Development and Applications in Neurobiology Li, J., Han, S., Xie, Q., Shuster, S. A., Li, H., Udeshi, N. D., Svinkina, T., Carey, D. K., Mani, D. R., Xu, C., Guajardo, R., Chon, U., Luginbuhl, D. J., McLaughlin, C. N., Takeo, Y. H., Li, T., Orlin, D., Hu, M. C., Kohani, S., Wu, B., Xie, A., Kaewsapsak, P., Murthy, S. E., Quake, S. R., Carr, S. A., Ting, A. Y., Luo, L. ELSEVIER. 2022: S71

    View details for DOI 10.1016/j.mcpro.2022.100382

    View details for Web of Science ID 000898188800091

  • Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code. Neuron Xie, Q., Li, J., Li, H., Udeshi, N. D., Svinkina, T., Orlin, D., Kohani, S., Guajardo, R., Mani, D. R., Xu, C., Li, T., Han, S., Wei, W., Shuster, S. A., Luginbuhl, D. J., Quake, S. R., Murthy, S. E., Ting, A. Y., Carr, S. A., Luo, L. 2022

    Abstract

    Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.

    View details for DOI 10.1016/j.neuron.2022.04.026

    View details for PubMedID 35613619

  • Reciprocal repulsions instruct the precise assembly of parallel hippocampal networks. Science (New York, N.Y.) Pederick, D. T., Lui, J. H., Gingrich, E. C., Xu, C., Wagner, M. J., Liu, Y., He, Z., Quake, S. R., Luo, L. 2021; 372 (6546): 1068-1073

    Abstract

    Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3Ten3, Lphn2Lphn2" rule instructed by reciprocal repulsions.

    View details for DOI 10.1126/science.abg1774

    View details for PubMedID 34083484

  • Temporal evolution of single-cell transcriptomes of Drosophila olfactory projection neurons. eLife Xie, Q., Brbic, M., Horns, F., Kolluru, S. S., Jones, R. C., Li, J., Reddy, A. R., Xie, A., Kohani, S., Li, Z., McLaughlin, C. N., Li, T., Xu, C., Vacek, D., Luginbuhl, D. J., Leskovec, J., Quake, S. R., Luo, L., Li, H. 2021; 10

    Abstract

    Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

    View details for DOI 10.7554/eLife.63450

    View details for PubMedID 33427646

  • Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators. Cell Li, J., Han, S., Li, H., Udeshi, N. D., Svinkina, T., Mani, D. R., Xu, C., Guajardo, R., Xie, Q., Li, T., Luginbuhl, D. J., Wu, B., McLaughlin, C. N., Xie, A., Kaewsapsak, P., Quake, S. R., Carr, S. A., Ting, A. Y., Luo, L. 2020

    Abstract

    Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed invivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

    View details for DOI 10.1016/j.cell.2019.12.029

    View details for PubMedID 31955847

  • Single-Cell Transcriptomes Reveal Diverse Regulatory Strategies for Olfactory Receptor Expression and Axon Targeting. Current biology : CB Li, H. n., Li, T. n., Horns, F. n., Li, J. n., Xie, Q. n., Xu, C. n., Wu, B. n., Kebschull, J. M., McLaughlin, C. N., Kolluru, S. S., Jones, R. C., Vacek, D. n., Xie, A. n., Luginbuhl, D. J., Quake, S. R., Luo, L. n. 2020

    Abstract

    The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, we identified 33 transcriptomic clusters for ORNs and mapped 20 to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.

    View details for DOI 10.1016/j.cub.2020.01.049

    View details for PubMedID 32059767

  • Cell-surface proteomic landscape of developing and mature olfactory projection neurons Li, J., Han, S., Li, H., Udeshi, N. D., Svinkina, T., Mani, D. R., Xu, C., Guajardo, R., Xie, Q., Li, T., Wu, B., Xie, A., Luginbuhl, D. J., Kaewsapsak, P., Quake, S. R., Carr, S. A., Ting, A. Y., Luo, L. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2019: S40

    View details for Web of Science ID 000516535000051

  • Transsynaptic Fish-lips signaling prevents misconnections between nonsynaptic partner olfactory neurons. Proceedings of the National Academy of Sciences of the United States of America Xie, Q., Wu, B., Li, J., Xu, C., Li, H., Luginbuhl, D. J., Wang, X., Ward, A., Luo, L. 2019

    Abstract

    Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the Drosophila olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the Drosophila olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.

    View details for DOI 10.1073/pnas.1905832116

    View details for PubMedID 31341080

  • Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene CELL Liu, X., Wu, H., Krzisch, M., Wu, X., Graef, J., Muffat, J., Hnisz, D., Li, C. H., Yuan, B., Xu, C., Li, Y., Vershkov, D., Cacace, A., Young, R. A., Jaenisch, R. 2018; 172 (5): 979-+

    Abstract

    Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.

    View details for DOI 10.1016/j.cell.2018.01.012

    View details for Web of Science ID 000425937100010

    View details for PubMedID 29456084

  • Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels. eLife Li, J. n., Guajardo, R. n., Xu, C. n., Wu, B. n., Li, H. n., Li, T. n., Luginbuhl, D. J., Xie, X. n., Luo, L. n. 2018; 7

    Abstract

    The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. Here, we examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in Drosophila. We found that different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the Drosophila olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.

    View details for PubMedID 30136927

  • RNA m(6)A methylation regulates the ultraviolet-induced DNA damage response NATURE Xiang, Y., Laurent, B., Hsu, C., Nachtergaele, S., Lu, Z., Sheng, W., Xu, C., Hen, H. C., Jian, O., Wang, S., Ling, D., Hsu, P., Zou, L., Jambhekar, A., IIe, C., Shi, Y. 2017; 543 (7646): 573-+

    Abstract

    Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

    View details for DOI 10.1038/nature21671

    View details for Web of Science ID 000397018000056

    View details for PubMedID 28297716

    View details for PubMedCentralID PMC5490984