Administrative Appointments

  • Co-Director, Stanford Down Syndrome Center (2004 - Present)

Honors & Awards

  • Beach Distinguished Lecture, Purdue University (2004)
  • Gian Tondury Prize, Swiss Science Foundation (1989)
  • A.K. Balls Award for Distinguished Research, Purdue University (1984)

Professional Education

  • Ph.D, Purdue University, Biochemistry (1984)
  • BA, Rutgers University, Biochemistry (1979)

Current Research and Scholarly Interests

What are the cellular and molecular mechanisms underlying synapse formation, stability, and elimination? This question is central to understanding both how the nervous system becomes wired during development, and how stable circuits, such as those thought to underlie the processes of learning and memory, are formed and maintained in the adult. To address these issues, our laboratory is studying synapse formation, stability and elimination at a variety of levels, e.g. from molecules to behavior. A major component of the lab is focused on the molecules that structurally define glutamatergic synapses, the major excitatory synapses of the nervous system. Here, we have used molecular strategies to identify and characterize many components of both the presynaptic active zone and the postsynaptic density. Our presynaptic studies are oriented towards a collection of active zone proteins, including Piccolo, Bassoon, RIM and Munc13, and their roles in active zone formation and function, with an emerging focus on molecular mechanisms of presynaptic forms of plasticity. Similarly, our work on postsynaptic proteins is focused on several classes of multidomain proteins, including PSD95/SAP90, SAP97, SAP102, ProSAP and SAPAPs, and their roles in assembling the postsynaptic density and establishing postsynaptic forms of plasticity via the directed trafficking of glutamate receptors. In addition to studying how these molecules are trafficked and recruited to nascent synapses, and their functional roles in synaptic plasticity, we are also examining mechanisms that determine their turnover and exchange rates within the synapse. We believe that these studies will be instrumental for understanding the development and function of neuronal circuits.

A new direction of the laboratory is to link changes in synaptic dynamics and plasticity to the cognitive deficits found in humans with genetic forms of mental retardation. Current efforts are directed towards understanding the root causes of cognitive impairment in individuals with Down syndrome. Here, we are employing mouse models of Down syndrome to address three questions. First, do neurons differentiate and form synaptic contacts at normal rates and numbers? Second, are there changes in the circuit properties of neuronal networks? Third, are there changes in neuronal excitability and if so, do these lead to changes in behavior and cognitive performance? Our studies reveal that neurons from these mice make synapses in normal numbers, but that their circuit properties are subtly altered. Of particular note, we find that the balance of excitation and inhibition in the brains of these mice is shifted such that there is too much inhibition. We are currently employing behavioral studies of these mice to examine if this is indeed the case and whether drugs that reduce inhibition lead to an increase in cognitive performance.

2013-14 Courses

Postdoctoral Advisees

Journal Articles

  • Short-term treatment with the GABAA receptor antagonist pentylenetetrazole produces a sustained pro-cognitive benefit in a mouse model of Down's syndrome. British journal of pharmacology Colas, D., Chuluun, B., Warrier, D., Blank, M., Wetmore, D. Z., Buckmaster, P., Garner, C. C., Heller, H. C. 2013; 169 (5): 963-973


    BACKGROUND AND PURPOSE: Down's syndrome (DS) is a common genetic cause of intellectual disability yet there are no drug therapies. Mechanistic studies in a model of DS (Ts65Dn mice) demonstrated that impaired cognitive function is due to excessive neuronal inhibitory tone. These deficits can be normalized by low doses of GABAA receptor (GABAA R) antagonists in adult animals. In this study, we explore the therapeutic potential of pentylenetetrazole (PTZ), a GABAA R antagonist which has a history of safe use in man. EXPERIMENTAL APPROACH: Long-term memory was assessed by the Novel Object Recognition (NOR) test in different cohorts of Ts65Dn mice after a delay following a short-term chronic treatment with PTZ. Seizure susceptibility, as an index of treatment safety, was studied by means of EEG, behavior and hippocampus morphology. EEG spectral analysis was used as a biometric of the treatment. RESULTS: PTZ has a broad therapeutic window ( ) that is >10-1000 fold below the seizure threshold for this drug, and chronic PTZ treatment does not lower the seizure threshold. Remarkably, short-term, low, chronic dose regimens of PTZ elicit long-lasting (>1week) normalization of cognitive function in young and aged mice. PTZ effectiveness is time of day dependent: cognitive performance improves when PTZ is delivered during the light (inactive) phase, but not during the dark (active) phase. Chronic PTZ treatment results in EEG power normalization. CONCLUSIONS: PTZ at very low dosage can be administered safely, produces long lasting cognitive improvements and has the potential of fulfilling an unmet therapeutic need in DS.

    View details for DOI 10.1111/bph.12169

    View details for PubMedID 23489250

  • Nanostraw-electroporation system for highly efficient intracellular delivery and transfection. ACS nano Xie, X., Xu, A. M., Leal-Ortiz, S., Cao, Y., Garner, C. C., Melosh, N. A. 2013; 7 (5): 4351-4358


    Nondestructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical, process. Here we demonstrate a simple nanoelectroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield cotransfection and sequential transfection.

    View details for DOI 10.1021/nn400874a

    View details for PubMedID 23597131

  • Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation EMBO JOURNAL Waites, C. L., Leal-Ortiz, S. A., Okerlund, N., Dalke, H., Fejtova, A., Altrock, W. D., Gundelfinger, E. D., Garner, C. C. 2013; 32 (7): 954-969


    The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.

    View details for DOI 10.1038/emboj.2013.27

    View details for Web of Science ID 000317040600007

    View details for PubMedID 23403927

  • Autism-Associated Mutations in ProSAP2/Shank3 Impair Synaptic Transmission and Neurexin-Neuroligin-Mediated Transsynaptic Signaling JOURNAL OF NEUROSCIENCE Arons, M. H., Thynne, C. J., Grabrucker, A. M., Li, D., Schoen, M., Cheyne, J. E., Boeckers, T. M., Montgomery, J. M., Garner, C. C. 2012; 32 (43): 14966-14978


    Mutations in several postsynaptic proteins have recently been implicated in the molecular pathogenesis of autism and autism spectrum disorders (ASDs), including Neuroligins, Neurexins, and members of the ProSAP/Shank family, thereby suggesting that these genetic forms of autism may share common synaptic mechanisms. Initial studies of ASD-associated mutations in ProSAP2/Shank3 support a role for this protein in glutamate receptor function and spine morphology, but these synaptic phenotypes are not universally penetrant, indicating that other core facets of ProSAP2/Shank3 function must underlie synaptic deficits in patients with ASDs. In the present study, we have examined whether the ability of ProSAP2/Shank3 to interact with the cytoplasmic tail of Neuroligins functions to coordinate pre/postsynaptic signaling through the Neurexin-Neuroligin signaling complex in hippocampal neurons of Rattus norvegicus. Indeed, we find that synaptic levels of ProSAP2/Shank3 regulate AMPA and NMDA receptor-mediated synaptic transmission and induce widespread changes in the levels of presynaptic and postsynaptic proteins via Neurexin-Neuroligin transsynaptic signaling. ASD-associated mutations in ProSAP2/Shank3 disrupt not only postsynaptic AMPA and NMDA receptor signaling but also interfere with the ability of ProSAP2/Shank3 to signal across the synapse to alter presynaptic structure and function. These data indicate that ASD-associated mutations in a subset of synaptic proteins may target core cellular pathways that coordinate the functional matching and maturation of excitatory synapses in the CNS.

    View details for DOI 10.1523/JNEUROSCI.2215-12.2012

    View details for Web of Science ID 000310523900013

    View details for PubMedID 23100419

  • Formation of Golgi-Derived Active Zone Precursor Vesicles JOURNAL OF NEUROSCIENCE Maas, C., Torres, V. I., Altrock, W. D., Leal-Ortiz, S., Wagh, D., Terry-Lorenzo, R. T., Fejtova, A., Gundelfinger, E. D., Ziv, N. E., Garner, C. C. 2012; 32 (32): 11095-11108


    Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1? associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.

    View details for DOI 10.1523/JNEUROSCI.0195-12.2012

    View details for Web of Science ID 000307640000028

    View details for PubMedID 22875941

  • RAE-1, a Novel PHR Binding Protein, Is Required for Axon Termination and Synapse Formation in Caenorhabditis elegans JOURNAL OF NEUROSCIENCE Grill, B., Chen, L., Tulgren, E. D., Baker, S. T., Bienvenut, W., Anderson, M., Quadroni, M., Jin, Y., Garner, C. C. 2012; 32 (8): 2628-2636


    Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

    View details for DOI 10.1523/JNEUROSCI.2901-11.2012

    View details for Web of Science ID 000300716600007

    View details for PubMedID 22357847

  • Synaptic Pathology of Down Syndrome SYNAPTIC PLASTICITY: DYNAMICS, DEVELOPMENT AND DISEASE Garner, C. C., Wetmore, D. Z. 2012; 970: 451-468


    Down syndrome is characterized by mild to moderate cognitive impairments that are caused by trisomy of chromosome 21. Several anatomical, behavioral, electrophysiological, and developmental abnormalities have been associated with Down syndrome. In this review, the current knowledge about the neurobiology of this disease and future perspectives of pharmacological treatments for this condition will be discussed.

    View details for DOI 10.1007/978-3-7091-0932-8_20

    View details for Web of Science ID 000303541700020

    View details for PubMedID 22351068

  • Use Dependence of Presynaptic Tenacity JOURNAL OF NEUROSCIENCE Fisher-Lavie, A., Zeidan, A., Stern, M., Garner, C. C., Ziv, N. E. 2011; 31 (46): 16770-16780


    Recent studies indicate that synaptic vesicles (SVs) are continuously interchanged among nearby synapses at very significant rates. These dynamics and the lack of obvious barriers confining synaptic vesicles to specific synapses would seem to challenge the ability of synapses to maintain a constant amount of synaptic vesicles over prolonged time scales. Moreover, the extensive mobilization of synaptic vesicles associated with presynaptic activity might be expected to intensify this challenge. Here we examined the ability of individual presynaptic boutons of rat hippocampal neurons to maintain their synaptic vesicle content, and the degree to which this ability is affected by continuous activity. We found that the synaptic vesicle content of individual boutons belonging to the same axons gradually changed over several hours, and that these changes occurred independently of activity. Intermittent stimulation for 1 h accelerated rates of vesicle pool size change. Interestingly, however, following stimulation cessation, vesicle pool size change rates gradually converged with basal change rates. Over similar time scales, active zones (AZs) exhibited substantial remodeling; yet, unlike synaptic vesicles, AZ remodeling was not affected by the stimulation paradigms used here. These findings indicate that enhanced activity levels can increase synaptic vesicle redistribution among nearby synapses, but also highlight the presence of forces that act to restore particular set points in terms of SV contents, and support a role for active zones in preserving such set points. These findings also indicate, however, that neither AZ size nor SV content set points are particularly stable, questioning the long-term tenacity of presynaptic specializations.

    View details for DOI 10.1523/JNEUROSCI.3384-11.2011

    View details for Web of Science ID 000307564200001

    View details for PubMedID 22090503

  • Piccolo Regulates the Dynamic Assembly of Presynaptic F-Actin JOURNAL OF NEUROSCIENCE Waites, C. L., Leal-Ortiz, S. A., Andlauer, T. F., Sigrist, S. J., Garner, C. C. 2011; 31 (40): 14250-14263


    Filamentous (F)-actin is a known regulator of the synaptic vesicle (SV) cycle, with roles in SV mobilization, fusion, and endocytosis. However, the molecular pathways that regulate its dynamic assembly within presynaptic boutons remain unclear. In this study, we have used shRNA-mediated knockdown to demonstrate that Piccolo, a multidomain protein of the active zone cytomatrix, is a key regulator of presynaptic F-actin assembly. Boutons lacking Piccolo exhibit enhanced activity-dependent Synapsin1a dispersion and SV exocytosis, and reduced F-actin polymerization and CaMKII recruitment. These phenotypes are rescued by stabilizing F-actin filaments and mimicked by knocking down Profilin2, another regulator of presynaptic F-actin assembly. Importantly, we find that mice with a targeted deletion of exon 14 from the Pclo gene, reported to lack >95% of Piccolo, continue to express multiple Piccolo isoforms. Furthermore, neurons cultured from these mice exhibit no defects in presynaptic F-actin assembly due to the expression of these isoforms at presynaptic boutons. These data reveal that Piccolo regulates neurotransmitter release by facilitating activity-dependent F-actin assembly and the dynamic recruitment of key signaling molecules into presynaptic boutons, and highlight the need for new genetic models with which to study Piccolo loss of function.

    View details for DOI 10.1523/JNEUROSCI.1835-11.2011

    View details for Web of Science ID 000295805500022

    View details for PubMedID 21976510

  • SAP97 directs NMDA receptor spine targeting and synaptic plasticity JOURNAL OF PHYSIOLOGY-LONDON Li, D., Specht, C. G., Waites, C. L., Butler-Munro, C., Leal-Ortiz, S., Foote, J. W., Genoux, D., Garner, C. C., Montgomery, J. M. 2011; 589 (18): 4491-4510


    SAP97 is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Alternative splicing of SAP97 transcripts gives rise to palmitoylated ?SAP97 and L27-domain containing ?SAP97 isoforms that differentially regulate the subsynaptic localization of GluR1 subunits of AMPA receptors. Here, we examined whether SAP97 isoforms regulate the mechanisms underlying long-term potentiation (LTP) and depression (LTD) and find that both ?- and ?-forms of SAP97 impair LTP but enhance LTD via independent isoform-specific mechanisms. Live imaging of ?- and ?SAP97 revealed that the altered synaptic plasticity was not due to activity-dependent changes in SAP97 localization or exchange kinetics. However, by recording from pairs of synaptically coupled hippocampal neurons, we show that ?SAP97 occludes LTP by enhancing the levels of postsynaptic AMPA receptors, while ?SAP97 blocks LTP by reducing the synaptic localization of NMDA receptors. Examination of the surface pools of AMPA and NMDA receptors indicates that ?SAP97 selectively regulates the synaptic pool of AMPA receptors, whereas ?SAP97 regulates the extrasynaptic pools of both AMPA and NMDA receptors. Knockdown of ?SAP97 increases the synaptic localization of both AMPA and NMDA receptors, showing that endogenous ?SAP97 restricts glutamate receptor expression at excitatory synapses. This isoform-dependent differential regulation of synaptic versus extrasynaptic pools of glutamate receptors will determine how many receptors are available for the induction and the expression of synaptic plasticity. Our data support a model wherein SAP97 isoforms can regulate the ability of synapses to undergo plasticity by controlling the surface distribution of AMPA and NMDA receptors.

    View details for DOI 10.1113/jphysiol.2011.215566

    View details for Web of Science ID 000295050800011

    View details for PubMedID 21768261

  • Presynaptic function in health and disease TRENDS IN NEUROSCIENCES Waites, C. L., Garner, C. C. 2011; 34 (6): 326-337


    Neurons communicate with one another at specialized contact sites called synapses, composed of pre- and postsynaptic compartments. Presynaptic compartments, or 'boutons', signal to the postsynaptic compartment by releasing chemical neurotransmitter in response to incoming electrical impulses. Recent studies link defects in the function of presynaptic boutons to the etiology of several neurodevelopmental and neurodegenerative diseases, including autism, schizophrenia and Alzheimer's disease. In this review, we describe five core functions of presynaptic boutons and the molecules that mediate these functions, focusing on a subset that are linked to human disease. We also discuss potential mechanisms through which the loss or alteration of these specific molecules could lead to defects in synaptic communication, neural circuit function and, ultimately, cognition and behavior.

    View details for DOI 10.1016/j.tins.2011.03.004

    View details for Web of Science ID 000292238300005

    View details for PubMedID 21596448

  • The Down Syndrome Critical Region Regulates Retinogeniculate Refinement JOURNAL OF NEUROSCIENCE Blank, M., Fuerst, P. G., Stevens, B., Nouri, N., Kirkby, L., Warrier, D., Barres, B. A., Feller, M. B., Huberman, A. D., Burgess, R. W., Garner, C. C. 2011; 31 (15): 5764-5776


    Down syndrome (DS) is a developmental disorder caused by a third chromosome 21 in humans (Trisomy 21), leading to neurological deficits and cognitive impairment. Studies in mouse models of DS suggest that cognitive deficits in the adult are associated with deficits in synaptic learning and memory mechanisms, but it is unclear whether alterations in the early wiring and refinement of neuronal circuits contribute to these deficits. Here, we show that early developmental refinement of visual circuits is perturbed in mouse models of Down syndrome. Specifically, we find excessive eye-specific segregation of retinal axons in the dorsal lateral geniculate nucleus. Indeed, the degree of refinement scales with defects in the "Down syndrome critical region" (DSCR) in a dose-dependent manner. We further identify Dscam (Down syndrome cell adhesion molecule), a gene within the DSCR, as a regulator of eye-specific segregation of retinogeniculate projections. Although Dscam is not the sole gene in the DSCR contributing to enhanced refinement in trisomy, Dscam dosage clearly regulates cell spacing and dendritic fasciculation in a specific class of retinal ganglion cells. Thus, altered developmental refinement of visual circuits that occurs before sensory experience is likely to contribute to visual impairment in individuals with Down syndrome.

    View details for DOI 10.1523/JNEUROSCI.6015-10.2011

    View details for Web of Science ID 000289472400026

    View details for PubMedID 21490218

  • Development of Novel Zn2+ Loaded Nanoparticles Designed for Cell-Type Targeted Drug Release in CNS Neurons: In Vitro Evidences PLOS ONE Grabrucker, A. M., Garner, C. C., Boeckers, T. M., Bondioli, L., Ruozi, B., Forni, F., Vandelli, M. A., Tosi, G. 2011; 6 (3)


    Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood-brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn(2+) ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn(2+). Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn(2+) to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.

    View details for DOI 10.1371/journal.pone.0017851

    View details for Web of Science ID 000288810500017

    View details for PubMedID 21448455

  • Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation EMBO JOURNAL Grabrucker, A. M., Knight, M. J., Proepper, C., Bockmann, J., Joubert, M., Rowan, M., Nienhaus, G. U., Garner, C. C., Bowie, J. U., Kreutz, M. R., Gundelfinger, E. D., Boeckers, T. M. 2011; 30 (3): 569-581


    Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity-dependent remodelling of excitatory synapses. Here, we report that Zn(2+) ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member-specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA-mediated knockdown, we could demonstrate that only the overexpression of zinc-sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn(2+) along with the knockdown of zinc-insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD-95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn(2+) is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity.

    View details for DOI 10.1038/emboj.2010.336

    View details for Web of Science ID 000287394200012

    View details for PubMedID 21217644

  • Brain-Delivery of Zinc-Ions as Potential Treatment for Neurological Diseases: Mini Review. Drug delivery letters Grabrucker, A. M., Rowan, M., Garner, C. C. 2011; 1 (1): 13-23


    Homeostasis of metal ions such as Zn(2+) is essential for proper brain function. Moreover, the list of psychiatric and neurodegenerative disorders involving a dysregulation of brain Zn(2+)-levels is long and steadily growing, including Parkinson's and Alzheimer's disease as well as schizophrenia, attention deficit and hyperactivity disorder, depression, amyotrophic lateral sclerosis, Down's syndrome, multiple sclerosis, Wilson's disease and Pick's disease. Furthermore, alterations in Zn(2+)-levels are seen in transient forebrain ischemia, seizures, traumatic brain injury and alcoholism. Thus, the possibility of altering Zn(2+)-levels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. Although the role of Zn(2+) in the brain has been extensively studied over the past decades, methods for controlled regulation and manipulation of Zn(2+) concentrations within the brain are still in their infancy. Since the use of dietary Zn(2+) supplementation and restriction has major limitations, new methods and alternative approaches are currently under investigation, such as the use of intracranial infusion of Zn(2+) chelators or nanoparticle technologies to elevate or decrease intracellular Zn(2+) levels. Therefore, this review briefly summarizes the role of Zn(2+) in psychiatric and neurodegenerative diseases and highlights key findings and impediments of brain Zn(2+)-level manipulation. Furthermore, some methods and compounds, such as metal ion chelation, redistribution and supplementation that are used to control brain Zn(2+)-levels in order to treat brain disorders are evaluated.

    View details for PubMedID 22102982

  • Emerging Pharmacotherapies for Neurodevelopmental Disorders JOURNAL OF DEVELOPMENTAL AND BEHAVIORAL PEDIATRICS Wetmore, D. Z., Garner, C. C. 2010; 31 (7): 564-581


    A growing and interdisciplinary translational neuroscience research effort for neurodevelopmental disorders (NDDs) is investigating the mechanisms of dysfunction and testing effective treatment strategies in animal models and, when possible, in the clinic. NDDs with a genetic basis have received particular attention. Transgenic animals that mimic genetic insults responsible for disease in man have provided insight about mechanisms of dysfunction, and, surprisingly, have shown that cognitive deficits can be addressed in adult animals. This review will present recent translational research based on animal models of genetic NDDs, as well as pharmacotherapeutic strategies under development to address deficits of brain function for Down syndrome, fragile X syndrome, Rett syndrome, neurofibromatosis-1, tuberous sclerosis, and autism. Although these disorders vary in underlying causes and clinical presentation, common pathways and mechanisms for dysfunction have been observed. These include abnormal gene dosage, imbalance among neurotransmitter systems, and deficits in the development, maintenance and plasticity of neuronal circuits. NDDs affect multiple brain systems and behaviors that may be amenable to drug therapies that target distinct deficits. A primary goal of translational research is to replace symptomatic and supportive drug therapies with pharmacotherapies based on a principled understanding of the causes of dysfunction. Based on this principle, several recently developed therapeutic strategies offer clear promise for clinical development in man.

    View details for DOI 10.1097/DBP.0b013e3181ee3833

    View details for Web of Science ID 000281561700007

    View details for PubMedID 20814256

  • Disruption of the interaction between myosin VI and SAP97 is associated with a reduction in the number of AMPARs at hippocampal synapses JOURNAL OF NEUROCHEMISTRY Nash, J. E., Appleby, V. J., Correa, S. A., Wu, H., Fitzjohn, S. M., Garner, C. C., Collingridge, G. L., Molnar, E. 2010; 112 (3): 677-690


    Myosin VI is an actin-based motor protein that is enriched at the postsynaptic density and appears to interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptors (AMPARs) via synapse associated protein 97 (SAP97). Here, we find that a Flag epitope-tagged dominant negative construct that inhibits the interaction between SAP97 and myosin VI (Flag-myoVI-DN) causes a dramatic reduction in the number of synapses and the surface expression of AMPARs in cultured hippocampal neurons. Furthermore, we find that Flag-myoVI-DN also prevents the rapid delivery of AMPARs to synapses that can be induced by the transient activation of N-methyl-d-aspartate receptors. The Flag-myoVI-DN induced decrease in surface AMPARs is not because of reduced AMPAR subunit protein synthesis. Using whole-cell recording, we show that Flag-myoVI-DN also prevents the activity-induced increase in miniature excitatory postsynaptic current frequency that is normally associated with recruitment of AMPARs to the cell surface at synaptic sites that lack these receptors (i.e. 'silent' synapses). Together, these results indicate that myosin VI/SAP97 plays an important role in trafficking and activity-dependent recruitment of AMPARs to synapses.

    View details for DOI 10.1111/j.1471-4159.2009.06480.x

    View details for Web of Science ID 000273396800009

    View details for PubMedID 19895665

  • Circadian Locomotor Rhythms Are Normal in Ts65Dn "Down Syndrome" Mice and Unaffected by Pentylenetetrazole JOURNAL OF BIOLOGICAL RHYTHMS Ruby, N. F., Fernandez, F., Zhang, P., Klima, J., Heller, H. C., Garner, C. C. 2010; 25 (1): 63-66


    Ts65Dn mice are used extensively as a model for Down syndrome. Recent studies have reported conflicting evidence as to whether these mice express circadian rhythms. The authors therefore recorded locomotor activity patterns from these animals while they were housed under a standard light-dark cycle, constant darkness (DD), and constant light (LL). Contrary to expectations, Ts65Dn mice had more robust circadian rhythms with slightly shorter periods compared with their wild-type littermates. They also exhibited increased rhythm period and marked activity suppression when moved from DD to LL (i.e., Aschoff's rule). Administration of the GABA(A) antagonist pentylenetetrazole did not influence any of these circadian parameters. Thus, locomotor activity is under strict circadian control in Ts65Dn mice, suggesting that their cognitive deficits and sleep disturbances are not due to dysfunctional circadian timing as proposed previously.

    View details for DOI 10.1177/0748730409356202

    View details for Web of Science ID 000273624700008

    View details for PubMedID 20075302

  • Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts JOURNAL OF NEUROSCIENCE Lucido, A. L., Sanchez, F. S., Thostrup, P., Kwiatkowski, A. V., Leal-Ortiz, S., Gopalakrishnan, G., Liazoghli, D., Belkaid, W., Lennox, R. B., Grutter, P., Garner, C. C., Colman, D. R. 2009; 29 (40): 12449-12466


    CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.

    View details for DOI 10.1523/JNEUROSCI.1381-09.2009

    View details for Web of Science ID 000270567400011

    View details for PubMedID 19812321

  • SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway NATURE NEUROSCIENCE Jeyifous, O., LWaites, C., Specht, C. G., Fujisawa, S., Schubert, M., Lin, E. I., Marshall, J., Aoki, C., de Silva, T., Montgomery, J. M., Garner, C. C., Green, W. N. 2009; 12 (8): 1011-U81


    Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

    View details for DOI 10.1038/nn.2362

    View details for Web of Science ID 000268396300015

    View details for PubMedID 19620977

  • Normal protein composition of synapses in Ts65Dn mice: a mouse model of Down syndrome JOURNAL OF NEUROCHEMISTRY Fernandez, F., Trinidad, J. C., Blank, M., Feng, D., Burlingame, A. L., Garner, C. C. 2009; 110 (1): 157-169


    Down syndrome (DS) is the most prevalent form of intellectual disability caused by the triplication of approximately 230 genes on chromosome 21. Recent data in Ts65Dn mice, the foremost mouse model of DS, strongly suggest that cognitive impairment in individuals with DS is a consequence of reduced synaptic plasticity because of chronic over-inhibition. It remains unclear however whether changes in plasticity are tied to global molecular changes at synapses, or are due to regional changes in the functional properties of synaptic circuits. One interesting framework for evaluating the activity state of the DS brain comes from in vitro studies showing that chronic pharmacological silencing of neuronal excitability orchestrates stereotyped changes in the protein composition of synaptic junctions. In the present study, we use proteomic strategies to evaluate whether synapses from the Ts65Dn cerebrum carry signatures characteristic of inactive cortical neurons. Our data reveal that synaptic junctions do not exhibit overt alterations in protein composition. Only modest changes in the levels of synaptic proteins and in their phosphorylation are observed. This suggests that subtle changes in the functional properties of specific synaptic circuits rather than large-scale homeostatic shifts in the expression of synaptic molecules contribute to cognitive impairment in people with DS.

    View details for DOI 10.1111/j.1471-4159.2009.06110.x

    View details for Web of Science ID 000266923700014

    View details for PubMedID 19453946

  • Cell autonomous defects in cortical development revealed by two-color chimera analysis MOLECULAR AND CELLULAR NEUROSCIENCE Kwiatkowski, A. V., Garner, C. C., Nelson, W. J., Gertler, F. B. 2009; 41 (1): 44-50


    A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based method to analyze cell autonomy by generating two-color chimeric mouse embryos. Ena/VASP-deficient mutant and control embryonic stem (ES) cells were labeled with different fluorescent chimeric proteins (EGFP and mCherry) that were modified to bind to the plasma membrane. These labeled ES cells were used to generate two-color chimeric embryos possessing two genetically distinct populations of cortical cells, permitting multiple aspects of neuronal morphogenesis to be analyzed and compared between the two cell populations. We observed little difference between the ability of control and Ena/VASP-deficient cells to contribute to cortical organization during development. In contrast, we observed axon fiber tracts originating from control neurons but not Ena/VASP-deficient neurons, indicating that loss of Ena/VASP causes a cell autonomous defect in cortical axon formation. This technique could be applied to determine other cell autonomous functions in different stages of cortical development.

    View details for DOI 10.1016/j.mcn.2009.01.008

    View details for Web of Science ID 000265715700005

    View details for PubMedID 19386231

  • Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon JOURNAL OF CELL BIOLOGY Fejtova, A., Davydova, D., Bischof, F., Lazarevic, V., Altrock, W. D., Romorini, S., Schoene, C., Zuschratter, W., Kreutz, M. R., Garner, C. C., Ziv, N. E., Gundelfinger, E. D. 2009; 185 (2): 341-355


    Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon-DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

    View details for DOI 10.1083/jcb.200807155

    View details for Web of Science ID 000265599200017

    View details for PubMedID 19380881

  • Synaptic SAP97 Isoforms Regulate AMPA Receptor Dynamics and Access to Presynaptic Glutamate JOURNAL OF NEUROSCIENCE Waites, C. L., Specht, C. G., Haertel, K., Leal-Ortiz, S., Genoux, D., Li, D., Drisdel, R. C., Jeyifous, O., Cheyne, J. E., Green, W. N., Montgomery, J. M., Garner, C. C. 2009; 29 (14): 4332-4345


    The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated alpha-isoform to the postsynaptic density (PSD) and the L27 domain-containing beta-isoform primarily to non-PSD, perisynaptic regions. Consequently, alpha- and betaSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs and, hence, their responsiveness to presynaptically released glutamate.

    View details for DOI 10.1523/JNEUROSCI.4431-08.2009

    View details for Web of Science ID 000265009600002

    View details for PubMedID 19357261

  • Exchange and Redistribution Dynamics of the Cytoskeleton of the Active Zone Molecule Bassoon JOURNAL OF NEUROSCIENCE Tsuriel, S., Fisher, A., Wittenmayer, N., Dresbach, T., Garner, C. C., Ziv, N. E. 2009; 29 (2): 351-358


    Presynaptic sites typically appear as varicosities (boutons) distributed along axons. Ultrastructurally, presynaptic boutons lack obvious physical barriers that separate them from the axon proper, yet activity-related and constitutive dynamics continuously promote the "reshuffling" of presynaptic components and even their dispersal into flanking axonal segments. How presynaptic sites manage to maintain their organization and individual characteristics over long durations is thus unclear. Conceivably, presynaptic tenacity might depend on the active zone (AZ), an electron-dense specialization of the presynaptic membrane, and particularly on the cytoskeletal matrix associated with the AZ (CAZ) that could act as a relatively stable "core scaffold" that conserves and dictates presynaptic organization. At present, however, little is known on the molecular dynamics of CAZ molecules, and thus, the factual basis for this hypothesis remains unclear. To examine the stability of the CAZ, we studied the molecular dynamics of the major CAZ molecule Bassoon in cultured hippocampal neurons. Fluorescence recovery after photobleaching and photoactivation experiments revealed that exchange rates of green fluorescent protein and photoactivatable green fluorescent protein-tagged Bassoon at individual presynaptic sites are very low (tau > 8 h). Exchange rates varied between boutons and were only slightly accelerated by stimulation. Interestingly, photoactivation experiments revealed that Bassoon lost from one synapse was occasionally assimilated into neighboring presynaptic sites. Our findings indicate that Bassoon is engaged in relatively stable associations within the CAZ and thus support the notion that the CAZ or some of its components might constitute a relatively stable presynaptic core scaffold.

    View details for DOI 10.1523/JNEUROSCI.4777-08.2009

    View details for Web of Science ID 000262442900006

    View details for PubMedID 19144835

  • Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis JOURNAL OF CELL BIOLOGY Leal-Ortiz, S., Waites, C. L., Terry-Lorenzo, R., Zamorano, P., Gundelfinger, E. D., Garner, C. C. 2008; 181 (5): 831-846


    Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II-dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.

    View details for Web of Science ID 000256593800011

    View details for PubMedID 18519737

  • Episodic-like memory in Ts65Dn, a mouse model of Down syndrome BEHAVIOURAL BRAIN RESEARCH Fernandez, F., Garner, C. C. 2008; 188 (1): 233-237


    Ts65Dn mice, like individuals with Down syndrome (DS), demonstrate a functional dissociation between explicit and implicit forms of memory, showing selective impairment in explicit or declarative learning tasks. Here, we explored Ts65Dn explicit memory deficits further by evaluating the ability of these mice to assimilate the temporal and spatial contexts under which previously novel objects had been encountered. We found that Ts65Dn mice could in fact form contextual representations of objects over the course of a few hours, contrary to their inability to discriminate object novelty over a more prolonged period of 24h. These results suggest that Ts65Dn mice might have particular difficulties in declarative tasks requiring long-term memory, presenting an especially important putative therapeutic target for pre-clinical and clinical DS research.

    View details for DOI 10.1016/j.bbr.2007.09.015

    View details for Web of Science ID 000253448200024

    View details for PubMedID 17950473

  • Caldendrin-Jacob: A protein liaison that couples NMDA receptor signalling to the nucleus PLOS BIOLOGY Dieterich, D. C., Karpova, A., Mikhaylova, M., Zdobnova, I., Koenig, I., Landwehr, M., Kreutz, M., Smalla, K., Richter, K., Landgraf, P., Reissner, C., Boeckers, T. M., Zuschratter, W., Spilker, C., Seidenbecher, C. I., Garner, C. C., Gundelfinger, E. D., Kreutz, M. R. 2008; 6 (2): 286-306


    NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

    View details for DOI 10.1371/journal.pbio.0060034

    View details for Web of Science ID 000254928400017

    View details for PubMedID 18303947

  • Molecular Mechanisms of Presynaptic Differentiation ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Jin, Y., Garner, C. C. 2008; 24: 237-262


    Information processing in the nervous system relies on properly localized and organized synaptic structures at the correct locations. The formation of synapses is a long and intricate process involving multiple interrelated steps. Decades of research have identified a large number of molecular components of the presynaptic compartment. In addition to neurotransmitter-containing synaptic vesicles, presynaptic terminals are defined by cytoskeletal and membrane specializations that allow highly regulated exo- and endocytosis of synaptic vesicles and that maintain precise registration with postsynaptic targets. Functional studies at multiple levels have revealed complex interactions between the transport of vesicular intermediates, the presynaptic cytoskeleton, growth cone navigation, and synaptic targets. With the advent of finer anatomical, physiological, and molecular tools, great insights have been gained toward the mechanistic dissection of functionally redundant processes controlling the specificity and dynamics of synapses. This review highlights the recent findings pertaining to the cellular and molecular regulation of presynaptic differentiation.

    View details for DOI 10.1146/annurev.cellbio.23.090506.123417

    View details for Web of Science ID 000260851500011

    View details for PubMedID 18588488

  • Over-inhibition: a model for developmental intellectual disability TRENDS IN NEUROSCIENCES Fernandez, F., Garner, C. C. 2007; 30 (10): 497-503


    Developmental intellectual disability (DID) is a daunting societal problem. Although tremendous progress has been made in defining the genetic causes of DID, therapeutic strategies remain limited. In particular, there is a marked absence of a unified approach to treating cognitive impairments associated with DID. Here, we suggest that the brain in many DID-related disorders is subject to a basic imbalance in neuronal activity, with an increased contribution of inhibition to neural circuits. This over-inhibition, in turn, is predicted to lead to deficits in synaptic plasticity and learning and memory. We further discuss possibilities for pharmacological intervention in DID, focusing on the concept of drug-induced 'therapeutic neuroadaptation' as a means of stably enhancing constitutive circuit excitability and cognition over time.

    View details for DOI 10.1016/i.tins.2007.07.005

    View details for Web of Science ID 000250679500003

    View details for PubMedID 17825437

  • Object recognition memory is conserved in Ts1Cje, a mouse model of Down syndrome NEUROSCIENCE LETTERS Fernandez, F., Garner, C. C. 2007; 421 (2): 137-141


    Ts1Cje and Ts65Dn are genetic mouse models of Down syndrome (DS). Like individuals with DS, these mice exhibit various hallmarks of hippocampal pathology, and deficits in hippocampal-based, declarative learning and memory tasks. Both spatial navigation and novel object recognition, two prototypical domains of declarative memory function, have been strongly characterized in the Ts65Dn DS model. Indeed, Ts65Dn mice show navigation problems in the Morris water maze, impaired alternation in a T-maze, and deficient working and reference memory in the radial arm maze task. They, likewise, show an inability to detect object novelty over time. In contrast to the Ts65Dn model, hippocampal-dependent cognition has been less well characterized in Ts1Cje. Although Ts1Cje mice have been found to exhibit spatial difficulties in the Morris water maze and reduced spontaneous alternation, their ability to process object-based information has never been examined. Here, we report that Ts1Cje mice perform normally in short-term and long-term novel object recognition tasks. The ability of Ts1Cje mice to detect object novelty, unlike Ts65Dn, may point to differences in the extent of hippocampal pathology in the two DS mouse models.

    View details for DOI 10.1016/j.neulet.2007.04.075

    View details for Web of Science ID 000248195900010

    View details for PubMedID 17566652

  • Antagonistic effects of TrkB and p75(NTR) on NMDA receptor currents in post-synaptic densities transplanted into Xenopus oocytes JOURNAL OF NEUROCHEMISTRY Sandoval, M., Sandoval, R., Thomas, U., Spilker, C., Smalla, K., Falcon, R., Marengo, J. J., Calderon, R., Saavedra, V., Heumann, R., Bronfman, F., Garner, C. C., Gundelfinger, E. D., Wyneken, U. 2007; 101 (6): 1672-1684


    Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are essential regulators of synaptic function in the adult CNS. A TrkB-mediated effect at excitatory synapses is enhancement of NMDA receptor (NMDA-R)-mediated currents. Recently, opposing effects of TrkB and the pan-neurotrophin receptor p75(NTR) on long-term synaptic depression and long-term potentiation have been reported in the hippocampus. To further study the regulation of NMDA-Rs by neurotrophin receptors in their native protein environment, we micro-transplanted rat forebrain post-synaptic densities (PSDs) into Xenopus oocytes. One-minute incubations of oocytes with BDNF led to dual effects on NMDA-R currents: either TrkB-dependent potentiation or TrkB-independent inhibition were observed. Pro-nerve growth factor, a ligand for p75(NTR) but not for TrkB, produced a reversible, dose-dependent, TrkB-independent and p75(NTR)-dependent inhibition of NMDA-Rs. Fractionation experiments showed that p75(NTR) is highly enriched in the PSD protein fraction. Immunoprecipitation and pull-down experiments further revealed that p75(NTR) is a core component of the PSD, where it interacts with the PDZ3 domain of the scaffolding protein SAP90/PSD-95. Our data provide striking evidence for a rapid inhibitory effect of p75(NTR) on NMDA-R currents that antagonizes TrkB-mediated NMDA-R potentiation. These opposing mechanisms might be present in a large proportion of forebrain synapses and may contribute importantly to synaptic plasticity.

    View details for DOI 10.1111/j.1471-4159.2007.04519.x

    View details for Web of Science ID 000247135300021

    View details for PubMedID 17394529

  • Pharmacotherapy for cognitive impairment in a mouse model of Down syndrome NATURE NEUROSCIENCE Fernandez, F., Morishita, W., Zuniga, E., Nguyen, J., Blank, M., Malenka, R. C., Garner, C. C. 2007; 10 (4): 411-413


    Ts65Dn mice, a model for Down syndrome, have excessive inhibition in the dentate gyrus, a condition that could compromise synaptic plasticity and mnemonic processing. We show that chronic systemic treatment of these mice with GABAA antagonists at non-epileptic doses causes a persistent post-drug recovery of cognition and long-term potentiation. These results suggest that over-inhibition contributes to intellectual disabilities associated with Down syndrome and that GABAA antagonists may be useful therapeutic agents for this disorder.

    View details for DOI 10.1038/nn1860

    View details for Web of Science ID 000245228600008

    View details for PubMedID 17322876

  • The functional nature of synaptic circuitry is altered in area CA3 of the hippocampus in a mouse model of Down's syndrome JOURNAL OF PHYSIOLOGY-LONDON Hanson, J. E., Blank, M., Valenzuela, R. A., Garner, C. C., Madison, D. V. 2007; 579 (1): 53-67


    Down's syndrome (DS) is the most common cause of mental retardation, and memory impairments are more severe in DS than in most if not all other causes of mental retardation. The Ts65Dn mouse, a genetic model of DS, exhibits phenotypes of DS, including memory impairments indicative of hippocampal dysfunction. We examined functional synaptic connectivity in area CA3 of the hippocampus of Ts65Dn mice using organotypic slice cultures as a model. We found reductions in multiple measures of synaptic function in both excitatory and inhibitory inputs to pyramidal neurons in CA3 of the Ts65Dn hippocampus. However, associational synaptic connections between pyramidal neurons were more abundant and more likely to be active rather than silent in the Ts65Dn hippocampus. Synaptic potentiation was normal in these associational connections. Decreased overall functional synaptic input onto pyramidal neurons expressed along with the specific hyperconnectivity of associational connections between pyramidal neurons will result in predictable alterations of CA3 network function, which may contribute to the memory impairments seen in DS.

    View details for DOI 10.1113/jphysiol.2006.114868

    View details for Web of Science ID 000244099400004

    View details for PubMedID 17158177

  • Synaptic protein dynamics in hibernation JOURNAL OF NEUROSCIENCE von der Ohe, C. G., Garner, C. C., Darian-Smith, C., Heller, H. C. 2007; 27 (1): 84-92


    Neurons in hibernating mammals exhibit a dramatic form of plasticity during torpor, with dendritic arbors retracting as body temperature cools and then regrowing rapidly as body temperature rises. In this study, we used immunohistochemical imaging and Western blotting of several presynaptic and postsynaptic proteins to determine the synaptic changes that accompany torpor and to investigate the mechanisms behind these changes. We show torpor-related alterations in synaptic protein localization that occur rapidly and uniformly across several brain regions in a temperature-dependent manner. Entry into torpor is associated with a 50-65% loss of synapses, as indicated by changes in the extent of colocalization of presynaptic and postsynaptic markers. We also show that the loss of synaptic protein clustering occurring during entry into torpor is not attributable to protein loss. These findings suggest that torpor-related changes in synapses stem from dissociation of proteins from the cytoskeletal active zone and postsynaptic density, creating a reservoir of proteins that can be quickly mobilized for rapid rebuilding of dendritic spines and synapses during the return to euthermia. A mechanism of neural plasticity based on protein dissociation rather than protein breakdown could explain the hibernator's capacity for large, rapid, and repeated microstructural changes, providing a fascinating contrast to neuropathologies that are dominated by protein breakdown and cell death.

    View details for DOI 10.1523/JNEUROSCI.4385-06.2007

    View details for Web of Science ID 000243254500011

    View details for PubMedID 17202475

  • A year of unprecedented progress in Down syndrome basic research MENTAL RETARDATION AND DEVELOPMENTAL DISABILITIES RESEARCH REVIEWS Reeves, R. H., Garner, C. C. 2007; 13 (3): 215-220


    The years 2006 and 2007 saw the publication of three new and different approaches to prevention or amelioration of Down syndrome effects on the brain and cognition. We describe the animal model systems that were critical to this progress, review these independent breakthrough studies, and discuss the implications for therapeutic approaches suggested by each.

    View details for DOI 10.1002/mrdd.20165

    View details for Web of Science ID 000250194000004

    View details for PubMedID 17910083

  • Synapse development: still looking for the forest, still lost in the trees CELL AND TISSUE RESEARCH Garner, C. C., Waites, C. L., Ziv, N. E. 2006; 326 (2): 249-262


    Synapse development in the vertebrate central nervous system is a highly orchestrated process occurring not only during early stages of brain development, but also (to a lesser extent) in the mature nervous system. During development, the formation of synapses is intimately linked to the differentiation of neuronal cells, the extension of their axons and dendrites, and the course wiring of the nervous system. Subsequently, the stabilization, elimination, and strengthening of synaptic contacts is coupled to the refinement of axonal and dendritic arbors, to the establishment of functionally meaningful connections, and probably also to the day-to-day acquisition, storage, and retrieval of memories, higher order thought processes, and behavioral patterns.

    View details for DOI 10.1007/s00441-006-0278-1

    View details for Web of Science ID 000240714000006

    View details for PubMedID 16909256

  • Ubiquitous and temperature-dependent neural plasticity in hibernators JOURNAL OF NEUROSCIENCE von der Ohe, C. G., Darian-Smith, C., Garner, C. C., Heller, H. C. 2006; 26 (41): 10590-10598


    Hibernating mammals are remarkable for surviving near-freezing brain temperatures and near cessation of neural activity for a week or more at a time. This extreme physiological state is associated with dendritic and synaptic changes in hippocampal neurons. Here, we investigate whether these changes are a ubiquitous phenomenon throughout the brain that is driven by temperature. We iontophoretically injected Lucifer yellow into several types of neurons in fixed slices from hibernating ground squirrels. We analyzed neuronal microstructure from animals at several stages of torpor at two different ambient temperatures, and during the summer. We show that neuronal cell bodies, dendrites, and spines from several cell types in hibernating ground squirrels retract on entry into torpor, change little over the course of several days, and then regrow during the 2 h return to euthermia. Similar structural changes take place in neurons from the hippocampus, cortex, and thalamus, suggesting a global phenomenon. Investigation of neural microstructure from groups of animals hibernating at different ambient temperatures revealed that there is a linear relationship between neural retraction and minimum body temperature. Despite significant temperature-dependent differences in extent of retraction during torpor, recovery reaches the same final values of cell body area, dendritic arbor complexity, and spine density. This study demonstrates large-scale and seemingly ubiquitous neural plasticity in the ground squirrel brain during torpor. It also defines a temperature-driven model of dramatic neural plasticity, which provides a unique opportunity to explore mechanisms of large-scale regrowth in adult mammals, and the effects of remodeling on learning and memory.

    View details for DOI 10.1523/JNEUROSCI.2874-06.2006

    View details for Web of Science ID 000241192800033

    View details for PubMedID 17035545

  • Local sharing as a predominant determinant of synaptic matrix molecular dynamics PLOS BIOLOGY Tsuriel, S., Geva, R., Zamorano, P., Dresbach, T., Boeckers, T., Gundelfinger, E. D., Garner, C. C., Ziv, N. E. 2006; 4 (9): 1572-1587


    Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins.

    View details for DOI 10.1371/journal.pbio.0040271

    View details for Web of Science ID 000240740900009

    View details for PubMedID 16903782

  • Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor EMBO JOURNAL Frese, K. K., Latorre, I. J., Chung, S. H., Caruana, G., Bernstein, A., Jones, S. N., Donehower, L. A., JUSTICE, M. J., Garner, C. C., Javier, R. T. 2006; 25 (6): 1406-1417


    The fact that several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on our results, we propose a model wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene.

    View details for DOI 10.1038/sj.emboj.7601030

    View details for Web of Science ID 000236737700023

    View details for PubMedID 16511562

  • Assembly of active zone precursor vesicles - Obligatory trafficking of presynaptic cytomatrix proteins bassoon and piccolo via a trans-Golgi compartment JOURNAL OF BIOLOGICAL CHEMISTRY Dresbach, T., Torres, V., Wittenmayer, N., Altrock, W. D., ZAMORANO, P., Zuschratter, W., Nawrotzki, R., Ziv, N. E., Garner, C. C., Gundelfinger, E. D. 2006; 281 (9): 6038-6047


    Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.

    View details for DOI 10.1074/jbc.M508784200

    View details for Web of Science ID 000235568900085

    View details for PubMedID 16373352

  • Transsynaptic signaling by postsynaptic synapse-associated protein 97 JOURNAL OF NEUROSCIENCE Regalado, M. P., Terry-Lorenzo, R. T., Waites, C. L., Garner, C. C., Malenka, R. C. 2006; 26 (8): 2343-2357


    The molecular mechanisms by which postsynaptic modifications lead to precisely coordinated changes in presynaptic structure and function are primarily unknown. To address this issue, we examined the presynaptic consequences of postsynaptic expression of members of the membrane-associated guanylate kinase family of synaptic scaffolding proteins. Postsynaptic expression of synapse-associated protein 97 (SAP97) increased presynaptic protein content and active zone size to a greater extent than comparable amounts of postsynaptic PSD-95 (postsynaptic density-95) or SAP102. In addition, postsynaptic expression of SAP97 enhanced presynaptic function, as measured by increased FM4-64 dye uptake. The structural presynaptic effects of postsynaptic SAP97 required ligand binding through two of its PDZ (PSD-95/Discs large/zona occludens-1) domains as well as intact N-terminal and guanylate kinase domains. Expression of SAP97 recruited a complex of additional postsynaptic proteins to synapses including glutamate receptor 1, Shank1a, SPAR (spine-associated RapGAP), and proSAP2. Furthermore, inhibition of several different transsynaptic signaling proteins including cadherins, integrins, and EphB receptor/ephrinB significantly reduced the presynaptic growth caused by postsynaptic SAP97. These results suggest that SAP97 may play a central role in the coordinated growth of synapses during development and plasticity by recruiting a complex of postsynaptic proteins that enhances presynaptic terminal growth and function via multiple transsynaptic molecular interactions.

    View details for DOI 10.1523/JNEUROSCI.5247-05.2006

    View details for Web of Science ID 000235515800024

    View details for PubMedID 16495462

  • Semaphorin 4B interacts with the post-synaptic density protein PSD-95/SAP90 and is recruited to synapses through a C-terminal PDZ-binding motif FEBS LETTERS Burkhardt, C., Muller, M., Badde, A., Garner, C. C., Gundelfinger, E. D., Puschel, A. W. 2005; 579 (17): 3821-3828


    The semaphorins are a large family of proteins that act as guidance signals for axons and dendrites. The class 4 semaphorins are integral membrane proteins that are widely expressed throughout the nervous system. Here, we show that a subclass of these semaphorins is characterized by a PDZ-binding motif at their carboxy-terminus. This sequence mediates the interaction with the post-synaptic density protein PSD-95/SAP90. Co-expression of Sema4B with PSD-95 in COS 7 cells results in the clustering of Sema4B. Sema4B co-localizes with PSD-95 at synaptic contacts between cultured hippocampal neurons. This synaptic localization depends on the presence of the PDZ-binding motif.

    View details for DOI 10.1016/j.febslet.2005.05.079

    View details for Web of Science ID 000230335600056

    View details for PubMedID 15978582

  • Neurabin/protein phosphatase-1 complex regulates dendritic spine morphogenesis and maturation MOLECULAR BIOLOGY OF THE CELL Terry-Lorenzo, R. T., Roadcap, D. W., Otsuka, T., Blanpied, T. A., Zamorano, P. L., Garner, C. C., Shenolikar, S., Ehlers, M. D. 2005; 16 (5): 2349-2362


    The majority of excitatory synapses in the mammalian brain form on filopodia and spines, actin-rich membrane protrusions present on neuronal dendrites. The biochemical events that induce filopodia and remodel these structures into dendritic spines remain poorly understood. Here, we show that the neuronal actin- and protein phosphatase-1-binding protein, neurabin-I, promotes filopodia in neurons and nonneuronal cells. Neurabin-I actin-binding domain bundled F-actin, promoted filopodia, and delayed the maturation of dendritic spines in cultured hippocampal neurons. In contrast, dimerization of neurabin-I via C-terminal coiled-coil domains and association of protein phosphatase-1 (PP1) with neurabin-I through a canonical KIXF motif inhibited filopodia. Furthermore, the expression of a neurabin-I polypeptide unable to bind PP1 delayed the maturation of neuronal filopodia into spines, reduced the synaptic targeting of AMPA-type glutamate (GluR1) receptors, and decreased AMPA receptor-mediated synaptic transmission. Reduction of endogenous neurabin levels by interference RNA (RNAi)-mediated knockdown also inhibited the surface expression of GluR1 receptors. Together, our studies suggested that disrupting the functions of a cytoskeletal neurabin/PP1 complex enhanced filopodia and impaired surface GluR1 expression in hippocampal neurons, thereby hindering the morphological and functional maturation of dendritic spines.

    View details for Web of Science ID 000228737400018

    View details for PubMedID 15743906

  • Subcellular redistribution of the synapse-associated proteins PSD-95 and SAP97 in animal models of Parkinson's disease and L-DOPA-induced dyskinesia. FASEB journal Nash, J. E., Johnston, T. H., Collingridge, G. L., Garner, C. C., Brotchie, J. M. 2005; 19 (6): 583-585


    Abnormalities in subcellular localization and interaction between receptors and their signaling molecules occur within the striatum in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). Synapse-associated proteins (SAPs), for example, PSD-95 and SAP97 organize the molecular architecture of synapses and regulate interactions between receptors and downstream-signaling molecules. Here, we show that expression and subcellular distribution of PSD-95 and SAP97 are altered in the striatum of unilateral 6-OHDA-lesioned rats following repeated vehicle (a model of PD) or L-DOPA administration (a model of L-DOPA-induced dyskinesia). Furthermore, following dopamine-depletion and development of behavioral deficits in Rotorod performance, indicative of parkinsonism, we observed a dramatic decrease in total striatal levels of PSD-95 and SAP97 (to 25.6 +/- 9.9% and 19.0 +/- 5.0% of control, respectively). The remaining proteins were redistributed from the synapse into vesicular compartments. L-DOPA (6.5mg/kg twice a day, 21 days) induced a rotational response, which became markedly enhanced with repeated treatment (day 1: -15.8+/-7.3 rotations cf day 21: 758.2+/-114.0 rotations). Post L-DOPA treatment, PSD-95 and SAP97 levels increased (367.4 +/- 43.2% and 159.9 +/- 9.5% from control values, respectively), with both being redistributed toward synaptic membranes from vesicular compartments. In situ hybridization showed that changes in total levels of PSD-95, but not SAP97, were accompanied by qualitatively similar changes in mRNA. These data highlight the potential role of abnormalities in the subcellular distribution of SAPs in the pathophysiology of a neurological disease.

    View details for PubMedID 15703272

  • Mechanisms of vertebrate synaptogenesis ANNUAL REVIEW OF NEUROSCIENCE Waites, C. L., Craig, A. M., Garner, C. C. 2005; 28: 251-274


    The formation of synapses in the vertebrate central nervous system is a complex process that occurs over a protracted period of development. Recent work has begun to unravel the mysteries of synaptogenesis, demonstrating the existence of multiple molecules that influence not only when and where synapses form but also synaptic specificity and stability. Some of these molecules act at a distance, steering axons to their correct receptive fields and promoting neuronal differentiation and maturation, whereas others act at the time of contact, providing positional information about the appropriateness of targets and/or inductive signals that trigger the cascade of events leading to synapse formation. In addition, correlated synaptic activity provides critical information about the appropriateness of synaptic connections, thereby influencing synapse stability and elimination. Although synapse formation and elimination are hallmarks of early development, these processes are also fundamental to learning, memory, and cognition in the mature brain.

    View details for DOI 10.1146/annurev.neuro.27.070203.144336

    View details for Web of Science ID 000231235700010

    View details for PubMedID 16022596

  • Cellular and molecular mechanisms of presynaptic assembly NATURE REVIEWS NEUROSCIENCE Ziv, N. E., Garner, C. C. 2004; 5 (5): 385-?

    View details for DOI 10.1038/nrn1370

    View details for Web of Science ID 000221158600014

    View details for PubMedID 15100721

  • MAGUKs in synapse assembly and function: an emerging view CELLULAR AND MOLECULAR LIFE SCIENCES Montgomery, J. M., Zamorano, P. L., Garner, C. C. 2004; 61 (7-8): 911-929


    Neuronal morphogenesis, synaptogenesis and synaptic plasticity are fundamental aspects of nervous system development. Much of our current understanding of how each of these processes contributes to the establishment and maintenance of neural circuitry has come from a molecular description of specific classes of key molecules. With regard to synapse assembly and function, a family of membrane-associated guanylate kinase homologs (MAGUKs) have emerged as central organizers of multicomponent protein signaling complexes. In particular MAGUKs appear to play fundamental roles in the transport, anchoring and signaling of specific subclasses of synaptic receptors and ion channels. In this review, we will focus on the role that subfamilies of MAGUKs play during the formation, maintenance and plasticity of the vertebrate central nervous system glutamatergic synapse.

    View details for DOI 10.1007/s00018-003-3364-5

    View details for Web of Science ID 000220982200015

    View details for PubMedID 15095012

  • Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors NATURE NEUROSCIENCE Ju, W., Morishita, W., Tsui, J., Gaietta, G., Deerinck, T. J., Adams, S. R., Garner, C. C., Tsien, R. Y., Ellisman, M. H., Malenka, R. C. 2004; 7 (3): 244-253


    Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. Here we examined the trafficking and synthesis of the GluR1 and GluR2 subunits using ReAsH-EDT(2) and FlAsH-EDT(2) staining. Activity blockade of rat cultured neurons increased dendritic GluR1, but not GluR2, levels. Examination of transected dendrites revealed that both AMPAR subunits were synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluR1 but not GluR2. In contrast, acute pharmacological manipulations increased dendritic synthesis of both subunits. AMPARs synthesized in dendrites were inserted into synaptic plasma membranes and, after activity blockade, the electrophysiological properties of native synaptic AMPARs changed in the manner predicted by the imaging experiments. In addition to providing a novel mechanism for synaptic modifications, these results point out the advantages of using FlAsH-EDT(2) and ReAsH-EDT(2) for studying the trafficking of newly synthesized proteins in local cellular compartments such as dendrites.

    View details for DOI 10.1038/nn1189

    View details for Web of Science ID 000189197900014

    View details for PubMedID 14770185

  • Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly JOURNAL OF NEUROSCIENCE Bresler, T., Shapira, M., Boeckers, T., Dresbach, T., Futter, M., Garner, C. C., Rosenblum, K., Gundelfinger, E. D., Ziv, N. E. 2004; 24 (6): 1507-1520


    The cellular mechanisms involved in the formation of the glutamatergic postsynaptic density (PSD) are mainly unknown. Previous studies have indicated that PSD assembly may occur in situ by a gradual recruitment of postsynaptic molecules, whereas others have suggested that the PSD may be assembled from modular transport packets assembled elsewhere. Here we used cultured hippocampal neurons and live cell imaging to examine the process by which PSD molecules from different layers of the PSD are recruited to nascent postsynaptic sites. GFP-tagged NR1, the essential subunit of the NMDA receptor, and ProSAP1/Shank2 and ProSAP2/Shank3, scaffolding molecules thought to reside at deeper layers of the PSD, were recruited to new synaptic sites in gradual manner, with no obvious involvement of discernible discrete transport particles. The recruitment kinetics of these three PSD molecules were remarkably similar, which may indicate that PSD assembly rate is governed by a common upstream rate-limiting process. In contrast, the presynaptic active zone (AZ) molecule Bassoon was observed to be recruited to new presynaptic sites by means of a small number of mobile packets, in full agreement with previous studies. These findings indicate that the assembly processes of PSDs and AZs may be fundamentally different.

    View details for DOI 10.1523/JNEUROSCI.3819-03.2004

    View details for Web of Science ID 000188896100029

    View details for PubMedID 14960624

  • Functional regions of the presynaptic cytomatrix protein Bassoon: significance for synaptic targeting and cytomatrix anchoring MOLECULAR AND CELLULAR NEUROSCIENCE Dresbach, T., Hempelmann, A., Spilker, C., Dieck, S. T., Altrock, W. D., Zuschratter, W., Garner, C. C., Gundelfinger, E. D. 2003; 23 (2): 279-291


    Exocytosis of neurotransmitter from synaptic vesicles is restricted to specialized sites of the presynaptic plasma membrane called active zones. A complex cytomatrix of proteins exclusively assembled at active zones, the CAZ, is thought to form a molecular scaffold that organizes neurotransmitter release sites. Here, we have analyzed synaptic targeting and cytomatrix association of Bassoon, a major scaffolding protein of the CAZ. By combining immunocytochemistry and transfection of cultured hippocampal neurons, we show that the central portion of Bassoon is crucially involved in synaptic targeting and CAZ association. An N-terminal region harbors a distinct capacity for N-myristoylation-dependent targeting to synaptic vesicle clusters, but is not incorporated into the CAZ. Our data provide the first experimental evidence for the existence of distinct functional regions in Bassoon and suggest that a centrally located CAZ targeting function may be complemented by an N-terminal capacity for targeting to membrane-bounded synaptic organelles.

    View details for DOI 10.1016/S1044-7431(03)00015-0

    View details for Web of Science ID 000183655700010

    View details for PubMedID 12812759

  • Synapse-associated protein-97 isoform-specific regulation of surface AMPA receptors and synaptic function in cultured neurons JOURNAL OF NEUROSCIENCE Rumbaugh, G., Sia, G. M., Garner, C. C., Huganir, R. L. 2003; 23 (11): 4567-4576


    Members of the synapse-associated protein-97 (SAP97) family of scaffold proteins have been implicated as central organizers of synaptic junctions to build macromolecular signaling complexes around specific postsynaptic neurotransmitter receptors. In this regard, SAP97 has been suggested to regulate the synaptic localization of glutamate receptor type 1 subunits of the AMPA-type glutamate receptors. To test this hypothesis directly, we assessed the effects of SAP97 overexpression on surface expression of synaptic AMPA receptors. We find that recombinant SAP97 not only becomes concentrated at synaptic junctions but also leads to an increase in synaptic AMPA receptors, spine enlargement, and an increase in miniature EPSC (mEPSC) frequency, indicating that SAP97 has both postsynaptic and presynaptic effects on synaptic transmission. Synaptic targeting of SAP97, increased surface AMPA receptors, and increased mEPSC frequency are dependent on the presence of specific alternatively spliced sequences in SAP97 that encode a protein 4.1 binding site. These results suggest that SAP97 can affect the synaptic recruitment of AMPA receptors and spine morphology and that these effects may be regulated by alternative splicing.

    View details for Web of Science ID 000183443700020

    View details for PubMedID 12805297

  • Interactions between Piccolo and the actin/dynamin-binding protein Abp1 link vesicle endocytosis to presynaptic active zones JOURNAL OF BIOLOGICAL CHEMISTRY Fenster, S. D., Kessels, M. M., Qualmann, B., Chung, W. J., NASH, J., Gundelfinger, E. D., Garner, C. C. 2003; 278 (22): 20268-20277


    Piccolo is a high molecular weight multi-domain protein shown to be a structural component of the presynaptic CAZ (cytoskeletal matrix assembled at active zones). These features indicate that Piccolo may act to scaffold proteins involved in synaptic vesicle endo- and exocytosis near their site of action. To test this hypothesis, we have utilized a functional cell-based endocytosis assay and identified the N-terminal proline-rich Q domain in Piccolo as a region that interferes with clathrin-mediated endocytosis. Utilizing the Piccolo Q domain as bait in a yeast two-hybrid screen, we have identified the F-actin-binding protein Abp1 (also called SH3P7 or HIP-55) as a potential binding partner for this domain. The physiological relevance of this interaction is supported by in vitro binding studies, colocalization in nerve terminals, in vivo recruitment studies, and immunoprecipitation experiments. Intriguingly, Abp1 binds to both F-actin and the GTPase dynamin and has been implicated in linking the actin cytoskeleton to clathrin-mediated endocytosis. Our results suggest that Piccolo, as a structural protein of the CAZ, may serve to localize Abp1 at active zones where it can actively participate in creating a functional connection between the dynamic actin cytoskeleton and synaptic vesicle recycling.

    View details for DOI 10.1074/jbc.M210792200

    View details for Web of Science ID 000183078000089

    View details for PubMedID 12654920

  • Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles NEURON Shapira, M., Zhai, R. G., Dresbach, T., Bresler, T., Torres, V. I., Gundelfinger, E. D., Ziv, N. E., Garner, C. C. 2003; 38 (2): 237-252


    Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.

    View details for Web of Science ID 000182498300013

    View details for PubMedID 12718858

  • The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina NEURON Dick, O., Dieck, S. T., Altrock, W. D., Ammermuller, J., WEILER, R., Garner, C. C., Gundelfinger, E. D., Brandstatter, J. H. 2003; 37 (5): 775-786


    The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.

    View details for Web of Science ID 000181452800007

    View details for PubMedID 12628168

  • Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon NEURON Altrock, W. D., Dieck, S. T., Sokolov, M., Meyer, A. C., Sigler, A., Brakebusch, C., Fassler, R., Richter, K., Boeckers, T. M., Potschka, H., Brandt, C., Loscher, W., Grimberg, D., Dresbach, T., Hempelmann, A., Hassan, H., Balschun, D., Frey, J. U., Brandstatter, J. H., Garner, C. C., Rosenmund, C., Gundelfinger, E. D. 2003; 37 (5): 787-800


    Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

    View details for Web of Science ID 000181452800008

    View details for PubMedID 12628169

  • The GIT family of proteins forms multimers and associates with the presynaptic cytomatrix protein Piccolo JOURNAL OF BIOLOGICAL CHEMISTRY Kim, S., Ko, J., Shin, H., Lee, J. R., Lim, C., Han, J. H., Altrock, W. D., Garner, C. C., Gundelfinger, E. D., Premont, R. T., Kaang, B. K., Kim, E. 2003; 278 (8): 6291-6300


    The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ADP-ribosylation factor small GTPases. A small region (approximately 150 amino acid residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins, including betaPIX, focal adhesion kinase, liprin-alpha, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, betaPIX, and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo- and heteromultimers through their C-terminal G-protein-coupled receptor kinase-binding domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHD-binding proteins including Piccolo and betaPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.

    View details for DOI 10.1074/jbc.M212287200

    View details for Web of Science ID 000181129400101

    View details for PubMedID 12473661

  • Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2 JOURNAL OF BIOLOGICAL CHEMISTRY Deshane, J., Garner, C. C., Sontheimer, H. 2003; 278 (6): 4135-4144


    Primary brain tumors (gliomas) have the unusual ability to diffusely infiltrate the normal brain thereby evading surgical treatment. Chlorotoxin is a scorpion toxin that specifically binds to the surface of glioma cells and impairs their ability to invade. Using a recombinant His-Cltx we isolated and identified the principal Cltx receptor on the surface of glioma cells as matrix metalloproteinase-2 (MMP-2). MMP-2 is specifically up-regulated in gliomas and related cancers, but is not normally expressed in brain. We demonstrate that Cltx specifically and selectively interacts with MMP-2 isoforms, but not with MMP-1, -3, and -9, which are also expressed in malignant glioma cells. Importantly, we show that the anti-invasive effect of Cltx on glioma cells can be explained by its interactions with MMP-2. Cltx exerts a dual effect on MMP-2: it inhibits the enzymatic activity of MMP-2 and causes a reduction in the surface expression of MMP-2. These findings suggest that Cltx is a specific MMP-2 inhibitor with significant therapeutic potential for gliomas and other diseases that invoke the activity of MMP-2.

    View details for DOI 10.1074/jbc.M205662200

    View details for Web of Science ID 000180869700080

    View details for PubMedID 12454020

  • Interaction of SAP97 with minus-end-directed actin motor myosin VI - Implications for AMPA receptor trafficking JOURNAL OF BIOLOGICAL CHEMISTRY Wu, H. J., Nash, J. E., ZAMORANO, P., Garner, C. C. 2002; 277 (34): 30928-30934


    SAP97 is a modular protein composed of three PDZ domains, an SH3 domain, and a guanylate kinase-like domain. It has been implicated functionally in the assembly and structural stability of synaptic junctions as well as in the trafficking, recruitment, and localization of specific ion channels and neurotransmitter receptors. The N terminus of SAP97 (S97N) has been shown to play a key role in the selection of binding partners and the localization of SAP97 at adhesion sites, as well as the clustering of ion channels in heterologous cells. Using the S97N domain as bait in a yeast two-hybrid screen, we identified the minus-end-directed actin-based motor, myosin VI, as an S97N binding partner. Moreover, in light membrane fractions prepared from rat brain, we found that myosin VI and SAP97 form a trimeric complex with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR1. These data suggest that SAP97 may serve as a molecular link between GluR1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors to and from the postsynaptic plasma membrane.

    View details for DOI 10.1074/jbc.M203735200

    View details for Web of Science ID 000177579800068

    View details for PubMedID 12050163

  • Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo INTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE Fenster, S. D., Garner, C. C. 2002; 20 (3-5): 161-171


    Piccolo belongs to a family of presynaptic cytoskeletal proteins likely to be involved in the assembly and function of presynaptic active zones as sites of neurotransmitter release. Given that abnormalities in the formation of synaptic junctions are thought to contribute to cognitive dysfunction during brain development, we have analyzed and compared the gene structure of the Piccolo gene, PCLO, from humans and mice and determined their chromosomal localization. A comparison of the deduced amino acid sequence of cDNA clones encoding Piccolo from human, mouse, rat and chicken reveals the presence of distinct homology domains. Only subsets of these are also present in the structurally related active zone protein Bassoon indicating that Piccolo and Bassoon perform related but distinct functions at active zones. Characterization of the PCLO gene reveals the presence of 25 coding exons spread over 380kb of genomic DNA. The human PCLO gene maps to 7q11.23-q21.3, a region of chromosome 7 implicated as a linkage site for autism and Williams Syndrome suggesting that alterations in the expression of Piccolo or the PCLO gene could contribute to developmental disabilities and mental retardation.

    View details for Web of Science ID 000178422900004

    View details for PubMedID 12175852

  • Molecular mechanisms of CNS synaptogenesis TRENDS IN NEUROSCIENCES Garner, C. C., Zhai, R. G., Gundelfinger, E. D., Ziv, N. E. 2002; 25 (5): 243-250


    Synapses of the mammalian CNS are asymmetric sites of cell-cell adhesion between nerve cells. They are designed to mediate the rapid and efficient transmission of signals from the presynaptic bouton of one neuron to the postsynaptic plasma membrane of a second neuron. Significant progress has been made in the characterization of the structural, functional and developmental assembly of CNS synapses. Recent progress has been made in understanding the molecular and cellular mechanisms that underlie synaptogenesis, in particular that of glutamatergic synapses of the CNS.

    View details for Web of Science ID 000175141300010

    View details for PubMedID 11972960

  • Priming plasticity. Nature Dobrunz, L. E., Garner, C. C. 2002; 415 (6869): 277-278

    View details for PubMedID 11796993

  • Localization of the presynaptic cytomatrix protein piccolo at ribbon and conventional synapses in the rat retina: Comparison with bassoon JOURNAL OF COMPARATIVE NEUROLOGY Dick, O., Hack, I., Altrock, W. D., Garner, C. C., Gundelfinger, E. D., Brandstatter, J. H. 2001; 439 (2): 224-234


    In recent years significant progress has been made in the elucidation of the molecular assembly of the postsynaptic density at synapses, whereas little is known as yet about the components of the presynaptic active zone. Piccolo and Bassoon, two structurally related presynaptic cytomatrix proteins, are highly concentrated at the active zones of both excitatory and inhibitory synapses in rat brain. In this study we used immunocytochemistry to examine the cellular and ultrastructural localization of Piccolo at synapses in the rat retina and compared it with that of Bassoon. Both proteins showed strong punctate immunofluorescence in the outer and the inner plexiform layers of the retina. They were found presynaptically at glutamatergic ribbon synapses and at conventional GABAergic and glycinergic synapses. Although the two proteins were coexpressed at all photoreceptor ribbon synapses and at some conventional amacrine cell synapses, at bipolar cell ribbon synapses only Piccolo was present. Our data demonstrate similarities but also differences in the molecular composition of the presynaptic apparatuses of the synapses in the retina, differences that may account for the functional differences observed between the ribbon and the conventional amacrine cell synapses and between the photoreceptor and the bipolar cell ribbon synapses in the retina.

    View details for Web of Science ID 000171301400008

    View details for PubMedID 11596050

  • Unwebbing the presynaptic web NEURON Zamorano, P. L., Garner, C. C. 2001; 32 (1): 3-6


    The release of neurotransmitter from nerve terminals occurs at a specialized region of the presynaptic plasma membrane called the active zone. A dense matrix of proteins associated with the active zone, called the presynaptic web, is thought to play a fundamental role in defining these neurotransmitter release sites. In this issue of Neuron, Phillips et al. have identified conditions for the biochemical purification of the presynaptic web and show that the web is comprised of proteins involved in the docking, fusion, and recycling of synaptic vesicles.

    View details for Web of Science ID 000171525200002

    View details for PubMedID 11604132

  • Principles of glutamatergic synapse formation: seeing the forest for the trees CURRENT OPINION IN NEUROBIOLOGY Ziv, N. E., Garner, C. C. 2001; 11 (5): 536-543


    General principles regarding glutamatergic synapse formation in the central nervous system are beginning to emerge. These principles concern the specific roles that dendrites and axons play in the induction of synaptic differentiation, the modes of presynaptic and postsynaptic assembly, the time course of synapse formation and maturation, and the roles of synaptic activity in these processes.

    View details for Web of Science ID 000171712000002

    View details for PubMedID 11595485

  • The dynamics of SAP90/PSD-95 recruitment to new synaptic junctions MOLECULAR AND CELLULAR NEUROSCIENCE Bresler, T., Ramati, Y., Zamorano, P. L., Zhai, R., Garner, C. C., Ziv, N. E. 2001; 18 (2): 149-167


    SAP90/PSD-95 is thought to be a central organizer of the glutamatergic synapse postsynaptic reception apparatus. To assess its potential role during glutamatergic synapse formation, we used GFP-tagged SAP90/PSD-95, time lapse confocal microscopy, and cultured hippocampal neurons to determine its dynamic recruitment into new synaptic junctions. We report that new SAP90/PSD-95 clusters first appeared at new axodendritic contact sites within 20-60 min of contact establishment. SAP90/PSD-95 clustering was rapid, with kinetics that fit a single exponential with a mean time constant of approximately 23 min. Most new SAP90/PSD-95 clusters were found juxtaposed to functional presynaptic boutons as determined by labeling with FM 4-64. No evidence was found for the existence of discrete transport particles similar to those previously reported to mediate presynaptic active zone cytoskeleton assembly. Instead, we found that SAP90/PSD-95 is recruited to nascent synapses from a diffuse dendritic cytoplasmic pool. Our findings show that SAP90/PSD-95 is recruited to nascent synaptic junctions early during the assembly process and indicate that its assimilation is fundamentally different from that of presynaptic active zone components.

    View details for Web of Science ID 000170896600003

    View details for PubMedID 11520177

  • Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97 JOURNAL OF BIOLOGICAL CHEMISTRY Mehta, S., Wu, H. J., Garner, C. C., Marshall, J. 2001; 276 (19): 16092-16099


    Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization.

    View details for Web of Science ID 000168623100072

    View details for PubMedID 11279111

  • Identification of a cis-acting dendritic targeting element in the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II EUROPEAN JOURNAL OF NEUROSCIENCE Blichenberg, A., Rehbein, M., Muller, R., Garner, C. C., Richter, D., Kindler, S. 2001; 13 (10): 1881-1888


    In mammalian neurons a selected group of mRNAs, including the transcript encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II, is found in dendrites. The molecular mechanisms underlying extrasomatic RNA trafficking are not well described. It is thought that dendritic transcripts contain cis-acting elements that direct their selective subcellular sorting. Here we report the identification of an extrasomatic targeting element in the 3' untranslated region of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. In primary hippocampal neurons, this 1200-nucleotide-spanning, cis-acting element is sufficient to mediate dendritic localization of chimeric reporter transcripts. The trafficking signal does not share any striking sequence similarity with a previously characterized dendritic targeting element in transcripts encoding the microtubule-associated protein 2. In dendrites of transfected primary neurons, recombinant RNAs form granules with an average diameter of 0.45 microm that may represent preferential RNA docking sites or multimolecular transport units. These findings imply that extrasomatic sorting of individual dendritic mRNAs involves at least partially distinct molecular mechanisms, as well as large trafficking complexes.

    View details for Web of Science ID 000168968100005

    View details for PubMedID 11403681

  • Proline-rich synapse-associated protein-1/cortactin binding protein 1 (ProSAP1/CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density ANNALS OF ANATOMY-ANATOMISCHER ANZEIGER Boeckers, T. M., Kreutz, M. R., Winter, C., Zuschratter, W., Smalla, K. H., Sanmarti-Vila, L., Wex, H., Langnaese, K., Bockmann, J., Garner, C. C., Gundelfinger, E. D. 2001; 183 (2): 101-101

    View details for Web of Science ID 000167916900003

    View details for PubMedID 11325055

  • The presynaptic cytomatrix of brain synapses CELLULAR AND MOLECULAR LIFE SCIENCES Dresbach, T., Qualmann, B., Kessels, M. M., Garner, C. C., Gundelfinger, E. D. 2001; 58 (1): 94-116


    Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.

    View details for Web of Science ID 000166988300007

    View details for PubMedID 11229820

  • Assembling the presynaptic active zone: A characterization of an active zone precursor vesicle NEURON Zhai, R. G., Vardinon-Friedman, H., Cases-Langhoff, C., Becker, B., Gundelfinger, E. D., Ziv, N. E., Garner, C. C. 2001; 29 (1): 131-143


    The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.

    View details for Web of Science ID 000166702700014

    View details for PubMedID 11182086

  • Intramolecular interactions regulate SAP97 binding to GKAP EMBO JOURNAL Wu, H. J., Reissner, C., Kuhlendahl, S., Coblentz, B., Reuver, S., Kindler, S., Gundelfinger, E. D., Garner, C. C. 2000; 19 (21): 5740-5751


    Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.

    View details for Web of Science ID 000165235000015

    View details for PubMedID 11060025

  • Membrane association of presynaptic cytomatrix protein bassoon BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Sanmarti-Vila, L., Dieck, S. T., Richter, K., Altrock, W., Zhang, L. X., Volknandt, W., Zimmermann, H., Garner, C. C., Gundelfinger, E. D., Dresbach, T. 2000; 275 (1): 43-46


    Components of the specialized cytomatrix at active zones of presynaptic nerve terminals are thought to be involved in organizing synaptic events such as immobilisation or translocation of synaptic vesicles and assemblingactive zone components. The 420-kDa non-transmembraneprotein Bassoon is a specific componentof the presynaptic cytomatrix that shares features with both cytoskeleton-associated and peripheral-membrane proteins. Using immunogold electron microscopy we show here that synapse associated Bassoon is distributed in a subregion of active zones. Using a biochemical assay we show that a fraction of Bassoon is membrane associated. Electron microscopy performed on the same biochemical fraction further revealed that Bassoon is associated with vesicular structures. Together these data suggest that at least a fraction of Bassoon is associated with a membraneous compartment in neurons.

    View details for DOI 10.1006/bbrc.2000.3256

    View details for Web of Science ID 000088945900009

    View details for PubMedID 10944438

  • PDZ domains in synapse assembly and signalling TRENDS IN CELL BIOLOGY Garner, C. C., NASH, J., Huganir, R. L. 2000; 10 (7): 274-280


    Synaptic junctions are highly specialized structures designed to promote the rapid and efficient transmission of signals from the presynaptic terminal to the postsynaptic membrane within the central nervous system. Proteins containing PDZ domains play a fundamental organizational role at both the pre- and postsynaptic plasma membranes. This review focuses on recent advances in our understanding of the mechanisms underlying the assembly of synapses in the central nervous system.

    View details for Web of Science ID 000087769300003

    View details for PubMedID 10856930

  • Assembly of new individual excitatory synapses: Time course and temporal order of synaptic molecule recruitment NEURON Friedman, H. V., Bresler, T., Garner, C. C., Ziv, N. E. 2000; 27 (1): 57-69


    Time-lapse microscopy, retrospective immunohistochemistry, and cultured hippocampal neurons were used to determine the time frame of individual glutamatergic synapse assembly and the temporal order in which specific molecules accumulate at new synaptic junctions. New presynaptic boutons capable of activity-evoked vesicle recycling were observed to form within 30 min of initial axodendritic contact. Clusters of the presynaptic active zone protein Bassoon were present in all new boutons. Conversely, clusters of the postsynaptic molecule SAP90/PSD-95 and glutamate receptors were found on average only approximately 45 min after such boutons were first detected. AMPA- and NMDA-type glutamate receptors displayed similar clustering kinetics. These findings suggest that glutamatergic synapse assembly can occur within 1-2 hr after initial contact and that presynaptic differentiation may precede postsynaptic differentiation.

    View details for Web of Science ID 000088503800010

    View details for PubMedID 10939331

  • Molecular determinants of presynaptic active zones CURRENT OPINION IN NEUROBIOLOGY Garner, C. C., Kindler, S., Gundelfinger, E. D. 2000; 10 (3): 321-327


    The presynaptic cytoskeletal matrix (cytomatrix) assembled at active zones has been implicated in defining neurotransmitter release sites. Munc13, Rim, Bassoon and Piccolo/Aczonin are recently identified presynaptic cytomatrix proteins. These multidomain proteins are thought to organize the exocytotic and endocytotic machinery precisely at active zones.

    View details for Web of Science ID 000087798000005

    View details for PubMedID 10851173

  • Temporal appearance of the presynaptic cytomatrix protein bassoon during synaptogenesis MOLECULAR AND CELLULAR NEUROSCIENCE Zhai, R., Olias, G., Chung, W. J., Lester, R. A., Dieck, S. T., Langnaese, K., Kreutz, M. R., Kindler, S., Gundelfinger, E. D., Garner, C. C. 2000; 15 (5): 417-428


    Bassoon is a 420-kDa presynaptic cytomatrix protein potentially involved in the structural organization of neurotransmitter release sites. In this study, we have investigated a possible role for Bassoon in synaptogenesis and in defining synaptic vesicle recycling sites. We find that it is expressed at early stages of neuronal differentiation in which it is selectively sorted into axons. As synaptogenesis begins, Bassoon clusters appear along dendritic profiles simultaneously with synaptotagmin I, sites of synaptic vesicle recycling, and the acquisition of functional excitatory and inhibitory synapses. A role for Bassoon in the assembly of excitatory and inhibitory synapses is supported by the colocalization of Bassoon clusters with clusters of GKAP and AMPA receptors as well as GABA(A) receptors. These data indicate that the recruitment of Bassoon is an early step in the formation of synaptic junctions.

    View details for Web of Science ID 000087487900001

    View details for PubMedID 10833299

  • Piccolo, a presynaptic zinc finger protein structurally related to bassoon NEURON Fenster, S. D., Chung, W. J., Zhai, R., Cases-Langhoff, C., Voss, B., Garner, A. M., Kaempf, U., Kindler, S., Gundelfinger, E. D., Garner, C. C. 2000; 25 (1): 203-214


    Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle-associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.

    View details for Web of Science ID 000085043200022

    View details for PubMedID 10707984

  • Identification of a cis-acting dendritic targeting element in MAP2 mRNAs JOURNAL OF NEUROSCIENCE Blichenberg, A., Schwanke, B., Rehbein, M., Garner, C. C., Richter, D., Kindler, S. 1999; 19 (20): 8818-8829


    In neurons, a limited number of mRNAs have been identified in dendritic processes, whereas other transcripts are restricted to the cell soma. Here we have investigated the molecular mechanisms underlying extrasomatic localization of mRNAs encoding microtubule-associated protein 2 (MAP2) in primary neuronal cultures. Vectors expressing recombinant mRNAs were introduced into hippocampal and sympathetic neurons using DNA transfection and microinjection protocols, respectively. Chimeric mRNAs containing the entire 3' untranslated region of MAP2 transcripts fused to a nondendritic reporter mRNA are detected in dendrites. In contrast, RNAs containing MAP2 coding and 5' untranslated regions or tubulin sequences are restricted to the cell soma. Moreover, 640 nucleotides from the MAP2 3' untranslated region (UTR) are both sufficient and essential for extrasomatic localization of chimeric mRNAs in hippocampal and sympathetic neurons. Thus, a cis-acting dendritic targeting element that is effective in two distinct neuronal cell types is contained in the 3' UTR of MAP2 transcripts. The observation of RNA granules in dendrites implies that extrasomatic transcripts seem to assemble into multimolecular complexes that may function as transport units.

    View details for Web of Science ID 000083072500015

    View details for PubMedID 10516301

  • Proline-rich synapse-associated proteins ProSAP1 and ProSAP2 interact with synaptic proteins of the SAPAP/GKAP family BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Boeckers, T. M., Winter, C., Smalla, K. H., Kreutz, M. R., Bockmann, J., Seidenbecher, C., Garner, C. C., Gundelfinger, E. D. 1999; 264 (1): 247-252


    We have recently isolated a novel proline-rich synapse-associated protein-1 (ProSAP1) that is highly enriched in postsynaptic density (PSD). A closely related multidomain protein, ProSAP2, shares a highly conserved PDZ (PSD-95/discs-large/ZO-1) domain (80% identity), a ppI domain that mediates the interaction with cortactin, and a C-terminal SAM (sterile alpha-motif) domain. In addition, ProSAP2 codes for five ankyrin repeats and a SH3 (Src homology 3) domain. Transcripts for both proteins are coexpressed in many regions of rat brain, but show a distinct expression pattern in the cerebellum. Using the PDZ domains of ProSAP1 and 2 as bait in the yeast two-hybrid system, we isolated several clones of the SAPAP/GKAP (SAP90/PSD-95-associated protein/guanylate kinase-associated protein) family. The association of the proteins was verified by coimmunoprecipitation and cotransfection in HEK cells. Therefore, proteins of the ProSAP family represent a novel link between SAP90/PSD-95 bound membrane receptors and the cytoskeleton at glutamatergic synapses of the central nervous system.

    View details for Web of Science ID 000083153500044

    View details for PubMedID 10527873

  • Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina EUROPEAN JOURNAL OF NEUROSCIENCE Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. 1999; 11 (10): 3683-3693


    Bassoon is a 420-kDa presynaptic protein which is highly concentrated at the active zones of nerve terminals of conventional synapses, both excitatory glutamatergic and inhibitory GABAergic, in rat brain. It is thought to be involved in the organization of the cytomatrix at the site of neurotransmitter release. In the retina, there are two structurally and functionally distinct types of synapses: ribbon and conventional synapses. Antibodies against bassoon were applied to sections of rat and rabbit retina. Strong punctate immunofluorescence was found in the outer and inner plexiform layers. Using pre- and post-embedding immunostaining and electron microscopy, bassoon was localized in the outer plexiform layer at ribbon synapses formed by rods and cones but was absent from basal synaptic contacts formed by cones. In the inner plexiform layer a different picture emerged. As in the brain, bassoon was found at conventional inhibitory GABAergic synapses, made by amacrine cells, but it was absent from the bipolar cell ribbon synapses. These data demonstrate differences in the molecular composition of the presynaptic apparatuses of outer and inner plexiform layer ribbon synapses. Thus, differential equipment with cytomatrix proteins may account for the functional differences observed between the two types of ribbon synapses in the retina.

    View details for Web of Science ID 000083149700032

    View details for PubMedID 10564375

  • Proline-rich synapse-associated protein-1/cortactin binding protein 1(ProSAP1/CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density JOURNAL OF NEUROSCIENCE Boeckers, T. M., Kreutz, M. R., Winter, C., Zuschratter, W., Smalla, K. H., Sanmarti-Vila, L., Wex, H., Langnaese, K., Bockmann, J., Garner, C. C., Gundelfinger, E. D. 1999; 19 (15): 6506-6518


    The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

    View details for Web of Science ID 000081648400029

    View details for PubMedID 10414979

  • Presynaptic cytomatrix protein bassoon is localized at both excitatory and inhibitory synapses of rat brain JOURNAL OF COMPARATIVE NEUROLOGY Righter, K., Langnaese, K., Kreutz, M. R., Olias, G., Zhai, R., Scheich, H., Garner, C. C., Gundelfinger, E. D. 1999; 408 (3): 437-448


    Bassoon is a 420-kDa protein specifically localized at the active zone of presynaptic nerve terminals. It is thought to be involved in the structural organization of the neurotransmitter release site. We studied the distribution of Bassoon transcripts and protein in rat brain and assessed which types of presynaptic terminals contain the protein. As shown by in situ hybridization, Bassoon transcripts are widely distributed in the brain and occur primarily in excitatory neurons. In addition, examples of gamma-aminobutyric acid (GABA)-ergic neurons expressing Bassoon are detected. At the light microscopic level, Bassoon immunoreactivity is found in synaptic neuropil regions throughout the brain, with the strongest expression in the hippocampus, the cerebellar cortex, and the olfactory bulb. Immunoelectron microscopy showed that Bassoon immunoreactivity is found in both asymmetric type 1 and symmetric type 2 synapses. Immunopositive asymmetric synapses include mossy fiber boutons and various spine and shaft synapses in the hippocampus and mossy fiber terminals and parallel fiber terminals in the cerebellum. Bassoon-containing symmetric synapses are observed, e.g., between basket and granule cells in the hippocampus, between Golgi cells and granule cells, and between basket cells and Purkinje cells in the cerebellum. Within synaptic terminals, Bassoon appears highly concentrated at sites opposite to postsynaptic densities. In cultured hippocampal neurons, Bassoon was found to colocalize with GABA(A) and glutamate (GluR1) receptors. These data indicate that Bassoon is a component of the presynaptic apparatus of both excitatory glutamatergic and inhibitory GABAergic synapses.

    View details for Web of Science ID 000080090200009

    View details for PubMedID 10340516

  • The presynaptic cytomatrix protein bassoon: Sequence and chromosomal localization of the human BSN gene GENOMICS Winter, C., tom Dieck, S., Boeckers, T. M., Bockmann, J., Kampf, U., Sanmarti-Vila, L., Langnaese, K., Altrock, W., Stumm, M., Soyke, A., Wieacker, P., Garner, C. C., Gundelfinger, E. D. 1999; 57 (3): 389-397


    Bassoon is a novel 420-kDa protein recently identified as a component of the cytoskeleton at presynaptic neurotransmitter release sites. Analysis of the rat and mouse sequences revealed a polyglutamine stretch in the C-terminal part of the protein. Since it is known for some proteins that abnormal amplification of such polyglutamine regions can cause late-onset neurodegeneration, we cloned and localized the human BASSOON gene (BSN). Phage clones spanning most of the open reading frame and the 3' untranslated region were isolated from a human genomic library and used for chromosomal localization of BSN to chromosome 3p21 by FISH. The localization was confirmed by PCR on rodent/human somatic cell hybrids; it is consistent with the localization of the murine Bsn gene at chromosome 9F. Sequencing revealed a polyglutamine stretch of only five residues in human, and PCR amplifications from 50 individuals showed no obvious length polymorphism in this region. Analysis of the primary structure of Bassoon and comparison to previous database entries provide evidence for a newly emerging protein family.

    View details for Web of Science ID 000080348000007

    View details for PubMedID 10329005

  • SAP90 binds and clusters kainate receptors causing incomplete desensitization NEURON Garcia, E. P., Mehta, S., Blair, L. A., Wells, D. G., Shang, J., Fukushima, T., Fallon, J. R., Garner, C. C., Marshall, J. 1998; 21 (4): 727-739


    The mechanism of kainate receptor targeting and clustering is still unresolved. Here, we demonstrate that members of the SAP90/PSD-95 family colocalize and associate with kainate receptors. SAP90 and SAP102 coimmunoprecipitate with both KA2 and GluR6, but only SAP97 coimmunoprecipitates with GluR6. Similar to NMDA receptors, GluR6 clustering is mediated by the interaction of its C-terminal amino acid sequence, ETMA, with the PDZ1 domain of SAP90. In contrast, the KA2 C-terminal region binds to, and is clustered by, the SH3 and GK domains of SAP90. Finally, we show that SAP90 coexpressed with GluR6 or GluR6/KA2 receptors alters receptor function by reducing desensitization. These studies suggest that the organization and electrophysiological properties of synaptic kainate receptors are modified by association with members of the SAP90/PSD-95 family.

    View details for Web of Science ID 000076697300017

    View details for PubMedID 9808460

  • Caldendrin, a novel neuronal calcium-binding protein confined to the somato-dendritic compartment JOURNAL OF BIOLOGICAL CHEMISTRY Seidenbecher, C. I., Langnaese, K., Sanmarti-Vila, L., Boeckers, T. M., Smalla, K. H., Sabel, B. A., Garner, C. C., Gundelfinger, E. D., Kreutz, M. R. 1998; 273 (33): 21324-21331


    Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.

    View details for Web of Science ID 000075386100086

    View details for PubMedID 9694893

  • Immunocytochemical localization of the synapse-associated protein SAP102 in the rat retina JOURNAL OF COMPARATIVE NEUROLOGY Koulen, P., Garner, C. C., Wassle, H. 1998; 397 (3): 326-336


    An elaborate network of transmitter receptors, synapse associated proteins (SAPs), and cytoskeletal elements, generally known as the postsynaptic density, is involved with efficient synaptic signaling. The localization of the synapse associated protein SAP102 was studied in the rat retina by using immunocytochemical methods. Immunofluorescence for SAP102 was most prominent in the inner plexiform layer (IPL). It had a punctate appearance, suggesting a synaptic clustering of SAP102 in the IPL. Electron microscopy by use of pre-embedding immunocytochemistry showed that SAP102 is concentrated in the IPL in processes which are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for SAP102. Double-labeling experiments were performed in order to find out whether SAP102 is involved with the clustering the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A). Only a fraction (approximately 23%) of the SAP102 clusters expressed NR2A, suggesting SAP102 is also associated with other subunits or receptors. Distinct SAP102 labeling was also present in horizontal cell processes in the outer plexiform layer (OPL), which are inserted as lateral elements into photoreceptor ribbon synapses (triads). The optic nerve fibre layer was also diffusely immunoreactive for SAP102. The postsynaptic aggregation of SAP102 at bipolar cell dyads and at photoreceptor triads suggests SAP102 is associated with the clustering of transmitter receptors. However, the labeling of the optic nerve fibre layer indicates additional functions of SAP102 in the retina.

    View details for Web of Science ID 000074479000002

    View details for PubMedID 9674560

  • Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane JOURNAL OF CELL SCIENCE Wu, H. J., Reuver, S. M., Kuhlendahl, S., Chung, W. J., Garner, C. C. 1998; 111: 2365-2376


    The synapse-associated protein SAP97 is a member of a novel family of cortical cytoskeletal proteins involved in the localization of ion channels at such membrane specializations as synaptic junctions. These multidomain proteins have binding sites for protein 4.1, GKAPs/SAPAPs, voltage- and ligand-gated ion channels and cell-adhesion molecules containing C-terminal T/SXV motifs. In this study, we evaluated the contribution of individual domains in SAP97 to its selective recruitment and attachment to the cortical cytoskeleton in epithelial cells. We find that the PDZ, SH3 and GK domains, as well as the I3 insert in SAP97, are not essential for subcellular targeting, though both PDZ1-2 domains and the I3 insert affect the efficiency of localization. Instead, we show that the first 65 amino acid residues in SAP97, which are absent from SAP90/PSD-95 and SAP102, direct the selective subcellular localization and can mediate at least one point of attachment of SAP97 to the cytoskeleton assembled at sites of cell-cell contact. Our data demonstrate that it is the sequences unique to SAP97 that direct its subcellular targeting to the epithelial lateral membrane.

    View details for Web of Science ID 000075758400008

    View details for PubMedID 9683631

  • SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit JOURNAL OF BIOLOGICAL CHEMISTRY Leonard, A. S., Davare, M. A., Horne, M. C., Garner, C. C., Hell, J. W. 1998; 273 (31): 19518-19524


    Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.

    View details for Web of Science ID 000075125200026

    View details for PubMedID 9677374

  • Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals JOURNAL OF CELL BIOLOGY Dieck, S. T., Sanmarti-Vila, L., Langnaese, K., Richter, K., Kindler, S., Soyke, A., Wex, H., Smalla, K. H., Kampf, U., Franzer, J. T., Stumm, M., Garner, C. C., Gundelfinger, E. D. 1998; 142 (2): 499-509


    The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

    View details for Web of Science ID 000075111300016

    View details for PubMedID 9679147

  • E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton JOURNAL OF CELL SCIENCE Reuver, S. M., Garner, C. C. 1998; 111: 1071-1080


    Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

    View details for Web of Science ID 000073668900005

    View details for PubMedID 9512503

  • Functional analysis of the guanylate kinase-like domain in the synapse-associated protein SAP97 EUROPEAN JOURNAL OF BIOCHEMISTRY Kuhlendahl, S., Spangenberg, O., Konrad, M., Kim, E., Garner, C. C. 1998; 252 (2): 305-313


    SAP97 is a membrane cytoskeletal protein localized at the presynaptic nerve terminals of type 1 asymmetric synapses. It has been implicated in the assembly of synapses and in particular in the localization and clustering of ion channels. The C-terminal GK domain of SAP97 shares a high degree of sequence similarity with low-molecular-mass guanylate kinases. These enzymes are involved in the guanine nucleotide metabolic cycle and in the maintenance of GTP/GDP pools required for example in Ras-mediated cell signaling. It has therefore been hypothesized that SAP97 plays an essential role in cellular signaling by regulating the guanine nucleotide pools at synaptic junctions. Here, we test this hypothesis by assessing whether the GK domain in SAP97 encodes an authentic guanylate kinase. We show that the GK domain in and of itself does not encode an active guanylate kinase, that it cannot be activated by its binding partner GKAP and that flanking regions are not acting as inhibitory regulators for enzymatic activity. Thus, it would appear that the GK domain of SAP97 is not involved in the metabolism of guanine nucleotides required for signaling events.

    View details for Web of Science ID 000072387700017

    View details for PubMedID 9523702

  • Synaptic clustering of the cell adhesion molecule fasciclin II by discs-large and its role in the regulation of presynaptic structure NEURON Thomas, U., Kim, E., Kuhlendahl, S., Koh, Y. H., Gundelfinger, E. D., Sheng, M., Garner, C. C., Budnik, V. 1997; 19 (4): 787-799


    The cell adhesion molecule Fasciclin II (FASII) is involved in synapse development and plasticity. Here we provide genetic and biochemical evidence that proper localization of FASII at type I glutamatergic synapses of the Drosophila neuromuscular junction is mediated by binding between the intracellular tSXV bearing C-terminal tail of FASII and the PDZ1-2 domains of Discs-Large (DLG). Moreover, mutations in fasII and/or dlg have similar effects on presynaptic ultrastructure, suggesting their functional involvement in a common developmental pathway. DLG can directly mediate a biochemical complex and a macroscopic cluster of FASII and Shaker K+ channels in heterologous cells. These results indicate a central role for DLG in the structural organization and downstream signaling mechanisms of cell adhesion molecules and ion channels at synapses.

    View details for Web of Science ID A1997YD18900007

    View details for PubMedID 9354326

  • Functional expression of rat synapse-associated proteins SAP97 and SAP102 in Drosophila dlg-1 mutants: Effects on tumor suppression and synaptic bouton structure MECHANISMS OF DEVELOPMENT Thomas, U., Phannavong, B., Muller, B., Garner, C. C., Gundelfinger, E. D. 1997; 62 (2): 161-174


    The synapse-associated proteins SAP97 and SAP102 are mammalian proteins that are structurally related to the Drosophila tumor suppressor protein DlgA. Previous analyses revealed that DlgA is essential for the integrity of epithelia and neuromuscular synapses. Here we show that synaptic bouton structure is severely affected in mutant larvae carrying the dlg-1(XI-2) allele. We have tested SAP97 and SAP102 for functional homology to DlgA by heterologous expression in Drosophila. Both SAP97 and SAP102 can suppress tumor formation in dlg-1 mutant flies and mimic DlgA at larval neuromuscular junctions. Neuronal expression of SAP97 or SAP102 is required for morphological restoration of synaptic boutons, indicating that presynaptic DlgA function is essential for establishing structurally intact motor nerve terminals at larval neuromuscular junctions.

    View details for Web of Science ID A1997WU47100005

    View details for PubMedID 9152008

  • Synaptic proteins and the assembly of synaptic junctions TRENDS IN CELL BIOLOGY Garner, C. C., Kindler, S. 1996; 6 (11): 429-433


    Synapses are highly specialized contact sites between neurons and their target cells where information in the form of chemical substances travels from a pre- to a postsynaptic cell. In the central nervous system of mammals, most nerve cells are innervated by functionally distinct types of synapses, each requiring a specific set of molecular constituents for proper function. Various molecular players that may be involved in the assembly of synaptic junctions have been identified recently.

    View details for Web of Science ID A1996VT35300006

    View details for PubMedID 15157514

  • Protein components of a rat brain synaptic junctional protein preparation MOLECULAR BRAIN RESEARCH Langnaese, K., Seidenbecher, C., Wex, H., Seidel, B., Hartung, K., APPELTAUER, U., Garner, A., Voss, B., Mueller, B., Garner, C. C., Gundelfinger, E. D. 1996; 42 (1): 118-122


    Antisera against a rat brain synaptic protein preparation, the postsynaptic density (PSD) fraction, were used to isolate cDNA clones by expression screening of a rat brain cDNA library. About one fifth of more than 200 analyzed cDNAs encoding potential synapse-associated proteins were previously unknown. Identifiable proteins include, among others, components of the pre- and postsynaptic cytoskeleton, synaptic vesicle proteins and several protein kinases and kinase substrates. This demonstrates that both pre- and postsynaptic elements purify with the PSD fraction.

    View details for Web of Science ID A1996VP33400014

    View details for PubMedID 8915587

  • Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux MOLECULAR BRAIN RESEARCH Laube, G., Roper, J., Pitt, J. C., Sewing, S., Kistner, U., Garner, C. C., Pongs, O., Veh, R. W. 1996; 42 (1): 51-61


    The Pinceau is a paintbrush-like network of cerebellar basket cell axon branchlets embracing the initial segment of the Purkinje cell axon. Its electrical activity contributes to the control of the cerebellar cortical output through the Purkinje cell axon by generating an inhibitory field effect. In addition to the structural features of the Pinceau, its repertoire of voltage-gated ion channels is likely to be an important aspect of this function. Therefore, we investigated the fine structural distribution of voltage-activated potassium (Kv1.1, Kv1.2, Kv3.4) and sodium channel proteins in the Pinceau. The ultrastructural localization of potassium channel subunits was compared to the distribution of synapse-associated protein 90 (SAP90), a protein capable to induce in vitro clustering of Kv1 proteins. With an improved preembedding technique including ultrasmall gold particles, silver enhancement and gold toning, we could show that antibodies recognizing Kv1.1, Kv1.2 and SAP90 are predominantly localized to septate-like junctions, which connect the basket cell axonal branchlets. Kv3.4 immunoreactivity is not concentrated in junctional regions but uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma. In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment. The results suggest that Kv1.1 and Kv1.2 form heterooligomeric delayed rectifier type Kv channels, being colocalized to septate-like junctions by interaction with SAP90.

    View details for Web of Science ID A1996VP33400007

    View details for PubMedID 8915580

  • Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102 JOURNAL OF BIOLOGICAL CHEMISTRY Lau, L. F., Mammen, A., Ehlers, M. D., Kindler, S., Chung, W. J., Garner, C. C., Huganir, R. L. 1996; 271 (35): 21622-21628


    Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl--aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.

    View details for Web of Science ID A1996VE47700096

    View details for PubMedID 8702950

  • SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo NEURON Muller, B. M., Kistner, U., Kindler, S., Chung, W. J., Kuhlendahl, S., Fenster, S. D., Lau, L. F., Veh, R. W., Huganir, R. L., Gundelfinger, E. D., Garner, C. C. 1996; 17 (2): 255-265


    Synapse-associated proteins (SAPs) are constituents of the pre- and postsynaptic submembraneous cytomatrix. Here, we present SAP102, a novel 102kDa SAP detected in dendritic shafts and spines of asymmetric type 1 synapses. SAP102 is enriched in preparations of synaptic junctions, where it biochemically behaves as a component of the cortical cytoskeleton. Antibodies directed against NMDA receptors coimmunoprecipitate SAP102 from rat brain synaptosomes. Recombinant proteins containing the carboxy-terminal tail of NMDA receptor subunit NR2B interact with SAP102 from rat brain homogenates. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. These data represent direct evidence that in vivo SAP102 is involved in linking NMDA receptors to the submembraneous cytomatrix associated with postsynaptic densities at excitatory synapses.

    View details for Web of Science ID A1996VE84000008

    View details for PubMedID 8780649

  • Juvenile and mature MAP2 isoforms induce distinct patterns of process outgrowth MOLECULAR BIOLOGY OF THE CELL Leclerc, N., Baas, P. W., Garner, C. C., Kosik, K. S. 1996; 7 (3): 443-455


    Microtubule-associated protein-2 (MAP2) is the most abundant MAP in neurons, where its distribution is restricted to the somatodendritic compartment. This molecule undergoes developmentally regulated alternative splicing, resulting in at least two isoforms, a juvenile isoform (termed MAP2c) and a mature isoform (MAP2), with greatly different molecular masses. Spodoptera frugiperda (Sf9) cell expression of the juvenile versus the mature MAP2 isoform generates two distinct patterns of process outgrowth. The smaller juvenile isoform induces multiple short thin processes. Mature MAP2 tends to induce single processes that are considerably thicker than those processes induced by juvenile MAP2. We found important differences in the variability of spacing between microtubules and the number of microtubules along the processes induced by MAP2c and mature MAP2. MAP2c showed variability with most microtubules spaced as closely as with tau, but some spaced as far apart as with mature MAP2. Over their length, the mature MAP2 processes demonstrate proximo-distal taper, which corresponds to a narrowing of the spacing between microtubules from 90 nm to 40 nm. Moreover, there is a decreased number of microtubules in mature MAP2-induced processes whereas in tau and MAP2-induced processes, the number of microtubules is constant along the length. Based on these observations, we conclude that MAP2 isoforms can serve as architectural elements by establishing specific morphological features of processes and specific arrangements of their microtubules.

    View details for Web of Science ID A1996TZ76100009

    View details for PubMedID 8868472

  • Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix EUROPEAN JOURNAL OF CELL BIOLOGY CASESLANGHOFF, C., Voss, B., Garner, A. M., APPELTAUER, U., Takei, K., Kindler, S., Veh, R. W., DeCamilli, P., Gundelfinger, E. D., Garner, C. C. 1996; 69 (3): 214-223


    In this study, we describe a novel 420 kDa protein, called Piccolo, found at a wide variety of adult rat brain synapses. High protein levels in the cerebellum, the olfactory bulb and the hippocampus were frequently observed to be associated with asymmetric type 1 synapses. Piccolo is selectively enriched in presynaptic terminals, but is not a component of synaptic vesicles (SVs). Immunogold electron microscopy revealed that Piccolo localizes to the amorphous material among SVs at the presynaptic plasma membrane. Biochemical studies showed that it is very tightly bound to this structure. Thus, we speculate that Piccolo is a structural component of the presynaptic cytomatrix which anchors SVs to the presynaptic plasmalemma.

    View details for Web of Science ID A1996UA00600002

    View details for PubMedID 8900486

  • MAP2a, an alternatively spliced variant of microtubule-associated protein 2 JOURNAL OF NEUROCHEMISTRY Chung, W. J., Kindler, S., Seidenbecher, C., Garner, C. C. 1996; 66 (3): 1273-1281


    MAP2, a dendritically localized microtubule-associated protein (MAP), consists of a pair of high molecular mass (280 kDa) polypeptides, MAP2a and MAP2b, and several low molecular mass (70 kDa) proteins called MAP2c. Although MAP2b and MAP2c have been shown to arise via alternative splicing. It was not clear whether MAP2a is also created by alternative splicing or by posttranslational modification. Using epitope peptide mapping, we have demonstrated that an element specific to MAP2a is situated at its N-terminal end. A cDNA clone from an adult rat brain library was found to contain an additional 246 nucleotides situated at the 5' end of the 9-kb MAP2 mRNA. Antibodies generated against the encoded protein sequence recognize specifically MAP2a in rat brain homogenates. Moreover, although MAP2a, like MAP2b, is found in dendrites and cell bodies, its temporal appearance and cell type-specific distribution in rat brain differs from MAP2b.

    View details for Web of Science ID A1996TV78600049

    View details for PubMedID 8769894

  • Molecular characterization of dendritically localized transcripts encoding MAP2 MOLECULAR BRAIN RESEARCH Kindler, S., Muller, R., Chung, W. J., Garner, C. C. 1996; 36 (1): 63-69


    Transcripts encoding high molecular weight (Hwt) isoforms of microtubule-associated protein 2 (MAP2) have been localized in the dendritic compartment of neurons. In contrast, nearly all other neuronal messages, including transcripts encoding low molecular weight (Lwt) MAP2 isoforms, are restricted to cell somas. The mechanisms underlying the dendritic localization of Hwt-MAP2 transcripts are not known. In non-neuronal systems, mRNAs, are localized via signal sequences situated in their 3' untranslated regions (3' UTRs). In this study, we have localized the putative dendritic targeting element (DTE) in Hwt-MAP2 mRNAs by comparing the nucleotide sequences of the somatically localized 6 kb Lwt-MAP2 transcripts with the dendritcally localized 9 kb messages. Our analysis shows that both 6 kb and 9 kb transcripts have identical 3' - and 5'- UTRs, precluding the possibility that the DTE lies in these regions. Within the coding region a single segment that is unique to 9 kb Hwt MAP2 transcripts was identified. These findings suggest that the DTE lies within the 4 kb RNA segment that encodes the projection domain of Hwt-MAP2.

    View details for Web of Science ID A1996TW86900008

    View details for PubMedID 9011766



    Studies on the identification and characterization of constituents of rat brain synaptic junctions have lead to the isolation of cDNA clones encoding segments of alpha-adducin. These and other studies suggest that adducin, a protein involved in promoting the assembly of actin and spectrin filaments at the plasma membrane, may play a role in dynamic assembly-disassembly processes underlying synaptic plasticity. In order to verify that brain alpha-adducin is indeed a constituent of synaptic structures, we have generated monoclonal antibodies against epitopes in the C-terminal region of alpha-adducin and have determined its spatial and sub-cellular distribution in postnatal day-30 rat brain. Alpha-adducin is found to be highly enriched in regions with high synapse densities of the hippocampus, corpus striatum, cerebral cortex and cerebellum. Immuno-electron microscopic analysis of peroxidase stained sections of the hippocampus and the cerebellum revealed that alpha-adducin is localized at distinct sub-cellular structures. In the CA1 and CA3 regions of the hippocampus alpha-adducin immunoreactivity is found in a distinct subset of dendrites and dendritic spines. In the molecular layer of the cerebellum, a distinct fraction of pre-synaptic terminals of parallel fiber terminals is labeled. In both cases the majority of synaptic structures does not contain adducin. Significant immunoreactivity is also detected in processes of glial cells both in the hippocampus and the cerebellum.

    View details for Web of Science ID A1995TH55100002

    View details for PubMedID 8624703



    cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.

    View details for Web of Science ID A1995TE58300078

    View details for PubMedID 7592978



    Synapses are highly specialized sites of cell-cell contact involved in signal transfer. The molecular mechanisms modulating the assembly and stability of synapses are unknown. We previously reported the identification of a 90 kDa synapse-associated protein, SAP90, that is localized at the presynaptic termini of inhibitory GABAergic synapses. SAP90 is a mosaic protein composed of three 90 amino acid residue repeats, an SH3 domain and a region homologous to guanylate kinases. SAP90 shares domain specific homology with a family of proteins involved in the assembly and possibly stability of sites of cell contact. These include the product of the lethal(1) discs-large-1 (dlgA) tumor suppressor gene and the zonula occludens proteins ZO-1, ZO-2. The further characterization of cDNA clones encoding components of synaptic junctions has lead to the identification of a 97 kDa protein, called SAP97, that exhibits a strong overall sequence similarity to SAP90. The present study was undertaken to determine the spatial distribution of SAP97, and to reveal further clues to the possible roles of these proteins in synapses. Light and immunoelectron microscopic analysis of the rat hippocampal formation revealed that SAP97 is localized in the presynaptic nerve termini of excitatory synapses. In other brain regions, SAP97 is found in and along bundles of unmyelinated axons. SAP97 is not restricted to the CNS, but is also present at the basal lateral membrane between a variety of epithelial cells. In cultured T84 cells, it is restricted to the cytoplasmic surface of the plasma membranes between adjacent cells, but not at the edges of cells lacking cell-cell contact suggesting a role for SAP97 in cell adhesion. These data suggest that members of the SAP90/SAP97 subfamily may be involved in the site specific assembly, stability or functions of membrane specialization at sites of cell-cell contact.

    View details for Web of Science ID A1995QM46800023

    View details for PubMedID 7891172

  • NUCLEOTIDE-BINDING BY THE SYNAPSE ASSOCIATED PROTEIN SAP90 FEBS LETTERS Kistner, U., Garner, C. C., Linial, M. 1995; 359 (2-3): 159-163


    The rat synapse associated protein SAP90 is a member of a superfamily of potential guanylate kinases localized at cell-cell contact sites. This superfamily includes the synapse associated protein SAP97, a close relative of SAP90, the Drosophila tumor suppressor gene product dlg-Ap, the mammalian zonula occludens proteins ZO-1 and ZO-2 and the erythrocyte protein p55. Here we show that SAP90 specifically binds GMP in the micromolar range while binding to ATP, GDP and ADP is at a much lower affinity (10-25 mM), whether or not binding is detected for other guanine and adenine nucleotides. No guanylate kinase activity of SAP90 was detected under our experimental conditions. The importance of the GMP binding capacity per se and an evolutionary role for conserving of the guanylate kinase domain in this superfamily are discussed.

    View details for Web of Science ID A1995QH11200015

    View details for PubMedID 7867790



    The apolipoprotein E type 4 allele is a susceptibility gene for late-onset Alzheimer's disease. Apolipoprotein E is found in neurons, some of which contain paired helical filaments made of the microtubule-associated protein tau. Previous studies have demonstrated that the apoE3 isoform, but not the apoE4 isoform, binds tau with high avidity. Because the microtubule-associated protein MAP2c also effects microtubule assembly and stability, we examined interactions between apoE isoforms and MAP2c. Similar to the tau-binding results, apoE3, but not apoE4, bound MAP2c. Binding was detectable down to 10(-9) M MAP2c and 10(-8) M apoE3. Isoform-specific interactions of apoE with the microtubule-associated proteins MAP2c and tau might affect intracellular maintenance of microtubules and could contribute to a time-dependent pathogenesis of Alzheimer's disease.

    View details for Web of Science ID A1994PY09300015

    View details for PubMedID 7891887

  • 4 REPEAT MAP2 ISOFORMS IN HUMAN AND RAT-BRAIN MOLECULAR BRAIN RESEARCH Kindler, S., Garner, C. C. 1994; 26 (1-2): 218-224


    In mammalian brain, variations in the primary structure of the characterized microtubule-associated protein 2 (MAP2) isoforms have only been observed in their projection domains whose length determines the spacing between neighboring microtubules. We now report that, as with MAP4 and tau, MAP2 isoforms containing four (4R) instead of three (3R) tandem repeats in their microtubule binding domains do exist in human and rat brain. The additional sequence, inserted between the first and second repeat of the 3R-MAP2 messages, appears on mRNAs encoding both high and low molecular weight (Hwt and Lwt) rat MAP2 variants. In contrast to the corresponding 3R-messages, 4R-Hwt MAP2 concentrations decrease during early postnatal rat brain development, while the amount of 4R-Lwt MAP2 messages remains constant. In general, 4R-/3R-MAP2 mRNA ratios appear to be low with the highest levels of 4R-messages found in the cerebellum.

    View details for Web of Science ID A1994PL65700027

    View details for PubMedID 7854050



    A novel synapse-associated protein, SAP90, accumulates around the axon hillock of Purkinje cells in rat cerebellum. By immuno-electron microscopy, SAP90 has been localized to the presynaptic termini of basket cells forming inhibitory, gamma-aminobutyric acid (GABA)ergic synapses onto Purkinje cell axon hillocks. The amino acid sequence for SAP90 has been deduced from the nucleotide sequence of a series of overlapping cDNA clones. SAP90 is related to the gene product encoded by the Drosophila tumor suppressor gene dlg-A. SAP90 and the dlg-A product share an overall sequence identity of 54%. Three distinct domains can be identified: (i) a potential cytoskeletal region consisting of three repeats of 90 amino acids in length, (ii) a domain with similarity to SH3, a putative regulatory motif found in the src family of non-receptor protein tyrosine kinases and several proteins associated with the cortical cytoskeleton, and (iii) a carboxyl-terminal domain homologous to yeast guanylate kinase. These features suggest a possible role for SAP90 in a guanine nucleotide-mediated signal transduction pathway at a subset of GABAergic synapses in the rat cerebellum.

    View details for Web of Science ID A1993KP88400004

    View details for PubMedID 7680343

  • MICROTUBULE-ASSOCIATED PROTEIN-1A AND PROTEIN-LC2 - 2 PROTEINS ENCODED IN ONE MESSENGER-RNA JOURNAL OF BIOLOGICAL CHEMISTRY Langkopf, A., Hammarback, J. A., Muller, R., Vallee, R. B., Garner, C. C. 1992; 267 (23): 16561-16566


    The deduced amino acid sequence for the filamentous microtubule-associated protein (MAP) 1A, thought to be involved in stabilizing the mature neuronal cytoskeleton, has been determined from a series of overlapping cDNA clones. Though previously described as biochemically and immunologically distinct from MAP1B, we now demonstrate that MAP1A is structurally related to MAP1B, a protein associated with neurite outgrowth and process plasticity. The two MAPs exhibit regional amino acid sequence similarities spanning their potential microtubule binding domains placing both into a new MAP family. The cDNA sequence encoding MAP1A was also found to encode one of its associated light chains (LC) called LC2. Both proteins are found on a single mRNA in the same open reading frame and are translated as a pre-MAP1A/LC2-protein. The topological relationship between MAP1A and LC2 coding sequences is, therefore, identical to that previously shown for MAP1B and LC1 (Hammarback, J. A., Obar, R. A., Hughes, S. M., and Vallee, R. B. (1991) Neuron 7, 129-139). Based on these and earlier results, we conclude that LC1 and LC2 are structurally related polypeptides generated from distinct MAP polyprotein precursors but free to exchange between the two MAPs.

    View details for Web of Science ID A1992JJ45800088

    View details for PubMedID 1379599



    Tau and MAP2 are two of the major microtubule-associated proteins in the vertebrate nervous system. They promote microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. In nerve cells immunohistochemistry shows complementary distributions, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites. Each protein consists of multiple isoforms that contain three or four homologous tandem repeats near the carboxy-terminus, which constitute microtubule-binding domains. In humans, tau consists of at least six isoforms of related amino acid sequences that are produced from a single gene by alternative mRNA splicing and that are expressed in a stage- and cell type-specific manner. Tau is also a component of the paired helical filaments associated with Alzheimer's disease and other disorders of the CNS. Rat MAP2 consists of at least three isoforms produced from a single gene: high molecular mass MAP2a and MAP2b, and low molecular mass MAP2c. MAP2c is expressed only during early development and has so far been seen only in axons; MAP2a appears to replace MAP2c, whereas MAP2b is expressed throughout life. Messenger RNAs for MAP2 of high molecular mass are expressed both in cell bodies and in dendrites, consistent with the dendritic localization of the corresponding protein isoforms.

    View details for Web of Science ID A1991FJ49900010

    View details for PubMedID 1713721



    Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.

    View details for Web of Science ID A1990EJ18800049

    View details for PubMedID 2174050



    cDNA clones encoding microtubule-associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10-kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11-kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse-hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap-1, and the MAP5 gene to chromosome 13, designated Mtap-5. MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.

    View details for Web of Science ID A1990DK37600020

    View details for PubMedID 2355215


    View details for Web of Science ID A1990DD51000040

    View details for PubMedID 2339070



    The most prominent microtubule-associated protein of the neuronal cytoskeleton is MAP2. In the brain it exists as a pair of high-molecular weight proteins, MAP2a and MAP2b, and a smaller form, MAP2c, which is particularly abundant in the developing brain. High-molecular weight MAP2 is expressed in dendrites, where its messenger RNA is also located, but is not found in axons; it has been shown to be present in fine filaments that crosslink dendritic microtubules. This correlates with the primary structure of high-molecular weight MAP2, which consists of a short carboxy-terminal tubulin-binding domain and a long amino-terminal arm, which forms a filamentous sidearm on reconstituted microtubules. Here we report that the high- and low-molecular weight forms of MAP2 are generated by alternative splicing and share the entire C-terminal tubulin-binding domain as well as a short N-terminal sequence. In contrast to high molecular weight MAP2, embryonic brain MAP2c lacks 1,342 amino acids from the filamentous sidearm domain. Furthermore, the mRNA for low molecular weight MAP2c is not present in dendrites, indicating that the dendritic targeting signal is specific for the high-molecular weight form.

    View details for Web of Science ID A1989AM35700059

    View details for PubMedID 2770869



    We have used cDNA probes specific for three of the major brain microtubule-associated proteins (MAPs), MAP1, MAP2, and MAP5, to study the timing of appearance, relative abundance, and intracellular compartmentalization of MAP gene transcripts in developing rat brain. The MAP1 probe hybridizes throughout the brain, in both grey and white matter. MAP2 mRNA is detected only in grey matter and appears in cerebral neurons only after they have ceased dividing and have migrated to the cortical plate. The MAP5 cDNA hybridizes throughout the embryonic brain, but by P12, MAP5 mRNA distribution is restricted to relatively immature areas. MAP2 mRNA, found in dendrites in the developing brain, persists in some adult dendrites. MAP5 mRNA, like beta-tubulin mRNA, is found only in the cell bodies of developing neurons, indicating that the protein must be transported from the soma into processes. MAP1 mRNA is found only in the proximal regions of cortical pyramidal cell dendrites in both developing and adult brain. The diverse distributions of MAP gene transcripts emphasize the importance of these proteins in generating heterogeneity of microtubule function and indicate that MAP compartmentalization within neurons is regulated in part by differential mRNA transport.

    View details for Web of Science ID A1989T802100006

    View details for PubMedID 2624748



    Monoclonal antibodies were used to explore the relationship between two similarly sized microtubule-associated proteins (MAPs), MAP1x and MAP5. Although the proteins detected by anti-MAP1x and anti-MAP5 co-migrate in SDS-polyacrylamide gels, the patterns of antigenic proteolytic fragments (epitope maps) derived from them were completely different. The results suggest either that MAP1x is more stable than MAP5 or that the MAP1x epitope is situated close to one end of the molecule and gives rise to a very short proteolytic fragment. Immunoprecipitation from brain supernatants with either antibody brought down protein that cross-reacted with the other antibody, indicating that individual molecules bearing both epitopes exist in brain. Peptide maps of the proteins immunoprecipitated with the two antibodies showed that they are closely similar. Despite these similarities, the two antibodies gave different staining patterns on sections of developing rat brain, anti-MAP5 staining both axons and dendrites whereas anti-MAP1x stained only axons. We conclude that the MAP5 and MAP1x molecules are very similar, and possibly identical. The difference in staining patterns with the two antibodies could be because there are two proteins present in brain, one in immature axons bearing both the MAP5 and MAP1x epitopes and another with a wider distribution bearing only the MAP5 epitope. Alternatively, there may be a single protein bearing both epitopes, with the MAP1x epitope being masked in neuronal dendrites and mature axons by covalent modification or inter-molecular binding.

    View details for Web of Science ID A1989R602400010

    View details for PubMedID 2927285



    For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.

    View details for Web of Science ID A1988R363900057

    View details for PubMedID 3200318



    We describe a method for isolating multiple cDNA clones coding for a set of related proteins from bacteriophage lambda expression libraries in a single screening with polyclonal antiserum. The antiserum is raised against a tissue sub-fraction containing the proteins of interest; in the example presented this was brain microtubules. Each antibody-positive clone from the lambda expression library is plaque-purified and then grown in contact with nitrocellulose membrane that becomes coated with protein synthesized from the cloned cDNA. Each filter, containing the protein produced by a single lambda cDNA clone, is then used to affinity-select clone-specific antibodies from the original polyclonal antiserum. The monospecific antibody for each cDNA clone can be used on Western blots to identify the protein that the cDNA encodes and also to stain tissue sections. Using this method we have, in a single screening, obtained: (1) multiple cDNA clones representing different regions of a single large protein, (2) cDNA clones representing several functionally related proteins (microtubule-associated proteins), and (3) cDNA clones related to a novel protein species for which neither biochemical nor immunological data have previously been available.

    View details for Web of Science ID A1988R610900026

    View details for PubMedID 2465208



    Brain microtubule-associated protein 2 (MAP2) consists of a pair of high molecular mass (280 kD) polypeptides, MAP2a and MAP2b, and a recently identified 70-kD protein, MAP2c, which is antigenically related to these high molecular mass MAP2's. Using cDNA clones we have analyzed the expression of these three proteins at the nucleic acid level. cDNA probes selective for the high molecular mass MAP2's a and b identified only a 9-kb mRNA, whereas a probe for sequence common to all three MAP2 isoforms, a, b, and c, recognized the 9-kb transcript and additionally a 6-kb mRNA. Southern blot analysis with cDNA probes indicated that there is only one MAP2 gene from which these two distinct mRNAs are derived. The 70-kD MAP2c protein is much more abundant in neurons of developing brain than those of adult tissues. Similarly the expression of the 6-kb MAP2c-related mRNA, is much greater in neonatal than adult rat brain, indicating that the developmental expression of MAP2 is determined by transcriptional regulation from a single MAP2 gene.

    View details for Web of Science ID A1988M615800025

    View details for PubMedID 3346325



    Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the tau MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.

    View details for Web of Science ID A1988L635500038

    View details for PubMedID 3121794



    The Escherichia coli aroF and aroG genes encode the tyrosine-sensitive and the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, respectively, two of the three isoenzymes that control carbon flow through the shikimate pathway. Transcription of aroF and aroG is repressed by the tyrR gene product complexed to tyrosine or phenylalanine, respectively. Constitutive aroF mutants with lesions linked to aroF were isolated. The nucleotide sequences in the regulatory regions of aroF from six such mutants and from the parental wild type strain were determined. The mutations were found in two 18-base pair imperfect palindromes, called aroFo1 and aroFo2, which are located upstream of the aroF transcription start by 61 and 113 base pairs, respectively. Nuclease S1 mapping and analysis of in vitro run-off transcripts identified the 5'-end of the aroF transcript 51 base pairs upstream of the aroF translation start. The -35 region of the aroF promoter overlaps aroFo1. The aroFo1 and aroFo2 sequences are homologous to a single 18-base pair DNA segment preceding the coding sequence of aroG.

    View details for Web of Science ID A1985ADZ0700090

    View details for PubMedID 2857723



    The translated sequence of aroF, the first structural gene of the tyrosine operon of Escherichia coli, has been determined. The 1068 nucleotides encode the 356 amino acids that form the subunit of the dimeric tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. The primary structure of this enzyme has been confirmed by automated Edman degradation of peptide fragments produced by cleavage with cyanogen bromide, limited trypsin digestion, Staphylococcus aureus strain V8 protease, or mild acid hydrolysis. The amino acid sequence of this enzyme is compared with the sequence of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, deduced from the aroG DNA sequence (Davies, W. D., and Davidson, B. E. (1982) Nucleic Acids Res. 10, 4045-4058).

    View details for Web of Science ID A1984TD64900050

    View details for PubMedID 6146618


    View details for Web of Science ID A1984TT99800011

    View details for PubMedID 6488212