G2Cdb::Gene report

Gene id
Gene symbol
Dlg4 (MGI)
Mus musculus
discs, large homolog 4 (Drosophila)
G00000029 (Homo sapiens)

Databases (10)

Curated Gene
OTTMUSG00000006024 (Vega mouse gene)
ENSMUSG00000020886 (Ensembl mouse gene)
13385 (Entrez Gene)
10 (G2Cdb plasticity & disease)
Gene Expression
NM_007864 (Allen Brain Atlas)
13385 (Genepaint)
dlg4 (gensat)
602887 (OMIM)
Marker Symbol
MGI:1277959 (MGI)
Protein Sequence
Q62108 (UniProt)

Synonyms (5)

  • Dlgh4
  • PSD-95
  • PSD95
  • SAP90
  • SAP90A

Alleles (7)

Allele Name Type Description Literature
A00000024 PSD95 GK truncation Dlg4 (PSD-95) guanylate kinase domain deletion  
A00000025 PSD95 ko knockout Dlg4 (PSD-95) knockout  

Literature (143)

Pubmed - g2c

  • Association of mouse Dlg4 (PSD-95) gene deletion and human DLG4 gene variation with phenotypes relevant to autism spectrum disorders and Williams' syndrome.

    Feyder M, Karlsson RM, Mathur P, Lyman M, Bock R, Momenan R, Munasinghe J, Scattoni ML, Ihne J, Camp M, Graybeal C, Strathdee D, Begg A, Alvarez VA, Kirsch P, Rietschel M, Cichon S, Walter H, Meyer-Lindenberg A, Grant SG and Holmes A

    Section on Behavioral Science and Genetics, Laboratory for Integrative Neuroscience, National Institute on Alcoholism and Alcohol Abuse, Rockville, MD 20852-9411, USA. michael.feyder@ki.se

    Objective: Research is increasingly linking autism spectrum disorders and other neurodevelopmental disorders to synaptic abnormalities ("synaptopathies"). PSD-95 (postsynaptic density-95, DLG4) orchestrates protein-protein interactions at excitatory synapses and is a major functional bridge interconnecting a neurexinneuroligin-SHANK pathway implicated in autism spectrum disorders.

    Method: The authors characterized behavioral, dendritic, and molecular phenotypic abnormalities relevant to autism spectrum disorders in mice with PSD-95 deletion (Dlg4⁻(/)⁻). The data from mice led to the identification of single-nucleotide polymorphisms (SNPs) in human DLG4 and the examination of associations between these variants and neural signatures of Williams' syndrome in a normal population, using functional and structural neuroimaging.

    Results: Dlg4⁻(/)⁻ showed increased repetitive behaviors, abnormal communication and social behaviors, impaired motor coordination, and increased stress reactivity and anxiety-related responses. Dlg4⁻(/)⁻ had subtle dysmorphology of amygdala dendritic spines and altered forebrain expression of various synaptic genes, including Cyln2, which regulates cytoskeletal dynamics and is a candidate gene for Williams' syndrome. A signifi-cant association was observed between variations in two human DLG4 SNPs and reduced intraparietal sulcus volume and abnormal cortico-amygdala coupling, both of which characterize Williams' syndrome.

    Conclusions: These findings demonstrate that DLG4 gene disruption in mice produces a complex range of behavioral and molecular abnormalities relevant to autism spectrum disorders and Williams' syndrome. The study provides an initial link between human DLG4 gene variation and key neural endophenotypes of Williams' syndrome and perhaps corticoamygdala regulation of emotional and social processes more generally.

    Funded by: Intramural NIH HHS: ZIA AA000411-07, ZIA AA000421-02, ZIA AA000421-03; Wellcome Trust

    The American journal of psychiatry 2010;167;12;1508-17

  • Neuroligin 1 is dynamically exchanged at postsynaptic sites.

    Schapitz IU, Behrend B, Pechmann Y, Lappe-Siefke C, Kneussel SJ, Wallace KE, Stempel AV, Buck F, Grant SG, Schweizer M, Schmitz D, Schwarz JR, Holzbaur EL and Kneussel M

    Center for Molecular Neurobiology (ZMNH), University of Hamburg Medical School, D-20251 Hamburg, Germany.

    Neuroligins are postsynaptic cell adhesion molecules that associate with presynaptic neurexins. Both factors form a transsynaptic connection, mediate signaling across the synapse, specify synaptic functions, and play a role in synapse formation. Neuroligin dysfunction impairs synaptic transmission, disrupts neuronal networks, and is thought to participate in cognitive diseases. Here we report that chemical treatment designed to induce long-term potentiation or long-term depression (LTD) induces neuroligin 1/3 turnover, leading to either increased or decreased surface membrane protein levels, respectively. Despite its structural role at a crucial transsynaptic position, GFP-neuroligin 1 leaves synapses in hippocampal neurons over time with chemical LTD-induced neuroligin internalization depending on an intact microtubule cytoskeleton. Accordingly, neuroligin 1 and its binding partner postsynaptic density protein-95 (PSD-95) associate with components of the dynein motor complex and undergo retrograde cotransport with a dynein subunit. Transgenic depletion of dynein function in mice causes postsynaptic NLG1/3 and PSD-95 enrichment. In parallel, PSD lengths and spine head sizes are significantly increased, a phenotype similar to that observed upon transgenic overexpression of NLG1 (Dahlhaus et al., 2010). Moreover, application of a competitive PSD-95 peptide and neuroligin 1 C-terminal mutagenesis each specifically alter neuroligin 1 surface membrane expression and interfere with its internalization. Our data suggest the concept that synaptic plasticity regulates neuroligin turnover through active cytoskeleton transport.

    Funded by: NINDS NIH HHS: NS060698, R01 NS060698, R01 NS060698-04

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;38;12733-44

  • In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93.

    Delint-Ramirez I, Fernández E, Bayés A, Kicsi E, Komiyama NH and Grant SG

    Genes to Cognition Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom.

    Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known whether the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CaMKIIalpha and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knock-outs of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A-containing NMDA receptor complexes to rafts. Deletion of the NR2A C terminus or the C-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.

    Funded by: Wellcome Trust: 066717

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;24;8162-70

  • The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment.

    Arbuckle MI, Komiyama NH, Delaney A, Coba M, Garry EM, Rosie R, Allchorne AJ, Forsyth LH, Bence M, Carlisle HJ, O'Dell TJ, Mitchell R, Fleetwood-Walker SM and Grant SG

    Centre for Neuroregeneration, The University of Edinburgh, Institute of Immunology and Infection, Ashworth Buildings, Kings Buildings, Edinburgh EH9 3JT, UK.

    Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

    Funded by: Medical Research Council; Wellcome Trust

    EMBO reports 2010;11;6;473-8

  • PSD-95 is essential for hallucinogen and atypical antipsychotic drug actions at serotonin receptors.

    Abbas AI, Yadav PN, Yao WD, Arbuckle MI, Grant SG, Caron MG and Roth BL

    Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

    Here, we report that postsynaptic density protein of 95 kDa (PSD-95), a postsynaptic density scaffolding protein, classically conceptualized as being essential for the regulation of ionotropic glutamatergic signaling at the postsynaptic membrane, plays an unanticipated and essential role in mediating the actions of hallucinogens and atypical antipsychotic drugs at 5-HT(2A) and 5-HT(2C) serotonergic G-protein-coupled receptors. We show that PSD-95 is crucial for normal 5-HT(2A) and 5-HT(2C) expression in vivo and that PSD-95 maintains normal receptor expression by promoting apical dendritic targeting and stabilizing receptor turnover in vivo. Significantly, 5-HT(2A)- and 5-HT(2C)-mediated downstream signaling is impaired in PSD-95(null) mice, and the 5-HT(2A)-mediated head-twitch response is abnormal. Furthermore, the ability of 5-HT(2A) inverse agonists to normalize behavioral changes induced by glutamate receptor antagonists is abolished in the absence of PSD-95 in vivo. These results demonstrate that PSD-95, in addition to the well known role it plays in scaffolding macromolecular glutamatergic signaling complexes, profoundly modulates metabotropic 5-HT(2A) and 5-HT(2C) receptor function.

    Funded by: NCRR NIH HHS: K26 RR000168, P51 RR000168, RR00168; NIDA NIH HHS: DA021420, R01 DA021420; NIGMS NIH HHS: T32 GM007250; NIMH NIH HHS: MH-73853, MH61887, R01 MH061887, R01 MH061887-07, R01 MH061887-08, R01 MH061887-09, R01 MH061887-10, R01 MH073853, R37 MH073853, U19 MH082441, U19 MH082441-01, U19 MH082441-010001, U19 MH082441-019003, U19 MH082441-02, U19 MH082441-020001, U19MH82441; NINDS NIH HHS: NS-19576, NS057311, R01 NS019576, R21 NS057311; Wellcome Trust

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;22;7124-36

  • PSD-95 uncouples dopamine-glutamate interaction in the D1/PSD-95/NMDA receptor complex.

    Zhang J, Xu TX, Hallett PJ, Watanabe M, Grant SG, Isacson O and Yao WD

    New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772, USA.

    Classical dopaminergic signaling paradigms and emerging studies on direct physical interactions between the D(1) dopamine (DA) receptor and the NMDA glutamate receptor predict a reciprocally facilitating, positive feedback loop. This loop, if not controlled, may cause concomitant overactivation of both D(1) and NMDA receptors, triggering neurotoxicity. Endogenous protective mechanisms must exist. Here, we report that PSD-95, a prototypical structural and signaling scaffold in the postsynaptic density, inhibits D(1)-NMDA receptor subunit 1 (NR1) NMDA receptor association and uncouples NMDA receptor-dependent enhancement of D(1) signaling. This uncoupling is achieved, at least in part, via a disinhibition mechanism by which PSD-95 abolishes NMDA receptor-dependent inhibition of D(1) internalization. Knockdown of PSD-95 immobilizes D(1) receptors on the cell surface and escalates NMDA receptor-dependent D(1) cAMP signaling in neurons. Thus, in addition to its role in receptor stabilization and synaptic plasticity, PSD-95 acts as a brake on the D(1)-NMDA receptor complex and dampens the interaction between them.

    Funded by: NCRR NIH HHS: K26 RR000168, P51 RR000168, RR00168; NIDA NIH HHS: DA021420, R01 DA021420; NINDS NIH HHS: NS057311, NS39793, P50 NS039793, P50 NS039793-09, R21 NS057311

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;9;2948-60

  • Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.

    Fernández E, Collins MO, Uren RT, Kopanitsa MV, Komiyama NH, Croning MD, Zografos L, Armstrong JD, Choudhary JS and Grant SG

    Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Cambridge, UK.

    The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K+ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage-dependent K+ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.

    Funded by: Wellcome Trust

    Molecular systems biology 2009;5;269

  • Opposing effects of PSD-93 and PSD-95 on long-term potentiation and spike timing-dependent plasticity.

    Carlisle HJ, Fink AE, Grant SG and O'Dell TJ

    Department of Physiology, David Geffen School of Medicine at UCLA, 53-231 Center for the Health Sciences, Box 951751, Los Angeles, CA 90095-1751, USA.

    The membrane-associated guanylate kinases (MAGUKs) PSD-95, PSD-93 and SAP102 are thought to have crucial roles in both AMPA receptor trafficking and formation of NMDA receptor-associated signalling complexes involved in synaptic plasticity. While PSD-95, PSD-93, and SAP102 appear to have similar roles in AMPA receptor trafficking, it is not known whether these MAGUKs also have functionally similar roles in synaptic plasticity. To explore this issue we examined several properties of basal synaptic transmission in the hippocampal CA1 region of PSD-93 and PSD-95 mutant mice and compared the ability of a number of different synaptic stimulation protocols to induce long-term potentiation (LTP) and long-term depression (LTD) in these mutants. We find that while both AMPA and NMDA receptor-mediated synaptic transmission are normal in PSD-93 mutants, PSD-95 mutant mice exhibit clear deficits in AMPA receptor-mediated transmission. Moreover, in contrast to the facilitation of LTP induction and disruption of LTD observed in PSD-95 mutant mice, PSD-93 mutant mice exhibit deficits in LTP and normal LTD. Our results suggest that PSD-95 has a unique role in AMPA receptor trafficking at excitatory synapses in the hippocampus of adult mice and indicate that PSD-93 and PSD-95 have essentially opposite roles in LTP, perhaps because these MAGUKs form distinct NMDA receptor signalling complexes that differentially regulate the induction of LTP by different patterns of synaptic activity.

    Funded by: NIMH NIH HHS: MH609197; Wellcome Trust

    The Journal of physiology 2008;586;24;5885-900

  • alpha-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus.

    Park CS, Elgersma Y, Grant SG and Morrison JH

    Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA. charles.park01@mssm.edu

    N-methyl-d-aspartate receptors (NMDARs) are critical determinants of bidirectional synaptic plasticity, however, studies of NMDAR function have been based primarily on pharmacological and electrophysiological manipulations, and it is still debated whether there are subunit-selective forms of long-term potentiation (LTP) and long-term depression (LTD). Here we provide ultrastructural analyses of axospinous synapses in cornu ammonis field 1 of hippocampus (CA1) stratum radiatum of transgenic mice with mutations to two key underlying postsynaptic density (PSD) proteins, postsynaptic density protein 95 (PSD-95) and the alpha-isoform of calcium-calmodulin-dependent protein kinase II (alphaCaMKII). Distribution profiles of synaptic proteins in these mice reveal very different patterns of subunit-specific NMDAR localization, which may be related to the divergent phenotypes of the two mutants. In PSD-95, Dlg, ZO-1/Dlg-homologous region (PDZ) 3-truncated mutant mice in which LTD could not be induced but LTP was found to be enhanced, we found a subtle, yet preferential displacement of synaptic N-methyl-d-aspartate receptor subunit 2B (NR2B) subunits in lateral regions of the synapse without affecting changes in the localization of N-methyl-d-aspartate receptor subunit 2A (NR2A) subunits. In persistent inhibitory alphaCaMKII Thr305 substituted with Asp in alpha-isoform of calcium-calmodulin kinase II (T305D) mutant mice with severely impaired LTP but stable LTD expression, we found a selective reduction of NR2A subunits at both the synapse and throughout the cytoplasm of the spine without any effect on the NR2B subunit. In an experiment of mutual exclusivity, neither PSD-95 nor alphaCaMKII localization was found to be affected by mutations to the corresponding PSD protein suggesting that they are functionally independent of the other in the regulation of NR2A- and NR2B-containing NMDARs preceding synaptic activity. Consequently, there may exist at least two distinct PSD-95 and alphaCaMKII-specific NMDAR complexes involved in mediating LTP and LTD through opposing signal transduction pathways in synapses of the hippocampus. The contrasting phenotypes of the PSD-95 and alphaCaMKII mutant mice further establish the prospect of an independent and, possibly, competing mechanism for the regulation of NMDAR-dependent bidirectional synaptic plasticity.

    Funded by: NIA NIH HHS: AG06647, R01 AG006647, R37 AG006647, R37 AG006647-20; Wellcome Trust

    Neuroscience 2008;151;1;43-55

  • Clustered gene expression changes flank targeted gene loci in knockout mice.

    Valor LM and Grant SG

    Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Cambridge, United Kingdom.

    Background: Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.

    Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.

    The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice.

    Funded by: Wellcome Trust

    PloS one 2007;2;12;e1303

  • Differential expression of two NMDA receptor interacting proteins, PSD-95 and SynGAP during mouse development.

    Porter K, Komiyama NH, Vitalis T, Kind PC and Grant SG

    Centre for Neuroscience Research, University of Edinburgh, Edinburgh UK.

    Patterns of neural activity mediated by N-methyl-D-aspartate (NMDA) receptors are known to play important roles in development of the central nervous system. However, the signalling pathways downstream from NMDA receptors that are critical for normal neuronal development are not yet clearly understood. NMDA receptors interact with various signalling proteins via scaffolding proteins, which are important in adult neuronal and behavioural plasticity. For example, the NR2B subunits of the NMDA receptor interact with postsynaptic density 95 (PSD-95), which in turn binds to synaptic ras GTPase-activating protein (SynGAP). Interestingly, the developmental phenotype of mice carrying null mutations in these genes differ. NR2B and SynGAP homozygote mice die within the first week of birth whereas PSD-95 homozygote mice survive to adulthood. We therefore examined the expression patterns of PSD-95 and SynGAP genes from embryonic stages to adult using lacZ (beta-galactosidase) marker gene knock-in mice. Dramatic changes of expression were observed throughout development in brain and other tissues. Although SynGAP binds PSD-95, both genes had distinct, as well as overlapping expression. SynGAP expression peaked at times of synaptogenesis and developmental plasticity in contrast to PSD-95, which was expressed throughout the brain from early embryonic stages. Furthermore, SynGAP showed a more spatially restricted pattern as illustrated by its restriction to forebrain in contrast to PSD-95, which was also found in mid- and hindbrain. These data support the model that synaptic signalling complexes are heterogeneous and individual components show temporal and spatial specificity during development.

    The European journal of neuroscience 2005;21;2;351-62

  • Separable features of visual cortical plasticity revealed by N-methyl-D-aspartate receptor 2A signaling.

    Fagiolini M, Katagiri H, Miyamoto H, Mori H, Grant SG, Mishina M and Hensch TK

    Neuronal Circuit Development, Institute of Physical and Chemical Research, RIKEN, Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

    How individual receptive field properties are formed in the maturing sensory neocortex remains largely unknown. The shortening of N-methyl-d-aspartate (NMDA) receptor currents by 2A subunit (NR2A) insertion has been proposed to delimit the critical period for experience-dependent refinement of circuits in visual cortex. In mice engineered to maintain prolonged NMDA responses by targeted deletion of NR2A, the sensitivity to monocular deprivation was surprisingly weakened but restricted to the typical critical period and delayed normally by dark rearing from birth. Orientation preference instead failed to mature, occluding further effects of dark rearing. Interestingly, a full ocular dominance plasticity (but not orientation bias) was selectively restored by enhanced inhibition, reflecting an imbalanced excitation in the absence of NR2A. Many of the downstream pathways involved in NMDA signaling are coupled to the receptor through a variety of protein-protein interactions and adaptor molecules. To further investigate a mechanistic dissociation of receptive field properties in the developing visual system, mice carrying a targeted disruption of the NR2A-associated 95-kDa postsynaptic density (PSD95) scaffolding protein were analyzed. Although the development and plasticity of ocular dominance was unaffected, orientation preference again failed to mature in these mice. Taken together, our results demonstrate that the cellular basis generating individual sensory response properties is separable in the developing neocortex.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;5;2854-9

  • SynGAP regulates ERK/MAPK signaling, synaptic plasticity, and learning in the complex with postsynaptic density 95 and NMDA receptor.

    Komiyama NH, Watabe AM, Carlisle HJ, Porter K, Charlesworth P, Monti J, Strathdee DJ, O'Carroll CM, Martin SJ, Morris RG, O'Dell TJ and Grant SG

    Division of Neuroscience, University of Edinburgh, Edinburgh EH8-9JZ, United Kingdom.

    At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(-/+)). In SynGAP(-/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(-/+) mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(-/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(-/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(-/+)/H-Ras(-)/- double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;22;9721-32

Pubmed - other

  • ApoE receptor 2 regulates synapse and dendritic spine formation.

    Dumanis SB, Cha HJ, Song JM, Trotter JH, Spitzer M, Lee JY, Weeber EJ, Turner RS, Pak DT, Rebeck GW and Hoe HS

    Department of Neuroscience, Georgetown University Medical Center, Washington, DC, United States of America.

    Background: Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation-processes critical for learning and memory.

    In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.

    These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.

    Funded by: NIA NIH HHS: AG026478, AG032330, AG032330-02S1, AG034253, P01 AG030128, R01 AG026478, R03 AG032330, R03 AG034253, R03 AG034253-02; NINDS NIH HHS: NS048085, R01 NS048085, R01 NS048085-05

    PloS one 2011;6;2;e17203

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95.

    Zhou L, Li F, Xu HB, Luo CX, Wu HY, Zhu MM, Lu W, Ji X, Zhou QG and Zhu DY

    Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing, China.

    Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS-PSD-95 interaction via overexpressing the N-terminal amino acid residues 1-133 of nNOS (nNOS-N(1-133)) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS-PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.

    Nature medicine 2010;16;12;1439-43

  • N-methyl-D-aspartate receptor subunit- and neuronal-type dependence of excitotoxic signaling through post-synaptic density 95.

    Fan J, Vasuta OC, Zhang LY, Wang L, George A and Raymond LA

    Graduate Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada.

    NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.

    Funded by: Canadian Institutes of Health Research: MOP-102517, MOP-129029

    Journal of neurochemistry 2010;115;4;1045-56

  • RNG105 deficiency impairs the dendritic localization of mRNAs for Na+/K+ ATPase subunit isoforms and leads to the degeneration of neuronal networks.

    Shiina N, Yamaguchi K and Tokunaga M

    Structural Biology Center, National Institute of Genetics, Research Organization of Information and Systems, and Department of Genetics, Sokendai, Mishima, Shizuoka 411-8540, Japan. nshiina@nibb.ac.jp

    mRNA transport and local translation in dendrites play key roles in use-dependent synaptic modification and in higher-order brain functions. RNG105, an RNA-binding protein, has previously been identified as a component of RNA granules that mediate dendritic mRNA localization and local translation. Here, we demonstrate that RNG105 knock-out in mice reduces the dendritic localization of mRNAs for Na+/K+ ATPase (NKA) subunit isoforms (i.e., α3, FXYD1, FXYD6, and FXYD7). The loss of dendritic mRNA localization is accompanied by the loss of function of NKA in dendrites without affecting the NKA function in the soma. Furthermore, we show that RNG105 deficiency affects the formation and maintenance of synapses and neuronal networks. These phenotypes are partly explained by an inhibition of NKA, which is known to influence synaptic functions as well as susceptibility to neurotoxicity. The present study first demonstrates the in vivo role of RNG105 in the dendritic localization of mRNAs and uncovers a novel link between dendritic mRNA localization and the development and maintenance of functional networks.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;38;12816-30

  • Cdk5-mediated phosphorylation of delta-catenin regulates its localization and GluR2-mediated synaptic activity.

    Poore CP, Sundaram JR, Pareek TK, Fu A, Amin N, Mohamed NE, Zheng YL, Goh AX, Lai MK, Ip NY, Pant HC and Kesavapany S

    Department of Biochemistry, Neurobiology Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    Cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation plays an important role in proper synaptic function and transmission. Loss of Cdk5 activity results in abnormal development of the nervous system accompanied by massive disruptions in cortical migration and lamination, therefore impacting synaptic activity. The Cdk5 activator p35 associates with delta-catenin, the synaptic adherens junction protein that serves as part of the anchorage complex of AMPA receptor at the postsynaptic membrane. However, the implications of Cdk5-mediated phosphorylation of delta-catenin have not been fully elucidated. Here we show that Cdk5-mediated phosphorylation of delta-catenin regulates its subcellular localization accompanied by changes in dendritic morphogenesis and synaptic activity. We identified two Cdk5 phosphorylation sites in mouse delta-catenin, serines 300 and 357, and report that loss of Cdk5 phosphorylation of delta-catenin increased its localization to the membrane. Furthermore, mutations of the serines 300 and 357 to alanines to mimic nonphosphorylated delta-catenin resulted in increased dendritic protrusions accompanied by increased AMPA receptor subunit GluR2 localization at the membrane. Consistent with these observations, loss of Cdk5 phosphorylation of delta-catenin increased the AMPA/NMDA ratio. This study reveals how Cdk5 phosphorylation of the synaptic mediator protein delta-catenin can alter its localization at the synapse to impact neuronal synaptic activity.

    Funded by: Intramural NIH HHS: Z99 NS999999

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;25;8457-67

  • AMPA receptor signaling through BRAG2 and Arf6 critical for long-term synaptic depression.

    Scholz R, Berberich S, Rathgeber L, Kolleker A, Köhr G and Kornau HC

    Center for Molecular Neurobiology (ZMNH), University of Hamburg, Hamburg, Germany.

    Central nervous system synapses undergo activity-dependent alterations to support learning and memory. Long-term depression (LTD) reflects a sustained reduction of the synaptic AMPA receptor content based on targeted clathrin-mediated endocytosis. Here we report a current-independent form of AMPA receptor signaling, fundamental for LTD. We found that AMPA receptors directly interact via the GluA2 subunit with the synaptic protein BRAG2, which functions as a guanine-nucleotide exchange factor (GEF) for the coat-recruitment GTPase Arf6. BRAG2-mediated catalysis, controlled by ligand-binding and tyrosine phosphorylation of GluA2, activates Arf6 to internalize synaptic AMPA receptors upon LTD induction. Furthermore, acute blockade of the GluA2-BRAG2 interaction and targeted deletion of BRAG2 in mature hippocampal CA1 pyramidal neurons prevents LTD in CA3-to-CA1 cell synapses, irrespective of the induction pathway. We conclude that BRAG2-mediated Arf6 activation triggered by AMPA receptors is the convergent step of different forms of LTD, thus providing an essential mechanism for the control of vesicle formation by endocytic cargo.

    Neuron 2010;66;5;768-80

  • Reduced cortical BDNF expression and aberrant memory in Carf knock-out mice.

    McDowell KA, Hutchinson AN, Wong-Goodrich SJ, Presby MM, Su D, Rodriguiz RM, Law KC, Williams CL, Wetsel WC and West AE

    Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Transcription factors are a key point of convergence between the cell-intrinsic and extracellular signals that guide synaptic development and brain plasticity. Calcium-response factor (CaRF) is a unique transcription factor first identified as a binding protein for a calcium-response element in the gene encoding brain-derived neurotrophic factor (Bdnf). We have now generated Carf knock-out (KO) mice to characterize the function of this factor in vivo. Intriguingly, Carf KO mice have selectively reduced expression of Bdnf exon IV-containing mRNA transcripts and BDNF protein in the cerebral cortex, whereas BDNF levels in the hippocampus and striatum remain unchanged, implicating CaRF as a brain region-selective regulator of BDNF expression. At the cellular level, Carf KO mice show altered expression of GABAergic proteins at striatal synapses, raising the possibility that CaRF may contribute to aspects of inhibitory synapse development. Carf KO mice show normal spatial learning in the Morris water maze and normal context-dependent fear conditioning. However they have an enhanced ability to find a new platform location on the first day of reversal training in the water maze and they extinguish conditioned fear more slowly than their wild-type littermates. Finally, Carf KO mice show normal short-term (STM) and long-term memory (LTM) in a novel object recognition task, but exhibit impairments during the remote memory phase of testing. Together, these data reveal novel roles for CaRF in the organization and/or function of neural circuits that underlie essential aspects of learning and memory.

    Funded by: Howard Hughes Medical Institute; NICHD NIH HHS: P30 HD018655, P30-HD18655; NIDA NIH HHS: R01 DA022202, R01 DA022202-04, R01-DA022202

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;22;7453-65

  • Effect of PSD-95/SAP90 and/or PSD-93/chapsyn-110 deficiency on the minimum alveolar anesthetic concentration of halothane in mice.

    Tao F, Skinner J, Yang Y and Johns RA

    Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Background: The authors have previously shown that the clinically relevant concentrations of inhalational anesthetics dose-dependently inhibit the postsynaptic density protein (PSD)-95, Dlg, and ZO-1 domain-mediated protein interactions between N-methyl-d-aspartate receptors and PSD-95/synaptic-associated protein (SAP) 90 or PSD-93/Chapsyn-110 and that the knockdown of spinal PSD-95/SAP90 significantly reduces the minimum alveolar concentration (MAC) for isoflurane in rats.

    Methods: The authors designed antisense oligodeoxynucleotides based on the mouse PSD-95/SAP90 and PSD-93/Chapsyn-110 messenger RNAs that correspond to their PSD-95, Dlg, and ZO-1 domain nucleotides and can specifically knock down the respective proteins. The authors intrathecally injected antisense oligodeoxynucleotides into wild-type and PSD-93/Chapsyn-110 knockout mice to investigate the effect of PSD-95/SAP90 and/or PSD-93/Chapsyn-110 deficiency on halothane anesthesia.

    Results: Both PSD-95/SAP90 and PSD-93/Chapsyn-110 antisense oligodeoxynucleotides caused a dose-dependent and significant reduction in the MAC of halothane in wild-type mice. The intrathecal injection of PSD-95/SAP90 antisense oligodeoxynucleotide at different doses (25 and 50 microg) reduced halothane MAC by 40 and 55%, respectively, and intrathecal injection of PSD-93/Chapsyn-110 antisense oligodeoxynucleotide at different doses (12 and 24 microg) reduced halothane MAC by 25 and 53%, respectively. The PSD-95/SAP90 antisense oligodeoxynucleotide showed similar effect on halothane MAC in wild-type and PSD-93/Chapsyn-110 knockout mice, suggesting that the combination of PSD-95/SAP90 knockdown with PSD-93/Chapsyn-110 deletion did not have an additive effect on halothane anesthesia.

    Conclusions: The current results indicate that PSD-95/SAP90 and PSD-93/Chapsyn-110 are involved in the molecular mechanisms of halothane anesthesia and that the functional role of PSD-95/SAP90 in halothane anesthesia is not enhanced after PSD-93/Chapsyn-110 deletion.

    Funded by: NIGMS NIH HHS: R01 GM049111

    Anesthesiology 2010;112;6;1444-51

  • DHHC5 interacts with PDZ domain 3 of post-synaptic density-95 (PSD-95) protein and plays a role in learning and memory.

    Li Y, Hu J, Höfer K, Wong AM, Cooper JD, Birnbaum SG, Hammer RE and Hofmann SL

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA.

    A family of integral membrane proteins containing a signature DHHC motif has been shown to display protein S-acyltransferase activity, modifying cysteine residues in proteins with fatty acids. The physiological roles of these proteins have largely been unexplored. Here we report that mice homozygous for a hypomorphic allele of a previously uncharacterized member, DHHC5, are born at half the expected rate, and survivors show a marked deficit in contextual fear conditioning, an indicator of defective hippocampal-dependent learning. DHHC5 is highly enriched in a post-synaptic density preparation and co-immunoprecipitates with post-synaptic density protein-95 (PSD-95), an interaction that is mediated through binding of the carboxyl terminus of DHHC5 and the PDZ3 domain of PSD-95. Immunohistochemistry demonstrated that DHHC5 is expressed in the CA3 and dentate gyrus in the hippocampus. These findings point to a previously unsuspected role for DHHC5 in post-synaptic function affecting learning and memory.

    Funded by: NINDS NIH HHS: NS36867, NS41930, R01 NS036867, R21 NS041930, R37 NS036867; Wellcome Trust: GR079491MA

    The Journal of biological chemistry 2010;285;17;13022-31

  • CKAMP44: a brain-specific protein attenuating short-term synaptic plasticity in the dentate gyrus.

    von Engelhardt J, Mack V, Sprengel R, Kavenstock N, Li KW, Stern-Bach Y, Smit AB, Seeburg PH and Monyer H

    Department of Clinical Neurobiology, University of Heidelberg, 6910 Heidelberg, Germany.

    CKAMP44, identified here by a proteomic approach, is a brain-specific type I transmembrane protein that associates with AMPA receptors in synaptic spines. CKAMP44 expressed in Xenopus oocytes reduced GluA1- and A2-mediated steady-state currents, but did not affect kainate- or N-methyl-D-aspartate (NMDA) receptor-mediated currents. Mouse hippocampal CA1 pyramidal neurons expressed CKAMP44 at low abundance, and overexpression of CKAMP44 led to stronger and faster AMPA receptor desensitization, slower recovery from desensitization, and a reduction in the paired-pulse ratio of AMPA currents. By contrast, dentate gyrus granule cells exhibited strong CKAMP44 expression, and CKAMP44 knockout increased the paired-pulse ratio of AMPA currents in lateral and medial perforant path-granule cell synapses. CKAMP44 thus modulates short-term plasticity at specific excitatory synapses.

    Science (New York, N.Y.) 2010;327;5972;1518-22

  • Synaptic clustering of PSD-95 is regulated by c-Abl through tyrosine phosphorylation.

    Perez de Arce K, de Arce KP, Varela-Nallar L, Farias O, Cifuentes A, Bull P, Couch BA, Koleske AJ, Inestrosa NC and Alvarez AR

    Laboratorio de Señalización Celular, Departmento de Biología Celular y Molecular, Pontificia Universidad Católica de Chile, 8331010 Santiago, Chile.

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95.

    Funded by: NINDS NIH HHS: NS39475, R01 NS039475, R01 NS039475-08

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;10;3728-38

  • TDP-43 is a developmentally regulated protein essential for early embryonic development.

    Sephton CF, Good SK, Atkin S, Dewey CM, Mayer P, Herz J and Yu G

    Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    TDP-43 is a DNA/RNA-binding protein implicated in multiple steps of transcriptional and post-transcriptional regulation of gene expression. Alteration of this multifunctional protein is associated with a number of neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin positive inclusions. Whereas a pathological link to neurodegenerative disorders has been established, the cellular and physiological functions of TDP-43 remain unknown. In this study, we show that TDP-43 is a nuclear protein with persistent high-level expression during embryonic development and with progressively decreased protein levels during postnatal development. In mice where the TDP-43 gene (Tardbp) was disrupted using a gene trap that carries a beta-galactosidase marker gene, heterozygous (Tardbp(+/-)) mice are fertile and healthy, but intercrosses of Tardbp(+/-) mice yielded no viable homozygotic null (Tardbp(-/-)) mice. Indeed, Tardbp(-/-) embryos die between 3.5 and 8.5 days of development. Tardbp(-/-) blastocysts grown in cell culture display abnormal expansion of their inner cell mass. The pattern of beta-galactosidase staining at E9.5 Tardbp(+/-) embryos is predominantly restricted to the neuroepithelium and remains prominent in neural progenitors at E10.5-12.5. TDP-43 is detected in spinal cord progenitors and in differentiated motor neurons as well as in the dorsal root ganglia at E12.5. Beta-galactosidase staining of tissues from adult Tardbp(+/-) mice shows widespread expression of TDP-43, including prominent levels in various regions of the central nervous system afflicted in neurodegenerative disorders. These results indicate that TDP-43 is developmentally regulated and indispensible for early embryonic development.

    Funded by: NHLBI NIH HHS: R37 HL063762; NIA NIH HHS: AG023104, R01 AG023104, R01 AG029547

    The Journal of biological chemistry 2010;285;9;6826-34

  • ADAM22, a Kv1 channel-interacting protein, recruits membrane-associated guanylate kinases to juxtaparanodes of myelinated axons.

    Ogawa Y, Oses-Prieto J, Kim MY, Horresh I, Peles E, Burlingame AL, Trimmer JS, Meijer D and Rasband MN

    Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.

    Clustered Kv1 K(+) channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments, and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 alpha subunits, and then used mass spectrometry to identify interacting proteins. We found that a disintegrin and metalloproteinase 22 (ADAM22) is a component of the Kv1 channel complex and that ADAM22 coimmunoprecipitates Kv1.2 and the membrane-associated guanylate kinases (MAGUKs) PSD-93 and PSD-95. When coexpressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, coexpression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel-interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that, like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K(+) channel complex that recruits MAGUKs to juxtaparanodes.

    Funded by: NCRR NIH HHS: P41 RR001614, P41RR001614, RR019934, S10 RR019934; NINDS NIH HHS: NS034383, NS044916, R01 NS034383, R01 NS034383-14, R01 NS044916, R37 NS034383, R37 NS044916, U24NS050606

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;3;1038-48

  • Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses.

    Medrihan L, Rohlmann A, Fairless R, Andrae J, Döring M, Missler M, Zhang W and Kilimann MW

    Center for Physiology, Georg-August University and DFG-Research Center of Molecular Physiology of the Brain, D-37073 Göttingen, Germany.

    The development of neuronal networks in the brain requires the differentiation of functional synapses. Neurobeachin (Nbea) was identified as a putative regulator of membrane protein trafficking associated with tubulovesicular endomembranes and postsynaptic plasma membranes. Nbea is essential for evoked transmission at neuromuscular junctions, but its role in the central nervous system has not been characterized. Here, we have studied central synapses of a newly generated gene-trap knockout (KO) mouse line at embryonic day 18, because null-mutant mice are paralysed and die perinatally. Although the overall brain architecture was normal, we identified major abnormalities of synaptic function in mutant animals. In acute slices from the brainstem, both spontaneous excitatory and inhibitory postsynaptic currents were clearly reduced and failure rates of evoked inhibitory responses were markedly increased. In addition, the frequency of miniature excitatory and both the frequency and amplitudes of miniature inhibitory postsynaptic currents were severely diminished in KO mice, indicating a perturbation of both action potential-dependent and -independent transmitter release. Moreover, Nbea appears to be important for the formation and composition of central synapses because the area density of mature asymmetric contacts in the fetal brainstem was reduced to 30% of wild-type levels, and the expression levels of a subset of synaptic marker proteins were smaller than in littermate controls. Our data demonstrate for the first time a function of Nbea at central synapses that may be based on its presumed role in targeting membrane proteins to synaptic contacts, and are consistent with the 'excitatory-inhibitory imbalance' model of autism where Nbea gene rearrangements have been detected in some patients.

    The Journal of physiology 2009;587;Pt 21;5095-106

  • Role of PSD95 in membrane association and catalytic activity of nNOSalpha in nitrergic varicosities in mice gut.

    Chaudhury A, He XD and Goyal RK

    Gastrointestinal Division, Veterans Affairs Boston Healthcare System and Harvard Medical School, Center for Swallowing and Motility Disorders, Boston, Massachusetts, USA.

    We have recently shown that membrane association of neuronal nitric oxide synthase-alpha (nNOSalpha) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSalpha. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSalpha, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSalpha was associated with PSD95 but not SAP97. nNOSalpha-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSalpha and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSalpha to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.

    Funded by: NIDDK NIH HHS: DK 062867

    American journal of physiology. Gastrointestinal and liver physiology 2009;297;4;G806-13

  • Fragile X mental retardation protein regulates the levels of scaffold proteins and glutamate receptors in postsynaptic densities.

    Schütt J, Falley K, Richter D, Kreienkamp HJ and Kindler S

    Institute for Human Genetics, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany.

    Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1-3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3'-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

    The Journal of biological chemistry 2009;284;38;25479-87

  • ASIC2 subunits target acid-sensing ion channels to the synapse via an association with PSD-95.

    Zha XM, Costa V, Harding AM, Reznikov L, Benson CJ and Welsh MJ

    Howard Hughes Medical Institute, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.

    Acid-sensing ion channel-1a (ASIC1a) mediates H(+)-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not coimmunoprecipitate with PSD-95. We found that ASIC2 and ASIC1a associated in brain, and through its interaction with PSD-95, ASIC2 increased ASIC1a localization in dendritic spines. Consistent with earlier work showing that acidic pH elevated spine [Ca(2+)](i) by activating ASIC1a, loss of ASIC2 decreased the percentage of spines responding to acid. Moreover, like a reduction of ASIC1a, the number of spine synapses fell in ASIC2(-/-) neurons. These results indicate that ASIC2 facilitates ASIC1a localization and function in dendritic spines and suggest that the two subunits work in concert to regulate neuronal function.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL076419, HL51670, P01 HL051670, P01 HL051670-119003, R01 HL076419-05; NIDDK NIH HHS: DK54759, P30 DK054759-109004

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;26;8438-46

  • PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b.

    Padányi R, Pászty K, Strehler EE and Enyedi A

    National Blood Center, Department of Molecular Cell Biology, Diószegi u. 64, H-1113 Budapest, Hungary.

    Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.

    Funded by: NINDS NIH HHS: NS51769, R01 NS051769, R01 NS051769-01A1, R01 NS051769-02

    Biochimica et biophysica acta 2009;1793;6;1023-32

  • PSD95beta regulates plasma membrane Ca2+ pump localization at the photoreceptor synapse.

    Aartsen WM, Arsanto JP, Chauvin JP, Vos RM, Versteeg I, Cardozo BN, Bivic AL and Wijnholds J

    Department of Neuromedical Genetics, The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), Meibergdreef 47, 1105 BA Amsterdam, The Netherlands.

    At the presynaptic plasma membrane of the photoreceptor the correct localization of the calcium extruder, plasma membrane Ca2+-ATPase (PMCA), is determined by a unique protein complex. Here, the role of two proteins within the complex; membrane palmitoylated protein 4 (MPP4) and postsynaptic density protein 95 (PSD95) is investigated in more details, using Mpp4 and Psd95 mutant mice. MPP4 deficiency results in the loss of both PMCA and PSD95 from the photoreceptor synapse. Truncation of the C-terminal part of MPP4 leads to a loss of PSD95 and mislocalization of PMCA, while truncation of the C-terminal part of PSD95 did not affect the localization of the complex members. Lentivirus-mediated molecular replacement strategy was used to selectively express either PSD95alpha or PSD95beta in wild type or Mpp4 mutant primary retinal explants. Silencing of the Psd95 gene resulted in the loss of presynaptic MPP4 and PMCA1. The plasma membrane localization of MPP4 and PMCA1 could be restored by the expression of PSD95beta. We conclude that both scaffold proteins PSD95beta and MPP4 are essential for the modulation of PMCA levels at the presynaptic plasma membrane and thereby influence the photoreceptor synaptic calcium handling.

    Molecular and cellular neurosciences 2009;41;2;156-65

  • TMEM16B, a novel protein with calcium-dependent chloride channel activity, associates with a presynaptic protein complex in photoreceptor terminals.

    Stöhr H, Heisig JB, Benz PM, Schöberl S, Milenkovic VM, Strauss O, Aartsen WM, Wijnholds J, Weber BH and Schulz HL

    Institut für Humangenetik, Universität Regensburg, 93053 Regensburg, Germany. heidi.stoehr@klinik.uni-regensburg.de

    Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca(2+)-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca(2+)-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca(2+)-dependent chloride channel in the photoreceptor synapse.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;21;6809-18

  • Synaptic removal of diacylglycerol by DGKzeta and PSD-95 regulates dendritic spine maintenance.

    Kim K, Yang J, Zhong XP, Kim MH, Kim YS, Lee HW, Han S, Choi J, Han K, Seo J, Prescott SM, Topham MK, Bae YC, Koretzky G, Choi SY and Kim E

    National Creative Research Initiative Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon, Republic of Korea.

    Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.

    Funded by: NCI NIH HHS: R01 CA095463, R01-CA95463; NIAID NIH HHS: R01 AI076357, R01 AI076357-01A1, R01 AI079088, R01 AI079088-01, R21 AI079873

    The EMBO journal 2009;28;8;1170-9

  • Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production.

    Hoey SE, Williams RJ and Perkinton MS

    King's College London, Wolfson Centre for Age-Related Diseases, London, United Kingdom.

    Altered amyloid precursor protein (APP) processing leading to increased production and oligomerization of Abeta may contribute to Alzheimer's disease (AD). Understanding how APP processing is regulated under physiological conditions may provide new insights into AD pathogenesis. Recent reports demonstrate that excitatory neural activity regulates APP metabolism and Abeta levels, although understanding of the molecular mechanisms involved is incomplete. We have investigated whether NMDA receptor activity regulates APP metabolism in primary cultured cortical neurons. We report that a pool of APP is localized to the postsynaptic compartment in cortical neurons and observed partial overlap of APP with both NR1 and PSD-95. NMDA receptor stimulation increased nonamyloidogenic alpha-secretase-mediated APP processing, as measured by a 2.5-fold increase in cellular alpha-C-terminal fragment (C83) levels after glutamate or NMDA treatment. This increase was blocked by the NMDA receptor antagonists d-AP5 and MK801 but not by the AMPA receptor antagonist CNQX or the L-type calcium channel blocker nifedipine, was prevented by chelation of extracellular calcium, and was blocked by the alpha-secretase inhibitor TAPI-1. Cotreatment of cortical neurons with bicuculline and 4-AP, which stimulates glutamate release and activates synaptic NMDA receptors, evoked an MK801-sensitive increase in C83 levels. Furthermore, NMDA receptor stimulation caused a twofold increase in the amount of soluble APP detected in the neuronal culture medium. Finally, NMDA receptor activity inhibited both Abeta1-40 release and Gal4-dependent luciferase activity induced by beta-gamma-secretase-mediated cleavage of an APP-Gal4 fusion protein. Altogether, these data suggest that calcium influx through synaptic NMDA receptors promotes nonamyloidogenic alpha-secretase-mediated APP processing.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;14;4442-60

  • The insulin receptor substrate of 53 kDa (IRSp53) limits hippocampal synaptic plasticity.

    Sawallisch C, Berhörster K, Disanza A, Mantoani S, Kintscher M, Stoenica L, Dityatev A, Sieber S, Kindler S, Morellini F, Schweizer M, Boeckers TM, Korte M, Scita G and Kreienkamp HJ

    Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany.

    IRSp53 is an essential intermediate between the activation of Rac and Cdc42 GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and paired-pulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-d-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm.

    The Journal of biological chemistry 2009;284;14;9225-36

  • Impaired maturation of dendritic spines without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the central nervous system.

    Barros CS, Calabrese B, Chamero P, Roberts AJ, Korzus E, Lloyd K, Stowers L, Mayford M, Halpain S and Müller U

    Department of Cell Biology, Institute of Childhood and Neglected Disease, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    Neuregulin-1 (NRG1) and its ErbB2/B4 receptors are encoded by candidate susceptibility genes for schizophrenia, yet the essential functions of NRG1 signaling in the CNS are still unclear. Using CRE/LOX technology, we have inactivated ErbB2/B4-mediated NRG1 signaling specifically in the CNS. In contrast to expectations, cell layers in the cerebral cortex, hippocampus, and cerebellum develop normally in the mutant mice. Instead, loss of ErbB2/B4 impairs dendritic spine maturation and perturbs interactions of postsynaptic scaffold proteins with glutamate receptors. Conversely, increased NRG1 levels promote spine maturation. ErbB2/B4-deficient mice show increased aggression and reduced prepulse inhibition. Treatment with the antipsychotic drug clozapine reverses the behavioral and spine defects. We conclude that ErbB2/B4-mediated NRG1 signaling modulates dendritic spine maturation, and that defects at glutamatergic synapses likely contribute to the behavioral abnormalities in ErbB2/B4-deficient mice.

    Funded by: NIDCD NIH HHS: R01 DC009413; NIMH NIH HHS: MH078833, R01 MH087823, U01 MH078833; NINDS NIH HHS: NS057096, NS37311, P30 NS057096, R01 NS037311, R01 NS046456

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;11;4507-12

  • Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.

    Ng D, Pitcher GM, Szilard RK, Sertié A, Kanisek M, Clapcote SJ, Lipina T, Kalia LV, Joo D, McKerlie C, Cortez M, Roder JC, Salter MW and McInnes RR

    Program in Developmental Biology, The Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.

    The N-methyl-D-aspartate receptor (NMDAR), a major excitatory ligand-gated ion channel in the central nervous system (CNS), is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1), a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.

    Funded by: Howard Hughes Medical Institute

    PLoS biology 2009;7;2;e41

  • N-methyl-D-aspartate receptor subunits are non-myosin targets of myosin regulatory light chain.

    Bajaj G, Zhang Y, Schimerlik MI, Hau AM, Yang J, Filtz TM, Kioussi C and Ishmael JE

    Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.

    Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the "GXXXR" portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.

    Funded by: NIAMS NIH HHS: R01AR054406; NIEHS NIH HHS: P30-ES000210

    The Journal of biological chemistry 2009;284;2;1252-66

  • Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development.

    Elias GM, Elias LA, Apostolides PF, Kriegstein AR and Nicoll RA

    Neuroscience Graduate Program, University of California, San Francisco, CA 94143, USA.

    The development of glutamatergic synapses involves changes in the number and type of receptors present at the postsynaptic density. To elucidate molecular mechanisms underlying these changes, we combine in utero electroporation of constructs that alter the molecular composition of developing synapses with dual whole-cell electrophysiology to examine synaptic transmission during two distinct developmental stages. We find that SAP102 mediates synaptic trafficking of AMPA and NMDA receptors during synaptogenesis. Surprisingly, after synaptogenesis, PSD-95 assumes the functions of SAP102 and is necessary for two aspects of synapse maturation: the developmental increase in AMPA receptor transmission and replacement of NR2B-NMDARs with NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95 expression fully rescues this deficit. This study demonstrates that SAP102 and PSD-95 regulate the synaptic trafficking of distinct glutamate receptor subtypes at different developmental stages, thereby playing necessary roles in excitatory synapse development.

    Funded by: NIMH NIH HHS: R37 MH038256

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;52;20953-8

  • Multiple molecular interactions determine the clustering of Caspr2 and Kv1 channels in myelinated axons.

    Horresh I, Poliak S, Grant S, Bredt D, Rasband MN and Peles E

    Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

    Clustering of Kv1 channels at the juxtaparanodal region (JXP) in myelinated axons depends on their association with the Caspr2/TAG-1 adhesion complex. The interaction between these channels and Caspr2 was suggested to depend on PDZ (PSD-95/Discs large/zona occludens-1) scaffolding proteins. Here, we show that at a subset of the JXP, PSD-93 colocalizes with Caspr2, K(+) channels and its related protein postsynaptic density protein-95 (PSD-95). The localization of PSD-93 and PSD-95 depends on the presence of Caspr2, as both scaffolding proteins failed to accumulate at the JXP in mice lacking either Caspr2 or TAG-1. In contrast, Caspr2 and K(+) channels still colocalized and associated in PSD-93, PSD-95 or double PSD-93/PSD-95 null mice. To directly evaluate the role of PDZ domain proteins in the function of Caspr2, we examined the ability of transgenic Caspr2 molecules lacking either their cytoplasmic domain (Caspr2dCT), or their PDZ-binding sequence (Caspr2dPDZ), to restore Kv1 channel clustering in Caspr2 null mice. We found that while Kv1 channels were distributed throughout internodes in nerves expressing Caspr2dCT, they were clustered at the JXP in axons expressing a full-length Caspr2 (Caspr2FL) or the Caspr2dPDZ transgene. Further proteomic analysis revealed that Caspr2 interacts with a distinct set of scaffolding proteins through its PDZ- and protein 4.1-binding sequences. These results demonstrate that while the molecular assembly of the JXP requires the cytoplasmic domain of Caspr2, its carboxy-terminal PDZ-binding motif is dispensable for Kv1 channel clustering. This mechanism is clearly distinct from the one operating at the axon initial segment, which requires PSD-93 for Kv1 channel clustering.

    Funded by: NINDS NIH HHS: NS044916, R01 NS044916, R01 NS044916-02, R01 NS044916-03, R01 NS044916-04, R01 NS044916-05, R37 NS044916

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;52;14213-22

  • Stable membrane expression of postsynaptic CaV1.2 calcium channel clusters is independent of interactions with AKAP79/150 and PDZ proteins.

    Di Biase V, Obermair GJ, Szabo Z, Altier C, Sanguesa J, Bourinet E and Flucher BE

    Department of Physiology and Medical Physics, Innsbruck Medical University, A-6020 Innsbruck, Austria.

    In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of Ca(V)1.2 and its association with upstream and downstream signaling proteins is important for efficient and specific signal transduction. Here we tested the hypothesis that A-kinase anchoring proteins (AKAPs) or PDZ-proteins are responsible for the targeting and anchoring of Ca(V)1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged Ca(V)1.2 demonstrated that clusters of membrane-incorporated Ca(V)1.2-HA were colocalized with AKAP79/150 but not with PSD-95 in the spines and shafts of dendrites. To disrupt the interactions with these scaffold proteins, we mutated known binding sequences for AKAP79/150 and PDZ proteins in the C terminus of Ca(V)1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant Ca(V)1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca(V)1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and alpha-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of Ca(V)1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca(V)1.2 calcium channels form signaling complexes apart from those of glutamate receptors and PSD-95. Their number and distribution in dendritic spines is not altered upon NMDA-induced disruption of the glutamate receptor signaling complex, and targeting and anchoring of Ca(V)1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.

    Funded by: Austrian Science Fund FWF: P 17806, P 17807, P 20059

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;51;13845-55

  • A differential developmental pattern of spinal interneuron apoptosis during synaptogenesis: insights from genetic analyses of the protocadherin-gamma gene cluster.

    Prasad T, Wang X, Gray PA and Weiner JA

    Department of Biology, The University of Iowa, Iowa City, IA 52242, USA.

    Although the role of developmental apoptosis in shaping the complement and connectivity of sensory and motoneurons is well documented, the extent to which cell death affects the 13 cardinal classes of spinal interneurons is unclear. Using a series of genetic manipulations in vivo, we demonstrate for the first time a differential pattern of developmental apoptosis in molecularly identified spinal interneuron populations, and implicate the adhesion molecule family encoded by the 22-member protocadherin-gamma (Pcdh-gamma) gene cluster in its control. In constitutive Pcdh-gamma null mouse embryos, many interneuron populations undergo increased apoptosis, but to differing extents: for example, over 80% of En1-positive V1 neurons are lost, whereas only 30% of Chx10-positive V2a neurons are lost and there is no reduction in the number of V1-derived Renshaw cells. We show that this represents an exacerbation of a normal, underlying developmental pattern: the extent of each population's decrease in Pcdh-gamma mutants is precisely commensurate both with the extent of its loss during normal embryogenesis and with the extent of its increase in Bax(-/-) mice, in which apoptosis is genetically blocked. Interneuron apoptosis begins during the first wave of synaptogenesisis in the spinal cord, occurring first among ventral populations (primarily between E14 and E17), and only later among dorsal populations (primarily after P0). Utilizing a new, conditional Pcdh-gamma mutant allele, we show that the gamma-Pcdhs can promote survival non-cell-autonomously: mutant neurons can survive if they are surrounded by normal neurons, and normal neurons can undergo apoptosis if they are surrounded by mutant neurons.

    Funded by: NINDS NIH HHS: R01 NS055272, R01 NS055272-01A2

    Development (Cambridge, England) 2008;135;24;4153-64

  • Localization and regional distribution of p23/TMP21 in the brain.

    Vetrivel KS, Kodam A, Gong P, Chen Y, Parent AT, Kar S and Thinakaran G

    Department of Neurobiology, The University of Chicago, Chicago, IL 60637, USA.

    Sequential processing of amyloid precursor protein by beta- and gamma-secretases generates Alzheimer's disease (AD)-associated beta-amyloid peptides. Recently it was reported that the transmembrane protein p23/TMP21 associates with gamma-secretase, and negatively regulates beta-amyloid production. Despite the link between p23 function and AD pathogenesis, the expression of p23 has not been examined in the brain. Here, we describe the detailed immunohistochemical characterization of p23 expression in rodent and human brain. We report that p23 is co-expressed with gamma-secretase subunits in select neuronal cell populations in rodent brain. Interestingly, the steady-state level of p23 in the brain is high during embryonic development and then declines after birth. Furthermore, the steady-state p23 levels are reduced in the brains of individuals with AD. We conclude that p23 is expressed in neurons throughout the brain and the decline in p23 expression during postnatal development may significantly contribute to enhanced beta-amyloid production in the adult brain.

    Funded by: NIA NIH HHS: AG019070, AG021495, R01 AG019070, R01 AG019070-07, R01 AG021495, R01 AG021495-06A1; NINDS NIH HHS: NS055223, R01 NS055223, R01 NS055223-01A2

    Neurobiology of disease 2008;32;1;37-49

  • Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain.

    Lugli G, Torvik VI, Larson J and Smalheiser NR

    Department of Psychiatry and Psychiatric Institute, University of Illinois at Chicago, Chicago, Illinois, USA.

    We have characterized the expression of microRNAs and selected microRNA precursors within several synaptic fractions of adult mouse forebrain, including synaptoneurosomes, synaptosomes and isolated post-synaptic densities (PSDs), using methods of microRNA microarray, real time qRT-PCR, Northern blotting and immunopurification using anti-PSD95 antibody. The majority of brain microRNAs (especially microRNAs known to be expressed in pyramidal neurons) are detectably expressed in synaptic fractions, and a subset of microRNAs is significantly enriched in synaptic fractions relative to total forebrain homogenate. MicroRNA precursors are also detectable in synaptic fractions at levels that are comparable to whole tissue. Whereas mature microRNAs are predominantly associated with soluble components of the synaptic fractions, microRNA precursors are predominantly associated with PSDs. For seven microRNAs examined, there was a significant correlation between the relative synaptic enrichment of the precursor and the relative synaptic enrichment of the corresponding mature microRNA. These findings support the proposal that microRNAs are formed, at least in part, via processing of microRNA precursors locally within dendritic spines. Dicer is expressed in PSDs but is enzymatically inactive until conditions that activate calpain cause its liberation; thus, we propose that synaptic stimulation may lead to local processing of microRNA precursors in proximity to the synapse.

    Funded by: NIDCD NIH HHS: DC 05793, R01 DC005793; NIMH NIH HHS: MH81099, R21 MH081099; NLM NIH HHS: LM07292, R01 LM007292

    Journal of neurochemistry 2008;106;2;650-61

  • Activation of beta-catenin signaling programs embryonic epidermis to hair follicle fate.

    Zhang Y, Andl T, Yang SH, Teta M, Liu F, Seykora JT, Tobias JW, Piccolo S, Schmidt-Ullrich R, Nagy A, Taketo MM, Dlugosz AA and Millar SE

    Department of Dermatology and Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    beta-Catenin signaling is required for hair follicle development, but it is unknown whether its activation is sufficient to globally program embryonic epidermis to hair follicle fate. To address this, we mutated endogenous epithelial beta-catenin to a dominant-active form in vivo. Hair follicle placodes were expanded and induced prematurely in activated beta-catenin mutant embryos, but failed to invaginate or form multilayered structures. Eventually, the entire epidermis adopted hair follicle fate, broadly expressing hair shaft keratins in place of epidermal stratification proteins. Mutant embryonic skin was precociously innervated, and displayed prenatal pigmentation, a phenomenon never observed in wild-type controls. Thus, beta-catenin signaling programs the epidermis towards placode and hair shaft fate at the expense of epidermal differentiation, and activates signals directing pigmentation and innervation. In transcript profiling experiments, we identified elevated expression of Sp5, a direct beta-catenin target and transcriptional repressor. We show that Sp5 normally localizes to hair follicle placodes and can suppress epidermal differentiation gene expression. We identified the pigmentation regulators Foxn1, Adamts20 and Kitl, and the neural guidance genes Sema4c, Sema3c, Unc5b and Unc5c, as potential mediators of the effects of beta-catenin signaling on pigmentation and innervation. Our data provide evidence for a new paradigm in which, in addition to promoting hair follicle placode and hair shaft fate, beta-catenin signaling actively suppresses epidermal differentiation and directs pigmentation and nerve fiber growth. Controlled downregulation of beta-catenin signaling is required for normal placode patterning within embryonic ectoderm, hair follicle downgrowth, and adoption of the full range of follicular fates.

    Funded by: NCI NIH HHS: P30 CA046592; NIAMS NIH HHS: R01 AR045973, R01 AR045973-08, R01 AR047709, R01 AR047709-01, R01 AR047709-02, R01 AR047709-03, R01 AR047709-03S1, R01 AR047709-04, R01 AR047709-05, R01 AR047709-05S1, R01 AR047709-06, R01 AR047709-07, R01 AR047709-08, R01-AR45973, R01-AR47709, T32 AR007465, T32 AR007465-22, T32 AR007465-23, T32 AR007465-24, T32 AR007465-25; NICHD NIH HHS: R01 HD053829, R01 HD053829-01A1, R01 HD053829-02, R01-HD053829; NIDCR NIH HHS: R01 DE015342, R01 DE015342-01A2, R01 DE015342-02, R01 DE015342-03, R01 DE015342-04, R01-DE015342

    Development (Cambridge, England) 2008;135;12;2161-72

  • Neuregulin signaling in neurons depends on ErbB4 interaction with PSD-95.

    Murphy SP and Bielby-Clarke K

    Institute of Cell Signalling, University of Nottingham, Nottingham, UK. seanm6@u.washington.edu

    Neuregulin-1 signaling molecules have various roles in the nervous system, including effects on neuronal migration and differentiation. In mouse cerebellar granule neurons the effects of neuregulin-1 (type I) are mediated by a homo-dimeric ErbB4 receptor, which can interact with the PSD-95 protein through a C-terminal motif. To determine whether this interaction is critical for signaling we introduced into granule cells a decoy peptide representing the C-terminal 20 amino acids of the mouse ErbB4 receptor. Exposure of unloaded cells to neuregulin-1 did not alter neurite density but increased average neurite length by 50%. In contrast, granule cell cultures loaded with the decoy peptide were unresponsive to the ligand. We conclude that the association of ErbB4 with PSD-95 is important for neuregulin-induced neuronal differentiation.

    Funded by: Biotechnology and Biological Sciences Research Council

    Brain research 2008;1207;32-5

  • The protocadherin-alpha family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse.

    Hasegawa S, Hamada S, Kumode Y, Esumi S, Katori S, Fukuda E, Uchiyama Y, Hirabayashi T, Mombaerts P and Yagi T

    KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.

    Olfactory sensory neurons (OSNs) that express the same odorant receptor project their axons to specific glomeruli in the main olfactory bulb. Protocadherin-alpha (Pcdha) proteins, diverse cadherin-related molecules that are encoded as a gene cluster, are highly concentrated in OSN axons and olfactory glomeruli. Here, we describe Pcdha mutant mice, in which the constant region of the Pcdha gene cluster has been deleted by gene targeting. The mutant mice show abnormal sorting of OSN axons into glomeruli. There are multiple, small, extraneous glomeruli for the odorant receptors M71 and MOR23. These abnormal patterns of M71 and MOR23 glomeruli persist until adulthood. Many M71 glomeruli, but apparently not MOR23 glomeruli, are heterogeneous in axonal innervation. Thus, Pcdha molecules are involved in coalescence of OSN axons into OR-specific glomeruli of the olfactory bulb.

    Molecular and cellular neurosciences 2008;38;1;66-79

  • Effect of disrupting N-methyl-d-aspartate receptor-postsynaptic density protein-95 interactions on the threshold for halothane anesthesia in mice.

    Tao F and Johns RA

    Department of Anesthesiology and Critical Care Medicine,Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Background: The authors' previous studies have shown that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PSD-95, Dlg, and ZO-1 (PDZ) domain-mediated protein interactions between postsynaptic density protein 95 (PSD-95) and N-methyl-d-aspartate receptors, and that the knockdown of spinal PSD-95 by intrathecal injection of PSD-95 antisense oligodeoxynucleotide significantly reduces the minimum alveolar anesthetic concentration for isoflurane in rats.

    Methods: The authors constructed a fusion peptide, Tat-PSD-95 PDZ2, comprising the second PDZ domain of PSD-95, which can specifically disrupt PSD-95 PDZ2-mediated protein interactions by binding to its interaction partner. By intraperitoneal injection of this fusion peptide into mice, the authors investigated the effect of disrupting the PSD-95 PDZ2-mediated protein interactions on the threshold for halothane anesthesia.

    Results: Systemically injected fusion peptide Tat-PSD-95 PDZ2 was delivered into the central nervous system, disrupted the protein-protein interactions between N-methyl-d-aspartate receptor NR2 subunits and PSD-95, and significantly reduced the minimum alveolar anesthetic concentration and righting reflex EC50 for halothane.

    Conclusions: By disrupting PSD-95 PDZ2 domain-mediated protein interactions, intraperitoneal injection of cell-permeant fusion peptide Tat-PSD-95 PDZ2 dose-dependently reduces the threshold for halothane anesthesia. These results suggest that PDZ domain-mediated protein interactions at synapses in the central nervous system might play an important role in the molecular mechanisms of halothane anesthesia.

    Funded by: NIGMS NIH HHS: GM049111, R01 GM049111, R01 GM049111-14; NINDS NIH HHS: NS44219, R01 NS044219

    Anesthesiology 2008;108;5;882-7

  • Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism.

    Jamain S, Radyushkin K, Hammerschmidt K, Granon S, Boretius S, Varoqueaux F, Ramanantsoa N, Gallego J, Ronnenberg A, Winter D, Frahm J, Fischer J, Bourgeron T, Ehrenreich H and Brose N

    Abteilung Molekulare Neurobiologie and Division für Klinische Neurowissenschaften, Max-Planck-Institut für Experimentelle Medizin, 37075 Göttingen, Germany.

    Autism spectrum conditions (ASCs) are heritable conditions characterized by impaired reciprocal social interactions, deficits in language acquisition, and repetitive and restricted behaviors and interests. In addition to more complex genetic susceptibilities, even mutation of a single gene can lead to ASC. Several such monogenic heritable ASC forms are caused by loss-of-function mutations in genes encoding regulators of synapse function in neurons, including NLGN4. We report that mice with a loss-of-function mutation in the murine NLGN4 ortholog Nlgn4, which encodes the synaptic cell adhesion protein Neuroligin-4, exhibit highly selective deficits in reciprocal social interactions and communication that are reminiscent of ASCs in humans. Our findings indicate that a protein network that regulates the maturation and function of synapses in the brain is at the core of a major ASC susceptibility pathway, and establish Neuroligin-4-deficient mice as genetic models for the exploration of the complex neurobiological disorders in ASCs.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;5;1710-5

  • IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for Arf6 that interacts with PSD-95 at postsynaptic density of excitatory synapses.

    Sakagami H, Sanda M, Fukaya M, Miyazaki T, Sukegawa J, Yanagisawa T, Suzuki T, Fukunaga K, Watanabe M and Kondo H

    Department of Anatomy, Kitasato University School of Medicine, Sagamihara 228-8555, Japan. sakagami@med.kitasato-u.ac.jp

    ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-d-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAa receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6.

    Neuroscience research 2008;60;2;199-212

  • Postsynaptic and differential localization to neuronal subtypes of protocadherin beta16 in the mammalian central nervous system.

    Junghans D, Heidenreich M, Hack I, Taylor V, Frotscher M and Kemler R

    Max-Planck Institute of Immunobiology, Department of Molecular Embryology, 79011 Freiburg, Germany.

    The formation of synapses is dependent on the expression of surface adhesion molecules that facilitate correct recognition, stabilization and function. The more than 60 clustered protocadherins (Pcdhalpha, Pcdhbeta and Pcdhgamma) identified in human and mouse have attracted considerable attention because of their clustered genomic organization and the potential role of alpha- and gamma-Pcdhs in allocating a neuronal surface code specifying synaptic connectivity. Here, we investigated whether beta-Pcdhs also contribute to these processes. By performing RT-PCR, we found a striking parallel onset of expression of many beta-Pcdhs around the onset of neurogenesis and wide expression in the central nervous system. We generated antibodies specific to Pcdhb16 and showed localization of Pcdhb16 protein in the adult mouse cerebellum, hippocampus and cerebral cortex. Analysing the mouse retina in detail revealed localization of Pcdhb16 to specific cell types and, importantly, subsets of synapses. We show that Pcdhb16 localizes predominantly to postsynaptic compartments and the comparison with Pcdhb22 implies differential localization and functions of individual beta-Pcdhs in the mammalian central nervous system. Moreover, we provide evidence for a role of beta-Pcdhs in the outer segments and connecting cilia of photoreceptors. Our data show for the first time that beta-Pcdhs also localize to specific neuronal subpopulations and synapses, providing support for the hypothesis that clustered Pcdhs are candidate genes for the specification of synaptic connectivity and neuronal networks.

    The European journal of neuroscience 2008;27;3;559-71

  • Interaction of brain somatostatin receptors with the PDZ domains of PSD-95.

    Christenn M, Kindler S, Schulz S, Buck F, Richter D and Kreienkamp HJ

    Institut für Zellbiochemie und klinische Neurobiologie, Martinistrasse 52, 20246 Hamburg, Germany.

    Using peptide affinity purification, we identified an interaction between somatostatin receptors SSTR4 and SSTR1 and PDZ domains 1 and 2 of the postsynaptic proteins postsynaptic density protein of 95kDa (PSD-95) and PSD-93. The existence of the SSTR4/PSD-95 complex was verified by coimmunoprecipitation from transfected cells and solubilized brain membranes. In neurons, dendritically localized SSTR4 partially colocalizes with postsynaptic PSD-95. As functional parameters of the receptor, such as coupling to potassium channels, are not affected by the interaction with PSD-95, the association may serve to localize the receptor to postsynaptic sites.

    FEBS letters 2007;581;27;5173-7

  • Defects in embryonic neurogenesis and initial synapse formation in the forebrain of the Ts65Dn mouse model of Down syndrome.

    Chakrabarti L, Galdzicki Z and Haydar TF

    Center for Neuroscience Research, Children's National Medical Center, Washington, DC 20010, USA.

    Trisomy 21, one of the most prevalent congenital birth defects, results in a constellation of phenotypes collectively termed Down syndrome (DS). Mental retardation and motor and sensory deficits are among the many debilitating symptoms of DS. Alterations in brain growth and synaptic development are thought to underlie the cognitive impairments in DS, but the role of early brain development has not been studied because of the lack of embryonic human tissue and because of breeding difficulties in mouse models of DS. We generated a breeding colony of the Ts65Dn mouse model of DS to test the hypothesis that early defects in embryonic brain development are a component of brain dysfunction in DS. We found substantial delays in prenatal growth of the Ts65Dn cerebral cortex and hippocampus because of longer cell cycle duration and reduced neurogenesis from the ventricular zone neural precursor population. In addition, the Ts65Dn neocortex remains hypocellular after birth and there is a lasting decrease in synaptic development beginning in the first postnatal week. These results demonstrate that specific abnormalities in embryonic forebrain precursor cells precede early deficits in synaptogenesis and may underlie the postnatal disabilities in Ts65Dn and DS. The early prenatal period is therefore an important new window for possible therapeutic amelioration of the cognitive symptoms in DS.

    Funded by: NICHD NIH HHS: P30HD40677, R01 HD057580

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;43;11483-95

  • Nicotine regulates multiple synaptic proteins by inhibiting proteasomal activity.

    Rezvani K, Teng Y, Shim D and De Biasi M

    Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.

    Ubiquitination regulates the degradation, membrane trafficking, and transcription of proteins. At mammalian synapses, the ubiquitin-proteasome system (UPS) influences synaptic transmission and plasticity. Nicotine also has the ability to affect synaptic function via mechanisms that remain partially unknown. We found that nicotine, at concentrations achieved by smokers, reduced proteasomal activity, produced accumulation of ubiquitinated synaptic proteins, and increased total protein levels. In particular, a 24 h exposure to nicotine decreased proteasome-dependent degradation of the alpha7 nicotinic acetylcholine receptor (nAChR) subunit, as indicated by the accumulation of ubiquitinated alpha7. The same nicotine treatment increased the levels of the AMPA glutamate receptor subunit GluR1, the NMDA receptor subunit NR2A, the metabotropic receptor mGluR1alpha, the plasticity factor Homer-1A, and the scaffolding postsynaptic density protein PSD-95, whereas the levels of another scaffolding protein, Shank, were reduced. These changes were associated with an inhibition of proteasomal chymotrypsin-like activity by nicotine. The nAChR antagonist mecamylamine was only partially able to block the effects of nicotine on the UPS, indicating that nAChR activation does not completely explain nicotine-induced inhibition of proteasomal catalytic activity. A competition binding assay suggested a direct interaction between nicotine and the 20S proteasome. These results suggest that the UPS might participate in nicotine-dependent synaptic plasticity.

    Funded by: NIDA NIH HHS: DA017173

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;39;10508-19

  • PX-RICS, a novel splicing variant of RICS, is a main isoform expressed during neural development.

    Hayashi T, Okabe T, Nasu-Nishimura Y, Sakaue F, Ohwada S, Matsuura K, Akiyama T and Nakamura T

    Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    In our previous study, we identified RICS, a novel beta-catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits.

    Genes to cells : devoted to molecular & cellular mechanisms 2007;12;8;929-39

  • BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation.

    Yoshii A and Constantine-Paton M

    Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, USA. ayoshii@mit.edu

    The N-methyl-D-aspartate receptor (NMDAR), brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95) and phosphatidylinositol 3-kinase (PI3K) have all been implicated in long-term potentiation. Here we show that these molecules are involved in a single pathway for synaptic potentiation. In visual cortical neurons in young rodents, the neurotrophin receptor TrkB is associated with PSD-95. When BDNF is applied to cultured visual cortical neurons, PSD-95-labeled synaptic puncta enlarge, and fluorescent recovery after photobleaching (FRAP) reveals increased delivery of green fluorescent protein-tagged PSD-95 to the dendrites. The recovery of fluorescence requires TrkB, signaling through PI3K and the serine-threonine kinase Akt, and an intact Golgi apparatus. Stimulation of NMDARs mimics the PSD-95 trafficking that is induced by BDNF but requires active BDNF and PI3K. Furthermore, local dendritic contact with a BDNF-coated microsphere induces PSD-95 FRAP throughout the dendrites of the stimulated neuron, suggesting that this mechanism induces rapid neuron-wide synaptic increases in PSD-95 and refinement whenever a few robust inputs activate the NMDAR-BDNF-PI3K pathway.

    Funded by: NEI NIH HHS: R01EY006039, R01EY014074

    Nature neuroscience 2007;10;6;702-11

  • Stage-specific association of apolipoprotein A-I and E in developing mouse retina.

    Kurumada S, Onishi A, Imai H, Ishii K, Kobayashi T and Sato SB

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

    Purpose: To characterize the intercellular lipid transport systems in differentiating retina.

    Methods: Developing mouse retinas were evaluated for the expression of apolipoproteins (apoE, apoA-I) by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). They were compared with changes in the lipid content and association of retinal proteins, such as postsynaptic density protein 95, glial fibrillary acidic protein, and cellular retinaldehyde-binding protein. Intraretinal distribution of apolipoproteins and their receptors was examined by immunofluorescence and in situ hybridization of prenatal and postnatal retinal sections. In vitro culture of dissociated cells was also examined.

    Results: Although apoE is known to be present in the mature retina, the neonatal retina remarkably expressed apoA-I mRNA and protein. This protein was present until postnatal day (P)3, and its putative receptor, scavenger receptor class B-I, was present until P5 to P7. This state subsequently exhibited a dramatic switchover to an apoE-rich one, in parallel with the stratification. Whereas apoE was synthesized at low levels until P7, apoE mRNA was clearly concentrated in Müller glia cells, which extended long apoE-bound processes to the plexuses and contours of photoreceptor cells. These acceptor cells expressed LDL receptor-related protein 1 as a putative receptor. ApoE genes were not transcribed in ganglion cells, though they were associated with a high level of the protein throughout the development. ApoE protein in ganglion cells initially appeared to be synthesized by astrocytes but later were observed to be supplied from an extraretinal space.

    Conclusions: The present results document several new aspects of apoA-I and apoE in the developing retina. The switchover of the lipoprotein systems runs a parallel course with the differentiation.

    Investigative ophthalmology & visual science 2007;48;4;1815-23

  • Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies.

    Cuthbert PC, Stanford LE, Coba MP, Ainge JA, Fink AE, Opazo P, Delgado JY, Komiyama NH, O'Dell TJ and Grant SG

    Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.

    Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. These in vivo data support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system.

    Funded by: NIMH NIH HHS: R01 MH060919, R01 MH060919-10; Wellcome Trust: 077155

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;10;2673-82

  • The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking.

    Bats C, Groc L and Choquet D

    Physiologie Cellulaire de la Synapse, UMR 5091 CNRS - Institut François Magendie, Université Bordeaux, Bordeaux 33077, France.

    Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an AMPAR auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate AMPAR number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin-PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.

    Neuron 2007;53;5;719-34

  • Synapse-specific regulation of AMPA receptor function by PSD-95.

    Béïque JC, Lin DT, Kang MG, Aizawa H, Takamiya K and Huganir RL

    Department of Neuroscience, Johns Hopkins University School of Medicine and Howard Hughes Medical Institute, Baltimore, MD 21205, USA.

    PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of "silent" synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDAR-mediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapse-specific role for PSD-95 in controlling synaptic function that is independent of spine morphology.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;51;19535-40

  • Redundant functions of RIM1alpha and RIM2alpha in Ca(2+)-triggered neurotransmitter release.

    Schoch S, Mittelstaedt T, Kaeser PS, Padgett D, Feldmann N, Chevaleyre V, Castillo PE, Hammer RE, Han W, Schmitz F, Lin W and Südhof TC

    Emmy Noether Research Group, Institute of Neuropathology, Department of Epileptology, University Bonn, Sigmund Freud Strasse 25, 53105 Bonn, Germany. susanne.schoch@uni-bonn.de

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

    The EMBO journal 2006;25;24;5852-63

  • Amyotrophic lateral sclerosis 2-deficiency leads to neuronal degeneration in amyotrophic lateral sclerosis through altered AMPA receptor trafficking.

    Lai C, Xie C, McCormack SG, Chiang HC, Michalak MK, Lin X, Chandran J, Shim H, Shimoji M, Cookson MR, Huganir RL, Rothstein JD, Price DL, Wong PC, Martin LJ, Zhu JJ and Cai H

    Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892-3707, USA.

    Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2(-/-) spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2(-/-) neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2(-/-) neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.

    Funded by: Intramural NIH HHS: Z01 AG000959-04, Z99 AG999999; NINDS NIH HHS: NS053570, NS34100, NS40014, NS52098, R01 NS034100, R01 NS040014, R01 NS052098, R01 NS053570

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;45;11798-806

  • Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability.

    Gavarini S, Bécamel C, Altier C, Lory P, Poncet J, Wijnholds J, Bockaert J and Marin P

    Centre National de la Recherche Scientifique Unité Mixte de Recherche 5203, Institut National de la Santé et de la Recherche Médicale, U661, Université Montpellier I, Université Montpellier II, Montpellier Cedex 5, France.

    PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.

    Molecular biology of the cell 2006;17;11;4619-31

  • Rapid redistribution of synaptic PSD-95 in the neocortex in vivo.

    Gray NW, Weimer RM, Bureau I and Svoboda K

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

    Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10-21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times tau(r) is approximately 22-63 min from P10-P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (tau(r) is approximately 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size.

    PLoS biology 2006;4;11;e370

  • Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins.

    Elias GM, Funke L, Stein V, Grant SG, Bredt DS and Nicoll RA

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.

    Trafficking of AMPA receptors (AMPA-Rs) to and from synapses controls the strength of excitatory synaptic transmission. However, proteins that cluster AMPA-Rs at synapses remain poorly understood. Here we show that PSD-95-like membrane-associated guanylate kinases (PSD-MAGUKs) mediate this synaptic targeting, and we uncover a remarkable functional redundancy within this protein family. By manipulating endogenous neuronal PSD-MAGUK levels, we find that both PSD-95 and PSD-93 independently mediate AMPA-R targeting at mature synapses. We also reveal unanticipated synapse heterogeneity as loss of either PSD-95 or PSD-93 silences largely nonoverlapping populations of excitatory synapses. In adult PSD-95 and PSD-93 double knockout animals, SAP-102 is upregulated and compensates for the loss of synaptic AMPA-Rs. At immature synapses, PSD-95 and PSD-93 play little role in synaptic AMPA-R clustering; instead, SAP-102 dominates. These studies establish a PSD-MAGUK-specific regulation of AMPA-R synaptic expression that establishes and maintains glutamatergic synaptic transmission in the mammalian central nervous system.

    Neuron 2006;52;2;307-20

  • PSD-95 is a negative regulator of the tyrosine kinase Src in the NMDA receptor complex.

    Kalia LV, Pitcher GM, Pelkey KA and Salter MW

    Division of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada.

    The tyrosine kinase Src upregulates the activity of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) and tyrosine phosphorylation of this receptor is critical for induction of NMDAR-dependent plasticity of synaptic transmission. A binding partner for Src within the NMDAR complex is the protein PSD-95. Here we demonstrate an interaction of PSD-95 with Src that does not require the well-characterized domains of PSD-95. Rather, we show binding to Src through a 12-amino-acid sequence in the N-terminal region of PSD-95, a region not previously known to participate in protein-protein interactions. This region interacts directly with the Src SH2 domain. Contrary to typical SH2 domain binding, the PSD-95-Src SH2 domain interaction is phosphotyrosine-independent. Binding of the Src-interacting region of PSD-95 inhibits Src kinase activity and reduces NMDAR phosphorylation. Intracellularly administering a peptide matching the Src SH2 domain-interacting region of PSD-95 depresses NMDAR currents in cultured neurons and inhibits induction of long-term potentiation in hippocampus. Thus, the PSD-95-Src SH2 domain interaction suppresses Src-mediated NMDAR upregulation, a finding that may be of broad importance for synaptic transmission and plasticity.

    The EMBO journal 2006;25;20;4971-82

  • Increased ethanol resistance and consumption in Eps8 knockout mice correlates with altered actin dynamics.

    Offenhäuser N, Castelletti D, Mapelli L, Soppo BE, Regondi MC, Rossi P, D'Angelo E, Frassoni C, Amadeo A, Tocchetti A, Pozzi B, Disanza A, Guarnieri D, Betsholtz C, Scita G, Heberlein U and Di Fiore PP

    Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milan, Italy. nina.offenhauser@ifom-ieo-campus.it

    Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

    Funded by: NIAAA NIH HHS: AA10035, AA13105

    Cell 2006;127;1;213-26

  • Reduced expression of neuropeptide genes in a genome-wide screen of a secretion-deficient mouse.

    Bouwman J, Spijker S, Schut D, Wächtler B, Ylstra B, Smit AB and Verhage M

    Department of Functional Genomics, Centre for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam (VUA) and VU Medical Centre (VUmc), Amsterdam, the Netherlands.

    Activity-dependent changes in synapses rely on functional changes in resident proteins and on gene expression. We addressed the relationship between synapse activity and the expression of synaptic genes by comparing RNA levels in the neocortex of normal mice versus secretion-deficient and therefore synaptically silent munc18-1 (mammalian homologue of Caenorhabditis elegans uncoordinated locomotion-18) null mutants, using microarray expression analysis, real-time quantitative PCR and northern blotting. We hypothesized that genes under the control of synaptic activity would be differentially expressed between mutants and controls. We found that few synaptic genes were differentially expressed. However, most neuropeptide genes with detectable expression on the microarray were differentially expressed, being expressed 3-20-fold higher in control cortex. Several other secreted proteins were also differentially expressed, but genes encoding their receptors and many other synaptic components were not. Differential expression was confirmed by real-time quantitative PCR analysis. In situ hybridization indicated that the difference in neuropeptide expression was uniform and not due to the loss of specific cells in the mutant. In primary sensory neurons, which do not depend on synaptic activity for their input, the differential expression of neuropeptides was not observed. These data argue against a general relationship between the activity of synapses and the expression of their resident proteins, but suggest a link between secretion and the expression of genes encoding the secreted products.

    Journal of neurochemistry 2006;99;1;84-96

  • Physical and functional interaction of Fyn tyrosine kinase with a brain-enriched Rho GTPase-activating protein TCGAP.

    Liu H, Nakazawa T, Tezuka T and Yamamoto T

    Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    Fyn, a member of the Src family of tyrosine kinases, is implicated in both brain development and adult brain function. In the present study, we identified a Rho GTPase-activating protein (GAP), TCGAP (Tc10/Cdc42 GTPase-activating protein), as a novel Fyn substrate. TCGAP interacted with Fyn and was phosphorylated by Fyn, with Tyr-406 in the GAP domain as a major Fyn-mediated phosphorylation site. Fyn suppressed the GAP activity of wild-type TCGAP but not the Y406F mutant of TCGAP in a phosphorylation-dependent manner, suggesting that Fyn-mediated Tyr-406 phosphorylation negatively regulated the TCGAP activity. In situ hybridization analyses showed that TCGAP mRNA was expressed prominently in both immature and adult mouse brain, with high levels in cortex, corpus striatum, hippocampus, and olfactory bulb. Overexpression of wild-type TCGAP in PC12 cells suppressed nerve growth factor-induced neurite outgrowth, whereas a GAP-defective mutant of TCGAP enhanced the neurite outgrowth. Nerve growth factor enhanced tyrosine phosphorylation of TCGAP through activation of Src family kinases. These results suggest that TCGAP is involved in Fyn-mediated regulation of axon and dendrite outgrowth.

    The Journal of biological chemistry 2006;281;33;23611-9

  • Signaling across the synapse: a role for Wnt and Dishevelled in presynaptic assembly and neurotransmitter release.

    Ahmad-Annuar A, Ciani L, Simeonidis I, Herreros J, Fredj NB, Rosso SB, Hall A, Brickley S and Salinas PC

    Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, England, UK.

    Proper dialogue between presynaptic neurons and their targets is essential for correct synaptic assembly and function. At central synapses, Wnt proteins function as retrograde signals to regulate axon remodeling and the accumulation of presynaptic proteins. Loss of Wnt7a function leads to defects in the localization of presynaptic markers and in the morphology of the presynaptic axons. We show that loss of function of Dishevelled-1 (Dvl1) mimics and enhances the Wnt7a phenotype in the cerebellum. Although active zones appear normal, electrophysiological recordings in cerebellar slices from Wnt7a/Dvl1 double mutant mice reveal a defect in neurotransmitter release at mossy fiber-granule cell synapses. Deficiency in Dvl1 decreases, whereas exposure to Wnt increases, synaptic vesicle recycling in mossy fibers. Dvl increases the number of Bassoon clusters, and like other components of the Wnt pathway, it localizes to synaptic sites. These findings demonstrate that Wnts signal across the synapse on Dvl-expressing presynaptic terminals to regulate synaptic assembly and suggest a potential novel function for Wnts in neurotransmitter release.

    Funded by: Wellcome Trust: 078764

    The Journal of cell biology 2006;174;1;127-39

  • The cytoplasmic domain of NrCAM binds to PDZ domains of synapse-associated proteins SAP90/PSD95 and SAP97.

    Dirks P, Thomas U and Montag D

    Neurogenetics Research Group, Leibniz Institute for Neurobiology, Brenneckestr. 6, D-39118 Magdeburg, Germany.

    NrCAM, a member of the L1 family of cell adhesion molecules, serves important functions during the development of the nervous system, e.g. in adhesion-dependent processes such as neurite outgrowth and axonal pathfinding. Complex homo- and heterophilic binding and several extracellular ligands of NrCAM have been described, but less is known about intracellular interaction partners. The cytoplasmic carboxy-terminus of NrCAM contains a typical sequence motif for binding to PDZ domains, making interactions with PDZ domain-containing scaffolding proteins quite conceivable. In this study, we identified specific interactions of the intracellular domain of NrCAM with class I PDZ domains of the membrane-associated guanylate kinases SAP90/PSD95 and SAP97. In contrast to NrCAM, the intracellular domains of the other mammalian L1 family molecules, e.g. L1, CHL1 and Neurofascin, did not interact with these PDZ domains. In transfected COS-7 cells, NrCAM-mediated recruitment of SAP97 to the plasma membrane was dependent on the PDZ binding motif. We show that NrCAM and SAP97 are colocalized, e.g. within photoreceptor terminals of the mammalian retina. In summary, our results confirm a functional PDZ domain binding motif at the carboxy-terminus of NrCAM and support potential functions of NrCAM during the assembly of highly organized protein complexes at the cell membrane.

    The European journal of neuroscience 2006;24;1;25-31

  • Expression and distribution of JNK/SAPK-associated scaffold protein JSAP1 in developing and adult mouse brain.

    Miura E, Fukaya M, Sato T, Sugihara K, Asano M, Yoshioka K and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.

    The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.

    Journal of neurochemistry 2006;97;5;1431-46

  • Neurone specific regulation of dendritic spines in vivo by post synaptic density 95 protein (PSD-95).

    Vickers CA, Stephens B, Bowen J, Arbuthnott GW, Grant SG and Ingham CA

    Department of Pre-Clinical Veterinary Sciences, (RDSVS) Summerhall, University of Edinburgh, Edinburgh. EH9 1QH, UK. Cathy.Vickers@ed.ac.uk

    Post synaptic density protein 95 (PSD-95) is a postsynaptic adaptor protein coupling the NMDA receptor to downstream signalling pathways underlying plasticity. Mice carrying a targeted gene mutation of PSD-95 show altered behavioural plasticity including spatial learning, neuropathic pain, orientation preference in visual cortical cells, and cocaine sensitisation. These behavioural effects are accompanied by changes in long-term potentiation of synaptic transmission. In vitro studies of PSD-95 signalling indicate that it may play a role in regulating dendritic spine structure. Here, we show that PSD-95 mutant mice have alterations in dendritic spine density in the striatum (a 15% decrease along the dendritic length) and in the hippocampus (a localised 40% increase) without changes in dendritic branch patterns or gross neuronal architecture. These changes in spine density were accompanied by altered expression of proteins known to interact with PSD-95, including NR2B and SAP102, suggesting that PSD-95 plays a role in regulating the expression and activation of proteins found within the NMDA receptor complex. Thus, PSD-95 is an important regulator of neuronal structure as well as plasticity in vivo.

    Funded by: Wellcome Trust

    Brain research 2006;1090;1;89-98

  • Compensation by tumor suppressor genes during retinal development in mice and humans.

    Donovan SL, Schweers B, Martins R, Johnson D and Dyer MA

    Department of Developmental Neurobiology, St, Jude Children's Research Hospital, Memphis, TN 38105, USA. stacy.donovan@stjude.org

    Background: The RB1 gene was the first tumor suppressor gene cloned from humans by studying genetic lesions in families with retinoblastoma. Children who inherit one defective copy of the RB1 gene have an increased susceptibility to retinoblastoma. Several years after the identification of the human RB1 gene, a targeted deletion of Rb was generated in mice. Mice with one defective copy of the Rb gene do not develop retinoblastoma. In this manuscript, we explore the different roles of the Rb family in human and mouse retinal development in order to better understand the species-specific difference in retinoblastoma susceptibility.

    Results: We found that the Rb family of proteins (Rb, p107 and p130) are expressed in a dynamic manner during mouse retinal development. The primary Rb family member expressed in proliferating embryonic retinal progenitor cells in mice is p107, which is required for appropriate cell cycle exit during retinogenesis. The primary Rb family member expressed in proliferating postnatal retinal progenitor cells is Rb. p130 protein is expressed redundantly with Rb in postmitotic cells of the inner nuclear layer and the ganglion cell layer of the mouse retina. When Rb is inactivated in an acute or chronic manner during mouse retinal development, p107 is upregulated in a compensatory manner. Similarly, when p107 is inactivated in the mouse retina, Rb is upregulated. No changes in p130 expression were seen when p107, Rb or both were inactivated in the developing mouse retina. In the human retina, RB1 was the primary family member expressed throughout development. There was very little if any p107 expressed in the developing human retina. In contrast to the developing mouse retina, when RB1 was acutely inactivated in the developing human fetal retina, p107 was not upregulated in a compensatory manner.

    Conclusion: We propose that intrinsic genetic compensation between Rb and p107 prevents retinoblastoma in Rb- or p107-deficient mice, but this compensation does not occur in humans. Together, these data suggest a model that explains why humans are susceptible to retinoblastoma following RB1 loss, but mice require both Rb and p107 gene inactivation.

    BMC biology 2006;4;14

  • Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

    Uchino S, Wada H, Honda S, Nakamura Y, Ondo Y, Uchiyama T, Tsutsumi M, Suzuki E, Hirasawa T and Kohsaka S

    Department of Neurochemistry, National Institute of Neuroscience, Kodaira, Tokyo, Japan.

    A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.

    Journal of neurochemistry 2006;97;4;1203-14

  • Mpp4 recruits Psd95 and Veli3 towards the photoreceptor synapse.

    Aartsen WM, Kantardzhieva A, Klooster J, van Rossum AG, van de Pavert SA, Versteeg I, Cardozo BN, Tonagel F, Beck SC, Tanimoto N, Seeliger MW and Wijnholds J

    Department of Neuromedical Genetics, The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands.

    Membrane-associated guanylate kinase (MAGUK) proteins function as scaffold proteins contributing to cell polarity and organizing signal transducers at the neuronal synapse membrane. The MAGUK protein Mpp4 is located in the retinal outer plexiform layer (OPL) at the presynaptic plasma membrane and presynaptic vesicles of photoreceptors. Additionally, it is located at the outer limiting membrane (OLM) where it might be involved in OLM integrity. In Mpp4 knockout mice, loss of Mpp4 function only sporadically causes photoreceptor displacement, without changing the Crumbs (Crb) protein complex at the OLM, adherens junctions or synapse structure. Scanning laser ophthalmology revealed no retinal degeneration. The minor morphological effects suggest that Mpp4 is a candidate gene for mild retinopathies only. At the OPL, Mpp4 is essential for correct localization of Psd95 and Veli3 at the presynaptic photoreceptor membrane. Psd95 labeling is absent of presynaptic membranes in both rods and cones but still present in cone basal contacts and dendritic contacts. Total retinal Psd95 protein levels are significantly reduced which suggests Mpp4 to be involved in Psd95 turnover, whereas Veli3 proteins levels are not changed. These protein changes in the photoreceptor synapse did not result in an altered electroretinograph. These findings suggest that Mpp4 coordinates Psd95/Veli3 assembly and maintenance at synaptic membranes. Mpp4 is a critical recruitment factor to organize scaffolds at the photoreceptor synapse and is likely to be associated with synaptic plasticity and protein complex transport.

    Human molecular genetics 2006;15;8;1291-302

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Maternal stress produces learning deficits associated with impairment of NMDA receptor-mediated synaptic plasticity.

    Son GH, Geum D, Chung S, Kim EJ, Jo JH, Kim CM, Lee KH, Kim H, Choi S, Kim HT, Lee CJ and Kim K

    School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.

    Stress in adulthood can have a profound effect on physiology and behavior, but the extent to which prolonged maternal stress affects the brain function of offspring when they are adult remains primarily unknown. In the present work, chronic immobilization stress to pregnant mice affected fetal growth and development. When pups born from stressed mice were reared to adulthood in an environment identical to that of nonstressed controls, several physiological parameters were essentially unaltered. However, spatial learning and memory was significantly impaired in the maternally stressed offspring in adulthood. Furthermore, electrophysiological examination revealed a significant reduction in NMDA receptor-mediated long-term potentiation in the CA1 area of hippocampal slices. Subsequent biochemical analysis demonstrated a substantial decrease in NR1 and NR2B subunits of the NMDA receptor in synapses of the hippocampus, and the interaction between these two subunits appeared to be reduced. These results suggest that prolonged maternal stress leads to long-lasting malfunction of the hippocampus, which extends to and is manifested in adulthood.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;12;3309-18

  • Regulation of dendritic branching and spine maturation by semaphorin3A-Fyn signaling.

    Morita A, Yamashita N, Sasaki Y, Uchida Y, Nakajima O, Nakamura F, Yagi T, Taniguchi M, Usui H, Katoh-Semba R, Takei K and Goshima Y

    Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.

    A member of semaphorin family, semaphorin3A (Sema3A), acts as a chemorepellent or chemoattractant on a wide variety of axons and dendrites in the development of the nervous systems. We here show that Sema3A induces clustering of both postsynaptic density-95 (PSD-95) and presynaptic synapsin I in cultured cortical neurons without changing the density of spines or filopodia. Neuropilin-1 (NRP-1), a receptor for Sema3A, is present on both axons and dendrites. When the cultured neurons are exposed to Sema3A, the cluster size of PSD-95 is markedly enhanced, and an extensive colocalization of PSD-95 and NRP-1 or actin-rich protrusion is seen. The effects of Sema3A on spine morphology are blocked by PP2, an Src type tyrosine kinase inhibitor, but not by the PP3, the inactive-related compound. In the cultured cortical neurons from fyn(-/-) mice, dendrites bear few spines, and Sema3A does not induce PSD-95 cluster formation on the dendrites. Sema3A and its receptor genes are highly expressed during the synaptogenic period of postnatal days 10 and 15. The cortical neurons in layer V, but not layer III, show a lowered density of synaptic bouton-like structure on dendrites in sema3A- and fyn-deficient mice. The neurons of the double-heterozygous mice show the lowered spine density, whereas those of single heterozygous mice show similar levels of the spine density as the wild type. These findings suggest that the Sema3A signaling pathway plays an important role in the regulation of dendritic spine maturation in the cerebral cortex neurons.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;11;2971-80

  • A novel family of adhesion-like molecules that interacts with the NMDA receptor.

    Wang CY, Chang K, Petralia RS, Wang YX, Seabold GK and Wenthold RJ

    Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027, USA.

    We have identified a novel family of synaptic adhesion-like molecules (SALMs). The family members, SALM1-SALM4, have a single transmembrane (TM) domain and contain extracellular leucine-rich repeats, an Ig C2 type domain, a fibronectin type III domain, and an intracellular postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 (PDZ) binding domain, which is present on all members except SALM4. SALM1 interacts with PSD-95, synapse-associated protein 102 (SAP102), and SAP97 based on coimmunoprecipitation of detergent-solubilized brain. Distribution studies show that SALM1 is present in synaptic membrane and postsynaptic density fractions but is also distributed in axons and dendrites. Transfection of hippocampal neurons for 4 d in vitro (DIV) with SALM1 more than doubles the dendritic lengths of neurons after 48 h, whereas transfection of neurons 14 DIV has no significant effect on neurite outgrowth. Overexpression of SALM1 in 14 DIV neurons recruits NMDA receptors (NR) and PSD-95 to dendritic puncta. This effect is dependent on the PDZ-binding domain of SALM1. SALM1 also enhances surface expression of transfected NR2A subunit. Immunoprecipitation of detergent-solubilized brain membranes with anti-SALM1 antibodies shows coimmunoprecipitation of NR1 and NR2 subunits. After transfection of heterologous cells with NR1 and NR2 cDNAs, through coimmunoprecipitation analyses, we find that SALM1 also interacts with the NMDA receptor NR1 subunit through its extracellular or TM1 domains.

    Funded by: Intramural NIH HHS

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;8;2174-83

  • Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors.

    Cai C, Li H, Rivera C and Keinänen K

    Department of Biological and Environmental Sciences, University of Helsinki, Finland.

    Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.

    The Journal of biological chemistry 2006;281;7;4267-73

  • Apolipoprotein E receptor 2 interactions with the N-methyl-D-aspartate receptor.

    Hoe HS, Pocivavsek A, Chakraborty G, Fu Z, Vicini S, Ehlers MD and Rebeck GW

    Department of Neuroscience and Physiology, Georgetown University Medical Center, Washington, DC 20057-1464, USA.

    In our previous studies we showed that apoE treatment of neurons activated ERK 1/2 signaling, and activation was blocked by treatment with inhibitors of the low density lipoprotein receptor family, the N-methyl-d-aspartate (NMDA) receptor antagonist MK 801, and calcium channel blockers. We hypothesized an interaction between the low density lipoprotein receptor family members and the NMDA receptor. In the present study, we confirmed through co-immunoprecipitation experiments an interaction between the apoE receptor, ApoEr2, and NMDAR1 through their extracellular domains. We also found that the PDZ1 domain of PSD95, a postsynaptic scaffolding protein, interacted with the C terminus of ApoEr2 via an alternatively spliced, intracellular exon. This interaction between ApoEr2 and PSD95 in neurons was modulated by NMDA receptor activation and an ApoEr2 ligand. We also found that the PDZ2 domain of PSD95 interacted with the NR2A and NR2B subunits of NMDA receptors. Full-length PSD95 increased cell surface levels of ApoEr2 and its cleavage, resulting in increases in secreted ApoEr2 and C-terminal fragments of ApoEr2. These studies suggest that ApoEr2 can form a multiprotein complex with NMDA receptor subunits and PSD95.

    The Journal of biological chemistry 2006;281;6;3425-31

  • Essential contribution of the ligand-binding beta B/beta C loop of PDZ1 and PDZ2 in the regulation of postsynaptic clustering, scaffolding, and localization of postsynaptic density-95.

    Nonaka M, Doi T, Fujiyoshi Y, Takemoto-Kimura S and Bito H

    Department of Biophysics, Kyoto University Graduate School of Science, Kyoto 606-8502, Japan.

    Postsynaptic density-95 (PSD-95), a PSD-95/Discs large/zona occludens-1 (PDZ) domain-containing scaffold protein, clusters many signaling molecules near NMDA-type glutamate receptors in the postsynaptic densities. Although the synaptic localization of PSD-95 requires palmitoylation of two cysteines at the N terminus and the presence of at least one PDZ domain, how the clustering of PSD-95 is initiated and regulated remains essentially unknown. To address this issue, we examined PSD-95 clustering in primary cultured hippocampal neurons expressing full-length PSD-95 mutant proteins lacking the ligand-binding ability of PDZ1, PDZ2, and/or PDZ3. The formation of either excitatory or inhibitory synapses was unaffected. Combinations of individual mutations, however, significantly reduced the PSD-95 clustering index, in an approximately additive manner. The sensitivity to 2-bromo-palmitate and latrunculin A, reagents known to affect PSD-95 turnover, was also augmented. Furthermore, the synaptic recruitment of a PSD-95 ligand, synaptic GTPase-activating protein (synGAP), was significantly impaired, whereas the clustering of other scaffolding proteins, such as Homer 1c, Shank/Synamon, and PSD-93/Chapsin-110 was spared. Intriguingly, overexpression of the PSD-95 PDZ1/2/3 mutants caused the PSD-95 clusters to localize away from the dendritic shaft, resulting in the formation of elongated spines, in an inverse correlation with the overall PDZ-ligand affinity. Expression of a mutant synGAP lacking the PDZ-binding motif replicated both the clustering and spine morphology phenotypes. In conclusion, the ligand-binding affinity of the PDZ domains of PSD-95, contributed in part via its interaction with the C-terminal end of synGAP, plays a critical role in titrating the synaptic clustering of PSD-95 and controlling its tight association with the PSD scaffold, thereby affecting synapse maturation.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;3;763-74

  • Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses.

    Almeida CG, Tampellini D, Takahashi RH, Greengard P, Lin MT, Snyder EM and Gouras GK

    Department of Neurology and Neuroscience, Laboratory of Alzheimer's Disease Neurobiology, Weill Medical College of Cornell University, 525 E 68th Street, NY 10021, USA.

    Synaptic dysfunction is increasingly viewed as an early manifestation of Alzheimer's disease (AD), but the cellular mechanism by which beta-amyloid (Abeta) may affect synapses remains unclear. Since cultured neurons derived from APP mutant transgenic mice secrete elevated levels of Abeta and parallel the subcellular Abeta accumulation seen in vivo, we asked whether alterations in synapses occur in this setting. We report that cultured Tg2576 APP mutant neurons have selective alterations in pre- and post-synaptic compartments compared to wild-type neurons. Post-synaptic compartments appear fewer in number and smaller, while active pre-synaptic compartments appear fewer in number and enlarged. Among the earliest changes in synaptic composition in APP mutant neurons were reductions in PSD-95, a protein involved in recruiting and anchoring glutamate receptor subunits to the post-synaptic density. In agreement, we observed early reductions in surface expression of glutamate receptor subunit GluR1 in APP mutant neurons. We provide evidence that Abeta is specifically involved in these alterations in synaptic biology, since alterations in PSD-95 and GluR1 are blocked by gamma-secretase inhibition, and since exogenous addition of synthetic Abeta to wild-type neurons parallels changes in synaptic PSD-95 and GluR1 observed in APP mutant neurons.

    Funded by: NIA NIH HHS: AG09464; NINDS NIH HHS: NS002037, NS045677

    Neurobiology of disease 2005;20;2;187-98

  • Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex.

    Olsen O, Moore KA, Fukata M, Kazuta T, Trinidad JC, Kauer FW, Streuli M, Misawa H, Burlingame AL, Nicoll RA and Bredt DS

    Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

    Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.

    The Journal of cell biology 2005;170;7;1127-34

  • Molecular constituents of neuronal AMPA receptors.

    Fukata Y, Tzingounis AV, Trinidad JC, Fukata M, Burlingame AL, Nicoll RA and Bredt DS

    Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

    Dynamic regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.

    Funded by: NIMH NIH HHS: R01 MH070957, R56 MH070957

    The Journal of cell biology 2005;169;3;399-404

  • Cellular and subcellular localization of EFA6C, a third member of the EFA6 family, in adult mouse Purkinje cells.

    Matsuya S, Sakagami H, Tohgo A, Owada Y, Shin HW, Takeshima H, Nakayama K, Kokubun S and Kondo H

    Division of Histology, Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai, Japan.

    EFA6C is a third member of the EFA6 family of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor 6 (ARF6). In this study, we first demonstrated that EFA6C indeed activated ARF6 more selectively than ARF1 by ARF pull-down assay. In situ hybridization histochemistry revealed that EFA6C mRNA was expressed predominantly in mature Purkinje cells and the epithelial cells of the choroid plexus in contrast to the ubiquitous expression of ARF6 mRNA throughout the brain. EFA6C mRNA was already detectable in the Purkinje cells at embryonic day 13, increased progressively during post-natal development and peaked during post-natal second week. In Purkinje cells, the immunoreactivity for EFA6C was localized particularly in the post-synaptic density as well as the plasma membranes of the cell somata, dendritic shafts and spines, while the immunoreactivity in their axon terminals in the deep cerebellar nuclei was very faint. These findings suggest that EFA6C may be involved in the regulation of the membrane dynamics of the somatodendritic compartments of Purkinje cells through the activation of ARF6.

    Journal of neurochemistry 2005;93;3;674-85

  • Phosphorylation state of postsynaptic density proteins.

    Trinidad JC, Thalhammer A, Specht CG, Schoepfer R and Burlingame AL

    Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

    The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.

    Funded by: NCRR NIH HHS: RR01614, RR14606

    Journal of neurochemistry 2005;92;6;1306-16

  • Control of excitatory and inhibitory synapse formation by neuroligins.

    Chih B, Engelman H and Scheiffele P

    Department of Physiology and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA.

    The normal function of neural networks depends on a delicate balance between excitatory and inhibitory synaptic inputs. Synapse formation is thought to be regulated by bidirectional signaling between pre- and postsynaptic cells. We demonstrate that members of the Neuroligin family promote postsynaptic differentiation in cultured rat hippocampal neurons. Down-regulation of neuroligin isoform expression by RNA interference results in a loss of excitatory and inhibitory synapses. Electrophysiological analysis revealed a predominant reduction of inhibitory synaptic function. Thus, neuroligins control the formation and functional balance of excitatory and inhibitory synapses in hippocampal neurons.

    Science (New York, N.Y.) 2005;307;5713;1324-8

  • Hippocampal synaptic modulation by the phosphotyrosine adapter protein ShcC/N-Shc via interaction with the NMDA receptor.

    Miyamoto Y, Chen L, Sato M, Sokabe M, Nabeshima T, Pawson T, Sakai R and Mori N

    Department of Molecular Genetics, National Institute for Longevity Sciences, Oobu 474-8522, Japan.

    N-Shc (neural Shc) (also ShcC), an adapter protein possessing two phosphotyrosine binding motifs [PTB (phosphotyrosine binding) and SH2 (Src homology 2) domains], is predominantly expressed in mature neurons of the CNS and transmits neurotrophin signals from the TrkB receptor to the Ras/mitogen-activated protein kinase (MAPK) pathway, leading to cellular growth, differentiation, or survival. Here, we demonstrate a novel role of ShcC, the modulation of NMDA receptor function in the hippocampus, using ShcC gene-deficient mice. In behavioral analyses such as the Morris water maze, contextual fear conditioning, and novel object recognition tasks, ShcC mutant mice exhibited superior ability in hippocampus-dependent spatial and nonspatial learning and memory. Consistent with this finding, electrophysiological analyses revealed that hippocampal long-term potentiation in ShcC mutant mice was significantly enhanced, with no alteration of presynaptic function, and the effect of an NMDA receptor antagonist on its expression in the mutant mice was notably attenuated. The tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B was also increased, suggesting that ShcC mutant mice have enhanced NMDA receptor function in the hippocampus. These results indicate that ShcC not only mediates TrkB-Ras/MAPK signaling but also is involved in the regulation of NMDA receptor function in the hippocampus via interaction with phosphotyrosine residues on the receptor subunits and serves as a modulator of hippocampal synaptic plasticity underlying learning and memory.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;7;1826-35

  • A role for myosin VI in postsynaptic structure and glutamate receptor endocytosis.

    Osterweil E, Wells DG and Mooseker MS

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA. emily.osterweil@yale.edu

    Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.

    Funded by: NIDDK NIH HHS: DK 25387, P01 DK055389, P01-DK55389, R01 DK025387, R37 DK025387, R56 DK025387; NINDS NIH HHS: K12 NS066274, NS66274-01

    The Journal of cell biology 2005;168;2;329-38

  • Analyses of murine postsynaptic density-95 identify novel isoforms and potential translational control elements.

    Bence M, Arbuckle MI, Dickson KS and Grant SG

    Division of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, UK.

    Postsynaptic density-95 (PSD-95) is an evolutionarily conserved synaptic adaptor protein that is known to bind many proteins including the NMDA receptor. This observation has implicated it in many NMDA receptor-dependent processes including spatial learning and synaptic plasticity. We have cloned and characterised the murine PSD-95 gene. In addition, we have identified two previously uncharacterised splice variants of the major murine PSD-95 transcript (PSD-95alpha): PSD-95alpha-2b results from an extension of exon 2 and PSD-95alpha-Delta18 from the temporal exclusion of exon 18. The presence of PSD-95alpha-2b sequences in other PSD-95 family members implicates this peptide stretch as functionally significant. Another potential transcript (PSD-95gamma) was also identified based on examination of EST databases. Immunoprecipitation assays demonstrate that proteins corresponding in size to PSD-95alpha-Delta18 and PSD-95gamma interact with the NMDA receptor, suggesting an important biological role for these isoforms. Finally, we have performed bioinformatics analyses of the PSD-95 mRNA untranslated regions, identifying multiple translational control elements that suggest protein production could be regulated post-transcriptionally. The variety of mRNA isoforms and regulatory elements identified provides for a high degree of diversity in the structure and function of PSD-95 proteins.

    Brain research. Molecular brain research 2005;133;1;143-52

  • Direct interaction of myosin regulatory light chain with the NMDA receptor.

    Amparan D, Avram D, Thomas CG, Lindahl MG, Yang J, Bajaj G and Ishmael JE

    Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, USA.

    NMDA receptors interact with a variety of intracellular proteins at excitatory synapses. In this paper we show that myosin regulatory light chain (RLC) isolated from mouse brain is a NMDA receptor-interacting protein. Myosin RLC bound directly to the C-termini of both NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) subunits, rendering the interaction of myosin RLC with NMDA receptors distinct from that of calmodulin which is considered a NR1-interacting protein. Myosin RLC co-localized with NR1 in the dendritic spines of isolated hippocampal neurons, and was co-immunoprecipitated from brain extracts in a complex with NR1, NR2A, NR2B, PSD-95, Adaptor protein-2 and myosin II heavy chain. The C0 region of NR1 was necessary and sufficient for binding myosin RLC. Ca2+/calmodulin, but not calmodulin alone, displaced recombinant myosin RLC from the carboxy tail of NR1 indicating that myosin RLC and Ca2+/calmodulin can compete for a common binding site on NR1 in vitro. Myosin RLC is the only known substrate for myosin regulatory light chain kinase, which has recently been shown to modulate NMDA receptor function in isolated hippocampal neurons. Our results suggest that an additional level of NMDA receptor regulation may be mediated via a direct interaction with a light chain of myosin II. Thus, myosin RLC-NMDA receptor interactions may contribute to the contractile and motile forces that are placed upon NMDA receptor subunits during changes associated with synaptic plasticity and neural morphogenesis.

    Funded by: NIEHS NIH HHS: ES00341, P30 ES00210

    Journal of neurochemistry 2005;92;2;349-61

  • Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins.

    Huang K, Yanai A, Kang R, Arstikaitis P, Singaraja RR, Metzler M, Mullard A, Haigh B, Gauthier-Campbell C, Gutekunst CA, Hayden MR and El-Husseini A

    Department of Psychiatry, Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

    In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.

    Neuron 2004;44;6;977-86

  • Identification of PSD-95 palmitoylating enzymes.

    Fukata M, Fukata Y, Adesnik H, Nicoll RA and Bredt DS

    Department of Physiology, University of California at San Francisco, San Francisco, California 94143, USA.

    Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.

    Neuron 2004;44;6;987-96

  • A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin.

    Prange O, Wong TP, Gerrow K, Wang YT and El-Husseini A

    Department of Psychiatry, Brain Research Centre, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

    Factors that control differentiation of presynaptic and postsynaptic elements into excitatory or inhibitory synapses are poorly defined. Here we show that the postsynaptic density (PSD) proteins PSD-95 and neuroligin-1 (NLG) are critical for dictating the ratio of excitatory-to-inhibitory synaptic contacts. Exogenous NLG increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory synaptic currents. In contrast, PSD-95 overexpression enhanced excitatory synapse size and miniature frequency, but reduced the number of inhibitory synaptic contacts. Introduction of PSD-95 with NLG augmented synaptic clustering of NLG and abolished NLG effects on inhibitory synapses. Interfering with endogenous PSD-95 expression alone was sufficient to reduce the ratio of excitatory-to-inhibitory synapses. These findings elucidate a mechanism by which the amounts of specific elements critical for synapse formation control the ratio of excitatory-to-inhibitory synaptic input.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;38;13915-20

  • Axon fasciculation defects and retinal dysplasias in mice lacking the immunoglobulin superfamily adhesion molecule BEN/ALCAM/SC1.

    Weiner JA, Koo SJ, Nicolas S, Fraboulet S, Pfaff SL, Pourquié O and Sanes JR

    Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA. joshua-weiner@uiowa.edu

    The immunoglobulin superfamily adhesion molecule BEN (other names include ALCAM, SC1, DM-GRASP, neurolin, and CD166) has been implicated in the control of numerous developmental and pathological processes, including the guidance of retinal and motor axons to their targets. To test hypotheses about BEN function, we disrupted its gene via homologous recombination and analyzed the resulting mutant mice. Mice lacking BEN are viable and fertile, and display no external morphological defects. Despite grossly normal trajectories, both motor and retinal ganglion cell axons fasciculated poorly and were occasionally misdirected. In addition, BEN mutant retinae exhibited evaginated or invaginated regions with photoreceptor ectopias that resembled the "retinal folds" observed in some human retinopathies. Together, these results demonstrate that BEN promotes fasciculation of multiple axonal populations and uncover an unexpected function for BEN in retinal histogenesis.

    Molecular and cellular neurosciences 2004;27;1;59-69

  • Genomic analysis of mouse retinal development.

    Blackshaw S, Harpavat S, Trimarchi J, Cai L, Huang H, Kuo WP, Weber G, Lee K, Fraioli RE, Cho SH, Yung R, Asch E, Ohno-Machado L, Wong WH and Cepko CL

    Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.

    The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs") were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.

    Funded by: NCI NIH HHS: P20 CA096470, P20 CA96470; NEI NIH HHS: EY08064, R01 EY008064

    PLoS biology 2004;2;9;E247

  • NMDA receptor GluRepsilon/NR2 subunits are essential for postsynaptic localization and protein stability of GluRzeta1/NR1 subunit.

    Abe M, Fukaya M, Yagi T, Mishina M, Watanabe M and Sakimura K

    Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan.

    In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2A and GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-C2') cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRzeta1-C2 and GluRzeta1-C2'. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 +/- 3.3% for GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-C1) was, although low in cerebellar content, also reduced to 12.7 +/- 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;33;7292-304

  • The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins.

    Bécamel C, Gavarini S, Chanrion B, Alonso G, Galéotti N, Dumuis A, Bockaert J and Marin P

    UPR CNRS 2580 and CNRS UMR 5101, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France.

    The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.

    The Journal of biological chemistry 2004;279;19;20257-66

  • Environmental enrichment results in cortical and subcortical changes in levels of synaptophysin and PSD-95 proteins.

    Nithianantharajah J, Levis H and Murphy M

    Department of Anatomy and Cell Biology, University of Melbourne, Melbourne, Victoria 3010, Australia.

    Experience-dependent plasticity is thought to involve selective change in pre-existing brain circuits, involving synaptic plasticity. One model for looking at experience-dependent plasticity is environmental enrichment (EE), where animals are exposed to a complex novel environment. Previous studies using electron microscopy showed that EE resulted in synaptic plasticity in the visual cortex and hippocampus. However, the areas in the brain that have been examined following EE have been limited. The present study quantified potential synaptic plasticity throughout the brains of C57BL/6 mice using an enzyme-linked immunosorbent assay (ELISA) for two synaptic proteins, synaptophysin and PSD-95. EE resulted in increased synaptophysin and PSD-95 levels through major brain regions, including anterior and posterior areas of the forebrain, hippocampus, thalamus, and hypothalamus. However, no changes in synaptophysin were detected in the cerebellum. These results demonstrate that EE results in an increase in levels of both pre- and post-synaptic proteins in multiple regions of the brain, and it is possible that such changes represent the underlying synaptic plasticity occurring in EE.

    Neurobiology of learning and memory 2004;81;3;200-10

  • Normal development and fertility of knockout mice lacking the tumor suppressor gene LRP1b suggest functional compensation by LRP1.

    Marschang P, Brich J, Weeber EJ, Sweatt JD, Shelton JM, Richardson JA, Hammer RE and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family. At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling. Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth. In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors. LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis. Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species. Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b. In addition, we have found several potential ligands that bind to the extracellular domain. Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.

    Funded by: NHLBI NIH HHS: HL20948, HL63762, P01 HL020948, R01 HL063762, R37 HL063762; NINDS NIH HHS: NS43408, R01 NS043408

    Molecular and cellular biology 2004;24;9;3782-93

  • Differential mRNA expression and protein localization of the SAP90/PSD-95-associated proteins (SAPAPs) in the nervous system of the mouse.

    Welch JM, Wang D and Feng G

    Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The supramolecular anchoring/signaling complex at the postsynaptic density of glutamatergic synapses has been proposed to play a key role in regulating synaptic function and plasticity. One class of proteins present in the complex is the SAP90/PSD-95-associated protein family (SAPAPs). The SAPAPs, identified by their direct interaction with PSD-95 family proteins, were initially proposed to function in the anchoring/signaling complex as linker proteins between glutamate receptor binding proteins and the cytoskeleton. However, recent studies have indicated that the SAPAPs also bind to signaling molecules and may thus have multiple roles at synapses. Four homologous genes encoding SAPAP proteins have been previously identified. As a first step toward understanding the physiological function of the SAPAPs, we have investigated in detail, at both the mRNA and protein levels, the localization of the individual SAPAP genes in the adult murine nervous system. We find that the SAPAP mRNAs are highly, yet differentially, expressed in many regions of the brain, including the hippocampus and cerebellum. Furthermore, SAPAP3 mRNA is targeted to dendrites, whereas SAPAP1, -2, and -4 mRNAs are detected mainly in cell bodies. The SAPAP proteins are localized at synapses in a manner consistent with mRNA expression. Surprisingly, in addition to glutamatergic synapse localization, antibody staining also reveals that the SAPAP proteins are localized at cholinergic synapses, including neuronal cholinergic synapses and the neuromuscular junction. Together, these results indicate that the SAPAPs are general components of excitatory synapses and that each of these proteins may perform a distinct function.

    Funded by: NINDS NIH HHS: R01 NS42609

    The Journal of comparative neurology 2004;472;1;24-39

  • Molecular determinants of synapsin targeting to presynaptic terminals.

    Gitler D, Xu Y, Kao HT, Lin D, Lim S, Feng J, Greengard P and Augustine GJ

    Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Although synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged synapsins in cultured hippocampal neurons. Whereas all synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, synapsin Ib scarcely targeted in neurons in which all synapsins were knocked-out. Coexpression of other synapsin isoforms significantly strengthened the targeting of synapsin Ib in knock-out neurons, indicating that heterodimerization is required for synapsin Ib to target. Truncation mutagenesis revealed that synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for synapsins to target to presynaptic terminals.

    Funded by: NIMH NIH HHS: MH 39327, MH 67044

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;14;3711-20

  • Role of Unc51.1 and its binding partners in CNS axon outgrowth.

    Tomoda T, Kim JH, Zhan C and Hatten ME

    Laboratory of Developmental Neurobiology, The Rockefeller University, New York, New York 10021-6399, USA.

    Previous studies showed that the serine/threonine kinase Unc51.1 is one of the earliest genes in neuronal differentiation and is required for granule cell axon formation. To examine the mechanism of Unc51.1 regulation of axon extension, we have identified two direct binding partners. The first, SynGAP, a negative regulator of Ras, is expressed within axons and growth cones of developing granule cells. Overexpression of SynGAP blocks neurite outgrowth by a mechanism that involves Ras-like GTPase cascade. The second binding partner is a PDZ domain-containing scaffolding protein, Syntenin, that binds Rab5 GTPase, the activity of which is attenuated by SynGAP. Thus, our results demonstrate that the Unc51.1-containing protein complex governs axon formation via Ras-like GTPase signaling and through regulation of the Rab5-mediated endocytic pathways within developing axons.

    Funded by: NINDS NIH HHS: NS39991, R01 NS039991

    Genes & development 2004;18;5;541-58

  • Identification of PSD-95 as a regulator of dopamine-mediated synaptic and behavioral plasticity.

    Yao WD, Gainetdinov RR, Arbuckle MI, Sotnikova TD, Cyr M, Beaulieu JM, Torres GE, Grant SG and Caron MG

    Howard Hughes Medical Institute Laboratories, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

    To identify the molecular mechanisms underlying psychostimulant-elicited plasticity in the brain reward system, we undertook a phenotype-driven approach using genome-wide microarray profiling of striatal transcripts from three genetic and one pharmacological mouse models of psychostimulant or dopamine supersensitivity. A small set of co-affected genes was identified. One of these genes encoding the synaptic scaffolding protein PSD-95 is downregulated in the striatum of all three mutants and in chronically, but not acutely, cocaine-treated mice. At the synaptic level, enhanced long-term potentiation (LTP) of the frontocortico-accumbal glutamatergic synapses correlates with PSD-95 reduction in every case. Finally, targeted deletion of PSD-95 in an independent line of mice enhances LTP, augments the acute locomotor-stimulating effects of cocaine, but leads to no further behavioral plasticity in response to chronic cocaine. Our findings uncover a previously unappreciated role of PSD-95 in psychostimulant action and identify a molecular and cellular mechanism shared between drug-related plasticity and learning.

    Funded by: NIDA NIH HHS: DA13511

    Neuron 2004;41;4;625-38

  • Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD-95.

    Wong W and Schlichter LC

    Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8, Canada.

    The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation. Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell. Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts. Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains. Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts. We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raft-associated. Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons. Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts. We then assessed the contribution of PSD-95 to the raft association of these channels. Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts. Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant. This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.

    The Journal of biological chemistry 2004;279;1;444-52

  • Quantification of synapse formation and maintenance in vivo in the absence of synaptic release.

    Bouwman J, Maia AS, Camoletto PG, Posthuma G, Roubos EW, Oorschot VM, Klumperman J and Verhage M

    Rudolf Magnus Institute for Neurosciences, University of Utrecht Medical Center, Utrecht, The Netherlands.

    Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.

    Neuroscience 2004;126;1;115-26

  • Intracellular domains of NMDA receptor subtypes are determinants for long-term potentiation induction.

    Köhr G, Jensen V, Koester HJ, Mihaljevic AL, Utvik JK, Kvello A, Ottersen OP, Seeburg PH, Sprengel R and Hvalby Ø

    Max-Planck-Institute for Medical Research, D-69120 Heidelberg, Germany. kohr@mpimf-heidelberg.mpg.de

    NMDA receptors (NMDARs) are essential for modulating synaptic strength at central synapses. At hippocampal CA3-to-CA1 synapses of adult mice, different NMDAR subtypes with distinct functionality assemble from NR1 with NR2A and/or NR2B subunits. Here we investigated the role of these NMDA receptor subtypes in long-term potentiation (LTP) induction. Because of the higher NR2B contribution in the young hippocampus, LTP of extracellular field potentials could be enhanced by repeated tetanic stimulation in young but not in adult mice. Similarly, NR2B-specific antagonists reduced LTP in young but only marginally in adult wild-type mice, further demonstrating that in mature CA3-to-CA1 connections LTP induction results primarily from NR2A-type signaling. This finding is also supported by gene-targeted mutant mice expressing C-terminally truncated NR2A subunits, which participate in synaptic NMDAR channel formation and Ca2+ signaling, as indicated by immunopurified synaptic receptors, postembedding immunogold labeling, and spinous Ca2+ transients in the presence of NR2B blockers. These blockers abolished LTP in the mutant at all ages, revealing that, without the intracellular C-terminal domain, NR2A-type receptors are deficient in LTP signaling. Without NR2B blockade, CA3-to-CA1 LTP was more strongly reduced in adult than young mutant mice but could be restored to wild-type levels by repeated tetanic stimulation. Thus, besides NMDA receptor-mediated Ca2+ influx, subtype-specific signaling is critical for LTP induction, with the intracellular C-terminal domain of the NR2 subunits directing signaling pathways with an age-dependent preference.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;34;10791-9

  • The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95.

    Todd PK, Mack KJ and Malter JS

    Department of Pathology, Neuroscience Training Program, and Waisman Center for Developmental Disabilities, University of Wisconsin, 1500 Highland Avenue, Madison, WI 53705, USA.

    Fragile X syndrome (FXS) is a common inherited cause of mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP is thought to regulate the translation of target mRNAs, including its own transcript. Here we show that the levels of FMRP are rapidly up-regulated in primary cortical neurons in response to the type-I metabotropic glutamate receptor (mGluR) agonist S-3,5-dihydrophenylglycine. These changes require new protein synthesis but not transcription and are specific to mGluR activation. We also demonstrate that the mRNA for PSD-95, a scaffolding protein involved in synaptic plasticity, contains a highly conserved canonical binding site for FMRP within its 3' UTR. Furthermore, PSD-95 is rapidly translated in response to S-3,5-dihydrophenylglycine. Finally, we show that these mGluR-dependent changes in PSD-95 expression are lost in neurons derived from FMRP knockout mice, a model of FXS. Taken together, these studies suggest that FMRP is required for mGluR-dependent translation of PSD-95 and provide insights into the pathophysiology of FXS.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14374-8

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Role of beta-catenin in synaptic vesicle localization and presynaptic assembly.

    Bamji SX, Shimazu K, Kimes N, Huelsken J, Birchmeier W, Lu B and Reichardt LF

    Howard Hughes Medical Institute and Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

    Cadherins and catenins are thought to promote adhesion between pre and postsynaptic elements in the brain. Here we show a role for beta-catenin in localizing the reserved pool of vesicles at presynaptic sites. Deletion of beta-catenin in hippocampal pyramidal neurons in vivo resulted in a reduction in the number of reserved pool vesicles per synapse and an impaired response to prolonged repetitive stimulation. This corresponded to a dispersion of vesicles along the axon in cultured neurons. Interestingly, these effects are not due to beta-catenin's involvement in cadherin-mediated adhesion or wnt signaling. Instead, beta-catenin modulates vesicle localization via its PDZ binding domain to recruit PDZ proteins such as Veli to cadherin at synapses. This study defines a specific role for cadherins and catenins in synapse organization beyond their roles in mediating cell adhesion.

    Funded by: NINDS NIH HHS: P01 NS016033, P01 NS016033-21

    Neuron 2003;40;4;719-31

  • Differential distribution of Rac1 and Rac3 GTPases in the developing mouse brain: implications for a role of Rac3 in Purkinje cell differentiation.

    Bolis A, Corbetta S, Cioce A and de Curtis I

    Cell Adhesion Unit, Department of Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.

    Rac3 is one of the three known Rac GTPases in vertebrates. Rac3 shows high sequence homology to Rac1, and its transcript is specifically expressed in the developing nervous system, where its localization and function are unknown. By using Rac3-specific antibodies, we show that the endogenous Rac3 protein is differentially expressed during mouse brain development, with a peak of expression at times of neuronal maturation and synaptogenesis. Comparison with Rac1 shows clear-cut differences in the overall distribution of the two GTPases in the developing brain, and in their subcellular distribution in regions of the brain where both proteins are expressed. At P7, Rac3 staining is particularly marked in the deep cerebellar nuclei and in the pons, where it shows a discontinuous distribution around the neuronal cell bodies, in contrast with the diffuse staining of Rac1. Rac3 does not evidently co-localize with pre- and post-synaptic markers, nor with GFAP-positive astrocytes, but it clearly co-localizes with actin filaments, and with the terminal portions of calbindin-positive Purkinje cell axons in the deep cerebellar nuclei. Our data implicate Rac3 in neuronal differentiation, and support a specific role of this GTPase in actin-mediated remodelling of Purkinje cell neuritic terminals at time of synaptogenesis.

    Funded by: Telethon: GGP02190

    The European journal of neuroscience 2003;18;9;2417-24

  • Slo2 sodium-activated K+ channels bind to the PDZ domain of PSD-95.

    Uchino S, Wada H, Honda S, Hirasawa T, Yanai S, Nakamura Y, Ondo Y and Kohsaka S

    Department of Neurochemistry, National Institute of Neuroscience, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.

    Slo2 channels are a type of sodium-activated K+ channels and possess a typical PDZ binding motif at the carboxy-terminal end. Thus, we investigated whether Slo2 channels bind to PSD-95, because it is well known that other types of K+ channels, voltage-gated and inward rectifier K+ channels, bind to PSD-95 via the PDZ binding motif and are involved in excitatory synaptic transmission. By using an extract prepared from cultured neocortical neurons, we demonstrated a biochemical interaction between mSlo2 channels and PSD-95, and a mutational analysis revealed that mSlo2 channels bound to the first PDZ domain of PSD-95 via the PDZ binding motif. To investigate the expression of mSlo2 protein in primary neocortical neurons, we raised anti-mSlo2 channel antibody and immunostained neocortical neurons. The immunocytochemical study showed that mSlo2 channels partly colocalized with PSD-95 in mouse neocortical neurons.

    Biochemical and biophysical research communications 2003;310;4;1140-7

  • Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

    Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, Johnson EM, Daniel DC, Kinoshita Y, Amini S and Gordon J

    Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, USA. kamel.khalili@temple.edu

    The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

    Molecular and cellular biology 2003;23;19;6857-75

  • RICS, a novel GTPase-activating protein for Cdc42 and Rac1, is involved in the beta-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling.

    Okabe T, Nakamura T, Nishimura YN, Kohu K, Ohwada S, Morishita Y and Akiyama T

    Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    Cadherin adhesion molecules are believed to be important for synaptic plasticity. beta-Catenin, which links cadherins and the actin cytoskeleton, is a modulator of cadherin adhesion and regulates synaptic structure and function. Here we show that beta-catenin interacts with a novel GTPase-activating protein, named RICS, that acts on Cdc42 and Rac1. The RICS-beta-catenin complex was found to be associated with N-cadherin, N-methyl-d-aspartate receptors, and postsynaptic density-95, and localized to the postsynaptic density. Furthermore, the GTPase-activating protein activity of RICS was inhibited by phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II. These results suggest that RICS is involved in the synaptic adhesion- and N-methyl-d-aspartate-mediated organization of cytoskeletal networks and signal transduction. Thus, RICS may regulate dendritic spine morphology and strength by modulating Rho GTPases.

    The Journal of biological chemistry 2003;278;11;9920-7

  • Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice.

    Garry EM, Moss A, Delaney A, O'Neill F, Blakemore J, Bowen J, Husi H, Mitchell R, Grant SG and Fleetwood-Walker SM

    Division of Preclinical Veterinary Sciences, R(D)SVS, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom.

    Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.

    Current biology : CB 2003;13;4;321-8

  • Gamma protocadherins are required for survival of spinal interneurons.

    Wang X, Weiner JA, Levi S, Craig AM, Bradley A and Sanes JR

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.

    Neuron 2002;36;5;843-54

  • Clustering of neuronal potassium channels is independent of their interaction with PSD-95.

    Rasband MN, Park EW, Zhen D, Arbuckle MI, Poliak S, Peles E, Grant SG and Trimmer JS

    Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

    Voltage-dependent potassium channels regulate membrane excitability and cell-cell communication in the mammalian nervous system, and are found highly localized at distinct neuronal subcellular sites. Kv1 (mammalian Shaker family) potassium channels and the neurexin Caspr2, both of which contain COOH-terminal PDZ domain binding peptide motifs, are found colocalized at high density at juxtaparanodes flanking nodes of Ranvier of myelinated axons. The PDZ domain-containing protein PSD-95, which clusters Kv1 potassium channels in heterologous cells, has been proposed to play a major role in potassium channel clustering in mammalian neurons. Here, we show that PSD-95 colocalizes precisely with Kv1 potassium channels and Caspr2 at juxtaparanodes, and that a macromolecular complex of Kv1 channels and PSD-95 can be immunopurified from mammalian brain and spinal cord. Surprisingly, we find that the high density clustering of Kv1 channels and Caspr2 at juxtaparanodes is normal in a mutant mouse lacking juxtaparanodal PSD-95, and that the indirect interaction between Kv1 channels and Caspr2 is maintained in these mutant mice. These data suggest that the primary function of PSD-95 at juxtaparanodes lies outside of its accepted role in mediating the high density clustering of Kv1 potassium channels at these sites.

    Funded by: NINDS NIH HHS: F32 NS010906, F32NS10906, R37 NS034383, R37NS34383

    The Journal of cell biology 2002;159;4;663-72

  • Cellular localization of the vesicular inhibitory amino acid transporter in the mouse and human retina.

    Jellali A, Stussi-Garaud C, Gasnier B, Rendon A, Sahel JA, Dreyfus H and Picaud S

    Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, INSERM EMI-99-18, Université Louis Pasteur, 1 Place de l'Hôpital, 67091 Strasbourg Cedex, France.

    Horizontal cells are classically thought to mediate lateral inhibition by gamma-aminobutyric acid (GABA)-transporter mediated release. In the mammalian retina, however, GABA uptake and cloned GABA transporter were not detected in horizontal cells. Furthermore, the vesicular inhibitory amino acid transporter (VIAAT or VGAT) that loads GABA and glycine into synaptic vesicles was reported recently to be expressed in horizontal cells. To further assess synaptic transmission in mammalian horizontal cells, we examined the subcellular distribution of VIAAT in mouse and human retina by confocal microscopy with specific cell markers. VIAAT was observed in the mouse outer plexiform layer as punctate structures that localized in calbindin-positive horizontal cells. These structures were in close apposition with synaptophysin-, PSD-95-, dystrophin-, and bassoon-immunopositive photoreceptor terminals, suggesting that VIAAT is localized in horizontal cell tips at photoreceptor terminals. VIAAT-positive puncta were also in apposition to lectin-labeled cone terminals or dendrites of PKCalpha-immunopositive rod bipolar cells, indicating that VIAAT is expressed in horizontal cell tips at both rod and cone terminals. By contrast, only a very few puncta were observed in the human outer plexiform layer, whereas the inner plexiform layer remained labeled as in the mouse retina. When using adult human retinal cells in culture, horizontal cells identified by parvalbumin immunostaining were found to contain VIAAT, either at their terminals or throughout the entire cell similarly as in syntaxin-immunopositive cells. These differences between human retinal tissue and cultured cells were attributed to VIAAT degradation in postmortem retinal tissue. VIAAT localization in mouse and human horizontal cells further support the role of inhibitory transmitters in lateral inhibition at the photoreceptor terminals.

    The Journal of comparative neurology 2002;449;1;76-87

  • Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins.

    Mok H, Shin H, Kim S, Lee JR, Yoon J and Kim E

    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A). Little is known, however, about how a defective KIF1B gene leads to CMT2A. Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90). KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments. KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain. In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM. These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;13;5253-8

  • Direct interaction of Frizzled-1, -2, -4, and -7 with PDZ domains of PSD-95.

    Hering H and Sheng M

    Center for Learning and Memory, Howard Hughes Medical Institute, RIKEN-MIT Neuroscience Research Center, and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue (E18-215), Cambridge, MA 02139, USA.

    In Drosophila, the frizzled gene plays a critical role in the establishment of tissue polarity, but the function of the Frizzled family of proteins in mammals is largely unknown. Recent evidence suggested that Frizzleds are receptors for the Wnt family of secreted glycoproteins which are involved in cell fate determination. However, it is unclear how Frizzled receptors transduce Wnt signals to intracellular signaling components. Here we show that the mouse Frizzled-1, -2, -4 and -7 can bind to proteins of the PSD-95 family, which are implicated in the assembly and localization of multiprotein signaling complexes in the brain. Moreover, PSD-95 can form a ternary complex with Frizzled-2 and the adenomatous polyposis coli protein, a negative regulator of Wnt signaling, suggesting that members of the PSD-95 family may serve to recruit intracellular signaling molecules of the Wnt/Frizzled pathway into the vicinity of the receptor.

    FEBS letters 2002;521;1-3;185-9

  • Microtubule-associated protein 1A is a modifier of tubby hearing (moth1).

    Ikeda A, Zheng QY, Zuberi AR, Johnson KR, Naggert JK and Nishina PM

    The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA.

    Once a mutation in the gene tub was identified as the cause of obesity, retinal degeneration and hearing loss in tubby mice, it became increasingly evident that the members of the tub gene family (tulps) influence maintenance and function of the neuronal cell lineage. Suggested molecular functions of tubby-like proteins include roles in vesicular trafficking, mediation of insulin signaling and gene transcription. The mechanisms through which tub functions in neurons, however, have yet to be elucidated. Here we report the positional cloning of an auditory quantitative trait locus (QTL), the modifier of tubby hearing 1 gene (moth1), whose wildtype alleles from strains AKR/J, CAST/Ei and 129P2/OlaHsd protect tubby mice from hearing loss. Through a transgenic rescue experiment, we verified that sequence polymorphisms in the neuron-specific microtubule-associated protein 1a gene (Mtap1a) observed in the susceptible strain C57BL/6J (B6) are crucial for the hearing-loss phenotype. We also show that these polymorphisms change the binding efficiency of MTAP1A to postsynaptic density molecule 95 (PSD95), a core component in the cytoarchitecture of synapses. This indicates that at least some of the observed polymorphisms are functionally important and that the hearing loss in C57BL/6J-tub/tub (B6-tub/tub) mice may be caused by impaired protein interactions involving MTAP1A. We therefore propose that tub may be associated with synaptic function in neuronal cells.

    Funded by: NEI NIH HHS: R01 EY016501, R01 EY016501-06; NIDCD NIH HHS: R01 DC004301, R01 DC004301-01, R01 DC005827, R01 DC005827-01, R03 DC004376, R03 DC004376-01A1; NIDDK NIH HHS: R01 DK046977, R01 DK046977-07, R01 DK046977-10

    Nature genetics 2002;30;4;401-5

  • The septin CDCrel-1 is dispensable for normal development and neurotransmitter release.

    Peng XR, Jia Z, Zhang Y, Ware J and Trimble WS

    Programmes in Cell Biology. Brain and Behavior, Hospital for Sick Children, Toronto, Ontario, Canada.

    Septins are GTPases required for the completion of cytokinesis in a variety of organisms, yet their role in this process is not known. Septins may have additional functions since the mammalian septin CDCrel-1 is predominantly expressed in the nervous system, a largely postmitotic tissue. While relatively little is known about the function of this protein, we have previously shown that it is involved in regulated secretion. In addition, the gene encoding this protein maps to a locus often deleted in velo-cardiofacial and DiGeorge syndromes, and CDCrel-1 has recently been shown to be a direct target of the E3 ubiquitin ligase activity of Parkin, a causative agent in autosomal recessive forms of Parkinson's disease. Here we show that CDCrel-1 expression rises at the time of synaptic maturation and that CDCrel-1 is present in a complex that includes the septins Nedd5 and CDC10. To investigate its function in the nervous system, we generated homozygotic CDCrel-1 null mice and showed that these mice appear normal with respect to synaptic properties and hippocampal neuron growth in vitro. Moreover, we found that while the expression of a number of synaptic proteins is not affected in the CDCrel-1 mutant mice, the expression of other septins is altered. Together, these data suggest that CDCrel-1 is not essential for neuronal development or function, and that changes in expression of other septins may account for its functional redundancy.

    Funded by: NHLBI NIH HHS: R01 HL069951, R01 HL069951-01

    Molecular and cellular biology 2002;22;1;378-87

  • Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95.

    Schultze W, Eulenburg V, Lessmann V, Herrmann L, Dittmar T, Gundelfinger ED, Heumann R and Erdmann KS

    Department of Molecular Neurobiochemistry, Ruhr-University Bochum, Bochum, Germany.

    Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

    Journal of neurochemistry 2001;78;3;482-9

  • Sema4c, a transmembrane semaphorin, interacts with a post-synaptic density protein, PSD-95.

    Inagaki S, Ohoka Y, Sugimoto H, Fujioka S, Amazaki M, Kurinami H, Miyazaki N, Tohyama M and Furuyama T

    Group of Neurobiology, School of Allied Health Sciences, Osaka University Faculty of Medicine, Yamadaoka 1-7, Suita-shi, Osaka, 565-0871, Japan. inagaki@sahs.med.osaka-u.ac.jp

    Semaphorins are known to act as chemorepulsive molecules that guide axons during neural development. Sema4C, a group 4 semaphorin, is a transmembrane semaphorin of unknown function. The cytoplasmic domain of Sema4C contains a proline-rich region that may interact with some signaling proteins. In this study, we demonstrate that Sema4C is enriched in the adult mouse brain and associated with PSD-95 isoforms containing PDZ (PSD-95/DLG/ZO-1) domains, such as PSD-95/SAP90, PSD-93/chapsin110, and SAP97/DLG-1, which are concentrated in the post-synaptic density of the brain. In the neocortex, S4C is enriched in the synaptic vesicle fraction and Triton X-100 insoluble post-synaptic density fraction. Immunostaining for Sema4C overlaps that for PSD-95 in superficial layers I-IV of the neocortex. In neocortical culture, S4C is colocalized with PSD-95 in neurons, with a dot-like pattern along the neurites. Sema4C thus may function in the cortical neurons as a bi-directional transmembrane ligand through interacting with PSD-95.

    The Journal of biological chemistry 2001;276;12;9174-81

  • The effects of aging on gene expression in the hypothalamus and cortex of mice.

    Jiang CH, Tsien JZ, Schultz PG and Hu Y

    Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row, San Diego, CA 92121, USA.

    A better understanding of the molecular effects of aging in the brain may help to reveal important aspects of organismal aging, as well as processes that lead to age-related brain dysfunction. In this study, we have examined differences in gene expression in the hypothalamus and cortex of young and aged mice by using high-density oligonucleotide arrays. A number of key genes involved in neuronal structure and signaling are differentially expressed in both the aged hypothalamus and cortex, including synaptotagmin I, cAMP-dependent protein kinase C beta, apolipoprotein E, protein phosphatase 2A, and prostaglandin D. Misregulation of these proteins may contribute to age-related memory deficits and neurodegenerative diseases. In addition, many proteases that play essential roles in regulating neuropeptide metabolism, amyloid precursor protein processing, and neuronal apoptosis are up-regulated in the aged brain and likely contribute significantly to brain aging. Finally, a subset of these genes whose expression is affected by aging are oppositely affected by exposure of mice to an enriched environment, suggesting that these genes may play important roles in learning and memory.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;4;1930-4

  • Calsyntenin-1, a proteolytically processed postsynaptic membrane protein with a cytoplasmic calcium-binding domain.

    Vogt L, Schrimpf SP, Meskenaite V, Frischknecht R, Kinter J, Leone DP, Ziegler U and Sonderegger P

    Institute of Biochemistry, Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, Zurich, CH-8057, Switzerland.

    In a screen for proteins released from synapse-forming spinal cord neurons, we found the proteolytically cleaved N-terminal fragment of a transmembrane protein localized in the postsynaptic membrane of both excitatory and inhibitory synapses. We termed this protein calsyntenin-1, because it binds synaptic Ca2+ with its cytoplasmic domain. By binding Ca2+, calsyntenin-1 may modulate Ca2+-mediated postsynaptic signals. Proteolytic cleavage of calsyntenin-1 in its extracellular moiety generates a transmembrane stump that is internalized and accumulated in the spine apparatus of spine synapses. Therefore, the synaptic Ca2+ modulation by calsyntenin-1 may be subject to regulation by extracellular proteolysis in the synaptic cleft. Thus, calsyntenin-1 may link extracellular proteolysis in the synaptic cleft and postsynaptic Ca2+ signaling.

    Molecular and cellular neurosciences 2001;17;1;151-66

  • Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction.

    Gotthardt M, Trommsdorff M, Nevitt MF, Shelton J, Richardson JA, Stockinger W, Nimpf J and Herz J

    Department of Molecular Genetics and Pathology, University of Texas Southwestern Medical Center, Dallas 75390-9046, USA.

    The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.

    Funded by: NHLBI NIH HHS: HL20948, HL63762, R37 HL063762

    The Journal of biological chemistry 2000;275;33;25616-24

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice.

    Kneussel M, Brandstätter JH, Laube B, Stahl S, Müller U and Betz H

    Department of Neurochemistry, Max-Planck-Institute for Brain Research, D-60528 Frankfurt/Main, Germany.

    The tubulin-binding protein gephyrin, which anchors the inhibitory glycine receptor (GlyR) at postsynaptic sites, decorates GABAergic postsynaptic membranes in various brain regions, and postsynaptic gephyrin clusters are absent from cortical cultures of mice deficient for the GABA(A) receptor gamma2 subunit. Here, we investigated the postsynaptic clustering of GABA(A) receptors in gephyrin knock-out (geph -/-) mice. Both in brain sections and cultured hippocampal neurons derived from geph -/- mice, synaptic GABA(A) receptor clusters containing either the gamma2 or the alpha2 subunit were absent, whereas glutamate receptor subunits were normally localized at postsynaptic sites. Western blot analysis and electrophysiological recording revealed that normal levels of functional GABA(A) receptors are expressed in geph -/- neurons, however the pool size of intracellular GABA(A) receptors appeared increased in the mutant cells. Thus, gephyrin is required for the synaptic localization of GlyRs and GABA(A) receptors containing the gamma2 and/or alpha2 subunits but not for the targeting of these receptors to the neuronal plasma membrane. In addition, gephyrin may be important for efficient membrane insertion and/or metabolic stabilization of inhibitory receptors at developing postsynaptic sites.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;21;9289-97

  • Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells.

    Shiraishi Y, Mizutani A, Bito H, Fujisawa K, Narumiya S, Mikoshiba K and Furuichi T

    Department of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

    A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;19;8389-400

  • Distinct spatiotemporal expression of mRNAs for the PSD-95/SAP90 protein family in the mouse brain.

    Fukaya M, Ueda H, Yamauchi K, Inoue Y and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.

    PSD-95 (SAP90), SAP102 and Chapsyn-110 (PSD-93) are members of the membrane-associated guanylate kinase family, and interact with N-methyl-D-aspartate (NMDA) receptor NR2A (GluRepsilon1) and NR2B (GluRepsilon2) subunits and with Shaker-type K+ channel subunits to cluster into a channel complex. In the present study, we examined their expression in developing and adult mouse brains by in situ hybridization with antisense oligonucleotide probes. PSD-95 and SAP102 mRNAs were prominently expressed at embryonic day 13 (E13) in the mantle zone of various brain regions, where NMDA receptor NR2B subunit mRNA is expressed at high levels. In the early postnatal period when active synaptogenesis takes place, both mRNAs became elevated and concentrated in the telencephalon and cerebellar granular layer, where NR2A and/or NR2B subunit mRNAs are abundantly expressed. Chapsyn-110 mRNA was, though at low levels, found over the mantle zone of embryonic brains, and the level was progressively increased in the telencephalon starting at perinatal stages. The spatial and temporal correlations in the brain in vivo suggest that the PSD-95/SAP90 protein family can interact with NMDA receptor subunits to cluster them into channel complex at both synaptic and non-synaptic sites before, during and after synaptogenic stages.

    Neuroscience research 1999;33;2;111-8

  • PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A.

    Tezuka T, Umemori H, Akiyama T, Nakanishi S and Yamamoto T

    Department of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

    Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-D-aspartate (NMDA) receptor function. In this study, we examined how Fyn participates in synaptic plasticity by analyzing the physical and functional interaction between Fyn and NMDA receptors. Results showed that tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, was reduced in fyn-mutant mice. NR2A was tyrosine-phosphorylated in 293T cells when coexpressed with Fyn. Therefore, NR2A would be a substrate for Fyn in vivo. Results also showed that PSD-95, which directly binds to and coclusters with NMDA receptors, promotes Fyn-mediated tyrosine phosphorylation of NR2A. Different regions of PSD-95 associated with NR2A and Fyn, respectively, and so PSD-95 could mediate complex formation of Fyn with NR2A. PSD-95 also associated with other Src-family PTKs, Src, Yes, and Lyn. These results suggest that PSD-95 is critical for regulation of NMDA receptor activity by Fyn and other Src-family PTKs, serving as a molecular scaffold for anchoring these PTKs to NR2A.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;2;435-40

  • Citron, a Rho-target, interacts with PSD-95/SAP-90 at glutamatergic synapses in the thalamus.

    Furuyashiki T, Fujisawa K, Fujita A, Madaule P, Uchino S, Mishina M, Bito H and Narumiya S

    Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8315, Japan.

    Proteins of the membrane-associated guanylate kinase family play an important role in the anchoring and clustering of neurotransmitter receptors in the postsynaptic density (PSD) at many central synapses. However, relatively little is known about how these multifunctional scaffold proteins might provide a privileged site for activity- and cell type-dependent specification of the postsynaptic signaling machinery. Rho signaling pathway has classically been implicated in mechanisms of axonal outgrowth, dendrogenesis, and cell migration during neural development, but its contribution remains unclear at the synapses in the mature CNS. Here, we present evidence that Citron, a Rho-effector in the brain, is enriched in the PSD fraction and interacts with PSD-95/synapse-associated protein (SAP)-90 both in vivo and in vitro. Citron colocalization with PSD-95 occurred, not exclusively but certainly, at glutamatergic synapses in a limited set of neurons, such as the thalamic excitatory neurons; Citron expression, however, could not be detected in the principal neurons of the hippocampus and the cerebellum in the adult mouse brain. In a heterologous system, Citron was shown to form a heteromeric complex not only with PSD-95 but also with NMDA receptors. Thus, Citron-PSD-95/SAP-90 interaction may provide a region- and cell type-specific link between the Rho signaling cascade and the synaptic NMDA receptor complex.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;1;109-18

  • Synapse formation by hippocampal neurons from agrin-deficient mice.

    Serpinskaya AS, Feng G, Sanes JR and Craig AM

    Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA.

    Agrin, a proteoglycan secreted by motoneurons, is a critical organizer of synaptic differentiation at skeletal neuromuscular junctions. Agrin is widely expressed in the nervous system so other functions seem likely, but none have been demonstrated. To test roles for agrin in interneuronal synapse formation, we studied hippocampi from mutant mice that completely lack the z+ splice form of agrin essential for neuromuscular differentiation and also exhibit severely ( approximately 90%) reduced levels of all agrin isoforms (M. Gautam et al., 1996, Cell 85, 525-535). The brains of neonatal homozygous agrin mutants were often smaller than those of heterozygous and wild-type littermates, but were morphologically and histologically indistinguishable. In particular, antibodies to pre- and postsynaptic components of glutamatergic synapses were similarly coaggregated at synaptic sites in both mutants and controls. Because mutants die at birth due to neuromuscular defects, we cultured neurons to assess later stages of synaptic maturation. In primary cultures, the agrin-deficient neurons formed MAP2-positive dendrites and tau-1-positive axons. Synaptic vesicle proteins, AMPA- and NMDA-type glutamate receptors, GABAA receptors, and the putative synapse-organizing proteins PSD-95, GKAP, and gephyrin formed numerous clusters at synaptic sites. Quantitatively, the number of SV2-labeled contacts per neuron at day 5 and the number of PSD-95 clusters per dendrite length at day 18 in culture showed no significant differences between genotypes. Furthermore, exogenous z+ agrin was unable to induce ectopic accumulation of components of central glutamatergic or GABAergic synapses as it does for neuromuscular cholinergic synapses. These results indicate that the z+ forms of agrin are dispensable for glutamatergic and GABAergic synaptic differentiation in the central nervous system.

    Funded by: NINDS NIH HHS: NS 33184; PHS HHS: 19195

    Developmental biology 1999;205;1;65-78

  • Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein.

    Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ and Grant SG

    Centre for Genome Research, University of Edinburgh, UK.

    Specific patterns of neuronal firing induce changes in synaptic strength that may contribute to learning and memory. If the postsynaptic NMDA (N-methyl-D-aspartate) receptors are blocked, long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission and the learning of spatial information are prevented. The NMDA receptor can bind a protein known as postsynaptic density-95 (PSD-95), which may regulate the localization of and/or signalling by the receptor. In mutant mice lacking PSD-95, the frequency function of NMDA-dependent LTP and LTD is shifted to produce strikingly enhanced LTP at different frequencies of synaptic stimulation. In keeping with neural-network models that incorporate bidirectional learning rules, this frequency shift is accompanied by severely impaired spatial learning. Synaptic NMDA-receptor currents, subunit expression, localization and synaptic morphology are all unaffected in the mutant mice. PSD-95 thus appears to be important in coupling the NMDA receptor to pathways that control bidirectional synaptic plasticity and learning.

    Funded by: Wellcome Trust

    Nature 1998;396;6710;433-9

  • CIPP, a novel multivalent PDZ domain protein, selectively interacts with Kir4.0 family members, NMDA receptor subunits, neurexins, and neuroligins.

    Kurschner C, Mermelstein PG, Holden WT and Surmeier DJ

    Department of Developmental Neurobiology, Saint Jude Children's Research Hospital, Memphis, Tennessee, 38105, USA. cornelia.kurschner@stjude.org

    We report a novel multivalent PDZ domain protein, CIPP (for channel-interacting PDZ domain protein), which is expressed exclusively in brain and kidney. Within the brain, the highest CIPP mRNA levels were found in neurons of the cerebellum, inferior colliculus, vestibular nucleus, facial nucleus, and thalamus. Furthermore, we identified the inward rectifier K+ (Kir) channel, Kir4.1 (also called "Kir1.2"), as a cellular CIPP ligand. Among several other Kir channels tested, only the closely related Kir4.2 (or "Kir1.3") also interacted with CIPP. In addition, specific PDZ domains within CIPP associated selectively with the C-termini of N-methyl-D-aspartate subtypes of glutamate receptors, as well as neurexins and neuroligins, cell surface molecules enriched in synaptic membranes. Thus, CIPP may serve as a scaffold that brings structurally diverse but functionally connected proteins into close proximity at the synapse. The functional consequences of CIPP expression on Kir4.1 channels were studied using whole-cell voltage clamp techniques in Kir4.1 transfected COS-7 cells. On average, Kir4.1 current densities were doubled by cotransfection with CIPP.

    Funded by: NCI NIH HHS: P30 CA21765

    Molecular and cellular neurosciences 1998;11;3;161-72

  • Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo.

    Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A, Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF, Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara JO and Seeburg PH

    Max-Planck Institute for Medical Research, Department of Molecular Neuroscience, Heidelberg, Germany.

    NMDA receptors, a class of glutamate-gated cation channels with high Ca2+ conductance, mediate fast transmission and plasticity of central excitatory synapses. We show here that gene-targeted mice expressing NMDA receptors without the large intracellular C-terminal domain of any one of three NR2 subunits phenotypically resemble mice made deficient in that particular subunit. Mice expressing the NR2B subunit in a C-terminally truncated form (NR2B(deltaC/deltaC) mice) die perinatally. NR2A(deltaC/deltaC) mice are viable but exhibit impaired synaptic plasticity and contextual memory. These and NR2C(deltaC/deltaC) mice display deficits in motor coordination. C-terminal truncation of NR2 subunits does not interfere with the formation of gateable receptor channels that can be synaptically activated. Thus, the phenotypes of our mutants appear to reflect defective intracellular signaling.

    Funded by: ACF HHS: AF05142; NIA NIH HHS: AG0093; OMHHE CDC HHS: IF32MN10521-01; Wellcome Trust; ...

    Cell 1998;92;2;279-89

  • Binding of neuroligins to PSD-95.

    Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, Takai Y, Rosahl TW and Südhof TC

    Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, 2-2-10, Murotani, Nishi-ku, Kobe, 651-22, Japan.

    PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins.

    Funded by: NIMH NIH HHS: R01-MH52804

    Science (New York, N.Y.) 1997;277;5331;1511-5

  • Human postsynaptic density-95 (PSD95): location of the gene (DLG4) and possible function in nonneural as well as in neural tissues.

    Stathakis DG, Hoover KB, You Z and Bryant PJ

    Developmental Biology Center, University of California at Irvine 92697-2275, USA. dgstatha@UCI.EDU

    We have determined the cDNA sequence, expression pattern, and chromosomal location of the human gene DLG4, encoding the postsynaptic density-95 (PSD95) protein. hPSD95 is a 723-amino-acid protein that is 99% identical to its rodent counterparts. This is the fourth human protein identified as showing significant similarity to the Drosophila tumor suppressor Dlg. These proteins constitute the DLG subfamily of the membrane-associated guanylate kinase protein family. The expression of DLG4 in neural tissue is consistent with the pattern observed for its rat homolog. However, DLG4 is also expressed in a wide range of nonneural tissues, suggesting that the protein may have additional roles in humans. Using radiation-hybrid mapping panels, we mapped the DLG4 locus to 17p13.1, a region associated with several diseases, the phenotypes of which are consistent with loss of PSD95 function.

    Funded by: NCI NIH HHS: P30 CA62203, R03 CA70557

    Genomics 1997;44;1;71-82

Gene lists (11)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000003 G2C Mus musculus Mouse clathrin Mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000004 G2C Mus musculus Mouse Synaptosome Mouse Synaptosome adapted from Collins et al (2006) 152
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000019 G2C Mus musculus Pocklington M1 Cluster 1 (mouse) from Pocklington et al (2006) 21
L00000050 G2C Mus musculus TAP-PSD-95-CORE TAP-PSD-95 pull-down core list 120
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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