G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
glutamate receptor, ionotropic, N-methyl D-aspartate 2A
G00000001 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000073102 (Vega human gene)
ENSG00000183454 (Ensembl human gene)
2903 (Entrez Gene)
2 (G2Cdb plasticity & disease)
GRIN2A (GeneCards)
138253 (OMIM)
Marker Symbol
HGNC:4585 (HGNC)
Protein Sequence
Q12879 (UniProt)

Diseases (3)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000179: Attention deficit hyperactivity disorder Y Y (14966475) Single nucleotide polymorphism (SNP) Y
D00000184: Huntington's disease Y Y (17018562) Single nucleotide polymorphism (SNP) Y
D00000184: Huntington's disease Y Y (15742215) Polymorphism (P) Y
D00000166: Schizophrenia Y Y (15774266) Repeat polymorphism (RP) Y
D00000166: Schizophrenia Y Y (12724619) Repeat polymorphism (RP) Y


  • Replication of twelve association studies for Huntington's disease residual age of onset in large Venezuelan kindreds.

    Andresen JM, Gayán J, Cherny SS, Brocklebank D, Alkorta-Aranburu G, Addis EA, US-Venezuela Collaborative Research Group, Cardon LR, Housman DE and Wexler NS

    Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. jma@mit.edu

    Background: The major determinant of age of onset in Huntington's disease is the length of the causative triplet CAG repeat. Significant variance remains, however, in residual age of onset even after repeat length is factored out. Many genetic polymorphisms have previously shown evidence of association with age of onset of Huntington's disease in several different populations.

    Objective: To replicate these genetic association tests in 443 affected people from a large set of kindreds from Venezuela.

    Methods: Previously tested polymorphisms were analysed in the HD gene itself (HD), the GluR6 kainate glutamate receptor (GRIK2), apolipoprotein E (APOE), the transcriptional coactivator CA150 (TCERG1), the ubiquitin carboxy-terminal hydrolase L1 (UCHL1), p53 (TP53), caspase-activated DNase (DFFB), and the NR2A and NR2B glutamate receptor subunits (GRIN2A, GRIN2B).

    Results: The GRIN2A single-nucleotide polymorphism explains a small but considerable amount of additional variance in residual age of onset in our sample. The TCERG1 microsatellite shows a trend towards association but does not reach statistical significance, perhaps because of the uninformative nature of the polymorphism caused by extreme allele frequencies. We did not replicate the genetic association of any of the other genes.

    Conclusions: GRIN2A and TCERG1 may show true association with residual age of onset for Huntington's disease. The most surprising negative result is for the GRIK2 (TAA)(n) polymorphism, which has previously shown association with age of onset in four independent populations with Huntington's disease. The lack of association in the Venezuelan kindreds may be due to the extremely low frequency of the key (TAA)(16) allele in this population.

    Journal of medical genetics 2007;44;1;44-50

  • Extended analyses support the association of a functional (GT)n polymorphism in the GRIN2A promoter with Japanese schizophrenia.

    Iwayama-Shigeno Y, Yamada K, Itokawa M, Toyota T, Meerabux JM, Minabe Y, Mori N, Inada T and Yoshikawa T

    Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama, Japan.

    Dysfunction of the N-methyl-D-aspartate (NMDA) type glutamate receptor has been proposed as a mechanism in the etiology of schizophrenia. Recently, we identified a variable (GT)n repeat in the promoter region of the NMDA NR2A subunit gene (GRIN2A), and showed its association with schizophrenia in a case-control study, together with a correlation between the length of the repeat and severity of chronic outcome. In this study, we extended our analyses, by increasing the number of case-control samples to a total of 672 schizophrenics and 686 controls, and excluded potential sample stratification effects. We confirmed the significant allelic association between the repeat polymorphism and disease (P = 0.011), and as in the previous study, we observed an over-representation of longer alleles in schizophrenia. These results suggest a probable genetic effect for the GRIN2A promoter (GT)n variation on the predisposition to schizophrenia in Japanese cohorts.

    Neuroscience letters 2005;378;2;102-5

  • NR2A and NR2B receptor gene variations modify age at onset in Huntington disease.

    Arning L, Kraus PH, Valentin S, Saft C, Andrich J and Epplen JT

    Department of Human Genetics, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany. larissa.arning@rub.de

    N -Methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity has been proposed to play a role in the pathogenesis of Huntington disease (HD), an autosomal dominantly inherited disorder associated with defined expansions in a stretch of perfect CAG repeats in the 5' part of the IT15 gene. The number of CAG repeat units is highly predictive for the age at onset (AO) in HD. However, AO is only modestly correlated with repeat length when the HD expansion range is in the high 30s or low 40s. Therefore, we investigated whether the genes for the different subunits composing the multimeric complexes of NMDA receptors (GRIN glutamate receptor, ionotropic, N-methyl-d-aspartate) represent candidates for modulating the AO of HD. In the studied cohort of 167 HD patients, the repeat range from 41 to 45 CAG units accounted for 30.8% of the variance in AO; 12.3% additional variance could be attributed to GRIN2B genotype variation and 4.5% to GRIN2A genotype variation. We conclude that these two genes, coding for NR2B and NR2A subtypes mainly expressed in the striatum, may influence the variability in AO of HD. Neuroprotective strategies for HD patients and persons at risk should be reconsidered in the light of these findings.

    Neurogenetics 2005;6;1;25-8

  • Follow-up of genetic linkage findings on chromosome 16p13: evidence of association of N-methyl-D aspartate glutamate receptor 2A gene polymorphism with ADHD.

    Turic D, Langley K, Mills S, Stephens M, Lawson D, Govan C, Williams N, Van Den Bree M, Craddock N, Kent L, Owen M, O'Donovan M and Thapar A

    Department of Psychological Medicine, University of Wales College of Medicine, Cardiff, UK.

    Attention deficit hyperactivity disorder (ADHD) is a childhood onset disorder, for which there is good evidence that genetic factors contribute to the aetiology. Recently reported linkage findings suggested evidence of a susceptibility locus on chromosome 16p13 (maximum LOD score of 4.2, P=5 x 10(-6)). The GRIN2A (glutamate receptor, ionotropic, N-methyl D-aspartate 2A) gene that encodes the N-methyl D-aspartate receptor subunit 2A (NMDA2A) maps to this region of linkage. As this is also a good functional candidate gene for ADHD, we undertook family-based association analysis in a sample of 238 families. We found significant evidence of association with a GRIN2A exon 5 polymorphism (chi(2)=5.7, P=0.01). Our data suggest that genetic variation in GRIN2A may confer increased risk for ADHD and that this, at least in part, might be responsible for the linkage result on 16p reported by Smalley et al. We conclude that replication is required and that further work examining for association of GRIN2A polymorphisms with ADHD is warranted.

    Molecular psychiatry 2004;9;2;169-73

  • A microsatellite repeat in the promoter of the N-methyl-D-aspartate receptor 2A subunit (GRIN2A) gene suppresses transcriptional activity and correlates with chronic outcome in schizophrenia.

    Itokawa M, Yamada K, Yoshitsugu K, Toyota T, Suga T, Ohba H, Watanabe A, Hattori E, Shimizu H, Kumakura T, Ebihara M, Meerabux JM, Toru M and Yoshikawa T

    Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako, Saitama, Japan.

    Hypofunction of the N-methyl-D-aspartate (NMDA) receptor has been hypothesized to underlie the pathophysiology of schizophrenia, based on the observation that non-competitive antagonists of the NMDA receptor, such as phencyclidine, induce schizophrenia-like symptoms. Mice lacking the NR2A subunit of the NMDA receptor complex are known to display abnormal behaviour, similar to schizophrenic symptoms. The expression of NR2A starts at puberty, a period corresponding to the clinical onset of schizophrenia. This evidence suggests that the NR2A (GRIN2A) gene may play a role in the development of schizophrenia and disease phenotypes. In this study, we performed a genetic analysis of this gene in schizophrenia. Analysis of the GRIN2A gene detected four single nucleotide polymorphisms, and a variable (GT)(n) repeat in the promoter region of the gene. A case-control study (375 schizophrenics and 378 controls) demonstrated evidence of an association between the repeat polymorphism and the disease (P = 0.05, Mann-Whitney test), with longer alleles overly represented in patients. An in-vitro promoter assay revealed a length dependent inhibition of transcriptional activity by the (GT)(n) repeat, which was consistent with a receptor binding assay in postmortem brains. Significantly, the score of symptom severity in chronic patients correlated with repeat size (P = 0.01, Spearman's Rank test). These results illustrate a genotype-phenotype correlation in schizophrenia and suggest that the longer (GT)(n) stretch may act as a risk-conferring factor that worsens chronic outcome by reducing GRIN2A levels in the brain.

    Pharmacogenetics 2003;13;5;271-8

Literature (124)

Pubmed - other

  • Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour.

    Taniguchi S, Nakazawa T, Tanimura A, Kiyama Y, Tezuka T, Watabe AM, Katayama N, Yokoyama K, Inoue T, Izumi-Nakaseko H, Kakuta S, Sudo K, Iwakura Y, Umemori H, Inoue T, Murphy NP, Hashimoto K, Kano M, Manabe T and Yamamoto T

    Division of Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.

    Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.

    The EMBO journal 2009;28;23;3717-29

  • Common genetic variation and the control of HIV-1 in humans.

    Fellay J, Ge D, Shianna KV, Colombo S, Ledergerber B, Cirulli ET, Urban TJ, Zhang K, Gumbs CE, Smith JP, Castagna A, Cozzi-Lepri A, De Luca A, Easterbrook P, Günthard HF, Mallal S, Mussini C, Dalmau J, Martinez-Picado J, Miro JM, Obel N, Wolinsky SM, Martinson JJ, Detels R, Margolick JB, Jacobson LP, Descombes P, Antonarakis SE, Beckmann JS, O'Brien SJ, Letvin NL, McMichael AJ, Haynes BF, Carrington M, Feng S, Telenti A, Goldstein DB and NIAID Center for HIV/AIDS Vaccine Immunology (CHAVI)

    Center for Human Genome Variation, Duke Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA.

    To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians.

    Funded by: CCR NIH HHS: HHSN261200800001C; Intramural NIH HHS; Medical Research Council: G0200585; NCI NIH HHS: HHSN261200800001E; NIAID NIH HHS: AI067854, U01 AI067854, U19 AI067854; PHS HHS: HHSN261200800001E

    PLoS genetics 2009;5;12;e1000791

  • Genetical genomic determinants of alcohol consumption in rats and humans.

    Tabakoff B, Saba L, Printz M, Flodman P, Hodgkinson C, Goldman D, Koob G, Richardson HN, Kechris K, Bell RL, Hübner N, Heinig M, Pravenec M, Mangion J, Legault L, Dongier M, Conigrave KM, Whitfield JB, Saunders J, Grant B, Hoffman PL and WHO/ISBRA Study on State and Trait Markers of Alcoholism

    Department of Pharmacology, University of Colorado, Denver, Aurora, CO, USA. boris.tabakoff@ucdenver.edu

    Background: We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

    Results: In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.

    Conclusion: Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL35018, P01 HL035018; NIAAA NIH HHS: AA006420, AA013162, AA013517-INIA, AA013522-INIA, AA016649-INIA, AA016663-INIA, AA16922, K01 AA016922, P50 AA006420, P60 AA006420, R01 AA013162, R24 AA013162, R24 AA015512, U01 AA013517, U01 AA013522, U01 AA016649, U01 AA016663, U24 AA013517, U24 AA013522, U24 AA015512; NIDDK NIH HHS: R01 DK100340; Wellcome Trust

    BMC biology 2009;7;70

  • Identification of new putative susceptibility genes for several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment.

    Gratacòs M, Costas J, de Cid R, Bayés M, González JR, Baca-García E, de Diego Y, Fernández-Aranda F, Fernández-Piqueras J, Guitart M, Martín-Santos R, Martorell L, Menchón JM, Roca M, Sáiz-Ruiz J, Sanjuán J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X, Carracedo A and Psychiatric Genetics Network Group

    CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.

    A fundamental difficulty in human genetics research is the identification of the spectrum of genetic variants that contribute to the susceptibility to common/complex disorders. We tested here the hypothesis that functional genetic variants may confer susceptibility to several related common disorders. We analyzed five main psychiatric diagnostic categories (substance-abuse, anxiety, eating, psychotic, and mood disorders) and two different control groups, representing a total of 3,214 samples, for 748 promoter and non-synonymous single nucleotide polymorphisms (SNPs) at 306 genes involved in neurotransmission and/or neurodevelopment. We identified strong associations to individual disorders, such as growth hormone releasing hormone (GHRH) with anxiety disorders, prolactin regulatory element (PREB) with eating disorders, ionotropic kainate glutamate receptor 5 (GRIK5) with bipolar disorder and several SNPs associated to several disorders, that may represent individual and related disease susceptibility factors. Remarkably, a functional SNP, rs945032, located in the promoter region of the bradykinin receptor B2 gene (BDKRB2) was associated to three disorders (panic disorder, substance abuse, and bipolar disorder), and two additional BDKRB2 SNPs to obsessive-compulsive disorder and major depression, providing evidence for common variants of susceptibility to several related psychiatric disorders. The association of BDKRB2 (odd ratios between 1.65 and 3.06) to several psychiatric disorders supports the view that a common genetic variant could confer susceptibility to clinically related phenotypes, and defines a new functional hint in the pathophysiology of psychiatric diseases.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;6;808-16

  • Pharmacogenetics of antipsychotic response in the CATIE trial: a candidate gene analysis.

    Need AC, Keefe RS, Ge D, Grossman I, Dickson S, McEvoy JP and Goldstein DB

    Center for Human Genome Variation, Institute for Genome Sciences & Policy, Duke University, Durham, NC 27708, USA.

    The Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) Phase 1 Schizophrenia trial compared the effectiveness of one typical and four atypical antipsychotic medications. Although trials such as CATIE present important opportunities for pharmacogenetics research, the very richness of the clinical data presents challenges for statistical interpretation, and in particular the risk that data mining will lead to false-positive discoveries. For this reason, it is both misleading and unhelpful to perpetuate the current practice of reporting association results for these trials one gene at a time, ignoring the fact that multiple gene-by-phenotype tests are being carried out on the same data set. On the other hand, suggestive associations in such trials may lead to new hypotheses that can be tested through both replication efforts and biological experimentation. The appropriate handling of these forms of data therefore requires dissemination of association statistics without undue emphasis on select findings. Here we attempt to illustrate this approach by presenting association statistics for 2769 polymorphisms in 118 candidate genes evaluated for 21 pharmacogenetic phenotypes. On current evidence it is impossible to know which of these associations may be real, although in total they form a valuable resource that is immediately available to the scientific community.

    Funded by: NIMH NIH HHS: N01 MH90001

    European journal of human genetics : EJHG 2009;17;7;946-57

  • Candidate gene analysis in an on-going genome-wide association study of attention-deficit hyperactivity disorder: suggestive association signals in ADRA1A.

    Elia J, Capasso M, Zaheer Z, Lantieri F, Ambrosini P, Berrettini W, Devoto M and Hakonarson H

    Department of Pediatrics, Division of Pulmonary Medicine, Center for Applied Genomics, The University of Pennsylvania, Philadelphia, Pennsylvania, USA. elia@email.chop.edu

    Objectives: Attention-deficit hyperactivity disorder (ADHD) is a highly heritable, common developmental disorder. Although a few confirmed associations have emerged from candidate gene studies, these have shown the same limitations that have become evident in the study of other complex diseases, often with inconsistent and nonreplicated results across different studies.

    Methods: In this report, 27 ADHD candidate genes were explored in greater depth using high-density tag single nucleotide polymorphism (SNP) genotyping. Association with 557 SNPs was tested using the transmission disequilibrium test in 270 nuclear pedigrees selected from an ongoing ADHD genetic study that includes all disease subtypes.

    Results: SNPs in seven genes including SLC1A3, SLC6A3, HTR4, ADRA1A, HTR2A, SNAP25, and COMT showed a nominal level of association with ADHD (P values <0.05), but none remained significant after a stringent correction for the total number of tests performed.

    Conclusion: The strongest signal emerged from SNPs in the promoter region (rs3808585) and in an intron (rs17426222, rs4732682, rs573514) of ADRA1A, all located within the same haplotype block. Some of the SNPs in HTR2A and COMT have already been reported by others, whereas other SNPs will need confirmation in independent samples.

    Funded by: NCRR NIH HHS: UL1-RR-024134; NIMH NIH HHS: K23MH066275-01

    Psychiatric genetics 2009;19;3;134-41

  • A common variant in DRD3 receptor is associated with autism spectrum disorder.

    de Krom M, Staal WG, Ophoff RA, Hendriks J, Buitelaar J, Franke B, de Jonge MV, Bolton P, Collier D, Curran S, van Engeland H and van Ree JM

    Department of Neuroscience and Pharmacology and Department of Child and Adolescent Psychiatry, Rudolph Magnus Institute of Neuroscience, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, The Netherlands.

    Background: The presence of specific and common genetic etiologies for autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD) was investigated for 132 candidate genes in a two-stage design-association study.

    Methods: 1,536 single nucleotide polymorphisms (SNPs) covering these candidate genes were tested in ASD (n = 144) and ADHD (n = 110) patients and control subjects (n = 404) from The Netherlands. A second stage was performed with those SNPs from Stage I reaching a significance threshold for association of p < .01 in an independent sample of ASD patients (n = 128) and controls (n = 124) from the United Kingdom and a Dutch ADHD (n = 150) and control (n = 149) sample.

    Results: No shared association was found between ASD and ADHD. However, in the first and second ASD samples and in a joint statistical analysis, a significant association between SNP rs167771 located in the DRD3 gene was found (joint analysis uncorrected: p = 3.11 x 10(-6); corrected for multiple testing and potential stratification: p = .00162).

    Conclusions: The DRD3 gene is related to stereotyped behavior, liability to side effects of antipsychotic medication, and movement disorders and may therefore have important clinical implications for ASD.

    Biological psychiatry 2009;65;7;625-30

  • Elevated levels of NR2A and PSD-95 in the lateral amygdala in depression.

    Karolewicz B, Szebeni K, Gilmore T, Maciag D, Stockmeier CA and Ordway GA

    Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA. bkarolewicz@psychiatry.umsmed.edu

    Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 13-14 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.

    Funded by: NCRR NIH HHS: P20 RR017701, P20 RR017701-067505, RR17701; NIMH NIH HHS: K02 MH002031, K02 MH002031-06, MH02031, MH46692, MH63187, MH67996, R01 MH046692, R01 MH046692-14A1, R01 MH063187, R01 MH063187-05, R01 MH067996, R01 MH067996-04

    The international journal of neuropsychopharmacology 2009;12;2;143-53

  • Genome-wide association study of smoking initiation and current smoking.

    Vink JM, Smit AB, de Geus EJ, Sullivan P, Willemsen G, Hottenga JJ, Smit JH, Hoogendijk WJ, Zitman FG, Peltonen L, Kaprio J, Pedersen NL, Magnusson PK, Spector TD, Kyvik KO, Morley KI, Heath AC, Martin NG, Westendorp RG, Slagboom PE, Tiemeier H, Hofman A, Uitterlinden AG, Aulchenko YS, Amin N, van Duijn C, Penninx BW and Boomsma DI

    Department of Biological Psychology, Center for Neurogenomic and Cognitive Research, VU University Amsterdam, The Netherlands. jm.vink@psy.vu.nl

    For the identification of genes associated with smoking initiation and current smoking, genome-wide association analyses were carried out in 3497 subjects. Significant genes that replicated in three independent samples (n = 405, 5810, and 1648) were visualized into a biologically meaningful network showing cellular location and direct interaction of their proteins. Several interesting groups of proteins stood out, including glutamate receptors (e.g., GRIN2B, GRIN2A, GRIK2, GRM8), proteins involved in tyrosine kinase receptor signaling (e.g., NTRK2, GRB14), transporters (e.g., SLC1A2, SLC9A9) and cell-adhesion molecules (e.g., CDH23). We conclude that a network-based genome-wide association approach can identify genes influencing smoking behavior.

    Funded by: NIMH NIH HHS: MH074027, MH077139, MH081802, R01 MH074027, R01 MH077139, R01 MH081802; Wellcome Trust

    American journal of human genetics 2009;84;3;367-79

  • SGK1 phosphorylation of IkappaB Kinase alpha and p300 Up-regulates NF-kappaB activity and increases N-Methyl-D-aspartate receptor NR2A and NR2B expression.

    Tai DJ, Su CC, Ma YL and Lee EH

    Graduate Institute of Life Sciences, National Defense Medical Center, Academia Sinica, Taipei 115, Taiwan.

    Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IkappaB kinase alpha (IKKalpha) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKalpha at Thr-23 and indirectly activates IKKalpha at Ser-180. Furthermore, SGK1 enhanced nuclear factor kappaB (NF-kappaB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKalpha at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKbeta, IKKbeta activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-kappaB. In addition, SGK1 increased the acetylation of NF-kappaB through phosphorylation of p300 at Ser-1834, and this also leads to NF-kappaB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKalpha and NF-kappaB phosphorylation as well as NF-kappaB acetylation and NF-kappaB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKalpha-NF-kappaB signaling pathway, we found that transfection of the IKKalpha double mutant (IKKalphaT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

    The Journal of biological chemistry 2009;284;7;4073-89

  • The integrity of the glycine co-agonist binding site of N-methyl-D-aspartate receptors is a functional quality control checkpoint for cell surface delivery.

    Kenny AV, Cousins SL, Pinho L and Stephenson FA

    School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.

    N-Methyl-D-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an approximately 3 x 10(4) decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D002753/1

    The Journal of biological chemistry 2009;284;1;324-33

  • The expression of NMDA receptor subunit mRNA in human chronic alcoholics.

    Ridge JP, Ho AM, Innes DJ and Dodd PR

    SMMS, University of Queensland, Brisbane, Queensland, Australia.

    Ethanol is a modulator at the N-methyl-d-aspartate class of glutamate receptors in the brain. In animal studies the receptor adapts to sustained ethanol exposure through altered expression of the subunits that make up the receptor complex. We used real-time RT-PCR normalized to GAPDH to assay NR1, NR2A, and NR2B subunit mRNA in superior frontal and primary motor cortex tissue obtained at autopsy from chronic alcoholics with and without co-morbid cirrhosis of the liver, and from matched controls. The expression of all three subunits was significantly lower in both areas of cirrhotic alcoholics than in the corresponding areas in both controls and alcoholics without co-morbid disease, who did not differ significantly from each other. The decrease was area-dependent when cases were partitioned by the 5-HTTLPR allele. Thus, polymorphisms in one gene can have a significant effect on the expression of a second, unrelated, gene. The expression of the N-methyl-d-aspartate glutamate receptor complex is under multifactorial control.

    Funded by: NIAAA NIH HHS: R24 AA012725, R28 AA012725

    Annals of the New York Academy of Sciences 2008;1139;10-9

  • Alterations in NMDA receptor subunit densities and ligand binding to glycine recognition sites are associated with chronic anxiety in Alzheimer's disease.

    Tsang SW, Vinters HV, Cummings JL, Wong PT, Chen CP and Lai MK

    Dementia Research Laboratory, Department of Clinical Research, Singapore General Hospital, Singapore.

    Glutamatergic deficits are established neuropathological features of Alzheimer's disease (AD) and are known to correlate with cognitive impairments. In contrast, the role of glutamatergic alterations in behavioral and psychological symptoms of dementia (BPSD) is unclear. There is considerable preclinical evidence for the importance of glycine recognition sites (GlyRS) of N-methyl-D-aspartate (NMDA) receptors in the regulation of anxiety behaviors. This study aimed to correlate several glutamatergic measures with chronic anxiety in AD. Twenty-one AD patients assessed by the Neuropsychiatric Inventory (NPI) were divided into low anxiety (LA) and high anxiety (HA) subgroups. GlyRS and NMDA channel were measured by brain homogenate binding with [(3)H]MDL105,519 and [(3)H]MK-801, respectively. Densities of NMDA receptor NR2A, NR2B and alternate spliced NR1 subunits were quantified by immunoblotting. We found that the binding affinity to GlyRS was significantly higher in HA compared to LA, and this higher GlyRS affinity correlated with selective reduction of NR2A density as well as with elevated anxiety scores. Our observations suggest a novel mechanism whereby subunit specific changes in the NMDA receptor complex may be linked to chronic anxiety in AD via effects on GlyRS function. We propose that NR2A and GlyRS should be further assessed as novel targets of behavioral pharmacotherapy in AD.

    Funded by: NIA NIH HHS: P50 AG016570, P50 AG016570-049003, P50 AG16570

    Neurobiology of aging 2008;29;10;1524-32

  • Systematic analysis of glutamatergic neurotransmission genes in alcohol dependence and adolescent risky drinking behavior.

    Schumann G, Johann M, Frank J, Preuss U, Dahmen N, Laucht M, Rietschel M, Rujescu D, Lourdusamy A, Clarke TK, Krause K, Dyer A, Depner M, Wellek S, Treutlein J, Szegedi A, Giegling I, Cichon S, Blomeyer D, Heinz A, Heath S, Lathrop M, Wodarz N, Soyka M, Spanagel R and Mann K

    Interdisciplinary Research Group Addiction, MRC-SGDP Center, Institute of Psychiatry at King's College, POB 080, London SE5 8AF, England. g.schumann@iop.kcl.ac.uk

    Context: Glutamatergic neurotransmission is implicated in alcohol-drinking behavior in animal models.

    Objective: To investigate whether genetic variations in glutamatergic neurotransmission genes, which are known to alter alcohol effects in rodents, contribute to the genetic basis of alcoholism in humans.

    Design: Association analysis of alcohol dependence and haplotype-tagging single nucleotide polymorphisms (SNPs) covering 10 glutamatergic genes. Resequencing of functional domains of these genes identified 204 SNPs. Haplotypes with a frequency of 5% or greater could be discriminated by 21 haplotype-tagging SNPs analyzed for association in 2 independent samples of alcohol-dependent adult patients and controls as well as adolescent trios.

    Setting: Four university medical centers in the south of Germany.

    Participants: One thousand three hundred thirty-seven patients and 1555 controls (study 1: 544 patients, 553 controls; study 2: 793 patients, 1002 controls). One hundred forty-four trios of 15-year-old adolescents assessed for risky drinking behavior.

    Genotype profiles for GLAST; N-methyl-d-aspartate-receptor subunits NR1, NR2A, and NR2B; MGLUR5; NNOS; PRKG2; CAMK4; the regulatory subunit of PI3K; and CREB were analyzed for association with alcohol dependence using multivariate statistical analysis. Risky adolescent drinking was tested using the transmission disequilibrium test.

    Results: Analysis of study 1 revealed that NR2A and MGLUR5 have the greatest relevance for human alcohol dependence among the genes selected with odds ratios of 2.35 and 1.69, respectively. Replication analysis in study 2 confirmed an association of alcohol dependence with NR2A (odds ratio, 2.01) but showed no association with MGLUR5. Combined analysis of study 1 and study 2 exhibited a more significant association on the Cochran-Mantel-Haenszel test (P < .001) for NR2A; NR2A was associated with positive family history, early onset of alcoholism, and maximum number of drinks in adults as well as risky drinking patterns in adolescents.

    Conclusion: Genetic variations in NR2A have the greatest relevance for human alcohol dependence among the glutamatergic genes selected for their known alteration of alcohol effects in animal models.

    Funded by: Department of Health

    Archives of general psychiatry 2008;65;7;826-38

  • Enhanced ethanol inhibition of recombinant N-methyl-D-aspartate receptors by magnesium: role of NR3A subunits.

    Jin C, Smothers CT and Woodward JJ

    Department of Neurosciences and Center for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, South Carolina.

    Background: The effects of ethanol on brain function are thought to be partly because of altered activity of ion channels that regulate synaptic activity. Results from previous studies from this lab and others have shown that ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) receptors, a calcium-permeable ion channel activated by the neurotransmitter glutamate. Factors that influence the acute sensitivity of NMDA receptors to ethanol may be critical in determining how neurons and neuronal networks respond to the presence of ethanol. In this study, we have examined the effect of physiologically relevant concentrations of magnesium on the ethanol sensitivity of recombinant NMDA receptors and how ethanol inhibition under these conditions is influenced by the NR3A subunit.

    Methods: Recombinant cDNAs encoding NMDA receptor subunits were expressed in human embryonic kidney 293 cells. Whole-cell patch-clamp electrophysiology was used to measure currents induced by rapid application of glutamate in the absence and presence of ethanol.

    Results: In magnesium-free recording solution, ethanol inhibited glutamate-mediated currents in cells transfected with NMDA receptor subunits. The magnitude of ethanol inhibition was significantly enhanced when recordings were carried out in media containing 1 mM magnesium. This effect was reversible and required magnesium-sensitive receptors. Magnesium did not enhance ethanol inhibition of glycine-activated NR1/NR3A/NR3B receptors. However, NR3A co-expression prevented the enhancement of ethanol's inhibitory effect on receptors composed of NR2A but not NR2B subunits.

    Conclusions: These results suggest that under physiological conditions, NR3A may be an important regulator of the acute ethanol sensitivity of brain NMDA receptors.

    Funded by: NIAAA NIH HHS: AA09986, R01 AA009986, R37 AA009986; PHS HHS: K0200238

    Alcoholism, clinical and experimental research 2008;32;6;1059-66

  • The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth.

    Kim MS, Chang X, Nagpal JK, Yamashita K, Baek JH, Dasgupta S, Wu G, Osada M, Woo JH, Westra WH, Trink B, Ratovitski EA, Moon C and Sidransky D

    Department of Otolaryngology, Head and Neck Cancer Research Division, Johns Hopkins University, Baltimore, MD, USA.

    N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

    Oncogene 2008;27;14;2045-54

  • The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells.

    Watanabe K, Kanno T, Oshima T, Miwa H, Tashiro C and Nishizaki T

    Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan.

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-d-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G(1) phase of cell cycling and decreased the proportion in the S/G(2) phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G(1) phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

    Biochemical and biophysical research communications 2008;367;2;487-90

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Analysis of NR3A receptor subunits in human native NMDA receptors.

    Nilsson A, Eriksson M, Muly EC, Akesson E, Samuelsson EB, Bogdanovic N, Benedikz E and Sundström E

    Division of Neurodegeneration and Neuroinflammation, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Stockholm, Sweden. Anna.Nilsson.2@ki.se

    NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2A/B-NR3A assemblies in adult human CNS. While NR1 and NR2A could only be efficiently solubilized by deoxycholate, NR3A was extracted by all detergents, suggesting that a large fraction is weakly anchored to cell membranes and other proteins. Using size exclusion chromatography we found that just as for NR1, a large fraction of NR3A exists as monomers and dimers, suggesting that these two glycine binding subunits behave similarly with regard to receptor assembly and trafficking.

    Funded by: NCRR NIH HHS: RR 00165; NIMH NIH HHS: MH 01994

    Brain research 2007;1186;102-12

  • Elevated GRIA1 mRNA expression in Layer II/III and V pyramidal cells of the DLPFC in schizophrenia.

    O'Connor JA and Hemby SE

    Molecular and Systems Pharmacology Graduate Program, Emory University School of Medicine, Atlanta, GA, United States.

    The functional integrity of the dorsolateral prefrontal cortex (DLPFC) is altered in schizophrenia leading to profound deficits in working memory and cognition. Growing evidence indicates that dysregulation of glutamate signaling may be a significant contributor to the pathophysiology mediating these effects; however, the contribution of NMDA and AMPA receptors in the mediation of this deficit remains unclear. The equivocality of data regarding ionotropic glutamate receptor alterations of subunit expression in the DLPFC of schizophrenics is likely reflective of subtle alterations in the cellular and molecular composition of specific neuronal populations within the region. Given previous evidence of Layer II/III and V pyramidal cell alterations in schizophrenia and the significant influence of subunit composition on NMDA and AMPA receptor function, laser capture microdissection combined with quantitative PCR was used to examine the expression of AMPA (GRIA1-4) and NMDA (GRIN1, 2A and 2B) subunit mRNA levels in Layer II/III and Layer V pyramidal cells in the DLPFC. Comparisons were made between individuals diagnosed with schizophrenia, bipolar disorder, major depressive disorder and controls (n=15/group). All subunits were expressed at detectable levels in both cell populations for all diseases as well as for the control group. Interestingly, GRIA1 mRNA was significantly increased in both cell types in the schizophrenia group compare to controls, while similar trends were observed in major depressive disorder (Layers II/III and V) and bipolar disorder (Layer V). These data suggest that increased GRIA1 subunit expression may contribute to schizophrenia pathology.

    Funded by: NIMH NIH HHS: MH074313, R01 MH074313, R01 MH074313-05

    Schizophrenia research 2007;97;1-3;277-88

  • NR2A and NR2B receptor gene variations modify age at onset in Huntington disease in a sex-specific manner.

    Arning L, Saft C, Wieczorek S, Andrich J, Kraus PH and Epplen JT

    Department of Human Genetics, Ruhr-University, 44780 Bochum, Germany. larissa.arning@rub.de

    In addition to the pathogenetic CAG repeat expansion other genetic factors play a significant role in determining age at onset (AO) in Huntington disease (HD), e.g. variations in the NR2A and NR2B glutamate receptor subunit genes (GRIN2A, GRIN2B). In order to expand these findings we fine-mapped a larger HD patient panel (n = 250) using densely spaced markers flanking the originally associated SNPs in GRIN2A and GRIN2B. In GRIN2A association fine-mapping based on eight additional SNPs confirmed intron 2 as the region of strongest association. In GRIN2B fine-mapping with seven additional SNPs consolidated C2664T as causal genetic variation. Gender stratification of patients revealed differences in the variability in AO attributable to the CAG repeat number and highly significant differences in the AO association with the C2664T and rs8057394/ rs2650427 variations. Addition of the corresponding genotype variations to the effect of CAG repeat lengths resulted in a significant increase of the R2 values only in females. The sex-specific effect for C2664T is underscored by differences in the genotype and allele frequencies observed for female versus male HD patients (P = 0.01) caused by decreased CC frequency in females. Overall, female HD patients homozygous for the CC genotype tended to have later AO compared to the other two genotypes. Stratification of the results by presumed menopausal status demonstrated that the significant findings were predominantly observed in pre-menopausal patients. We speculate that altered hormone levels herald protective effects of this genotype. Together, GRIN2A and GRIN2B genotype variations explain 7.2% additional variance in AO for HD.

    Human genetics 2007;122;2;175-82

  • Mutations at F637 in the NMDA receptor NR2A subunit M3 domain influence agonist potency, ion channel gating and alcohol action.

    Ren H, Salous AK, Paul JM, Lipsky RH and Peoples RW

    Department of Biomedical Sciences, Marquette University, Milwaukee, WI 53201-1881, USA.

    NMDA receptors are important molecular targets of ethanol action in the CNS. Previous studies have identified a site in membrane-associated domain 3 (M3) of the NR1 subunit and two sites in M4 of the NR2A subunit that influence alcohol action; the sites in NR2A M4 also regulate ion channel gating. The purpose of this study was to determine whether mutations at the site in the NR2A subunit corresponding to the NR1 M3 site influence alcohol action and ion channel gating.

    We investigated the effects of mutations at phenylalanine (F) 637 of the NR2A subunit using whole-cell and single-channel patch-clamp electrophysiological recording in transiently-transfected HEK 293 cells.

    Mutations at F637 in the NR2A subunit altered peak and steady-state glutamate EC(50) values, maximal steady-state to peak current ratios (I(ss):I(p)), mean open time, and ethanol IC(50) values. Differences in glutamate potency among the mutants were not due to changes in desensitization. Ethanol IC(50) values were significantly correlated with glutamate EC(50) values, but not with maximal I(ss):I(p) or mean open time. Ethanol IC(50) values were linearly and inversely related to molecular volume of the substituent.

    These results demonstrate that NR2A(F637) influences NMDA receptor affinity, ion channel gating, and ethanol sensitivity. The changes in NMDA receptor affinity are likely to be the result of altered ion channel gating. In contrast to the cognate site in the NR1 subunit, the action of ethanol does not appear to involve occupation of a critical volume at NR2A(F637).

    Funded by: NIAAA NIH HHS: AA15203-01A1, R01 AA015203

    British journal of pharmacology 2007;151;6;749-57

  • A three amino acid tail following the TM4 region of the N-methyl-D-aspartate receptor (NR) 2 subunits is sufficient to overcome endoplasmic reticulum retention of NR1-1a subunit.

    Yang W, Zheng C, Song Q, Yang X, Qiu S, Liu C, Chen Z, Duan S and Luo J

    Department of Neurobiology, Institute for Neuroscience, Zhejiang University School of Medicine, Hangzhou 3100058, China.

    The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2Delta2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2Delta2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2Delta2 subunits were cotransfected with either the NR1-4a or NR1-1a(AAA) mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2BDelta5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface.

    The Journal of biological chemistry 2007;282;12;9269-78

  • Disruption of interdomain interactions in the glutamate binding pocket affects differentially agonist affinity and efficacy of N-methyl-D-aspartate receptor activation.

    Maier W, Schemm R, Grewer C and Laube B

    Max-Planck-Institute for Brain Research, Frankfurt 60528, Germany.

    In ionotropic glutamate receptors, agonist binding occurs in a conserved clam shell-like domain composed of the two lobes D1 and D2. Docking of glutamate into the binding cleft promotes rotation in the hinge region of the two lobes, resulting in closure of the binding pocket, which is thought to represent a prerequisite for channel gating. Here, we disrupted D1D2 interlobe interactions in the NR2A subunit of N-methyl-d-aspartate (NMDA) receptors through systematic mutation of individual residues and studied the influence on the activation kinetics of currents from NR1/NR2 NMDA receptors heterologously expressed in HEK cells. We show that the mutations affect differentially glutamate binding and channel gating, depending on their location within the binding domain, mainly by altering k(off) and k(cl), respectively. Whereas impaired stability of glutamate in its binding site is the only effect of mutations on one side of the ligand binding pocket, close to the hinge region, alterations in gating are the predominant consequence of mutations on the opposite side, at the entrance of the binding pocket. A mutation increasing D1D2 interaction at the entrance of the pocket resulted in an NMDA receptor with an increased open probability as demonstrated by single channel and whole cell kinetic analysis. Thus, the results indicate that agonist-induced binding domain closure is itself a complex process, certain aspects of which are coupled either to binding or to gating. Specifically, we propose that late steps of domain closure, in kinetic terms, represent part of channel gating.

    The Journal of biological chemistry 2007;282;3;1863-72

  • Replication of twelve association studies for Huntington's disease residual age of onset in large Venezuelan kindreds.

    Andresen JM, Gayán J, Cherny SS, Brocklebank D, Alkorta-Aranburu G, Addis EA, US-Venezuela Collaborative Research Group, Cardon LR, Housman DE and Wexler NS

    Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. jma@mit.edu

    Background: The major determinant of age of onset in Huntington's disease is the length of the causative triplet CAG repeat. Significant variance remains, however, in residual age of onset even after repeat length is factored out. Many genetic polymorphisms have previously shown evidence of association with age of onset of Huntington's disease in several different populations.

    Objective: To replicate these genetic association tests in 443 affected people from a large set of kindreds from Venezuela.

    Methods: Previously tested polymorphisms were analysed in the HD gene itself (HD), the GluR6 kainate glutamate receptor (GRIK2), apolipoprotein E (APOE), the transcriptional coactivator CA150 (TCERG1), the ubiquitin carboxy-terminal hydrolase L1 (UCHL1), p53 (TP53), caspase-activated DNase (DFFB), and the NR2A and NR2B glutamate receptor subunits (GRIN2A, GRIN2B).

    Results: The GRIN2A single-nucleotide polymorphism explains a small but considerable amount of additional variance in residual age of onset in our sample. The TCERG1 microsatellite shows a trend towards association but does not reach statistical significance, perhaps because of the uninformative nature of the polymorphism caused by extreme allele frequencies. We did not replicate the genetic association of any of the other genes.

    Conclusions: GRIN2A and TCERG1 may show true association with residual age of onset for Huntington's disease. The most surprising negative result is for the GRIK2 (TAA)(n) polymorphism, which has previously shown association with age of onset in four independent populations with Huntington's disease. The lack of association in the Venezuelan kindreds may be due to the extremely low frequency of the key (TAA)(16) allele in this population.

    Journal of medical genetics 2007;44;1;44-50

  • Significant linkage and association between a functional (GT)n polymorphism in promoter of the N-methyl-D-aspartate receptor subunit gene (GRIN2A) and schizophrenia.

    Tang J, Chen X, Xu X, Wu R, Zhao J, Hu Z and Xia K

    Institute of Mental Health, The Second Xiangya Hospital, Central South University, 139 Renmin Road, Changsha Hunan 410011, PR China.

    Dysfunction of the N-methyl-d-aspartate (NMDA) type glutamate receptor has been proposed as a mechanism in the etiology of schizophrenia, based on the observation that non-competitive antagonists of the NMDA receptor, such as phencyclidine, induce schizophrenia-like symptoms. Previous study identified a variable (GT)n polymorphism in the promoter region of the N-methyl-d-aspartate (NMDA) subunit gene (GRIN2A), and showed its association with schizophrenia in a case-control study, together with a correlation between the length of the repeat and severity of chronic outcome. Our present study was aimed at confirming the association of the (GT)n polymorphism of GRIN2A promoter with schizophrenia using 122 Han Chinese sib-pair families. Non-parametric linkage analysis and transmission/disequilibrium test (TDT) were undertaken using the GENEHUNTER, v2.1. In non-parametric linkage analysis, suggestive linkage was found for the (GT)n polymorphism (NPL=2.77, P=0.002902). The TDT was significant for (GT)n polymorphism and that the (GT)23 was preferentially transmitted to schizophrenia-affected children (T/NT: 123:72, chi(2)=13.34, P=0.000260). Our results indicate that the (GT)n polymorphism in the promoter of GRIN2A gene may play a significant role in the etiology of schizophrenia among our samples.

    Neuroscience letters 2006;409;1;80-2

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • 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

  • The human immunodeficiency virus-1 protein transactivator of transcription up-regulates N-methyl-D-aspartate receptor function by acting at metabotropic glutamate receptor 1 receptors coexisting on human and rat brain noradrenergic neurones.

    Longordo F, Feligioni M, Chiaramonte G, Sbaffi PF, Raiteri M and Pittaluga A

    Pharmacology and Toxicology Section, Department of Experimental Medicine, University of Genova, Genova, Italy.

    We investigated the effects of the human immunodeficiency virus-1 transactivator of transcription (Tat) on the release of norepinephrine (NE) from human and rat brain synaptosomes. Tat could not evoke directly release of [3H]NE. In the presence of Tat (1 nM), N-methyl-D-aspartate (NMDA) concentrations unable to release (human synaptosomes) or slightly releasing (rat synaptosomes) [3H]NE became very effective. The NMDA/Tat-evoked release depends on NMDA receptors (NMDARs) since it was abolished by MK-801 (dizocilpine). Tat binding at NMDARs was excluded. The NMDA-induced release of [3H]NE in the presence of glycine was further potentiated by Tat. The release evoked by NMDA/glycine/Tat depends on metabotropic glutamate receptor 1 (mGluR1) activation, since it was halved by mGluR1 antagonists. Tat seems to act at the glutamate recognition site of mGluR1. Recently, Tat was shown to release [3H]acetylcholine from human cholinergic terminals; here, we demonstrate that this effect is also mediated by presynaptic mGluR1. The peptide sequence Tat41-60, but not Tat61-80, mimicked Tat. Phospholipase C, protein kinase C, and cytosolic tyrosine kinase are involved in the NMDA/glycine/Tat-evoked [3H]NE release. To conclude, Tat can represent a potent pathological agonist at mGlu1 receptors able to release acetylcholine from human cholinergic terminals and up-regulate NMDARs mediating NE release from human and rat noradrenergic terminals.

    The Journal of pharmacology and experimental therapeutics 2006;317;3;1097-105

  • Effects of SDF-1alpha and gp120IIIB on apoptotic pathways in SK-N-SH neuroblastoma cells.

    Geeraerts T, Deiva K, M'sika I, Salim H, Héry C and Tardieu M

    Laboratoire Immunologie antivirale systémique et cérébrale, INSERM EMI 0109, Faculté de médecine Paris-Sud, 63 rue Gabriel Péri, 94 276 Le Kremlin Bicêtre, France. thgeeraerts@hotmail.com

    CXCR4, a chemokine receptor constitutively expressed in the brain, binds both ligands, the chemokine SDF-1alpha and the HIV envelope glycoprotein gp120(IIIB). There seem to be intracellular differences between the neuronal apoptosis induced by SDF-1alpha and that induced by gp120(IIIB), but the apoptotic pathways involved have not been compared in human neuronal cells. In this study, we characterized the apoptotic intracellular pathways activated by neurotoxic concentrations of SDF-1alpha and gp120(IIIB) in human neuroblastoma cells SK-N-SH. SDF-1alpha (10 nM) and gp120(IIIB) (2 nM) induced similar levels of apoptosis after 24 h of incubation (49 +/- 4% and 48 +/- 3%, respectively, of the neurons were apoptotic). SDF1alpha-induced apoptosis was completely abolished by the inhibition of Src phosphorylation by PP2. Exposure to SDF-1alpha (10 nM) triggered an increase in Src phosphorylation, with a maximum after 20 min of incubation (1.80 +/- 0.24 times higher than control, P = 0.01). NMDA calcium flux was enhanced only if cells were incubated with SDF-1alpha for 20 min before applying NMDA. By contrast, gp120(IIIB)-induced apoptosis was not affected by the inhibition of Src phosphorylation. Moreover, gp120(IIIB) enhanced NMDA calcium flux immediately, without modifying Src phosphorylation status. Finally, levels of phospho-JNK increased following exposure to gp120(IIIB) (by a factor of 1.46 +/- 0.4 at 120 min, P = 0.03), but not after exposure to SDF-1alpha. Thus, SDF-1alpha and gp120(IIIB) induced a similar level of neuronal apoptosis, but by activating different intracellular pathways. SDF-1alpha enhanced NMDA activity indirectly via Src phosphorylation, whereas gp120(IIIB) probably activated the NMDA receptor directly and phosphorylated JNK.

    Neuroscience letters 2006;399;1-2;115-20

  • Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.

    Stiffler MA, Grantcharova VP, Sevecka M and MacBeath G

    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.

    One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

    Funded by: NIGMS NIH HHS: 1 R01 GM072872-01, 5 T32 GM07598-25, R01 GM072872, R01 GM072872-04, T32 GM007598

    Journal of the American Chemical Society 2006;128;17;5913-22

  • Significant association between the genetic variations in the 5' end of the N-methyl-D-aspartate receptor subunit gene GRIN1 and schizophrenia.

    Zhao X, Li H, Shi Y, Tang R, Chen W, Liu J, Feng G, Shi J, Yan L, Liu H and He L

    Bio-X Center, Shanghai Jiao Tong University, China.

    Background: N-methyl-D-aspartate (NMDA) receptors play important roles in many neurophysiological processes. Evidence from previous studies indicate that NMDA receptors contribute to the pathophysiology of schizophrenia. Two NMDA receptor subunit genes, GRIN1 and GRIN2A, are both good candidate genes for schizophrenia.

    Method: We genotyped five single nucleotide polymorphisms (SNPs) in GRIN1 and two in GRIN2A in 2455 Han Chinese subjects, including population- and family-based samples, and performed case-control and transmission disequilibrium test (TDT) analyses. A microsatellite in GRIN2A was genotyped in population-based samples and a Mann-Whitney U test was performed.

    Results: A highly significant association was detected at the 5' end of GRIN1. Analyses of single variants and multiple-locus haplotypes indicate that the association is mainly generated by rs11146020 (case-control study: p = .0000013, odds ratio = .61, 95% confidence interval .50-.74; TDT: p = .0019, T/NT = 79/123). No association was found in the GRIN2A polymorphisms.

    Conclusions: Our results provide support for the hypothesis that NMDA receptors are an important factor in schizophrenia. Moreover, rs11146020 is located in 5' untranslated region where several functional elements have been found. Hence, the SNP is a potential candidate in altering risk for schizophrenia and worthy of further replication and functional study.

    Biological psychiatry 2006;59;8;747-53

  • HIV tat and neurotoxicity.

    King JE, Eugenin EA, Buckner CM and Berman JW

    Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

    HIV tat is the transactivator of HIV-1, supporting efficient viral replication by stabilizing the transcription of viral genes. Tat can be released from HIV-infected cells and alter several functions in uninfected cells. In the brain, tat induces neuronal dysfunction/toxicity, even though neurons cannot be directly infected with HIV, resulting in CNS pathology, such as the dementia and encephalitis associated with NeuroAIDS. This review discusses the most recent data addressing tat-induced neurotoxicity and integrates these new findings in the context of NeuroAIDS.

    Funded by: NIAID NIH HHS: AI-051519; NIGMS NIH HHS: 5 T32 GM007288; NIMH NIH HHS: K01 MH076679, MH0702297, MH52974; NINDS NIH HHS: NS07098, NS11920

    Microbes and infection 2006;8;5;1347-57

  • Analysis of correlation between serum D-serine levels and functional promoter polymorphisms of GRIN2A and GRIN2B genes.

    Iwayama Y, Hashimoto K, Nakajima M, Toyota T, Yamada K, Shimizu E, Itokawa M, Hoshika A, Iyo M and Yoshikawa T

    Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako, Saitama, Japan.

    D-Serine is an endogenous coagonist that increases the opening of N-methyl-D-aspartate (NMDA)-type glutamate receptor channels. We previously reported a reduction of D-serine serum levels in schizophrenia, supporting the disease hypothesis of NMDA receptor-mediated hypo-neurotransmission. The serum levels of D-serine are thought to reflect brain d-serine content. It is important to understand whether there is a direct link between the altered D-serine levels and NMDA receptor expression in vivo or whether these are independent processes. Two polymorphisms are known to regulate the expression of NMDA receptor subunit genes: (GT)(n) (rs3219790) in the promoter region of the NR2A subunit gene (GRIN2A) and -200T > G (rs1019385) in the NR2B gene (GRIN2B). These polymorphisms are also reported to be associated with schizophrenia. Therefore, we examined the correlation between these two polymorphisms and d-serine serum levels in mentally healthy controls, schizophrenics and the combined group. We observed no significant genotype-phenotype correlations in any of the sample groups. However, analyses of larger sample numbers and the detection of additional polymorphisms that affect gene expression are needed before we can conclude that NMDA receptor expression and serum levels of d-serine, if involved in schizophrenia pathophysiology, are independent and additive events.

    Neuroscience letters 2006;394;2;101-4

  • Subunit assembly of N-methyl-d-aspartate receptors analyzed by fluorescence resonance energy transfer.

    Qiu S, Hua YL, Yang F, Chen YZ and Luo JH

    Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310031, China.

    N-methyl-d-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function.

    The Journal of biological chemistry 2005;280;26;24923-30

  • Candidate-gene screening and association analysis at the autism-susceptibility locus on chromosome 16p: evidence of association at GRIN2A and ABAT.

    Barnby G, Abbott A, Sykes N, Morris A, Weeks DE, Mott R, Lamb J, Bailey AJ, Monaco AP and International Molecular Genetics Study of Autism Consortium

    Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

    Autism is a highly heritable neurodevelopmental disorder whose underlying genetic causes have yet to be identified. To date, there have been eight genome screens for autism, two of which identified a putative susceptibility locus on chromosome 16p. In the present study, 10 positional candidate genes that map to 16p11-13 were examined for coding variants: A2BP1, ABAT, BFAR, CREBBP, EMP2, GRIN2A, MRTF-B, SSTR5, TBX6, and UBN1. Screening of all coding and regulatory regions by denaturing high-performance liquid chromatography identified seven nonsynonymous changes. Five of these mutations were found to cosegregate with autism, but the mutations are not predicted to have deleterious effects on protein structure and are unlikely to represent significant etiological variants. Selected variants from candidate genes were genotyped in the entire International Molecular Genetics Study of Autism Consortium collection of 239 multiplex families and were tested for association with autism by use of the pedigree disequilibrium test. Additionally, genotype frequencies were compared between 239 unrelated affected individuals and 192 controls. Patterns of linkage disequilibrium were investigated, and the transmission of haplotypes across candidate genes was tested for association. Evidence of single-marker association was found for variants in ABAT, CREBBP, and GRIN2A. Within these genes, 12 single-nucleotide polymorphisms (SNPs) were subsequently genotyped in 91 autism trios (one affected individual and two unaffected parents), and the association was replicated within GRIN2A (Fisher's exact test, P<.0001). Logistic regression analysis of SNP data across GRIN2A and ABAT showed a trend toward haplotypic differences between cases and controls.

    Funded by: NCRR NIH HHS: M01 RR006022, MO1 RR06022; NICHD NIH HHS: U19 HD035482, U19 HD35482; NIMH NIH HHS: K02 MH01389, K05 MH01196; Telethon: GGP030227; Wellcome Trust

    American journal of human genetics 2005;76;6;950-66

  • Extended analyses support the association of a functional (GT)n polymorphism in the GRIN2A promoter with Japanese schizophrenia.

    Iwayama-Shigeno Y, Yamada K, Itokawa M, Toyota T, Meerabux JM, Minabe Y, Mori N, Inada T and Yoshikawa T

    Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama, Japan.

    Dysfunction of the N-methyl-D-aspartate (NMDA) type glutamate receptor has been proposed as a mechanism in the etiology of schizophrenia. Recently, we identified a variable (GT)n repeat in the promoter region of the NMDA NR2A subunit gene (GRIN2A), and showed its association with schizophrenia in a case-control study, together with a correlation between the length of the repeat and severity of chronic outcome. In this study, we extended our analyses, by increasing the number of case-control samples to a total of 672 schizophrenics and 686 controls, and excluded potential sample stratification effects. We confirmed the significant allelic association between the repeat polymorphism and disease (P = 0.011), and as in the previous study, we observed an over-representation of longer alleles in schizophrenia. These results suggest a probable genetic effect for the GRIN2A promoter (GT)n variation on the predisposition to schizophrenia in Japanese cohorts.

    Neuroscience letters 2005;378;2;102-5

  • Ethanol potentiates HIV-1 gp120-induced apoptosis in human neurons via both the death receptor and NMDA receptor pathways.

    Chen W, Tang Z, Fortina P, Patel P, Addya S, Surrey S, Acheampong EA, Mukhtar M and Pomerantz RJ

    Center for Human Virology and Biodefense, Division of Infectious Diseases and Environmental Medicine, Thomas Jefferson University, 1020 Locust Street, Suite 329, Philadelphia, PA 19107, USA.

    Neuronal loss is a hallmark of AIDS dementia syndromes. Human immunodeficiency virus type I (HIV-1)-specific proteins may induce neuronal apoptosis, but the signal transduction of HIV-1 gp120-induced, direct neuronal apoptosis remains unclear. Ethanol (EtOH) is considered to be an environmental co-factor in AIDS development. However, whether EtOH abuse in patients with AIDS increases neuronal dysfunction is still uncertain. Using pure, differentiated, and post-mitotic NT2.N-derived human neurons, we investigated the mechanisms of HIV-1 and/or EtOH-related direct neuronal injury and the molecular interactions between HIV-1-specific proteins and EtOH. It was demonstrated that NT2.N neurons were susceptible to HIV-1 Bal (R5-tropic strain) gp120-induced direct cell death. Of importance, EtOH induced cell death in human neurons in a clinically-relevant dose range and EtOH strongly potentiated HIV-1 gp120-induced neuronal injury at low and moderate concentrations. Furthermore, this potentiation of neurotoxicity could be blocked by N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B) antagonists. We analyzed human genomic profiles in these human neurons, using Affymetrix genomics technology, to elucidate the apoptotic pathways involved in HIV-1- and EtOH-related neurodegeneration. Our findings indicated significant over-expression of selected apoptosis functional genes. Significant up-regulation of TRAF5 gene expression may play an essential role in triggering potentiation by EtOH of HIV-1 gp120-induced neuronal apoptosis at early stages of interaction. These studies suggested that two primary apoptotic pathways, death receptor (extrinsic) and NMDA receptor (intrinsic)-related programmed cell-death pathways, are both involved in the potentiation by EtOH of HIV-1 gp120-induced direct human neuronal death. Thus, these data suggest rationally-designed, molecular targets for potential anti-HIV-1 neuroprotection.

    Funded by: NIAAA NIH HHS: AA13849; NIAID NIH HHS: AI46149; NINDS NIH HHS: NS41864, NS44513

    Virology 2005;334;1;59-73

  • NR2A and NR2B receptor gene variations modify age at onset in Huntington disease.

    Arning L, Kraus PH, Valentin S, Saft C, Andrich J and Epplen JT

    Department of Human Genetics, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany. larissa.arning@rub.de

    N -Methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity has been proposed to play a role in the pathogenesis of Huntington disease (HD), an autosomal dominantly inherited disorder associated with defined expansions in a stretch of perfect CAG repeats in the 5' part of the IT15 gene. The number of CAG repeat units is highly predictive for the age at onset (AO) in HD. However, AO is only modestly correlated with repeat length when the HD expansion range is in the high 30s or low 40s. Therefore, we investigated whether the genes for the different subunits composing the multimeric complexes of NMDA receptors (GRIN glutamate receptor, ionotropic, N-methyl-d-aspartate) represent candidates for modulating the AO of HD. In the studied cohort of 167 HD patients, the repeat range from 41 to 45 CAG units accounted for 30.8% of the variance in AO; 12.3% additional variance could be attributed to GRIN2B genotype variation and 4.5% to GRIN2A genotype variation. We conclude that these two genes, coding for NR2B and NR2A subtypes mainly expressed in the striatum, may influence the variability in AO of HD. Neuroprotective strategies for HD patients and persons at risk should be reconsidered in the light of these findings.

    Neurogenetics 2005;6;1;25-8

  • Small CTD phosphatases function in silencing neuronal gene expression.

    Yeo M, Lee SK, Lee B, Ruiz EC, Pfaff SL and Gill GN

    Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

    Neuronal gene transcription is repressed in non-neuronal cells by the repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) complex. To understand how this silencing is achieved, we examined a family of class-C RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) phosphatases [small CTD phosphatases (SCPs) 1 to 3], whose expression is restricted to non-neuronal tissues. We show that REST/NRSF recruits SCPs to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. Phosphatase-inactive forms of SCP interfere with REST/NRSF function and promote neuronal differentiation of P19 stem cells. Likewise, small interfering RNA directed to the single Drosophila SCP unmasks neuronal gene expression in S2 cells. Thus, SCP activity is an evolutionarily conserved transcriptional regulator that acts globally to silence neuronal genes.

    Funded by: NIDDK NIH HHS: DK13149; NINDS NIH HHS: NS37116

    Science (New York, N.Y.) 2005;307;5709;596-600

  • Subunit-specific regulation of NMDA receptor endocytosis.

    Lavezzari G, McCallum J, Dewey CM and Roche KW

    National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    At excitatory synapses, both NMDA and AMPA receptors are localized to the postsynaptic density (PSD). However, unlike AMPA receptors, synaptic NMDA receptors are stable components of the PSD. Even so, surface-expressed NMDA receptors undergo endocytosis, which is more robust early in development and declines during synaptic development. We investigated the subunit-specific contributions to NMDA receptor endocytosis, specifically defining the endocytic motifs and endocytic pathways preferred by the NR2A and NR2B subunits. We find that NR2A and NR2B have distinct endocytic motifs encoded in their distal C termini and that these interact with clathrin adaptor complexes with differing affinities. We also find that NR2A and NR2B sort into different intracellular pathways after endocytosis, with NR2B preferentially trafficking through recycling endosomes. In mature cultures, we find that NR2B undergoes more robust endocytosis than NR2A, consistent with previous studies showing that NR2A is more highly expressed at stable synaptic sites. Our findings demonstrate fundamental differences between NR2A and NR2B that help clarify developmental changes in NMDA receptor trafficking and surface expression.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;28;6383-91

  • Glutamate receptor, ionotropic, N-methyl D-aspartate 2A (GRIN2A) gene as a positional candidate for attention-deficit/hyperactivity disorder in the 16p13 region.

    Adams J, Crosbie J, Wigg K, Ickowicz A, Pathare T, Roberts W, Malone M, Schachar R, Tannock R, Kennedy JL and Barr CL

    Cell and Molecular Biology Division, Toronto Western Research Institute, University Health Network, Toronto, ON, Canada.

    The glutamate system may be involved in the development of attention-deficit/hyperactivity disorder (ADHD) based on animal models and the role of N-methyl-D-aspartate receptors (NMDAR) in cognition and motor processes. A follow-up study of the first genome scan for ADHD identified significant evidence for linkage to the 16p13 region. The glutamate receptor, ionotropic, N-methyl D-aspartate 2A (GRIN2A) gene that encodes the 2A subunit of the NMDA receptor, resides in this region and a recent study has reported an association between this gene and ADHD. We tested for linkage between the alleles and haplotypes of four polymorphisms at the GRIN2A locus and ADHD in our sample of 183 nuclear families with 229 affected children. In contrast to previous findings, we did not identify any evidence for a relationship of these markers and ADHD. Owing to the role of GRIN2A in aspects of cognition, we investigated the relationship of this gene to the cognitive phenotypes of inhibitory control, verbal short-term memory and verbal working memory. There was no significant evidence of linkage between GRIN2A and these phenotypes. While the results were not significant in our sample, the previous association finding suggests that further study of this gene is warranted.

    Molecular psychiatry 2004;9;5;494-9

  • Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors.

    Papadakis M, Hawkins LM and Stephenson FA

    Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.

    A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.

    The Journal of biological chemistry 2004;279;15;14703-12

  • Follow-up of genetic linkage findings on chromosome 16p13: evidence of association of N-methyl-D aspartate glutamate receptor 2A gene polymorphism with ADHD.

    Turic D, Langley K, Mills S, Stephens M, Lawson D, Govan C, Williams N, Van Den Bree M, Craddock N, Kent L, Owen M, O'Donovan M and Thapar A

    Department of Psychological Medicine, University of Wales College of Medicine, Cardiff, UK.

    Attention deficit hyperactivity disorder (ADHD) is a childhood onset disorder, for which there is good evidence that genetic factors contribute to the aetiology. Recently reported linkage findings suggested evidence of a susceptibility locus on chromosome 16p13 (maximum LOD score of 4.2, P=5 x 10(-6)). The GRIN2A (glutamate receptor, ionotropic, N-methyl D-aspartate 2A) gene that encodes the N-methyl D-aspartate receptor subunit 2A (NMDA2A) maps to this region of linkage. As this is also a good functional candidate gene for ADHD, we undertook family-based association analysis in a sample of 238 families. We found significant evidence of association with a GRIN2A exon 5 polymorphism (chi(2)=5.7, P=0.01). Our data suggest that genetic variation in GRIN2A may confer increased risk for ADHD and that this, at least in part, might be responsible for the linkage result on 16p reported by Smalley et al. We conclude that replication is required and that further work examining for association of GRIN2A polymorphisms with ADHD is warranted.

    Molecular psychiatry 2004;9;2;169-73

  • The human immunodeficiency virus type-1 transcription factor Tat produces elevations in intracellular Ca2+ that require function of an N-methyl-D-aspartate receptor polyamine-sensitive site.

    Self RL, Mulholland PJ, Nath A, Harris BR and Prendergast MA

    Department of Psychology, University of Kentucky, 115 Kastle Hall, Lexington, KY 40506-0044, USA.

    Human immunodeficiency virus type-1 (HIV-1) infection is commonly associated with neuronal loss, as well as, cognitive and motor deficits collectively termed HIV-1-associated dementia (HAD). Function of the HIV-1 transcription factor Tat, activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, and subsequent rapid rises in free intracellular Ca2+ have been implicated in the development of this neurological disorder. However, the role of specific NMDA receptor modulatory sites in mediating effects of Tat has not been examined. The present studies examined the ability of two variants of Tat protein (1-100 nM), Tat 1-72 and Tat 1-86, to produce rapid rises in intracellular Ca2+ in organotypic slice cultures of rat hippocampus. Further, these studies evaluated the role of an NMDA receptor polyamine-sensitive site in mediating Tat-induced elevations in intracellular Ca2+. Brief exposure (10 min) to each variant of Tat protein (>1 nM) markedly increased levels of intracellular Ca2+ in each region of the hippocampus to as much as 145% of controls. In contrast, exposure of cultures to a deletion mutant of Tat protein devoid of amino acids 31-61 (Tat Delta31-61) did not produce changes in intracellular Ca2+ levels. Most significantly, exposure to the NMDA receptor antagonist dizocilpine (MK801 20 microM) and the polyamine site antagonist arcaine (10 microM) significantly attenuated increases in intracellular Ca2+ levels when co-administered with either the Tat 1-72 or Tat 1-86 amino acid variant of Tat. Thus, exposure of the hippocampus to Tat produces increases in intracellular Ca2+ levels that require function of an NMDA receptor polyamine-sensitive site and this may well contribute to the neurotoxic effects of HIV-1 infection. Polyamine-sensitive portions of this receptor may then represent novel therapeutic targets in the pharmacologic treatment of HAD-related neurotoxicity.

    Brain research 2004;995;1;39-45

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor.

    Sharp CD, Hines I, Houghton J, Warren A, Jackson TH, Jawahar A, Nanda A, Elrod JW, Long A, Chi A, Minagar A and Alexander JS

    Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932, USA.

    l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.

    American journal of physiology. Heart and circulatory physiology 2003;285;6;H2592-8

  • The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1.

    Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE and Medina I

    Howard Hughes Medical Institute, Children's Hospital, 1309 Enders Building, 320 Longwood Avenue, Boston, MA 02115, USA.

    The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.

    Neuron 2003;40;4;775-84

  • CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction.

    Gardoni F, Mauceri D, Fiorentini C, Bellone C, Missale C, Cattabeni F and Di Luca M

    Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy. Fabrizio.Gardoni@unimi.it

    Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

    The Journal of biological chemistry 2003;278;45;44745-52

  • PDZ Domain-mediated interaction of interleukin-16 precursor proteins with myosin phosphatase targeting subunits.

    Bannert N, Vollhardt K, Asomuddinov B, Haag M, König H, Norley S and Kurth R

    Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany. BannertN@rki.de

    The cytokine interleukin-16 is generated by posttranscriptional cleavage by caspase-3 of two large precursor isoforms. The smaller protein of 67 kDa (pro-IL-16) is expressed in cells of the immune system and contains three PDZ (postsynaptic density/disc large/zona occludens-1) domains, whereas the larger 141-kDa neuronal variant (npro-IL-16) has two additional PDZ domains in its N-terminal extension that interact with neuronal ion channels. Using the yeast two-hybrid approach we have identified three closely related myosin phosphatase targeting subunits, MYPT1, MYPT2, and MBS85, as binding partners of the IL-16 precursor proteins. These interactions were verified using pull-down assays, coimmunoprecipitations, and plasmon resonance experiments. Binding requires the intact PDZ2 domain of pro-IL-16 and highly related C-terminal regions in the ligands consisting of a short leucine zipper and an indispensable serine at the -1 position, suggesting a novel unconventional PDZ binding mode. Pro-IL-16 and the myosin phosphatase targeting subunits colocalize along actomyosin filaments and stress fibers in transfected COS-7 cells. By modulating and targeting the catalytic phosphatase subunit to its substrates, MYPT1, MYPT2, and MBS85 regulate various contractile processes in muscle and non-muscle cells. Our findings indicate an involvement of the IL-16 precursor molecules in myosin-based contractile processes, most likely in cell motility, providing a functional link to the chemotactic activity of the mature cytokine. Alternatively, an intracellular complex of npro-IL-16, ion channels, and components of myosin motors in neurons suggests a role in protein targeting.

    The Journal of biological chemistry 2003;278;43;42190-9

  • Activation of peripheral NMDA receptors contributes to human pain and rat afferent discharges evoked by injection of glutamate into the masseter muscle.

    Cairns BE, Svensson P, Wang K, Hupfeld S, Graven-Nielsen T, Sessle BJ, Berde CB and Arendt-Nielsen L

    Department of Anesthesia, Harvard Medical School/Children's Hospital, Boston, Massachusetts 02115, USA.

    Peripheral N-methyl-d-aspartate (NMDA) receptors are found in deep tissues and may play a role in deep tissue pain. Injection of the endogenous NMDA receptor agonist glutamate into the masseter muscle excites deep craniofacial afferent fibers in rats and evokes pain in human subjects. It is not clear whether peripheral NMDA receptors play a role in these effects of glutamate. Accordingly, the effect of NMDA on afferent activity as well as the effect of locally administered NMDA receptor antagonists on glutamate-evoked afferent discharges in acutely anesthetized rats and muscle pain in human subjects was examined. Injection of NMDA into the masseter muscle evoked afferent discharges in a concentration-related manner. It was found that the NMDA receptor antagonists 2-amino-5-phosphonvalerate (APV, 10 mM), ketamine (10 mM), and dextromethorphan (40 mM) significantly decreased glutamate-evoked afferent discharges. The effects of APV and ketamine, but not dextromethorphan, were selective for glutamate-evoked afferent discharges and did not affect hypertonic saline-evoked afferent discharges. In human experiments, it was found that 10 mM ketamine decreased glutamate-evoked muscle pain but had no effect on hypertonic saline-evoked muscle pain. These results indicate that injection of glutamate into the masseter muscle evokes afferent discharges in rats and muscle pain in humans in part through activation of peripheral NMDA receptors. It is conceivable that activation of peripheral NMDA receptors may contribute to masticatory muscle pain and that peripherally acting NMDA receptor antagonists could prove to be effective analgesics for this type of pain.

    Journal of neurophysiology 2003;90;4;2098-105

  • Lithium reduced N-methyl-D-aspartate receptor subunit 2A tyrosine phosphorylation and its interactions with Src and Fyn mediated by PSD-95 in rat hippocampus following cerebral ischemia.

    Ma J and Zhang GY

    Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002 Jiangsu, PR China.

    Recently, the neuroprotective effects of lithium against excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors have been demonstrated. Since brain ischemia results in NMDA receptor over-excitation and Src family protein tyrosine kinase-mediated tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) enhances NMDA receptor activity, we examined the effects of lithium on tyrosine phosphorylation of NR2A and its interactions with Src and Fyn (two members of the Src family of protein tyrosine kinases) mediated by PSD-95 (postsynaptic density protein 95 kDa) after 6 h of reperfusion following 15 min of ischemia (I/R), which was induced by occlusion of the four vessels in Sprague-Dawley rats. After abdominal injection of LiCl (2 mg/kg) for 7 days, the data showed that together with the significant decrease in I/R-induced tyrosine phosphorylation of NR2A, the interactions of NR2A with Src and Fyn mediated by PSD-95 were also decreased significantly. However, lithium pretreatment did not alter the total protein levels of NR2A, Src, Fyn and PSD-95. These results suggest that the inhibition of NR2A tyrosine phosphorylation and its interactions with Src and Fyn mediated by PSD-95 may contribute to the lithium-induced downregulation of NMDA receptor function and provide neuroprotection against excitotoxicity.

    Neuroscience letters 2003;348;3;185-9

  • Expression of NMDA receptor NR1, NR2A and NR2B subunit mRNAs during development of the human hippocampal formation.

    Law AJ, Weickert CS, Webster MJ, Herman MM, Kleinman JE and Harrison PJ

    Department of Psychiatry, University of Oxford, Neurosciences Building, Warneford Hospital, Oxford OX3 7JX, UK. amanda.law@psych.ox.ac.uk

    The N-methyl-d-aspartate receptor plays a critical role in the formation and maintenance of synapses during brain development. In the rodent, changes in subunit expression and assembly of the heteromeric receptor complex accompany these maturational processes. However, little is known about N-methyl-d-aspartate receptor subunit expression during human brain development. We used in situ hybridization to examine the distribution and relative abundance of NR1, NR2A and NR2B subunit messenger ribonucleic acids in the hippocampal formation and adjacent cortex of 34 human subjects at five stages of life (neonate, infant, adolescent, young adult and adult). At all ages, the three messenger ribonucleic acids were expressed in all subfields, predominantly by pyramidal neurons, granule cells and polymorphic hilar cells. However, their abundance varied across ontogeny. Levels of NR1 messenger ribonucleic acid in CA4, CA3 and CA2 subfields were significantly lower in the neonate than all other age groups. In the dentate gyrus, subiculum and parahippocampal gyrus, NR2B messenger ribonucleic acid levels were higher in the neonate than in older age groups. NR2A messenger ribonucleic acid levels remained constant, leading to an age-related increase in NR2A/2B transcript ratio. We conclude that N-methyl-d-aspartate receptor subunit messenger ribonucleic acids are differentially expressed during postnatal development of the human hippocampus, with a pattern similar but not identical to that seen in the rodent. Changes in subunit composition may thus contribute to maturational differences in human hippocampal N-methyl-d-aspartate receptor function, and to their role in the pathophysiology of schizophrenia and other neurodevelopmental disorders.

    The European journal of neuroscience 2003;18;5;1197-205

  • Impaired NMDA receptor-mediated postsynaptic function and blunted NMDA receptor-dependent persistent pain in mice lacking postsynaptic density-93 protein.

    Tao YX, Rumbaugh G, Wang GD, Petralia RS, Zhao C, Kauer FW, Tao F, Zhuo M, Wenthold RJ, Raja SN, Huganir RL, Bredt DS and Johns RA

    Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA. ytau@jhmi.edu

    Modification of synaptic NMDA receptor (NMDAR) expression influences NMDAR-mediated synaptic function and associated persistent pain. NMDARs directly bind to a family of membrane-associated guanylate kinases (MAGUKs) that regulate surface and synaptic NMDAR trafficking in the CNS. We report here that postsynaptic density-93 protein (PSD-93), a postsynaptic neuronal MAGUK, is expressed abundantly in spinal dorsal horn and forebrain, where it colocalizes and interacts with NMDAR subunits NR2A and NR2B. Targeted disruption of the PSD-93 gene reduces not only surface NR2A and NR2B expression but also NMDAR-mediated excitatory postsynaptic currents and potentials, without affecting surface AMPA receptor expression or its synaptic function, in the regions mentioned above. Furthermore, mice lacking PSD-93 exhibit blunted NMDAR-dependent persistent pain induced by peripheral nerve injury or injection of Complete Freund's Adjuvant, although they display intact nociceptive responsiveness to acute pain. PSD-93 appears to be important for NMDAR synaptic targeting and function and to be a potential biochemical target for the treatment of persistent pain.

    Funded by: NIGMS NIH HHS: R01 GM 49111; NINDS NIH HHS: NS 44219, NS360017; PHS HHS: 10833, 38680

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;17;6703-12

  • Human immunodeficiency virus type 1 Tat protein directly activates neuronal N-methyl-D-aspartate receptors at an allosteric zinc-sensitive site.

    Song L, Nath A, Geiger JD, Moore A and Hochman S

    Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada.

    The human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat is neurotoxic and may be involved in the neuropathogenesis of HIV-1 dementia, in part via N-methyl-D-aspartate (NMDA) receptor activation. Here, in acutely isolated rat hippocampal neurons, Tat evoked inward currents reversing near 0 mV, with a negative slope conductance region characteristic of NMDA receptor activation. Although the NMDA receptor antagonist ketamine blocked Tat's actions, competitive glutamate- and glycine-binding site antagonists were ineffective (AP-5 and 5,7-dichlorokynurenate, respectively). Evidence for Tat acting at a distinct modulatory site on the NR1 subunit of NMDA receptors was provided by findings that 1 microM Zn(2+) abolished Tat-evoked responses in all neurons tested. Thus, Tat appears to excite neurons via direct activation of the NMDA receptor at an allosteric Zn(2+)-sensitive site.

    Funded by: NINDS NIH HHS: R01 NS39253-01

    Journal of neurovirology 2003;9;3;399-403

  • A microsatellite repeat in the promoter of the N-methyl-D-aspartate receptor 2A subunit (GRIN2A) gene suppresses transcriptional activity and correlates with chronic outcome in schizophrenia.

    Itokawa M, Yamada K, Yoshitsugu K, Toyota T, Suga T, Ohba H, Watanabe A, Hattori E, Shimizu H, Kumakura T, Ebihara M, Meerabux JM, Toru M and Yoshikawa T

    Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako, Saitama, Japan.

    Hypofunction of the N-methyl-D-aspartate (NMDA) receptor has been hypothesized to underlie the pathophysiology of schizophrenia, based on the observation that non-competitive antagonists of the NMDA receptor, such as phencyclidine, induce schizophrenia-like symptoms. Mice lacking the NR2A subunit of the NMDA receptor complex are known to display abnormal behaviour, similar to schizophrenic symptoms. The expression of NR2A starts at puberty, a period corresponding to the clinical onset of schizophrenia. This evidence suggests that the NR2A (GRIN2A) gene may play a role in the development of schizophrenia and disease phenotypes. In this study, we performed a genetic analysis of this gene in schizophrenia. Analysis of the GRIN2A gene detected four single nucleotide polymorphisms, and a variable (GT)(n) repeat in the promoter region of the gene. A case-control study (375 schizophrenics and 378 controls) demonstrated evidence of an association between the repeat polymorphism and the disease (P = 0.05, Mann-Whitney test), with longer alleles overly represented in patients. An in-vitro promoter assay revealed a length dependent inhibition of transcriptional activity by the (GT)(n) repeat, which was consistent with a receptor binding assay in postmortem brains. Significantly, the score of symptom severity in chronic patients correlated with repeat size (P = 0.01, Spearman's Rank test). These results illustrate a genotype-phenotype correlation in schizophrenia and suggest that the longer (GT)(n) stretch may act as a risk-conferring factor that worsens chronic outcome by reducing GRIN2A levels in the brain.

    Pharmacogenetics 2003;13;5;271-8

  • Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102.

    Seabold GK, Burette A, Lim IA, Weinberg RJ and Hell JW

    Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.

    The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102.

    Funded by: NIA NIH HHS: AG17502; NINDS NIH HHS: NS35563, NS39444

    The Journal of biological chemistry 2003;278;17;15040-8

  • 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

  • Quantitation of NMDA receptor NR2 mRNA transcripts in human brain by competitive RT-PCR.

    Hynd MR, Scott HL and Dodd PR

    Department of Biochemistry, University of Queensland, Brisbane, Australia.

    The NMDA-selective ionotropic receptor constitutes one of the three principal classes of L-glutamate receptors within the mammalian brain. It plays key roles in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Lab. Invest., 68 (1993) 372-387]. NMDA receptors exist as multimeric complexes comprising proteins from two families, NR1 and NR2(A-D) [J. Biol. Chem., 271 (1996) 15669-15674]. Studies on recombinant receptors have revealed that while homomeric NR2 receptors are non-functional, co-expression of an NR1 with an NR2 subunit modulates the efficacy of the resulting channel [Nature, 357 (1992) 70-74]. The RT-PCR assay we describe here was developed to allow quantitation of all hNR2 transcripts in a single-tube PCR assay. Each hNR2 isoform is quantified on the basis of standard curves in which a known amount of synthetic ribonucleic acid competitor is co-amplified against total RNA. The protocol has been applied to the quantitation of hNR2 mRNA levels in autopsy brain. Used in conjunction with a method for the quantitation of hNR1 transcripts [Brain Res. Protoc., in press], a complete analysis of NMDA receptor mRNA expression can be obtained.

    Brain research. Brain research protocols 2003;11;1;67-79

  • Activation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn-PSD95-NR2A complex after transient brain ischemia.

    Hou XY, Zhang GY, Yan JZ, Chen M and Liu Y

    Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu 221002, China.

    Recent studies have indicated that tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) by Src family kinases (Src, Fyn, etc.) up-regulates NMDA receptors activity and postsynaptic density protein 95 kDa (PSD95) may mediate the regulation. To investigate whether the above processes are involved in brain ischemia-induced enhancement of NMDA receptors function, we examined the effects of transient (15 min) brain ischemia followed by reperfusion on interactions involving Fyn, NR2A and PSD95 in rat hippocampus by co-immunoprecipitation. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague-Dawley rats. Association between Fyn and NR2A increased immediately after brain ischemia and the increase was maintained for at least 24 h during followed reperfusion, up to about 1.7-1.8-fold relative to sham-groups. The 15-min reperfusion after brain ischemia induced enhanced co-immunoprecipitation of PSD95, Fyn and NR2A with one another. The associations of PSD95 with Fyn and NR2A increased at 0-24 h, 0-1 h of reperfusion, up to 6.9- and 2.1-fold relative to sham groups, respectively. Inhibiting activation of NMDA receptors or L-type voltage-gated calcium channels (L-VGCC) by ketamine or nifedipine attenuated the above increases of associations. These results suggest that stimulation of NMDA receptors and L-VGCC facilitates formation of a ternary complex: Fyn-PSD95-NR2A during transient brain ischemia followed by reperfusion, which may result in potentiation of NMDA receptor function and contribute to ischemic neuronal cell death.

    Brain research 2002;955;1-2;123-32

  • Neurotoxic effects of the human immunodeficiency virus type-1 transcription factor Tat require function of a polyamine sensitive-site on the N-methyl-D-aspartate receptor.

    Prendergast MA, Rogers DT, Mulholland PJ, Littleton JM, Wilkins LH, Self RL and Nath A

    Department of Psychology, University of Kentucky, 115 Kastle Hall, Lexington, KY 40506-0044, USA. prender@pop.uky.edu

    Human immunodeficiency virus type-I (HIV-1) infection is often associated with neuronal loss in cortical and subcortical regions that may manifest as motor dysfunction and dementia. The function of the HIV-1 transcription protein Tat and subsequent activation of N-methyl-D-aspartate receptors (NMDAr) have been implicated in this form of neurodegeneration. However, it is unclear if Tat interacts directly with the NMDAr and the role of specific NMDAr subunit composition in mediating effects of Tat is also unclear. The present studies examined the ability of HIV-1 Tat1-72 protein (10 pM-1.0 microM) to displace [3H]MK-801 binding and to attenuate spermidine-induced potentiation of this binding in rat brain homogenate comprised of cerebellum, hippocampus, and cerebral cortex. The role of NMDAr polyamine-site function in the neurotoxic effects of Tat was determined using organotypic hippocampal slice cultures. Binding of [3H]MK-801 in adult rat brain homogenate was not reduced by Tat at concentrations below 1 microM. Tat potently inhibited the potentiation of [3H]MK-801 binding produced by co-exposure of membranes to the NMDAr co-agonist spermidine (IC(50)=3.74 nM). In hippocampal explants, Tat produced neurotoxicity in the CA3 and CA1 pyramidal cell layers, as well as in the dentate gyrus, that was significantly reduced by co-exposure to MK-801 (20 microM) and the NMDAr polyamine-site antagonist arcaine (10 microM). Exposure to the HIV-1 Tat deletion mutant (Tatdelta31-61) did not produce neurotoxicity in hippocampal explants. These data suggest that the neurotoxic effects of HIV-1 Tat are mediated, in part, by direct interactions with a polyamine-sensitive site on the NMDAr that positively modulates the function of this receptor.

    Brain research 2002;954;2;300-7

  • Association of NR3A with the N-methyl-D-aspartate receptor NR1 and NR2 subunits.

    Al-Hallaq RA, Jarabek BR, Fu Z, Vicini S, Wolfe BB and Yasuda RP

    Department of Pharmacology, Georgetown University Medical Center, Washington DC, USA.

    The NR3A subunit of the N-methyl-D-aspartate receptor has been shown to form glutamatergic receptor complexes with NR1 and NR2 subunits and excitatory glycinergic receptor complexes with NR1 alone. We developed an antibody to NR3A and, using quantitative immunoblotting techniques, determined the degree of association between the NR3A subunit and the NR1 and NR2 subunits as well as changes in these associations during development. NR3A expression peaks between postnatal days 7 and 10 in the cortex, midbrain, and hippocampus and reaches higher maximal expression levels in these areas than in the olfactory bulb and cerebellum. Immunoprecipitation experiments with an anti-NR1 antibody demonstrated that the majority of NR3A is associated with NR1 in postnatal day 10 rat cortex (80 +/- 8%), decreasing by half (38 +/- 4%) in the adult rat cortex. Using the anti-NR3A antibody in immunoprecipitation studies, we find that 9.7 +/- 0.8% of NR1, 8.7 +/- 1.8% of NR2A, and 5.0 +/- 0.6% of NR2B are associated with NR3A at postnatal day 10. These values decrease by about half in adult rat cortex. The results of this study demonstrate that NR3A is expressed, distributed, and associated with other subunits in a manner that supports its role in synaptic transmission throughout the rat brain, perhaps playing different roles during development.

    Funded by: NIAAA NIH HHS: AA11284; NINDS NIH HHS: NS36246

    Molecular pharmacology 2002;62;5;1119-27

  • Selective binding of synapse-associated protein 97 to GluR-A alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionate receptor subunit is determined by a novel sequence motif.

    Cai C, Coleman SK, Niemi K and Keinänen K

    Department of Biosciences, Division of Biochemistry, University of Helsinki, Helsinki FIN-00014, Finland.

    A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position -9 to -11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the -2 position). Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.

    The Journal of biological chemistry 2002;277;35;31484-90

  • N-methyl-D-aspartate receptor subunit NR2A and NR2B messenger RNA levels are altered in the hippocampus and entorhinal cortex in Alzheimer's disease.

    Bi H and Sze CI

    Xian Jiao-Tong University, Xian, 710061, People's Republic of China.

    The N-methyl-D-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in Alzheimer's disease (AD), especially in brain regions known to be important in memory. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the messenger RNA (mRNA) levels of the NMDA receptor subunits NR1, NR2A, and NR2B in the hippocampus and entorhinal cortex of postmortem brain samples from nine clinically well-characterized AD patients and nine aged controls. Cerebellum, a site minimally affected by AD, was also chosen for comparison assessment. Results showed decreased levels of the NR2 mRNAs in AD brains compared to controls. Reductions of NR2A (46.2%, p<0.01) and NR2B (43.2%, p<0.0001) mRNA levels were identified in the entorhinal cortex. Reductions of NR2A (41.4%, p<0.05) and NR2B (40.6%, p=0.058) mRNA levels were found in the hippocampus. NR1 mRNA levels were similar in all three brain regions in both AD and controls. No significant changes of subunit NR2A and NR2B mRNA levels were identified in the cerebellum. Postmortem delay (PMD), tissue storage time, brain weight, or age of the subjects did not affect these changes. These data suggest that alterations in NMDA receptor subunits, especially the NR2A and NR2B, may be important in AD, particularly in neuronal populations that underlie impaired learning and memory.

    Journal of the neurological sciences 2002;200;1-2;11-8

  • Sequence determinants on the NR2A and NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII.

    Mayadevi M, Praseeda M, Kumar KS and Omkumar RV

    Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuam, Kerala-695014, India.

    Calcium/calmodulin-dependent protein kinase type II (CaMKII) and NMDA-type glutamate receptor (NMDAR) are neuronal proteins involved in learning and memory. CaMKII binds to the NR2B subunit of NMDAR in more than one mode, a stable association involving a noncatalytic site on CaMKII and an enzyme-substrate mode of interaction by its catalytic site. The latter binding results in phosphorylation of serine-1303 on NR2B. We have investigated this binding by studying the kinetics of phosphorylation of synthetic peptides harboring nested sequences of the phosphorylation site motif. We find that residues 1292-1297 of NR2B enhance the affinity of the catalytic site-mediated binding of CaMKII to the minimal phosphorylation site motif, 1298-1308 of NR2B, as evident from measurements of K(m) values for phosphorylation. However, CaMKII shows decreased affinity towards the closely related NR2A subunit due to an -Ile-Asn- motif present as a natural insertion in the analogous sequence on NR2A.

    Biochimica et biophysica acta 2002;1598;1-2;40-5

  • Immunohistochemical localization of N-methyl-D-aspartate receptor subunits in the adult murine hippocampal formation: evidence for a unique role of the NR2D subunit.

    Thompson CL, Drewery DL, Atkins HD, Stephenson FA and Chazot PL

    School of Biological and Biomedical Sciences, University of Durham, Science Research Laboratories, South Road, Durham, UK. c.l.thompson@durham.ac.uk

    NMDA receptors were immunopurified from adult mouse forebrain and screened by immunoblotting. NR1 was co-associated with NR2A, NR2B and NR2D but not NR2C, nor was NR2C detected in adult mouse hippocampal membranes. The anatomical distribution of NR1, 2A, 2B and 2D was mapped in the adult murine hippocampal formation. NR1-like immunoreactivity was localised to cell bodies of pyramidal neurons, granule cells and hilar cells of the dentate gyrus. Apical dendrites of the CA subfields and hilar cells were also immunopositive. NR2A- and NR2B-like immunoreactivity essentially co-localised with that of NR1 implying co-assembly of all three subunits in this brain structure. NR2D-like immunoreactivity was distinct, being totally excluded from pyramidal, granule and hilar cell bodies. Strong, punctate staining was restricted to the oriens layer of CA1 and the stratum lucidum of CA3 consistent with labelling of presynaptic receptors. Less intense staining was also observed in the internal third of the molecular layer of the dentate gyrus.

    Brain research. Molecular brain research 2002;102;1-2;55-61

  • Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102.

    Lim IA, Hall DD and Hell JW

    Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.

    PDZ domains typically interact with the very carboxyl terminus of their binding partners. Type 1 PDZ domains usually require valine, leucine, or isoleucine at the very COOH-terminal (P(0)) position, and serine or threonine 2 residues upstream at P(-2). We quantitatively defined the contributions of carboxyl-terminal residues to binding selectivity of the prototypic interactions of the PDZ domains of postsynaptic density protein 95 (PSD-95) and its homolog synapse-associated protein 90 (SAP102) with the NR2b subunit of the N-methyl-d-aspartate-type glutamate receptor. Our studies indicate that all of the last five residues of NR2b contribute to the binding selectivity. Prominent were a requirement for glutamate or glutamine at P(-3) and for valine at P(0) for high affinity binding and a preference for threonine over serine at P(-2), in the context of the last 11 residues of the NR2b COOH terminus. This analysis predicts a COOH-terminal (E/Q)(S/T)XV consensus sequence for the strongest binding to the first two PDZ domains of PSD-95 and SAP102. A search of the human genome sequences for proteins with a COOH-terminal (E/Q)(S/T)XV motif yielded 50 proteins, many of which have not been previously identified as PSD-95 or SAP102 binding partners. Two of these proteins, brain-specific angiogenesis inhibitor 1 and protein kinase Calpha, co-immunoprecipitated with PSD-95 and SAP102 from rat brain extracts.

    Funded by: NIA NIH HHS: AG00213; NIDDK NIH HHS: DK07759; NINDS NIH HHS: R01-NS35563

    The Journal of biological chemistry 2002;277;24;21697-711

  • Further characterization of the molecular interaction between PSD-95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating.

    Rutter AR, Freeman FM and Stephenson FA

    Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, London, UK.

    Coexpression of PSD-95(c-Myc) with NR1-1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3 H]MK801 binding similar to that previously described for l-glutamate. The inhibition constants (K (I) s) for the binding of l-glutamate and glycine to NR1-1a/NR2A determined by [3 H]CGP 39653 and [3 H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1-1a/NR2A receptors coexpressed +/- PSD-95(c-Myc). The increased EC(50) for l-glutamate enhancement of [3 H]MK801 binding was also found for NR1-2a/NR2A and NR1-4b/NRA receptors thus the altered EC(50) is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1-4b but not the NR1-1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1-4b and NR1-4b/NR2A expression was enhanced by PSD-95(c-Myc) but whole cell enzyme-linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD-95(c-Myc) has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C-terminal E(T/S)XV PSD-95 binding motifs.

    Journal of neurochemistry 2002;81;6;1298-307

  • Cloning and characterization of a novel NMDA receptor subunit NR3B: a dominant subunit that reduces calcium permeability.

    Matsuda K, Kamiya Y, Matsuda S and Yuzaki M

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105, USA.

    We report the cloning and characterization of a novel NMDA receptor subunit cDNA, which encodes a predicted polypeptide of 1003 amino acids. Phylogenic analysis indicates that this new subunit is most closely related to NR3A. Therefore, we term it NR3B. Important functional domains of glutamate receptors, such as the ligand-binding domain, the channel pore, and the channel gate, are conserved in NR3B. NR3B mRNA was expressed highly in pons, midbrain, medulla, and the spinal cord, but at low levels in the forebrain and the cerebellum. Although NR3A mRNA expression decreases sharply after the second postnatal weeks, NR3B mRNA expression levels in whole brain were constant during postnatal development and into adult. Coimmunoprecipitation analysis showed that NR3B could form NMDA receptor complex with NR1a and NR2A subunits in heterologous cells. Although expression of NR3B alone did not reconstitute functional NMDA receptors, coexpression of NR3B reduced the Ca(2+) permeability of glutamate-induced currents in cells expressing NR1a and NR2A. These results indicate that NR3B is a dominant modulatory subunit that can modify the function of NMDA receptors. Since high Ca(2+) permeability of NMDA receptors is thought to be a key feature for NMDA receptors to play critical roles in neurodevelopment, synaptic plasticity, and neuronal death, NR3B may contribute to the regulation of these physiological and pathological processes.

    Funded by: NCI NIH HHS: CA 21765; NINDS NIH HHS: NS 36925

    Brain research. Molecular brain research 2002;100;1-2;43-52

  • Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors.

    Krupp JJ, Vissel B, Thomas CG, Heinemann SF and Westbrook GL

    Vollum Institute, Oregon Health & Science University, L474, 3181 SW Sam Jackson Park Rd., Portland OR 97201, USA. johannes.krup@astrazeneca.com

    Phosphatase IIb (calcineurin, CaN) can reduce N-methyl-D-aspartate (NMDA) synaptic responses by enhancing glycine-independent desensitization. We examined the action of CaN on desensitization in recombinant NMDA receptors comprised of NMDA receptor 1 (NR1) and NR2A subunits. The C-terminus of NR2A, but not NR1, was critical for modulation of desensitization by CaN. Alanine-scanning mutagenesis indicated that serines 900 and 929 in NR2A altered desensitization, as did inhibition of tyrosine phosphatases. Our data suggest that dephosphorylation-dependent regulation of the C-terminus of NR2A increases desensitization of NMDA receptors, providing an additional mechanism for modulation of synaptic signals.

    Funded by: NIMH NIH HHS: MH46613; NINDS NIH HHS: NS28709

    Neuropharmacology 2002;42;5;593-602

  • Intracellular domains of NR2 alter calcium-dependent inactivation of N-methyl-D-aspartate receptors.

    Vissel B, Krupp JJ, Heinemann SF and Westbrook GL

    Molecular Neurobiology Laboratory, the Salk Institute, La Jolla, California, USA.

    At central excitatory synapses, the transient elevation of intracellular calcium reduces N-methyl-D-aspartate (NMDA) receptor activity. Such 'calcium-dependent inactivation' is mediated by interactions of calcium/calmodulin and alpha-actinin with the C terminus of NMDA receptor 1 (NR1) subunit. However, inactivation is also NR2-subunit specific, because it occurs in NR2A- but not NR2C-containing receptors. We examined the molecular basis for NR2-subunit specificity using chimeric and mutated NMDA receptor subunits expressed in HEK293 cells. We report that the intracellular loop immediately distal to the pore-forming P-loop M2 (M2-3 loop), as well as a short region in the C terminus, are involved in NR2-subunit specificity. Within the M2-3 loop, substitution of residue 619 in NR2A (valine) for the corresponding NR2C residue (isoleucine) reduced inactivation without affecting calcium permeability of the channel. In contrast, a Q620E mutation in NR2A reduced the relative calcium permeability without altering inactivation. Mutation of three serine/threonine residues in the M2-3 loop also reduced inactivation, as did substitution of the intracellular C terminus of NR2A for NR2C. We speculate that the M2-3 loop of NR2 modulates calcium-dependent inactivation by interacting with the NR1 C terminus, a region known to be essential for inactivation.

    Funded by: NIMH NIH HHS: MH46613; NINDS NIH HHS: NS28709

    Molecular pharmacology 2002;61;3;595-605

  • Turnover analysis of N-methyl-D-aspartate receptor subunit NR1 protein in PC12 cells.

    Vazhappilly R and Sucher NJ

    Biotechnology Research Institute and Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, SAR, Hong Kong, China.

    The post-translational fate of N-methyl-D-aspartate receptor (NMDAR) subunit NR1 was characterized in PC12 cells using pulse-chase labeling, block of protein synthesis by cyclohexamide and deglycosylation by endoglycosidase H. Metabolic labeling of NR1 protein indicated a biphasic degradation of NR1 protein with half-lives of 1.6 and 16.1 h for a rapidly (78%) and a slowly (22%) degrading population. Immunoprecipitation of NR1 following the block of protein synthesis by cyclohexamide revealed that the rapidly and slowly degrading pools mainly consisted of the NR1 splice variants NR1-4a and NR1-2a. Sensitivity of NR1 protein to deglycosylation by endoglycosidase H indicated the presence of an immature form of NR1 that was retained in the endoplasmic reticulum. PC12 cells serve as a useful model for the elucidation of translational and post-translational mechanisms of NMDAR expression.

    Neuroscience letters 2002;318;3;153-7

  • Determination of the genomic structure and mutation screening in schizophrenic individuals for five subunits of the N-methyl-D-aspartate glutamate receptor.

    Williams NM, Bowen T, Spurlock G, Norton N, Williams HJ, Hoogendoorn B, Owen MJ and O'Donovan MC

    Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK.

    The glutamatergic system is the major excitatory neurotransmitter system in the CNS. Glutamate receptors, and in particular N-methyl-D-aspartate (NMDA) receptors, have been proposed as mediators of many common neuropsychiatric phenotypes including cognition, psychosis, and degeneration. We have reconstructed the genomic structure of all five genes encoding NMDA receptors in silico. We screened each for sequence variation and estimated the allele frequencies of all detected SNPs in pooled samples of 184 UK Caucasian schizophrenics and 184 UK Caucasian blood donor controls. Only a single non-synonymous polymorphism was found indicating extreme selection pressure. The rarity of non-synonymous changes suggests that such variants are unlikely to make a common contribution to common phenotypes. We found a further 26 polymorphisms within exonic or adjacent intronic sequences. The minor alleles of most of these have a relatively high frequency (63% above 0.2). These SNPs will therefore be suitable for studying neuropsychiatric phenotypes that are putatively related to NMDA dysfunction. Pooled analysis provided no support for association between any of the GRIN genes and schizophrenia.

    Funded by: Medical Research Council: G9309834, G9810900

    Molecular psychiatry 2002;7;5;508-14

  • HIV-1 Tat causes apoptotic death and calcium homeostasis alterations in rat neurons.

    Bonavia R, Bajetto A, Barbero S, Albini A, Noonan DM and Schettini G

    Pharmacology and Neuroscience, National Cancer Research Institute, c/o Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genoa, Italy.

    We investigated the role of the HIV-1 protein Tat in AIDS-associated dementia, by studying its toxicity on rat cortical and hippocampal neurons in vitro. We evaluated the involvement of astroglial cells and of caspase transduction pathway in determining Tat toxicity. Here we report that synthetic Tat(1-86) induced apoptotic death on cultured rat neurons in a time-dependent manner that was not influenced by glial coculture, and that was abolished by blocking caspase transduction pathway. A microfluorimetric analysis on the Tat excitatory properties on neurons, and its effect on intracellular calcium concentrations, revealed that Tat(1-86) induced increase in cytoplasmic free calcium concentrations in rat hippocampal and cortical neurons. This effect required extracellular calcium and was differently reduced by voltage dependent calcium channel blockers and both NMDA and non-NMDA glutamate receptors antagonists. Furthermore, we observed that Tat(1-86)-treated neurons showed increased sensitivity to the glutamate excitotoxicity. Thus, the Tat-induced neuronal injury seems to occur through a direct interaction of the protein with neurons, requires activation of caspases, and is likely to derive from Tat(1-86)-induced calcium loads and disruption of glutamatergic transmission.

    Biochemical and biophysical research communications 2001;288;2;301-8

  • Regulation of NMDA receptors by cyclin-dependent kinase-5.

    Li BS, Sun MK, Zhang L, Takahashi S, Ma W, Vinade L, Kulkarni AB, Brady RO and Pant HC

    Laboratory of Neurochemistry, Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.

    Members of the N-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;22;12742-7

  • NMDA receptor activation results in tyrosine phosphorylation of NMDA receptor subunit 2A(NR2A) and interaction of Pyk2 and Src with NR2A after transient cerebral ischemia and reperfusion.

    Liu Y, Zhang G, Gao C and Hou X

    Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, China.

    Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor. Several tyrosine kinases are involved in this process. In this study, effect of ischemia and reperfusion (I/R) on tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and the interaction of two tyrosine kinases, Src and Pyk2, with NR2A was investigated. Four-vessel occlusion was used to produce transient (15 min) cerebral ischemia in SD rats. Tyrosine phosphorylation of NR2A in hippocampus was enhanced after 15 min of reperfusion and reached its peak level at 6 h of reperfusion. The increase sustained for at least 24 h. Src and Pyk2 co-immunoprecipitated with NR2A and the binding increased after I/R, which also reached a peak at 6 h of reperfusion. Besides, Src and Pyk2 were activated after I/R. These increases were prevented by ketamine, a selective NMDA receptor antagonist, which was administered to the SD rats 20 min before ischemia. Moreover, Src and Pyk2 coprecipitated with each other. These data show that NR2A, Src and Pyk2 might form a protein complex in vivo and the interaction suggests a possible mechanism of signal transduction in the postischemic hippocampus.

    Brain research 2001;909;1-2;51-8

  • HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity.

    Haughey NJ, Nath A, Mattson MP, Slevin JT and Geiger JD

    Laboratory of Neurosciences, National Institute on Aging, Baltimore, Maryland, USA.

    Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.

    Funded by: NINDS NIH HHS: NS/MH39253, NS39184

    Journal of neurochemistry 2001;78;3;457-67

  • Identification of molecular determinants that are important in the assembly of N-methyl-D-aspartate receptors.

    Meddows E, Le Bourdelles B, Grimwood S, Wafford K, Sandhu S, Whiting P and McIlhinney RA

    Medical Research Council Anatomical Neuropharmacology Unit, Mansfield Road, Oxford OX1 3TH.

    To determine which domains of the N-methyl-d-aspartate (NMDA) receptor are important for the assembly of functional receptors, a number of N- and C-terminal truncations of the NR1a subunit have been produced. Truncations containing a complete ligand binding domain bound glycine antagonist and gave binding constants similar to those of the native subunit, suggesting they were folding to form antagonist binding sites. Since NR2A is not transported to the cell surface unless it is associated with NR1 (McIlhinney, R. A. J., Le Bourdellès, B., Tricuad, N., Molnar, E., Streit, P., and Whiting, P. J. (1998) Neuropharmacology 37, 1355-1367), surface expression of NR2A can be used to monitor the association of the subunits. There was progressive loss of NR2A cell surface expression as the N terminus of NR1a was shortened, with complete loss when truncated beyond residue 380. Removal of the C terminus and/or the last transmembrane domain did not affect NR2A surface expression. Similar results were obtained in co-immunoprecipitation experiments. The oligomerization status of the co-expressed NR1a constructs and NR2A subunits was investigated using a non-denaturing gel electrophoresis system (blue native-polyacrylamide gel electrophoresis) and sucrose density gradient centrifugation. The blue native-polyacrylamide gel electrophoresis system also showed that the NR1a subunits could form a homodimer, which was confirmed using soluble constructs of the NR1a subunit. Together these results suggest the residues N-terminal of residue 380 are important for the association of NR2A with NR1a and that the complete N-terminal domain of the NR1a subunit is required for oligomerization with NR2A.

    The Journal of biological chemistry 2001;276;22;18795-803

  • Protein kinase C activation modulates alpha-calmodulin kinase II binding to NR2A subunit of N-methyl-D-aspartate receptor complex.

    Gardoni F, Bellone C, Cattabeni F and Di Luca M

    Institute of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy. fabrizio.gardoni@unimi.it

    The N-methyl-d-aspartate (NMDA) receptor subunits NR2 possess extended intracellular C-terminal domains by which they can directly interact with a large number of postsynaptic density (PSD) proteins involved in synaptic clustering and signaling. We have previously shown that PSD-associated alpha-calmodulin kinase II (alphaCaMKII) binds with high affinity to the C-terminal domain of the NR2A subunit. Here, we show that residues 1412-1419 of the cytosolic tail of NR2A are critical for alphaCaMKII binding, and we identify, by site directed mutagenesis, PKC-dependent phosphorylation of NR2A(Ser(1416)) as a key mechanism in inhibiting alphaCaMKII-binding and promoting dissociation of alphaCaMKII.NR2A complex. In addition, we show that stimulation of PKC activity in hippocampal slices either with phorbol esters or with the mGluRs specific agonist trans-1-amino-1,3- cyclopentanedicarboxylic acid (t-ACPD) decreases alphaCaMKII binding to NMDA receptor complex. Thus, our data provide clues on understanding the molecular basis of a direct cross-talk between alphaCaMKII and PKC pathways in the postsynaptic compartment.

    Funded by: Telethon: 946

    The Journal of biological chemistry 2001;276;10;7609-13

  • Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor.

    Gardoni F, Schrama LH, Kamal A, Gispen WH, Cattabeni F and Di Luca M

    Institute of Pharmacological Sciences, University of Milan, 20133 Milan, Italy. fabrizio.gardoni@unimi.it

    NMDA receptor, Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), and postsynaptic density 95 (PSD-95) are three major components of the PSD fraction. Both alphaCaMKII and PSD-95 have been shown previously to bind NR2 subunits of the NMDA receptor complex. The nature and mechanisms of targeting to the NMDA receptor subunits are, however, not completely understood. Here we report that the C-terminal NR2A(S1389-V1464) sequence was sufficient to guarantee the association of both native and recombinant alphaCaMKII and PSD-95. PSD-95(54-256) was able to compete with the binding of both native and recombinant alphaCaMKII to the NR2A C-tail. Accordingly, alphaCaMKII(1-325) competes with both the native PSD-95 and the native kinase itself for the binding to NR2A. In addition, Ser/Ala1289 and Ser/Asp1289 point mutations on the unique CaMKII phosphosite of NR2A did not significantly influence the binding of native alphaCaMKII and PSD-95 to the NR2A C-tail. Finally, the association-dissociation of alphaCaMKII and PSD-95 to and from the NR2A C-tail was significantly modulated by activation of NMDA receptor achieved by either pharmacological tools or long-term potentiation induction, underlining the importance of dynamic and reciprocal interactions of NMDA receptor, alphaCaMKII, and PSD-95 in hippocampal synaptic plasticity.

    Funded by: Telethon: 946

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;5;1501-9

  • Src-mediated tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate receptors protects from calpain-mediated truncation of their C-terminal domains.

    Bi R, Rong Y, Bernard A, Khrestchatisky M and Baudry M

    Neuroscience Program, University of Southern California, Los Angeles, California 90089-2520, USA.

    Src-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunits has been shown to modify the functional properties of N-methyl-d-aspartate receptors. Moreover, calpain-mediated truncation of N-methyl-d-aspartate receptor subunits has been found to alter the structure of the receptors. In the present study, we first used immunoprecipitation with a variety of antibodies against N-methyl-d-aspartate receptor subunits and anti-phosphotyrosine antibodies to show that tyrosine-phosphorylated subunits of N-methyl-d-aspartate receptor are protected against calpain-mediated truncation of their C-terminal domains. A GST fusion protein containing the C-terminal domain of NR2A was used to identify the calpain cutting sites in the C-terminal domain. One site was identified at residues 1278-1279, corresponding to one of the preferred calpain truncation sites. This site is adjacent to a consensus sequence for Src-mediated tyrosine phosphorylation, and Src-mediated tyrosine phosphorylation of the GST-NR2A C-terminal fusion protein also inhibited calpain-mediated truncation of the fusion protein. We propose that phosphorylation of NR2 subunits and the resulting inhibition of calpain-mediated truncation of their C-terminal domains provide for the stabilization of the N-methyl-d-aspartate receptors in postsynaptic structures.

    Funded by: NIA NIH HHS: AG-14751

    The Journal of biological chemistry 2000;275;34;26477-83

  • Nonreceptor tyrosine protein kinase pp60c-src in spatial learning: synapse-specific changes in its gene expression, tyrosine phosphorylation, and protein-protein interactions.

    Zhao W, Cavallaro S, Gusev P and Alkon DL

    Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. zhaow@ninds.nih.gov

    c-src is a nonreceptor tyrosine protein kinase that is highly concentrated in synaptic regions, including synaptic vesicles and growth cones. Here, we report that the mRNA signal of pp60c-src is widely distributed in the rat brain with particularly high concentrations in the hippocampus. After spatial maze learning, up-regulation of c-src mRNA was observed in the CA3 region of the hippocampus, which was accompanied by increases in pp60c-src protein in hippocampal synaptosomal preparations. Training also triggered an increase in c-src protein tyrosine kinase activity that was correlated with its tyrosine dephosphorylation in the synaptic membrane fraction. After training, pp60c-src from hippocampus showed enhanced interactions with synaptic proteins such as synapsin I, synaptophysin, and the type 2 N-methyl-d-aspartate receptor, as well as the cytoskeletal protein actin. The association of pp60c-src with insulin receptor in the synaptic membrane fraction, however, was temporally decreased after training. Furthermore, in vitro results showed that Ca(2+) and protein kinase C might be involved in the regulation of protein-protein interactions of pp60c-src. These results suggest, therefore, that pp60c-src participates in the regulation of hippocampal synaptic activity during learning and memory.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;14;8098-103

  • 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

  • The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits.

    Hironaka K, Umemori H, Tezuka T, Mishina M and Yamamoto T

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

    Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning. Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade. To study molecular mechanisms underlying GluRdelta2-dependent motor learning, we employed yeast two-hybrid screening to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG. PTPMEG is a family member of band 4.1 domain-containing protein-tyrosine phosphatases and is expressed prominently in brain. Here, we showed by in situ hybridization analysis that the PTPMEG mRNA was enriched in mouse thalamus and Purkinje cells. We also showed that PTPMEG interacted with GluRdelta2 as well as with N-methyl-d-aspartate receptor GluRepsilon1 in cultured cells and in brain. PTPMEG bound to the putative C-terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain. Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner. Thus, we conclude that PTPMEG associates directly with GluRdelta2 and GluRepsilon1. Moreover, our data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation.

    The Journal of biological chemistry 2000;275;21;16167-73

  • A developmental change in NMDA receptor-associated proteins at hippocampal synapses.

    Sans N, Petralia RS, Wang YX, Blahos J, Hell JW and Wenthold RJ

    Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA. sansn@nidcd.nih.gov

    The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

    Funded by: NINDS NIH HHS: NS 35563

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;3;1260-71

  • Evidence of HIV type 1 glycoprotein 120 binding to recombinant N-methyl-D-aspartate receptor subunits expressed in a baculovirus system.

    Xin KQ, Hamajima K, Hattori S, Cao XR, Kawamoto S and Okuda K

    Department of Bacteriology, Yokohama City University School of Medicine, Japan.

    Activation of the N-methyl-D-aspartate (NMDA) receptor by HIV-1 envelope glycoprotein 120 (gp120) is thought to represent at least one of the pathways causing neuronal damage in AIDS patients. In the present study, recombinant gp120 binding to NMDA receptor subunits expressed in a baculovirus system was examined by immunocytochemistry and a binding assay, using horseradish peroxidase (HRP)-conjugated and 125I-labeled recombinant gp120, respectively. We found that recombinant gp120 binds to Sf21 cells expressing epsilon1/zeta1 or epsilon2/zeta1 combined NMDA receptor subunits, but not to Sf21 cells infected with mock virus or Sf21 cells expressing a single epsilon1, epsilon2, or zeta1 NMDA receptor subunit. The binding was strongly blocked by unlabeled recombinant gp120, monoclonal anti-HIV-1 gp160 antibody, and a mixture of anti-epsilon1/epsilon2 and anti-zeta1 antibodies. The same results were obtained by flow cytometric analysis. These data suggest that HIV-1 gp120 may directly bind to the NMDA receptor. This evidence enhances our understanding of the mechanism of HIV-1-induced neuronal damage in AIDS patients.

    AIDS research and human retroviruses 1999;15;16;1461-7

  • Biochemical evidence for the co-association of three N-methyl-D-aspartate (NMDA) R2 subunits in recombinant NMDA receptors.

    Hawkins LM, Chazot PL and Stephenson FA

    Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.

    Functional characterization of wild-type and mutant cloned N-methyl-D-aspartate (NMDA) receptors has been used to deduce their subunit stoichiometry and quaternary structure. However, the results reported from different groups have been at variance and are thus inconclusive. This study has employed a biochemical approach to determine the number of NMDA R2 (NR2) subunits/receptor together with the NMDA R1 (NR1)/NR2 subunit ratio of both cloned and native NMDA receptors. Thus, human embryonic kidney 293 cells were transfected with the NR1-1a and NR2A NMDA receptor subunits in combination with both FLAG- and c-Myc epitope-tagged NR2B subunits. The expressed receptors were detergent-extracted and subjected to double immunoaffinity purification using anti-NR2A and anti-FLAG antibody immunoaffinity columns in series. Immunoblotting of the double immunopurified NR2A/NR2B(FLAG)-containing material demonstrated the presence of anti-NR1, anti-NR2A, anti-FLAG, and, more important, anti-c-Myc antibody immunoreactivities. The presence of anti-c-Myc antibody immunoreactivity in the double immunoaffinity-purified material showed the co-assembly of three NR2 subunits, i.e. NR2A/NR2B(FLAG)/NR2B(c-Myc), within the same NMDA receptor complex. Control experiments excluded the possibility that the co-immunopurification of the three NR2 subunits was an artifact of the solubilization procedure. These results, taken together with those previously described that showed two NR1 subunits/oligomer, suggest that the NMDA receptor is at least pentameric.

    The Journal of biological chemistry 1999;274;38;27211-8

  • Neuronal interleukin-16 (NIL-16): a dual function PDZ domain protein.

    Kurschner C and Yuzaki M

    Department of Developmental Neurobiology, Saint Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

    Interleukin (IL)-16 is a proinflammatory cytokine that has attracted widespread attention because of its ability to block HIV replication. We describe the identification and characterization of a large neuronal IL-16 precursor, NIL-16. The N-terminal half of NIL-16 constitutes a novel PDZ domain protein sequence, whereas the C terminus is identical with splenocyte-derived mouse pro-IL-16. IL-16 has been characterized only in the immune system, and the identification of NIL-16 marks a previously unsuspected connection between the immune and the nervous systems. NIL-16 is a cytosolic protein that is detected only in neurons of the cerebellum and the hippocampus. The N-terminal portion of NIL-16 interacts selectively with a variety of neuronal ion channels, which is similar to the function of many other PDZ domain proteins that serve as intracellular scaffolding proteins. Among the NIL-16-interacting proteins is the class C alpha1 subunit of a mouse brain calcium channel (mbC alpha1). The C terminus of NIL-16 can be processed by caspase-3, resulting in the release of secreted IL-16. Furthermore, in cultured cerebellar granule neurons undergoing apoptosis, NIL-16 proteolysis parallels caspase-3 activation. Cerebellar granule neurons express the IL-16 receptor CD4. Exposure of these cells to IL-16 induces expression of the immediate-early gene, c-fos, via a signaling pathway that involves tyrosine phosphorylation. This suggests that IL-16 provides an autocrine function in the brain. Therefore, we hypothesize that NIL-16 is a dual function protein in the nervous system that serves as a secreted signaling molecule as well as a scaffolding protein.

    Funded by: NCI NIH HHS: P30 CA21765

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;18;7770-80

  • The effect of transient global ischemia on the interaction of Src and Fyn with the N-methyl-D-aspartate receptor and postsynaptic densities: possible involvement of Src homology 2 domains.

    Takagi N, Cheung HH, Bissoon N, Teves L, Wallace MC and Gurd JW

    Division of Life Sciences, University of Toronto at Scarborough, West Hill, Ontario, Canada.

    Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.

    Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 1999;19;8;880-8

  • Glucocorticoid modulation of gp120-induced effects on calcium-dependent degenerative events in primary hippocampal and cortical cultures.

    Howard SA, Nakayama AY, Brooke SM and Sapolsky RM

    Department of Biological Sciences, Stanford University, California 94305, USA.

    The HIV coat protein gp120 has been implicated in damaging the nervous system and may play a role in AIDS-related dementia complex. The glycoprotein triggers the release of a glutamatergic agent from infected microglia and macrophages, causing NMDA receptor- and calcium-dependent excitotoxic damage to neurons. We have previously shown that glucocorticoids, the adrenal steroids secreted during stress, worsen gp120 neurotoxicity and calcium mobilization in various brain regions. This study explores events down-stream of gp120-induced calcium mobilization, specifically, generation of reactive oxygen species (ROS) and subsequent lipid peroxidation, destruction of the cytoskeleton through spectrin proteolysis, and the glucocorticoid modulation of these events in primary hippocampal cultures. We observe that 200 pM gp120 causes a significant accumulation of ROS, including superoxide, and of lipid peroxidation. Counter to our predictions, pretreatment with the glucocorticoid corticosterone (CORT) did not worsen the effects of gp120 on ROS accumulation, but did increase lipid peroxidation. We also observed that neither gp120 alone nor gp120 plus CORT caused detectable proteolysis of the cytoskeletal protein spectrin, whose breakdown has been shown to be a damaging consequence of calcium excess in other models of necrotic neuronal injury.

    Funded by: NIMH NIH HHS: R01 MH53814

    Experimental neurology 1999;158;1;164-70

  • Ras-specific exchange factor GRF: oligomerization through its Dbl homology domain and calcium-dependent activation of Raf.

    Anborgh PH, Qian X, Papageorge AG, Vass WC, DeClue JE and Lowy DR

    Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892, USA.

    The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other organs, possess an ionomycin-dependent activation of Erk mitogen-activated protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature 376:524-527, 1995; N. P. Fam et al., Mol. Cell. Biol. 17:1396-1406, 1996). Each GRF protein contains a Dbl homology (DH) domain. A yeast two-hybrid screen was used to identify polypeptides that associate with the DH domain of GRF1. In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacent ilimaquinone domain. Deletion analysis verified that the two-hybrid interaction required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interaction, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction. Oligomers between GRF1 and GRF2 were detected in a rat brain extract, and forced expression of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers. Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in oligomer formation, while GRF1 containing the DH cluster mutations formed homo-oligomers with an efficiency similar to that of wild type. Compared to wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH cluster mutant was reduced, while the L263Q mutant was inactive. Both mutants were impaired in their ability to mediate ionomycin-dependent Erk activity in 293T cells. In the absence of ionomycin, 293T cells expressing wild-type GRF1 contained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further increase in the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cells GRF1 mediates at least two pathways for Raf activation: one a constitutive signal that is mainly Ras-dependent, and one an ionomycin-induced signal that cooperates with the constitutive signal without further augmenting the level of GTP-Ras.

    Molecular and cellular biology 1999;19;7;4611-22

  • Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97.

    Bassand P, Bernard A, Rafiki A, Gayet D and Khrestchatisky M

    Université René Descartes (Paris V), INSERM U-29, Paris, France.

    The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are encoded by distinct genes. In the rat brain, four C-terminal variants of the NR1 subunit (NR1-1 to NR1-4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A-D). Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits. The NR2B subunit interacts with post-synaptic density protein 95 (PSD-95), SAP97 and members of the membrane-associated guanylate-like kinase (MAGUK) family of proteins. This interaction occurs through the binding of the C-terminal tSXV intracellular motif of the NR2B subunit to the N-terminal PDZ (PSD-95, discs-large, ZO-1) domains of the PSD-95 and SAP97 proteins. Both NR1-3 and NR1-4 also display a consensus C-terminal tSXV motif. Using the two-hybrid genetic system in yeast and site-directed mutagenesis, we compared the binding of the NR2A, NR1-3 and NR1-4 tSXV motifs with the PDZ domains of PSD-95 and SAP97. The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD-95 and SAP97, the NR1-3 and NR1-4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD-95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs. Using immunohistochemistry, we also compared the distribution of the PSD-95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.

    The European journal of neuroscience 1999;11;6;2031-43

  • 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

  • Tat, a human immunodeficiency virus-1-derived protein, augments excitotoxic hippocampal injury in neonatal rats.

    Wang P, Barks JD and Silverstein FS

    Department of Pediatrics, University of Michigan, Ann Arbor 48109-0646, USA.

    To test the hypothesis that the human immunodeficiency virus-1-derived Tat protein may cause neuronal damage in the CNS, we evaluated the neurotoxicity of recombinant human immunodeficiency virus-1-derived Tat in vivo in seven-day-old rats. The intrinsic neurotoxicity of Tat (250 ng-1 microg) and the effects of direct intra-hippocampal co-infusion of Tat with N-methyl-D-aspartate were assessed. Extent of injury in the lesioned hippocampus was evaluated five days later, based on histopathology and morphometric measurements of hippocampal volume. To confirm that any observed neurotoxic effects were attributable to Tat bioactivity, all experiments included controls that received equal amounts of heat-treated (boiled) Tat. Intra-hippocampal injection of Tat, alone, elicited minimal focal tissue damage immediately adjacent to the injection track, and no hippocampal atrophy. Co-injection of Tat (500 ng) with N-methyl-D-aspartate (5 nmol, threshold excitotoxic dose) doubled the severity of hippocampal injury, quantified by comparison of bilateral hippocampal volumes, in comparison with animals that received heat-treated Tat or saline co-injections; in animals that received injections of N-methyl-D-aspartate (5 nmol) in combination with saline, heat-treated Tat, or Tat [mean(+/-S.E.M.) % volume loss values in the lesioned hippocampus were: 11(+/-3), 11(+/-3), and 26(+/-3), respectively (P<0.002, ANOVA)]. Co-injection of 100 ng Tat with 5 nmol N-methyl-D-aspartate exacerbated the severity of excitotoxic injury to a similar extent, whereas co-injection of 20 ng Tat had no effect on N-methyl-D-aspartate-mediated injury. Treatment with the N-methyl-D-aspartate antagonist 3-((RS)-2-carboxypiperazin4-yl)-propyl-1-phosphonic acid (20 mg/kg) markedly attenuated hippocampal injury resulting from co-injection of 100 ng Tat with N-methyl-D-aspartate [mean(+/-S.E.M.) % volume loss in lesioned hippocampus: 0.1(+/-2) in 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid-treated vs 19(+/-3) in controls, P<0.001, ANOVA]. Co-injection of Tat had no effect on N-methyl-D-aspartate-mediated striatal damage or on alpha-amino-3-hydroxy-5-methylisoxazole-4-pro hippocampal damage. These data support the hypothesis that locally released Tat could exert neurotoxic effects, mediated by N-methyl-D-aspartate receptor activation, in vivo in the immature brain.

    Funded by: NINDS NIH HHS: NS 31054

    Neuroscience 1999;88;2;585-97

  • Mechanisms of prionSc- and HIV-1 gp120 induced neuronal cell death.

    Schröder HC, Perovic S, Kavsan V, Ushijima H and Müller WE

    Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Mainz, Germany.

    In vitro experiments revealed that the scrapie prion protein, PrP(Sc), as well as the PrP fragment PrP106-126, and the HIV-1 coat protein gp120 induce apoptosis of rat cortical neurons. The toxic effect displayed by PrP and gp120 could be blocked by NMDA receptor antagonists. Treatment of neuronal cells with PrP106-126 resulted in a drop of intracellular glutathione level and changes in the level of Bcl-2. Evidence is presented that gp120 causes an activation of phospholipase A2, resulting in the increased release of arachidonic acid, which may in turn sensitize the NMDA receptor.

    Neurotoxicology 1998;19;4-5;683-8

  • HIV-1 Tat induces neuronal death via tumor necrosis factor-alpha and activation of non-N-methyl-D-aspartate receptors by a NFkappaB-independent mechanism.

    New DR, Maggirwar SB, Epstein LG, Dewhurst S and Gelbard HA

    Department of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, New York, 14642, USA.

    Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system may result in neuronal apoptosis in vulnerable brain regions, including cerebral cortex and basal ganglia. The mechanisms for neuronal loss are likely to be multifactorial and indirect, since HIV-1 productively infects brain-resident macrophages and microglia but does not cause cytolytic infection of neurons in the central nervous system. HIV-1 infection of macrophages and microglia leads to production and release of diffusible factors that result in neuronal cell death, including the HIV-1 regulatory protein Tat. We demonstrate in this report that recombinant Tat1-86 and Tat peptides containing the basic region induce neuronal apoptosis in approximately 50% of vulnerable neurons in both rat and human neuronal cultures, and this apoptotic cell death is mediated by release of the pro-inflammatory cytokine tumor necrosis factor alpha, and by activation of glutamate receptors of the non-N-methyl-D-aspartate subtype. Finally, we show that Tat-induced apoptosis of human neuronal cell cultures occurs in the absence of activation of the transcription factor NFkappaB. These findings further define cellular pathways activated by Tat, that dysregulate production of tumor necrosis factor alpha, and lead to activation of glutamate receptors and neuronal death during HIV-1 infection of the central nervous system.

    Funded by: NIMH NIH HHS: P01 MH57556; NINDS NIH HHS: F32-NS10307

    The Journal of biological chemistry 1998;273;28;17852-8

  • Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition.

    Zheng F, Gingrich MB, Traynelis SF and Conn PJ

    Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. fzheng@emory.edu

    Activation of the tyrosine kinase Src potentiates NMDA-receptor currents, which is thought to be necessary for induction of hippocampal long-term potentiation. Although the carboxy(C)-terminal domain of the NR2A subunit contains potential tyrosine phosphorylation sites, the mechanisms by which Src modulates synaptic plasticity and NMDA receptor currents is not fully understood. Here we present evidence from NR1 mutants and splice variants that Src potentiates NMDA-receptor currents by reducing the tonic inhibition of receptors composed of NR1 and NR2A subunits by extracellular zinc. Using site-directed mutagenesis, we have identified three C-terminal tyrosine residues of NR2A that are required for Src's modulation of the zinc sensitivity of NMDA receptors. Our data link two modulatory sites of NMDA receptors that were previously thought to be independent.

    Nature neuroscience 1998;1;3;185-91

  • 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

  • Localization of the human NMDAR2D receptor subunit gene (GRIN2D) to 19q13.1-qter, the NMDAR2A subunit gene to 16p13.2 (GRIN2A), and the NMDAR2C subunit gene (GRIN2C) to 17q24-q25 using somatic cell hybrid and radiation hybrid mapping panels.

    Kalsi G, Whiting P, Bourdelles BL, Callen D, Barnard EA and Gurling H

    Molecular Psychiatry Laboratory, UCL Medical School, Windeyer Building, 46 Cleveland Street, London, W1P 6DB, United Kingdom.

    Genomics 1998;47;3;423-5

  • Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+.

    Wollmuth LP, Kuner T and Sakmann B

    Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany. wollmuth@sunny.mpimf-Heidelberg.mpg.de

    1. The voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1- and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In wild-type channels, the voltage dependence (delta) of the block Mg2+ was concentration dependent with values of delta of integral of 0.82 in 0.07 mM and higher concentrations. Under bionic conditions with high extracellular Mg2+ and K+ as the reference ion, Mg2+ weakly permeated the channel. Over intermediate potentials (approximately -60 to -10 mV), this weak permeability had no apparent effect on the block but at potentials negative to approximately -60mV, it attenuated the extent and voltage dependence of the block. 3. Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg2+ permeability. 4. In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block Mg2+ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to integral of 0.57 in 0.7 mM Mg2+. 5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0.7 mM Mg2+, the voltage dependence of the block was reduced to 0.50 (glycine), 0.53 (serine) and 0.46 (glutamine). 6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg2+ permeability suggesting that these adjacent asparagines form a barrier for inward Mg2+ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg2+ blocks mutant channels. 8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg2+. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N-site asparagine. The block may involve binding of Mg2+ to these residues.

    The Journal of physiology 1998;506 ( Pt 1);13-32

  • 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

  • The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    Gurd JW and Bissoon N

    Division of Life Sciences, University of Toronto at Scarborough, West Hill, Ontario, Canada.

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

    Journal of neurochemistry 1997;69;2;623-30

  • DAP-1, a novel protein that interacts with the guanylate kinase-like domains of hDLG and PSD-95.

    Satoh K, Yanai H, Senda T, Kohu K, Nakamura T, Okumura N, Matsumine A, Kobayashi S, Toyoshima K and Akiyama T

    Department of Oncogene Research, Institute for Microbial Diseases, Osaka University, Suita, Japan.

    Background: The human homologue of the Drosophila discs large tumour suppressor protein (hDLG) and closely related proteins such as postsynaptic density protein 95 kDa (PSD-95) are associated with N-methyl-D-aspartate receptors (NMDA-R) and Shaker-type K+ channels, and are thought to be involved in their clustering.

    Results: We have identified a protein named DAP-1 that binds to the guanylate kinase-like domains of hDLG and PSD-95. DAP-1 was found to associate with hDLG, PSD-95, NMDA-R and adenomatous polyposis coli protein (APC). Furthermore, we found that DAP-1 is specifically expressed in the brain and colocalizes with PSD-95 and APC in mouse cerebellum. We also found that DAP-1 is colocalized with PSD-95 and NMDA-R at the synapses in cultured rat hippocampal neurons.

    Conclusion: Our findings suggest that DAP-1 may play several roles in the molecular organization of synapses and neuronal cell signalling by interacting with hDLG and PSD-95, which in turn are associated with receptors, ion channels and APC.

    Genes to cells : devoted to molecular & cellular mechanisms 1997;2;6;415-24

  • Central nervous system expression of HIV-1 Gp120 activates the hypothalamic-pituitary-adrenal axis: evidence for involvement of NMDA receptors and nitric oxide synthase.

    Raber J, Toggas SM, Lee S, Bloom FE, Epstein CJ and Mucke L

    Department of Neuropharmacology, Scripps Research Institute, La Jolla, California 92037, USA.

    The impact of HIV-1 expression in the brain on the development of AIDS is unknown. In the present study, we examined the hypothalamic-pituitary-adrenal (HPA) axis in a transgenic model in which expression of the HIV-1 envelope glycoprotein gp120 induced central nervous system (CNS) damage similar to that seen in HIV-1-infected patients. Compared with nontransgenic littermates, gp120 transgenic mice showed significant increases in plasma corticosterone and adrenocorticotrophic hormone (ACTH) levels and pituitary ACTH content. To determine whether this activation of the HPA axis could be mediated by ACTH secretagogues, the effect of recombinant gp120 on the release of these factors from hypothalamic slices was investigated in vitro. Recombinant gp120 induced release of the ACTH secretagogue arginine vasopressin from nontransgenic hypothalamic slices in a calcium-dependent fashion. This effect was inhibited by antagonists of N-methyl-D-aspartate (NMDA) receptors or of nitric oxide synthase (NOS), suggesting a role for NMDA receptor stimulation and NOS activity. Further evidence for a role of free radicals was obtained from bigenic mice coexpressing gp120 and the free radical scavenger human copper/zinc superoxide dismutase which showed normal corticosterone levels. This might relate to superoxide dismutase-mediated scavenging of superoxides generated by NOS. These findings demonstrate that CNS expression of a viral envelope protein can activate the HPA axis and thereby alter peripheral levels of immunomodulatory hormones.

    Funded by: NIA NIH HHS: AG08938; NIMH NIH HHS: MH47680; NINDS NIH HHS: NS34602

    Virology 1996;226;2;362-73

  • Cloning and functional characterization of human heteromeric N-methyl-D-aspartate receptors.

    Hess SD, Daggett LP, Crona J, Deal C, Lu CC, Urrutia A, Chavez-Noriega L, Ellis SB, Johnson EC and Veliçelebi G

    SIBIA Neurosciences, Inc., La Jolla, California, USA.

    Human cDNAs encoding N-methyl-D-aspartate receptor type (NMDAR)1A, NMDAR2A and NMDAR2B subunits were cloned and receptors encoded by these cDNAs were functionally expressed by injection of the respective mRNAs in Xenopus oocytes. The pharmacological properties of recombinant human N-methyl-D-aspartate (NMDA) receptors were characterized by profiling two agonists and four antagonists at both the NMDA and glycine sites in voltage-clamped oocytes. NMDA, glycine and D-serine were significantly more potent at human NMDAR (hNMDAR)1A/2B receptors than at nNMDAR1A/2A, whereas there was no detectable subtype-dependent difference in the potency of glutamate. Of the NMDA-site antagonists tested, CGP 43487 and 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonate exhibited 5.8- and 3.9-fold greater potency, respectively, at hNMDAR1A/2A receptors than at hNMDAR1A/2B. Of the four glycine-site competitive antagonists tested, L-689,560 displayed 5-fold greater potency at hNMDAR1A/2A, whereas 5,7-dichlorokynurenic acid, HA-966 and CGP 58411 did not discriminate between hNMDAR1A/2A and hNMDAR1A/2B. Receptors resulting from injection of hNMDAR1A, hNMDAR2A and hNMDAR2B transcripts in a 1:1:1 ratio were indistinguishable from hNMDAR1A/2B receptors in terms of their sensitivity to NMDA, glycine, D-serine, CGS 19755 and CGP 40116. Ifenprodil was approximately 350-fold more potent at hNMDAR1A/2B than at hNMDAR1A/2A receptors. Ifenprodil sensitivities of receptors formed in oocytes injected with a constant amount of hNMDAR1A mRNA but varying ratios of hNMDAR2A or hNMDAR2B mRNAs were compared. The receptors expressed at a 10:1 ratio of 2A:2B transcripts displayed an ifenprodil sensitivity that would be predicted for a population in which 51% was represented by hNMDAR(1A)2(2A)3 complexes. Our results underscore the need for subtype-selective compounds acting at novel sites to sufficiently probe the pharmacological differences between NMDA receptor subtypes formed by different subunit combinations.

    The Journal of pharmacology and experimental therapeutics 1996;278;2;808-16

  • Human brain N-methyl-D-aspartate receptors regulating noradrenaline release are positively modulated by HIV-1 coat protein gp120.

    Pittaluga A, Pattarini R, Severi P and Raiteri M

    Istituto di Farmacologia e Farmacognosia, University of Genoa, Italy.

    Objective: To investigate the effect of HIV-1 gp120 on the function of glutamate receptors of the N-methyl-D-aspartate (NMDA) type in the human brain.

    Design: The monitoring of neurotransmitter release from superfused isolated nerve endings is widely recognized as a technique appropriate for the study of neurotransmitter release and to attribute a precise localization to the site(s) of action of drugs able to modulate release.

    Methods: Synaptosomes (pinched-off nerve endings) were prepared from fresh human brain tissue samples removed during neurosurgery, labelled with [3H]-noradrenaline and superfused at a rate of 0.5 ml/min with NMDA in the presence of gp41, gp160, gp120 or the V3 loop, with or without NMDA receptor antagonists. Fractions of superfusate were collected and measured for radioactivity.

    Results: NMDA elicited a glycine-sensitive release of [3H]-noradrenaline from human brain synaptosomes. HIV-1 gp120 potentiated the NMDA (1 mM)-evoked [3H]-noradrenaline release (maximal effect approximately 110% at 1 nM). The release elicited by NMDA plus gp120 was prevented by the classical NMDA receptor antagonists dizocilpine or 7-chlorokynurenic acid, as well as by memantine. The potentiation by gp120 of the NMDA-evoked [3H]-noradrenaline release was mimicked by gp160 but not by gp41. The effect of gp120 was retained by the V3 loop. Finally, gp120 reversed (1 nM) and surmounted (10nM) the antagonism by 10 microM 7-chlorokynurenate of the NMDA-evoked [3H]-noradrenaline release.

    Conclusion: gp 120 binds directly through the V3 loop at noradrenergic axon terminals in human brain neocortex and may alter the function of presynaptic NMDA receptors mediating regulation of noradrenaline release.

    AIDS (London, England) 1996;10;5;463-8

  • Direct cytotoxicity of HIV-1 envelope protein gp120 on human NT neurons.

    Wu P, Price P, Du B, Hatch WC and Terwilliger EF

    Division of Hematology/Oncology, New England Deaconess Hospital, MA 02215, USA.

    A new in vitro system comprising a pure population of neurons, human NT cells, was used to characterize the direct neurotoxic effect of HIV-1 envelope protein gp120. Cytotoxicity was monitored by a quantitative assay after exposure to recombinant gp120 in the presence or absence of other reagents. Treatment of mature NT neurons with various doses of gp120 for 24 h caused a decrease of up to 27% in the number of viable cells. This neurotoxicity was abolished by co-treatment with either D-2-amino-5-phosphonopentanoic acid (APV), MK801 or nimodipine, or by culturing cells in a Ca(2+)-free environment. Taken together, these data indicate that gp120 exerts a direct neurotoxic effect by acting through NMDA receptors and Ca2+ channels.

    Funded by: NHLBI NIH HHS: P01 HL43520

    Neuroreport 1996;7;5;1045-9

  • Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases.

    Niethammer M, Kim E and Sheng M

    Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02114, USA.

    Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;7;2157-63

  • Expression of Ca2+/calmodulin-dependent protein kinase types II and IV, and reduced DNA synthesis due to the Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl piperazine) in small cell lung carcinoma.

    Williams CL, Phelps SH and Porter RA

    Molecular Pharmacology Laboratory, Guthrie Research Institute, Sayre, PA 18840, USA.

    Because changes in intracellular Ca2+ affect progression through the mitotic cell cycle, we investigated the role of Ca2+-binding proteins in regulating cell cycle progression. Evidence was found demonstrating that the activation of Ca2+/calmodulin-dependent protein kinase (CaM kinase) inhibits cell cycle progression in small cell lung carcinoma (SCLC) cells. We also demonstrated that SCLC cells express both CaM kinase type II (CaMKII) and CaM kinase type IV (CaMKIV). Five independent SCLC cell lines expressed proteins reactive with antibody to the CaMKII beta subunit, but none expressed detectable proteins reactive with antibody to the CaMKII alpha subunit. All SCLC cell lines tested expressed both the alpha and beta isoforms of CaMKIV. Immunoprecipitation of CaMKII from SCLC cells yielded multiple proteins that autophosphorylated in the presence of Ca2+ / calmodulin. Autophosphorylation was inhibited by the CaMKII(281-302) peptide, which corresponds to the CaMKII autoinhibitory domain, and by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), a specific CaM kinase antagonist. Influx of Ca2+ through voltage-gated Ca2+ channels stimulated phosphorylation of CaMKII in SCLC cells, and this was inhibited by KN-62. Incubation of SCLC cells of KN-62 potently inhibited DNA synthesis, and slowed progression through S phase. Similar anti-proliferative effects of KN-62 occurred in SK-N-SH human neuroblastoma cells, which express both CaMKII and CaMKIV, and in K562 human chronic myelogenous leukemia cells, which express CaMKII but not CaMKIV. The expression of both CaMKII and CaMKIV by SCLC cells, and the sensitivity of these cells to the anti-proliferative effects of KN-62, suggest a role for CaM kinase in regulating SCLC proliferation.

    Funded by: NCI NIH HHS: CA52471

    Biochemical pharmacology 1996;51;5;707-15

  • Prevention of HIV-1 gp120-induced neuronal damage in the central nervous system of transgenic mice by the NMDA receptor antagonist memantine.

    Toggas SM, Masliah E and Mucke L

    Department of Neuropharmacology, Scripps Research Institute, La Jolla, CA 92037, USA.

    To investigate the in vivo role of NMDA receptor stimulation in HIV-1-related CNS neurotoxicity, we evaluated the neuroprotective potential of the NMDA receptor antagonist memantine in transgenic mice which have gp120-induced CNS damage. Brains of mice treated chronically with memantine and of untreated controls were analysed for structural damage by laser scanning confocal microscopy of sections immunolabeled for microtubule-associated protein-2 (MAP-2) and synaptophysin. Qualitative and quantitative analysis of confocal images revealed that memantine treatment substantially decreased neuropathology in gp120 transgenic mice; this included statistically significant improvements in both dendritic and presynaptic terminal density. These results provide in vivo evidence that gp120 can activate neurotoxic pathways that can ultimately result in aberrant NMDA receptor stimulation and neuronal damage in the CNS. They also suggest that clinically tolerated NMDA receptor antagonists may be useful in the prevention of neuronal damage in HIV-1-infected patients.

    Funded by: NIMH NIH HHS: MH452941, MH47680; NINDS NIH HHS: NS33056; ...

    Brain research 1996;706;2;303-7

  • HIV-1 gp120-induced neurotoxicity to midbrain dopamine cultures.

    Bennett BA, Rusyniak DE and Hollingsworth CK

    Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, NC 27157-1083, USA.

    HIV-1-associated cognitive/motor dysfunction is a frequent neurological complication of acquired immunodeficiency syndrome (AIDS) and has been termed AIDS dementia complex (ADC). The HIV-1 envelope glycoprotein gp120 has been implicated in producing brain injury associated with ADC. The purpose of the present study was to determine if gp120-induced neurotoxicity is associated with damage to dopaminergic systems. Exposure of rat midbrain dopamine cultures to gp120 for 3 days reduced the ability of dopaminergic cells to transport this amine and also resulted in a reduction in dopamine neuron process length while it did not alter either dopamine cell number or the total number of neuronal cells. These detrimental effects of gp120 were prevented by an NMDA receptor antagonist (MK-801) or by preincubation with anti-gp120 antibody. These results suggest that dopaminergic neuronal damage may contribute to the manifestations of AIDS dementia complex.

    Funded by: NIDA NIH HHS: DA05073; NIDDK NIH HHS: 5T35DK-07400-14

    Brain research 1995;705;1-2;168-76

  • The coat protein gp120 of HIV-1 inhibits astrocyte uptake of excitatory amino acids via macrophage arachidonic acid.

    Dreyer EB and Lipton SA

    Department of Ophthalmology, Harvard Medical School, Boston, MA 02115, USA.

    The human immunodeficiency virus coat protein gp120 injures central mammalian neurons both in vitro and in vivo, and this observation may contribute, at least in part, to the neurological dysfunction associated with the acquired immunodeficiency syndrome. Recent work suggests that gp120 mediates neuronal damage predominantly via an indirect route involving activation of brain macrophages. We have previously shown that the stimulation of N-methyl-D-aspartate receptors by excitatory amino acids is essential for the neuronal injury observed with gp120. Here we show that gp120 impairs astrocyte uptake of excitatory amino acids and the excess glutamate thus engendered may contribute to the increased neuronal damage. We also studied the mechanism whereby gp120 inhibits the uptake of excitatory amino acids by astrocytes. We present data suggesting that at least one pathway involves a direct effect of gp120 on macrophages, which in turn release arachidonic acid, a known inhibitor of excitatory amino acid uptake by astrocytes. Our findings suggest that the observed effects on glia and neurons of gp120 may be secondary, at least in part, to its initial activation of macrophages.

    Funded by: NEI NIH HHS: EY09024, EY10009; NICHD NIH HHS: HD29587

    The European journal of neuroscience 1995;7;12;2502-7

  • HIV-1 envelope proteins gp120 and gp160 potentiate NMDA-induced [Ca2+]i increase, alter [Ca2+]i homeostasis and induce neurotoxicity in human embryonic neurons.

    Lannuzel A, Lledo PM, Lamghitnia HO, Vincent JD and Tardieu M

    Laboratoire de Neurovirologie et Neuroimmunologie, UFR Kremlin-Bicêtre, Université Paris XI, Le Kremlin-Bicêtre, France.

    The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N-methyl-D-aspartate (NMDA)-induced current were recorded from neurons maintained in culture for 10-30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca2+ concentration ([Ca2+])i, gp120 and gp160 (20-250 pM) potentiated the large rises in [Ca2+]i induced by 50 microM NMDA. The potentiation of NMDA-induced Ca2+ responses required the presence of Ca2+ in the medium, and was abolished by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca2+ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20-250 pM gp120 or gp160 resulted in an increase in [Ca2+]i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca2+]i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immunofluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and

    The European journal of neuroscience 1995;7;11;2285-93

  • Developmental rearrangements of cortical glutamate-NMDA receptor binding sites in late human gestation.

    Andersen DL, Tannenberg AE, Burke CJ and Dodd PR

    Royal Brisbane Hospital Research Foundation, Australia.

    NMDA-preferring glutamate receptor biding sites were characterized using the site-selective ligand [3H]MK801, in synaptic membranes prepared from cerebral cortex tissue obtained postmortem from human infants who had died with minimal neurological and neuropathological impairment between 22 and 42 weeks' gestation. It proved necessary to modify the assay protocol used with adult tissue before reliable data could be obtained. In the four cortical region studied (prefrontal, motor, occipital, temporal), [3H]MK801 bound to a single class of sites which showed significant variations in affinity only in motor cortex. The density of [3H]MK801 binding sites (calculated at constant affinity) showed marked increases in all cortical regions over this period. The extent to which glutamate could enhance [3H]MK801 binding became significantly lower in prefrontal and motor cortex as gestation progressed, so that at term, little activation was apparent. In occipital and temporal cortex, this parameter was low throughout late gestation. The evidence suggests that Glutamate-NMDA binding sites may undergo structural rearrangements which alter their ability to interact with ligands during the later stages of human gestation, and that such changes are regionally variable.

    Brain research. Developmental brain research 1995;88;2;178-85

  • Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95.

    Kornau HC, Schenker LT, Kennedy MB and Seeburg PH

    Center for Molecular Biology (ZMBH), University of Heidelberg, Germany.

    The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses.

    Funded by: NINDS NIH HHS: NS-28710

    Science (New York, N.Y.) 1995;269;5231;1737-40

  • Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase.

    Corasaniti MT, Melino G, Navarra M, Garaci E, Finazzi-Agrò A and Nisticò G

    Faculty of Pharmacy, University of Reggio Calabria, Cantanzaro, Italy.

    The cytotoxic effects of the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 were studied in human CHP100 neuroblastoma cell cultures. Incubation of neuroblastoma cultures with gp120 (1 pM-10 nM) induces cell death which is not concentration-related. The significant cell death evoked by 10 pM gp120 was prevented by neutralization of the viral protein with a monoclonal anti-gp120 (IgG) antibody. In addition, gp120-induced cytotoxicity was inhibited by [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] (CGP37849; 100 microM), [(+/-)-3R*, 4as*, 6R*, 8aR*-6-(phosphonomethyl) decahydro-isoquinoline-3-carboxylic acid] (LY274614; 100 microM), MK801 (dizocilpine; 200 nM) and 7-chloro kynurenic acid (100 microM), selective antagonists of the NMDA receptor complex; by contrast, (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100 microM), a non-NMDA antagonist, was ineffective. Prevention of the lethality elicited by the HIV-1 coat protein was also obtained by incubating neuroblastoma cells with gp120 in Ca(2+)-free medium. The lethal effects induced by gp120 involve activation of L-arginine-nitric oxide (NO) pathway since these were prevented by haemoglobin (10 microM), a NO-trapping agent, and by D-arginine (1 mM), the less active enantiomer of the endogenous precursor of NO synthesis. Cytoprotection was also afforded by N omega-nitro-L-arginine methyl ester (L-NAME; 200 microM), an inhibitor of NO synthase, and this was reversed by L-arginine (1 mM). Interestingly, indomethacin and flufenamic acid (10 microM), two inhibitors of cyclooxygenase, protected neuroblastoma cells from death induced by gp120. Furthermore, indomethacin prevented the neuroblastoma cell death evoked by exposure of cultures to sodium nitroprusside (SNP; 0.2-1.6 mM), a NO donor. Finally significant cytotoxic effects were observed after incubation of neuroblastoma cells with prostaglandin E2 (0.1-10 microM). In conclusion, the present data suggest that death of human CHP100 neuroblastoma cells in culture produced by gp120 involves NO and PGE2 production.

    Neurodegeneration : a journal for neurodegenerative disorders, neuroprotection, and neuroregeneration 1995;4;3;315-21

  • Human immunodeficiency virus type 1 tat activates non-N-methyl-D-aspartate excitatory amino acid receptors and causes neurotoxicity.

    Magnuson DS, Knudsen BE, Geiger JD, Brownstone RM and Nath A

    Department of Physiology, University of Manitoba, Winnipeg, Canada.

    The human immunodeficiency virus type 1 (HIV-1) protein Tat is known to be released from HIV-1-infected cells. We show that micromolar concentrations of Tat depolarized young rat and adult human neurons. In addition, Tat, at similar concentrations, was toxic to human fetal neurons in culture. Tat-induced responses were insensitive to the Na+ channel blocker tetrodotoxin, suggesting a direct effect of Tat on neurons. Tat-induced depolarizations and cytotoxicity were blocked by the excitatory amino acid antagonist kynurenate. The N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate had little effect on Tat-induced depolarizations but did provide protection from Tat neurotoxicity. These results suggest that Tat, released from HIV-1-infected cells, may be an important mediator of neurotoxicity observed in HIV-1 encephalopathy.

    Annals of neurology 1995;37;3;373-80

  • Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit.

    Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, Yagi T, Aizawa S, Inoue Y, Sugiyama H et al.

    Department of Neuropharmacology, Niigata University, Japan.

    The NMDA (N-methyl-D-aspartate) receptor channel is important for synaptic plasticity, which is thought to underlie learning, memory and development. The NMDA receptor channel is formed by at least two members of the glutamate receptor (GluR) channel subunit families, the GluR epsilon (NR2) and GluR zeta (NR1) subunit families. The four epsilon subunits are distinct in distribution, properties and regulation. On the basis of the Mg2+ sensitivity and expression patterns, we have proposed that the epsilon 1 (NR2A) and epsilon 2 (NR2B) subunits play a role in synaptic plasticity. Here we show that targeted disruption of the mouse epsilon 1 subunit gene resulted in significant reduction of the NMDA receptor channel current and long-term potentiation at the hippocampal CA1 synapses. The mutant mice also showed a moderate deficiency in spatial learning. These results support the notion that the NMDA receptor channel-dependent synaptic plasticity is the cellular basis of certain forms of learning.

    Nature 1995;373;6510;151-5

  • Human N-methyl-D-aspartate receptor modulatory subunit hNR2A: cloning and sequencing of the cDNA and primary structure of the protein.

    Foldes RL, Adams SL, Fantaske RP and Kamboj RK

    Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.

    Several cDNA clones encoding the human N-methyl-D-aspartate receptor modulatory subunit hNR2A, were isolated from human hippocampus and fetal brain libraries. DNA sequence analysis revealed overlapping clones permitting the reconstruction of full-length hNR2A cDNA. The hNR2A cDNA demonstrated an 88-89% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequence of hNR2A would encode a 1464-aa protein that has a 95.2% identity with the rodent NR2A subunits.

    Biochimica et biophysica acta 1994;1223;1;155-9

  • Transmembrane topology of the glutamate receptor subunit GluR6.

    Roche KW, Raymond LA, Blackstone C and Huganir RL

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

    Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system. These receptors are divided into alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate receptors based on pharmacological and electrophysiological characteristics. Ionotropic receptor subunits are integral membrane proteins that have been proposed to have a large extracellular ligand-binding N-terminal domain, four hydrophobic transmembrane domains, and an extracellular C-terminal domain. In this study we have shown that both AMPA receptor subunits (GluR1-4) and kainate receptor subunits (GluR6/7) are glycosylated in adult rat brain; however, the kainate receptor subunits are glycosylated to a greater extent. Examination of the sequences of AMPA and kainate receptors revealed that kainate receptors have several additional consensus sites for N-linked glycosylation; interestingly, one of these is located in the proposed major intracellular loop of the receptor subunits. To test the proposed transmembrane topology model for these receptors, we have used site-specific mutagenesis of the GluR6 subunit to remove the consensus glycosylation site located within the proposed intracellular loop. Mutagenesis of this site demonstrates that it is glycosylated in transiently transfected human embryonic kidney cells, which express functional kainate receptors. Since N-linked glycosylation has only been found to occur on extracellular domains of plasma membrane proteins, these results suggest that the proposed transmembrane topology model for the glutamate receptor subunits is incorrect. Combining these results with other recent data, we have proposed an alternative transmembrane topology model.

    Funded by: NIGMS NIH HHS: GM-07309

    The Journal of biological chemistry 1994;269;16;11679-82

  • Changing subunit composition of heteromeric NMDA receptors during development of rat cortex.

    Sheng M, Cummings J, Roldan LA, Jan YN and Jan LY

    Howard Hughes Medical Institute, University of California, San Francisco 94143-0724.

    Activation of the N-methyl-D-aspartate (NMDA) receptor is important for certain forms of activity-dependent synaptic plasticity, such as long-term potentiation (reviewed in ref. 1), and the patterning of connections during development of the visual system (reviewed in refs 2, 3). Several subunits of the NMDA receptor have been cloned: these are NMDAR1 (NR1), and NMDAR2A, 2B, 2C and 2D (NR2A-D). Based on heterologous co-expression studies, it is inferred that NR1 encodes an essential subunit of NMDA receptors and that functional diversity of NMDA receptors in vivo is effected by differential incorporation of subunits NR2A-NR2D. Little is known, however, about the actual subunit composition or heterogeneity of NMDA receptors in the brain. By co-immunoprecipitation with subunit-specific antibodies, we present here direct evidence that NMDA receptors exist in rat neocortex as heteromeric complexes of considerable heterogeneity, some containing both NR2A and NR2B subunits. A progressive alteration in subunit composition seen postnatally could contribute to NMDA-receptor variation and changing synaptic plasticity during cortical development.

    Nature 1994;368;6467;144-7

  • Chromosomal localization of the epsilon 1, epsilon 3 and zeta 1 subunit genes of the human NMDA receptor channel.

    Takano H, Onodera O, Tanaka H, Mori H, Sakimura K, Hori T, Kobayashi H, Mishina M and Tsuji S

    Department of Neurology, Niigata University, Japan.

    Partial complementary DNAs for the epsilon 1, epsilon 3 and zeta 1 subunits of the human N-methyl-D-aspartate (NMDA) receptor channel were cloned using the corresponding mouse subunit cDNA clones as probes. Genomic DNA clones for the human epsilon 1, epsilon 3 and zeta 1 subunit genes were isolated using the corresponding partial cDNA clones as probes. By the fluorescence in situ hybridization, we mapped the genes for the epsilon 1, epsilon 3 and zeta 1 subunits of the human NMDA receptor channel to chromosomes 16p13, 17q25 and 9q34, respectively.

    Biochemical and biophysical research communications 1993;197;2;922-6

  • Heteromeric NMDA receptors: molecular and functional distinction of subtypes.

    Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B and Seeburg PH

    Center for Molecular Biology, University of Heidelberg, Germany.

    The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

    Science (New York, N.Y.) 1992;256;5060;1217-21

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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