G2Cdb::Gene report

Gene id
G00002098
Gene symbol
GRIN1 (HGNC)
Species
Homo sapiens
Description
glutamate receptor, ionotropic, N-methyl D-aspartate 1
Orthologue
G00000849 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000020976 (Vega human gene)
Gene
ENSG00000176884 (Ensembl human gene)
2902 (Entrez Gene)
1 (G2Cdb plasticity & disease)
GRIN1 (GeneCards)
Literature
138249 (OMIM)
Marker Symbol
HGNC:4584 (HGNC)
Protein Sequence
Q05586 (UniProt)

Diseases (5)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000166: Schizophrenia Y Y (11109007) Single nucleotide polymorphism (SNP) N
D00000166: Schizophrenia Y Y (11326295) Single nucleotide polymorphism (SNP) N
D00000166: Schizophrenia Y Y (11326295) Deletion (D) N
D00000166: Schizophrenia Y Y (11326295) Microinsertion/deletion (MI/D) N
D00000166: Schizophrenia Y Y (12210277) Microdeletion/insertion (MD/I) N
D00000166: Schizophrenia Y Y (12363394) Single nucleotide polymorphism (SNP) N
D00000166: Schizophrenia Y Y (12454527) Single nucleotide polymorphism (SNP) N
D00000170: Bipolar disorder Y Y (12610658) Single nucleotide polymorphism (SNP) Y
D00000166: Schizophrenia Y Y (12679240) Single nucleotide polymorphism (SNP) Y
D00000166: Schizophrenia Y Y (12707933) Single nucleotide polymorphism (SNP) N
D00000162: Alcoholism Y Y (14573320) Single nucleotide polymorphism (SNP) Y
D00000193: Alzheimer's disease Y Y (15030408) Splice variant (SpVar) Y
D00000163: Alcoholism withdrawal-induced seizures Y Y (15635650) Single nucleotide polymorphism (SNP) ?
D00000162: Alcoholism Y Y (15635650) Single nucleotide polymorphism (SNP) N
D00000166: Schizophrenia Y Y (15841096) Single nucleotide polymorphism (SNP) ?
D00000170: Bipolar disorder Y Y (16969270) Single nucleotide polymorphism (SNP) N
D00000170: Bipolar disorder Y Y (16969270) Microsatellite polymorphism (MSP) N

References

  • No association between genetic variants at the GRIN1 gene and bipolar disorder in a German sample.

    Georgi A, Jamra RA, Schumacher J, Becker T, Schmael C, Deschner M, Höfels S, Wulff M, Schwarz M, Klopp N, Illig T, Propping P, Cichon S, Nöthen MM, Rietschel M and Schulze TG

    Central Institute of Mental Health, Mannheim, Germany.

    Disturbed glutamatergic neurotransmission has been implicated in the pathogenesis of schizophrenia and bipolar disorder, with the N-methy-D-aspartate receptors being in the focus of research. The NR1 subunit, which is encoded by the gene GRIN1, plays a key role in the functionality of N-methy-D-aspartate receptors. We tested the association between GRIN1 and bipolar disorder in a sample of German descent, consisting of 306 bipolar disorder patients and 319 population-based controls. No significant association was found. In accordance with our recent findings, we hypothesized that restricting case definition to individuals with a history of persecutory delusions might clarify the relationship between bipolar disorder and GRIN1. This stratified analysis did not yield any significant association either. Our results do not support an association of the GRIN1 gene with bipolar disorder in the German population.

    Psychiatric genetics 2006;16;5;183-4

  • An association study of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B) in schizophrenia with universal DNA microarray.

    Qin S, Zhao X, Pan Y, Liu J, Feng G, Fu J, Bao J, Zhang Z and He L

    Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 230030, PR China.

    Dysfunction of the N-methyl-D-aspartate (NMDA) receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of several antagonists and on observation of genetic animal models. To conduct association analysis of the NMDA receptors in the Chinese population, we examined 16 reported SNPs across the NMDA receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B), five of which were identified in the Chinese population. In this study, we combined universal DNA microarray and ligase detection reaction (LDR) for the purposes of association analysis, an approach we considered to be highly specific as well as offering a potentially high throughput of SNP genotyping. The association study was performed using 253 Chinese patients with schizophrenia and 140 Chinese control subjects. No significant frequency differences were found in the analysis of the alleles but some were found in the haplotypes of the GRIN2B gene. The interactions between the GRIN1 and GRIN2B genes were evaluated using the multifactor-dimensionality reduction (MDR) method, which showed a significant genetic interaction between the G1001C in the GRIN1 gene and the T4197C and T5988C polymorphisms in the GRIN2B gene. These findings suggest that the combined effects of the polymorphisms in the GRIN1 and GRIN2B genes might be involved in the etiology of schizophrenia.European Journal of Human Genetics (2005) 13, 807-814. doi:10.1038/sj.ejhg.5201418 Published online 20 April 2005.

    European journal of human genetics : EJHG 2005;13;7;807-14

  • GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal.

    Rujescu D, Soyka M, Dahmen N, Preuss U, Hartmann AM, Giegling I, Koller G, Bondy B, Möller HJ and Szegedi A

    Department of Psychiatry, University of Munich, Munich, Germany. Dan.Rujescu@psy.med.uni-muenchen.de

    N-Methyl-D-aspartate (NMDA) receptors, members of the glutamate receptor channel superfamily, are generally inhibited by alcohol. The expression and alternative splicing of the obligatory NR1 subunit is altered by alcohol exposure, emphasizing the involvement of the NR1 subunit, which is coded by the GRIN1 gene, in alcohol-mediated effects. We performed an association study in patients with alcohol dependence with the GRIN1 locus. Two independent case control samples consisting of a total of 442 alcohol-dependent patients and 442 unrelated controls were included. There was no overall difference in allele or genotype frequency between patients and controls. However, the 2108A allele and A-containing genotypes were over-represented in the patients with a history of withdrawal-induced seizures when compared to healthy volunteers (allele: chi(2) = 5.412, df = 1, P = 0.020) or an independent sample of patients without a history of seizures (allele: chi(2) = 4.185, df = 1, P = 0.041). Age at onset, years of alcohol dependence, and a history of delirium tremens did not differ between genotype or allele groups. These findings support the hypothesis that the GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal. This novel finding warrants replication.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2005;133B;1;85-7

  • Selective loss of NMDA receptor NR1 subunit isoforms in Alzheimer's disease.

    Hynd MR, Scott HL and Dodd PR

    Department of Biochemistry, University of Queensland, Australia.

    Previous work had shown that the ratio of NMDA receptor NR1 subunit mRNA transcripts containing an N-terminal splice cassette to those that do not is markedly lower in regions of the Alzheimer's disease (AD) brain that are susceptible to pathological damage, compared with spared regions in the same cases or homotropic regions in controls. To elucidate the origins of this difference in proportionate expression, we measured the absolute levels of each of the eight NR1 transcripts by quantitative internally standardized RT-PCR assay. Expression of transcripts with the cassette was strongly attenuated in susceptible regions of Alzheimer's brain, whereas expression of non-cassette transcripts differed little from that in controls. The expression of other NR1 splice variants was not associated with pathology relevant to disease status, although some combinations of splice cassettes were well maintained in AD cases. The population profile of NR1 transcripts in occipital cortex differed from the profiles in other brain regions studied. Western analysis confirmed that the expression of protein isoforms containing the N-terminal peptide was very low in susceptible areas of the Alzheimer's brain. Cells that express NR1 subunits with the N-terminal cassette may be selectively vulnerable to toxicity in AD.

    Journal of neurochemistry 2004;89;1;240-7

  • Polymorphisms in the N-methyl-D-aspartate receptor 1 and 2B subunits are associated with alcoholism-related traits.

    Wernicke C, Samochowiec J, Schmidt LG, Winterer G, Smolka M, Kucharska-Mazur J, Horodnicki J, Gallinat J and Rommelspacher H

    Department of Clinical Neurobiology, University Hospital Benjamin Franklin, Free University of Berlin, Berlin, Germany.

    Background: This study examined the hypothesis that allelic variants of the ionotropic glutamatergic N-methyl-D-aspartate receptor (NMDAR) are associated with vulnerability to alcoholism and some related traits.

    Methods: We investigated the silent G2108A and C2664T polymorphisms of the NMDAR1 and the NMDAR2B genes, respectively. The case control study included 367 alcoholic and 335 control subjects of German origin. The family-based study comprised 81 Polish alcoholic patients and their parents using the transmission disequilibrium test.

    Results: The genotype frequencies of the NMDAR1 polymorphism differed significantly between control and alcoholic subjects. This difference was also observed in more homogenous subgroups of alcoholic subjects with vegetative withdrawal syndrome and Cloninger type 1. Patients with a history of delirium tremens or seizures during withdrawal showed a significantly increased prevalence of the A allele. Genotyping of the NMDAR2B polymorphism revealed a significantly reduced T allele in Cloninger type 2 alcoholics and in patients reporting an early onset compared with control subjects. Our family-based study for NMDAR2B, revealed a trend to a preferred transmission of the C allele by the fathers, and families with early-onset patients contributed most to this trend.

    Conclusions: These results suggest that variants in NMDAR genes are associated with alcoholism and related traits.

    Biological psychiatry 2003;54;9;922-8

  • N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia: TDT and case-control analyses.

    Martucci L, Wong AH, Trakalo J, Cate-Carter T, Wong GW, Macciardi FM and Kennedy JL

    Neurogenetics Section, CAMH, Clarke Division, University of Toronto, Toronto, Ontario, Canada.

    The N-methyl-d-aspartate glutamate receptors (NMDAR) act in the CNS as regulators of the release of neurotransmitters such as dopamine, noradrenaline, acetylcholine, and GABA. It has been suggested that a weakened glutamatergic tone increases the risk of sensory overload and of exaggerated responses in the monoaminergic system, which is consistent with the symptomatology of schizophrenia. We studied two silent polymorphisms in GRIN1. GRIN1/1 is a G/C substitution localized on the 5' untranslated region; GRIN1/10 is an A/G substitution localized in exon 6 of GRIN1. Minor allele frequencies in our sample were calculated to be 0.05 and 0.2 respectively. We genotyped 86 nuclear families and 91 ethnically matched case-control pairs. Both samples were collected from the Toronto area. We tested the hypothesis that GRIN1 polymorphisms were associated with schizophrenia using the transmission disequilibrium test (TDT) and comparing allele frequencies between cases and controls. The results are as follows: GRIN1/1: chi(2) = 2.19, P = 0.14; GRIN1/10: chi(2) = 1.5, P = 0.22. For the case-control sample: GRIN1/1: chi(2) = 0.013, P = 0.908; GRIN1/10: chi(2) = 0.544, P = 0.461. No significant results were obtained. Haplotype analyses showed a borderline significant result for the 2,1 haplotype (chi(2) = 3.86, P-value = 0.049). An analysis of variance (ANOVA) to evaluate the association between genetic makeup and age at onset was performed, with no significant results: GRIN1/1, F[df = 2] = 0.42, P-value = 0.659; GRIN1/10, F[df = 2] = 0.16, P-value = 0.853. We are currently collecting additional samples to increase the power of the analyses.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2003;119B;1;24-7

  • Association between the G1001C polymorphism in the GRIN1 gene promoter region and schizophrenia.

    Begni S, Moraschi S, Bignotti S, Fumagalli F, Rillosi L, Perez J and Gennarelli M

    Genetics Unit, IRCCS S. Giovanni di Dio, Fatebenefratelli, Brescia, Italy

    Background: The GRIN1 gene plays a fundamental role in many brain functions, and its involvement in the pathogenesis of the schizophrenia has been widely investigated. Non-synonymous polymorphisms have not been identified in the coding regions. To investigate the potential role of GRIN1 in the susceptibility to schizophrenia, we analyzed the G1001C polymorphism located in the promoter region in a case-control association study.

    Methods: The G1001C polymorphism allele distribution was analyzed in a sample of 139 Italian schizophrenic patients and 145 healthy control subjects by a polymerase chain reaction amplification followed by digestion with a restriction endonuclease.

    Results: We found that the C allele may alter a consensus sequence for the transcription factor NF-kappa B and that its frequency was higher in patients than in control subjects (p =.0085). The genotype distribution also was different, with p =.034 (if C allele dominant, p =.0137, odds ratio 2.037, 95% confidence interval 1.1502-3.6076).

    Conclusions: The association reported in this study suggests that the GRIN1 gene is a good candidate for the susceptibility to schizophrenia.

    Biological psychiatry 2003;53;7;617-9

  • Evidence that the N-methyl-D-aspartate subunit 1 receptor gene (GRIN1) confers susceptibility to bipolar disorder.

    Mundo E, Tharmalingham S, Neves-Pereira M, Dalton EJ, Macciardi F, Parikh SV, Bolonna A, Kerwin RW, Arranz MJ, Makoff AJ and Kennedy JL

    Neurogenetics Section, Centre for Addiction and Mental Health (CAMH), Department of Psychiatry, University of Toronto, Clarke Site R-31, Toronto, Ontario, Canada M5T 1R8.

    There is evidence for the involvement of glutamatergic transmission in the pathogenesis of major psychoses. The two most commonly used mood stabilizers (ie lithium and valproate) have been found to act via the N-methyl-D-aspartate receptor (NMDAR), suggesting a specific role of NMDAR in the pathogenesis of bipolar disorder (BP). The key subunit of the NMDAR, named NMDA-1 receptor, is coded by a gene located on chromosome 9q34.3 (GRIN1). We tested for the presence of linkage disequilibrium between the GRIN1 (1001-G/C, 1970-A/G, and 6608-G/C polymorphisms) and BP. A total of 288 DSM-IV Bipolar I, Bipolar II, or schizoaffective disorder, manic type, probands with their living parents were studied. In all, 73 triads had heterozygous parents for the 1001-G/C polymorphism, 174 for the 1970-A/G, and 48 for the 6608-G/C. These triads were suitable for the final analyses, that is, the transmission disequilibrium test (TDT) and the haplotype-TDT. For the 1001-G/C and the 6608-G/C polymorphisms, we found a preferential transmission of the G allele to the affected individuals (chi(2)=4.765, df=1, P=0.030 and chi(2)= 8.395, df=1, P=0.004, respectively). The 1001G-1970A-6608A and the 1001G-1970A-6608G haplotypes showed the strongest association with BP (global chi(2)=14.12, df=4, P=0.007). If these results are replicated there could be important implications for the involvement of the GRIN1 in the pathogenesis of BP. The role of the gene variants in predicting the response to mood stabilizers in BP should also be investigated.

    Molecular psychiatry 2003;8;2;241-5

  • Systematic mutation analysis of the human glutamate receptor, ionotropic, N-methyl-D-aspartate 1 gene(GRIN1) in schizophrenic patients.

    Hung CC, Chen HY and Chen CH

    Institute of Human Genetics, Tzu-Chi University, Hualien City, Taiwan. cchen@mail.tcu.edu.tw

    Schizophrenia is a severe, complex mental disorder with unknown etiology. Abnormal glutamate neurotransmission has been proposed as one of the hypotheses of the pathogenesis of schizophrenia. Mohn recently reported that transgenic mice with the reduced glutamate receptor, ionotropic, -methyl-D-aspartate 1 gene (GRIN1) (formerly referred to as NMDAR1) expression display schizophrenia-like behaviors, which can be ameliorated by antipsychotic drug treatment. Their report promoted us to examine whether mutations in the human GRIN1 gene may convey genetic susceptibility to schizophrenia. To test this possibility, we systematically screened mutations in the promoter region and in all the exons of the human GRIN1 gene in a cohort of Chinese schizophrenic patients from Taiwan. Using single-strand conformation polymorphism analysis and autosequencing, we identified two single nucleotide polymorphisms, designated g.-1140G>A and g.-855G>C, respectively, at the 5'-untranslated region of the human GRIN1 gene. Genetic association study, however, revealed no association of these two single nucleotide polymorphisms with schizophrenia in our patients. Besides, no other mutations of the human GRIN1 gene were detected in this study. Our data suggest that the human GRIN1 gene may not contribute substantially to the genetic etiology of schizophrenia in our population.

    Psychiatric genetics 2002;12;4;225-30

  • Polymorphism analysis of the upstream region of the human N-methyl-D-aspartate receptor subunit NR1 gene (GRIN1): implications for schizophrenia.

    Tani A, Kikuta R, Itoh K, Joo A, Shibata H, Ninomiya H, Tashiro N and Fukumaki Y

    Division of Disease Genes, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.

    Dysfunction of the gene for the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor (GRIN1) has been implicated in the pathogenesis of schizophrenia. In support of this hypothesis are behavioral abnormalities reminiscent of schizophrenia in mice with an attenuated expression of the NR1 subunit receptor and the reduced level of NR1 mRNA in postmortem brains of patients with schizophrenia. We screened single nucleotide polymorphisms (SNPs) in the upstream region between +51 and -941 from the translation initiation codon of GRIN1 and identified 17 SNPs, 10 of which were located within the region containing the Sp1 motif and the GSG motifs. As genotyping of 191-196 Japanese patients with schizophrenia and 202-216 controls revealed no significant association between schizophrenia and the SNPs in the upstream region of GRIN1, these SNPs apparently do not play a critical role in the pathogenesis of schizophrenia in the Japanese population.

    Schizophrenia research 2002;58;1;83-6

  • Alteration of branch site consensus sequence and enhanced pre-mRNA splicing of an NMDAR1 intron not associated with schizophrenia.

    Hammond L, Castanotto D, Rice SR, Nimgaonkar VL, Wirshing DA, Rossi JJ, Heston LL and Sobell JL

    Division of Molecular Medicine, City of Hope National Medical Center, Duarte, California 91010-0269, USA.

    Aberrant splicing of pre-mRNA is recognized to account for a significant minority of disease-causing mutations. The N-methyl-D-aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African-American male near a weak putative branch-site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in-frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer-extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African-American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease.

    American journal of medical genetics 2002;114;6;631-6

  • Identification of single nucleotide polymorphisms (SNPs) and other sequence changes and estimation of nucleotide diversity in coding and flanking regions of the NMDAR1 receptor gene in schizophrenic patients.

    Rice SR, Niu N, Berman DB, Heston LL and Sobell JL

    Division of Molecular Medicine, City of Hope National Medical Center, Duarte, CA, USA.

    Glutamatergic dysregulation has been hypothesized to play a role in schizophrenia. The N-methyl-D-aspartate (NMDA) type of glutamate receptor especially is of interest because, in addition to binding sites for glutamate and glycine, a necessary co-agonist, this receptor also contains noncompetitive binding sites for the psychotomimetics phencyclidine (PCP), MK-801, and ketamine. PCP-induced psychosis has been a useful disease model in that both the positive as well as the negative symptomatologies seen in schizophrenia are observed. Recently, a mouse deficient in expression of the NR1 subunit gene (NMDAR1) of the heteromeric receptor has been developed and shown to display aberrant behaviors, with reduced social and sexual interactions as well as increased stereotypic motor activity. In an extensive examination of the NMDAR1 gene in our laboratory in approximately 100 chronic schizophrenic patients, 28 unique sequence changes were identified, including eight single nucleotide polymorphisms (SNPs) in the 5' untranslated region (5'UTR), six SNPs in coding regions (cSNPs), eleven intronic SNPs, two intronic deletions of 7 and 30 bp, and an intronic microinsertion/deletion. With the exception of one previously reported cSNP, all of the identified changes were novel. The frequency of polymorphisms differed significantly by ethnicity and several appeared to be in linkage disequilibrium. None of the changes appeared likely to be of functional significance, thus suggesting that changes in the genomic NMDAR1 are unlikely to contribute to the etiology of schizophrenia. Estimates of nucleotide diversity are comparable to those observed in studies of other genes.

    Funded by: NIMH NIH HHS: MH54232

    Molecular psychiatry 2001;6;3;274-84

  • Mutation analysis of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia.

    Sakurai K, Toru M, Yamakawa-Kobayashi K and Arinami T

    Department of Medical Genetics, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305-8575, Ibaraki-ken, Japan.

    Dysfunction of N-methyl-D-aspartate (NMDA) type ionotropic glutamate receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of the antagonist phencyclidine (PCP) and observation that mice expressing low levels of the N-methyl-D-aspartate receptor NR1 subunit exhibit behavioral alterations that may be ameliorated by neuroleptic drugs. Based on the hypothesis that some schizophrenic patients have functionally deficient mutation(s) of the gene encoding N-methyl-D-aspartate receptor NR1 subunit (GRIN1), we screened 48 Japanese patients with schizophrenia for mutations in the coding region of the GRIN1 gene. Four variants, IVS2-22T>C, IVS2-12G>A, IVS4-34C>T, and 1719G/A (Pro516Pro), were identified. No non-synonymous mutation was detected. No significant association was suggested by case-control comparisons. Results indicate that genomic variations of the GRIN1 gene are not likely to be involved substantially in the etiology of schizophrenia.

    Neuroscience letters 2000;296;2-3;168-70

Literature (132)

Pubmed - human_disease

  • No association between genetic variants at the GRIN1 gene and bipolar disorder in a German sample.

    Georgi A, Jamra RA, Schumacher J, Becker T, Schmael C, Deschner M, Höfels S, Wulff M, Schwarz M, Klopp N, Illig T, Propping P, Cichon S, Nöthen MM, Rietschel M and Schulze TG

    Central Institute of Mental Health, Mannheim, Germany.

    Disturbed glutamatergic neurotransmission has been implicated in the pathogenesis of schizophrenia and bipolar disorder, with the N-methy-D-aspartate receptors being in the focus of research. The NR1 subunit, which is encoded by the gene GRIN1, plays a key role in the functionality of N-methy-D-aspartate receptors. We tested the association between GRIN1 and bipolar disorder in a sample of German descent, consisting of 306 bipolar disorder patients and 319 population-based controls. No significant association was found. In accordance with our recent findings, we hypothesized that restricting case definition to individuals with a history of persecutory delusions might clarify the relationship between bipolar disorder and GRIN1. This stratified analysis did not yield any significant association either. Our results do not support an association of the GRIN1 gene with bipolar disorder in the German population.

    Psychiatric genetics 2006;16;5;183-4

  • GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal.

    Rujescu D, Soyka M, Dahmen N, Preuss U, Hartmann AM, Giegling I, Koller G, Bondy B, Möller HJ and Szegedi A

    Department of Psychiatry, University of Munich, Munich, Germany. Dan.Rujescu@psy.med.uni-muenchen.de

    N-Methyl-D-aspartate (NMDA) receptors, members of the glutamate receptor channel superfamily, are generally inhibited by alcohol. The expression and alternative splicing of the obligatory NR1 subunit is altered by alcohol exposure, emphasizing the involvement of the NR1 subunit, which is coded by the GRIN1 gene, in alcohol-mediated effects. We performed an association study in patients with alcohol dependence with the GRIN1 locus. Two independent case control samples consisting of a total of 442 alcohol-dependent patients and 442 unrelated controls were included. There was no overall difference in allele or genotype frequency between patients and controls. However, the 2108A allele and A-containing genotypes were over-represented in the patients with a history of withdrawal-induced seizures when compared to healthy volunteers (allele: chi(2) = 5.412, df = 1, P = 0.020) or an independent sample of patients without a history of seizures (allele: chi(2) = 4.185, df = 1, P = 0.041). Age at onset, years of alcohol dependence, and a history of delirium tremens did not differ between genotype or allele groups. These findings support the hypothesis that the GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal. This novel finding warrants replication.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2005;133B;1;85-7

  • Association between the G1001C polymorphism in the GRIN1 gene promoter region and schizophrenia.

    Begni S, Moraschi S, Bignotti S, Fumagalli F, Rillosi L, Perez J and Gennarelli M

    Genetics Unit, IRCCS S. Giovanni di Dio, Fatebenefratelli, Brescia, Italy

    Background: The GRIN1 gene plays a fundamental role in many brain functions, and its involvement in the pathogenesis of the schizophrenia has been widely investigated. Non-synonymous polymorphisms have not been identified in the coding regions. To investigate the potential role of GRIN1 in the susceptibility to schizophrenia, we analyzed the G1001C polymorphism located in the promoter region in a case-control association study.

    Methods: The G1001C polymorphism allele distribution was analyzed in a sample of 139 Italian schizophrenic patients and 145 healthy control subjects by a polymerase chain reaction amplification followed by digestion with a restriction endonuclease.

    Results: We found that the C allele may alter a consensus sequence for the transcription factor NF-kappa B and that its frequency was higher in patients than in control subjects (p =.0085). The genotype distribution also was different, with p =.034 (if C allele dominant, p =.0137, odds ratio 2.037, 95% confidence interval 1.1502-3.6076).

    Conclusions: The association reported in this study suggests that the GRIN1 gene is a good candidate for the susceptibility to schizophrenia.

    Biological psychiatry 2003;53;7;617-9

  • Polymorphism analysis of the upstream region of the human N-methyl-D-aspartate receptor subunit NR1 gene (GRIN1): implications for schizophrenia.

    Tani A, Kikuta R, Itoh K, Joo A, Shibata H, Ninomiya H, Tashiro N and Fukumaki Y

    Division of Disease Genes, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.

    Dysfunction of the gene for the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor (GRIN1) has been implicated in the pathogenesis of schizophrenia. In support of this hypothesis are behavioral abnormalities reminiscent of schizophrenia in mice with an attenuated expression of the NR1 subunit receptor and the reduced level of NR1 mRNA in postmortem brains of patients with schizophrenia. We screened single nucleotide polymorphisms (SNPs) in the upstream region between +51 and -941 from the translation initiation codon of GRIN1 and identified 17 SNPs, 10 of which were located within the region containing the Sp1 motif and the GSG motifs. As genotyping of 191-196 Japanese patients with schizophrenia and 202-216 controls revealed no significant association between schizophrenia and the SNPs in the upstream region of GRIN1, these SNPs apparently do not play a critical role in the pathogenesis of schizophrenia in the Japanese population.

    Schizophrenia research 2002;58;1;83-6

  • Identification of single nucleotide polymorphisms (SNPs) and other sequence changes and estimation of nucleotide diversity in coding and flanking regions of the NMDAR1 receptor gene in schizophrenic patients.

    Rice SR, Niu N, Berman DB, Heston LL and Sobell JL

    Division of Molecular Medicine, City of Hope National Medical Center, Duarte, CA, USA.

    Glutamatergic dysregulation has been hypothesized to play a role in schizophrenia. The N-methyl-D-aspartate (NMDA) type of glutamate receptor especially is of interest because, in addition to binding sites for glutamate and glycine, a necessary co-agonist, this receptor also contains noncompetitive binding sites for the psychotomimetics phencyclidine (PCP), MK-801, and ketamine. PCP-induced psychosis has been a useful disease model in that both the positive as well as the negative symptomatologies seen in schizophrenia are observed. Recently, a mouse deficient in expression of the NR1 subunit gene (NMDAR1) of the heteromeric receptor has been developed and shown to display aberrant behaviors, with reduced social and sexual interactions as well as increased stereotypic motor activity. In an extensive examination of the NMDAR1 gene in our laboratory in approximately 100 chronic schizophrenic patients, 28 unique sequence changes were identified, including eight single nucleotide polymorphisms (SNPs) in the 5' untranslated region (5'UTR), six SNPs in coding regions (cSNPs), eleven intronic SNPs, two intronic deletions of 7 and 30 bp, and an intronic microinsertion/deletion. With the exception of one previously reported cSNP, all of the identified changes were novel. The frequency of polymorphisms differed significantly by ethnicity and several appeared to be in linkage disequilibrium. None of the changes appeared likely to be of functional significance, thus suggesting that changes in the genomic NMDAR1 are unlikely to contribute to the etiology of schizophrenia. Estimates of nucleotide diversity are comparable to those observed in studies of other genes.

    Funded by: NIMH NIH HHS: MH54232

    Molecular psychiatry 2001;6;3;274-84

  • Mutation analysis of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia.

    Sakurai K, Toru M, Yamakawa-Kobayashi K and Arinami T

    Department of Medical Genetics, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305-8575, Ibaraki-ken, Japan.

    Dysfunction of N-methyl-D-aspartate (NMDA) type ionotropic glutamate receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of the antagonist phencyclidine (PCP) and observation that mice expressing low levels of the N-methyl-D-aspartate receptor NR1 subunit exhibit behavioral alterations that may be ameliorated by neuroleptic drugs. Based on the hypothesis that some schizophrenic patients have functionally deficient mutation(s) of the gene encoding N-methyl-D-aspartate receptor NR1 subunit (GRIN1), we screened 48 Japanese patients with schizophrenia for mutations in the coding region of the GRIN1 gene. Four variants, IVS2-22T>C, IVS2-12G>A, IVS4-34C>T, and 1719G/A (Pro516Pro), were identified. No non-synonymous mutation was detected. No significant association was suggested by case-control comparisons. Results indicate that genomic variations of the GRIN1 gene are not likely to be involved substantially in the etiology of schizophrenia.

    Neuroscience letters 2000;296;2-3;168-70

Pubmed - other

  • 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; NIDDK NIH HHS: R01 DK100340; Wellcome Trust

    BMC biology 2009;7;70

  • Disruption of glutamate receptors at Shank-postsynaptic platform in Alzheimer's disease.

    Gong Y, Lippa CF, Zhu J, Lin Q and Rosso AL

    Department of Neurology, Drexel University College of Medicine, 245 N 15th Street, Philadelphia, PA 19102, USA. Yuesong.Gong@drexelmed.edu

    Synaptic loss underlies the memory deficit of Alzheimer's disease (AD). The molecular mechanism is elusive; however, excitatory synapses organized by the postsynaptic density (PSD) have been used as targets for AD treatment. To identify pathological entities at the synapse in AD, synaptic proteins were screened by quantitative proteomic profiling. The critical proteins were then selected for immunoblot analysis. The glutamate receptors N-methyl-d-aspartate (NMDA) receptor 1 and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2 (GluR2) were substantially lost; specifically, the loss of GluR2 was up to 40% at PSD in AD. Shank proteins, the organizers of these glutamate receptors at excitatory synapses, were dramatically altered in AD. The level of Shank2 was increased, whereas the protein level of Shank3 was decreased. Further, the Shank3 protein was modified by ubiquitin, indicating that abnormal activity of the ubiquitin-proteasome system may lead to Shank3 degradation in AD. Our findings suggest that disruption of glutamate receptors at the Shank-postsynaptic platform could contribute to destruction of the PSD which underlies the synaptic dysfunction and loss in AD.

    Funded by: NCRR NIH HHS: 1S10RR01782-01A1; NIA NIH HHS: 1R21AG031388, R21 AG031388, R21 AG031388-01

    Brain research 2009;1292;191-8

  • Novel environment and GABA agonists alter event-related potentials in N-methyl-D-aspartate NR1 hypomorphic and wild-type mice.

    Bodarky CL, Halene TB, Ehrlichman RS, Banerjee A, Ray R, Hahn CG, Jonak G and Siegel SJ

    Department of Psychiatry, University of Pennsylvania, Philadelphia, USA.

    Clinical and experimental data suggest dysregulation of N-methyl-d-aspartate receptor (NMDAR)-mediated glutamatergic pathways in schizophrenia. The interaction between NMDAR-mediated abnormalities and the response to novel environment has not been studied. Mice expressing 5 to 10% of normal N-methyl-d-aspartate receptor subunit 1 (NR1) subunits [NR1(neo)(-/-)] were compared with wild-type littermates for positive deflection at 20 ms (P20) and negative deflection at 40 ms (N40) auditory event-related potentials (ERPs). Groups were tested for habituation within and across five testing sessions, with novel environment tested during a sixth session. Subsequently, we examined the effects of a GABA(A) positive allosteric modulator (chlordiazepoxide) and a GABA(B) receptor agonist (baclofen) as potential interventions to normalize aberrant responses. There was a reduction in P20, but not N40 amplitude within each habituation day. Although there was no amplitude or gating change across habituation days, there was a reduction in P20 and N40 amplitude and gating in the novel environment. There was no difference between genotypes for N40. Only NR1(neo)(-/-) mice had reduced P20 in the novel environment. Chlordiazepoxide increased N40 amplitude in wild-type mice, whereas baclofen increased P20 amplitude in NR1(neo)(-/-) mice. As noted in previous publications, the pattern of ERPs in NR1(neo)(-/-) mice does not recapitulate abnormalities in schizophrenia. In addition, reduced NR1 expression does not influence N40 habituation but does affect P20 in a novel environment. Thus, the pattern of P50 (positive deflection at 50 ms) but not N100 (negative deflection at 100 ms) in human studies may relate to subjects' reactions to unfamiliar environments. In addition, NR1 reduction decreased GABA(A) receptor-mediated effects on ERPs while causing increased GABA(B) receptor-mediated effects. Future studies will examine changes in GABA receptor subunits after reductions in NR1 expression.

    Funded by: NCI NIH HHS: P50 CA084718, P50-CA084718; NIDA NIH HHS: 5R01-DA02321002; NIMH NIH HHS: P50 MH064045, P50-MH064045, R01 MH080718, R01-MH080718

    The Journal of pharmacology and experimental therapeutics 2009;331;1;308-18

  • Case-control association study of 65 candidate genes revealed a possible association of a SNP of HTR5A to be a factor susceptible to bipolar disease in Bulgarian population.

    Yosifova A, Mushiroda T, Stoianov D, Vazharova R, Dimova I, Karachanak S, Zaharieva I, Milanova V, Madjirova N, Gerdjikov I, Tolev T, Velkova S, Kirov G, Owen MJ, O'Donovan MC, Toncheva D and Nakamura Y

    Laboratory for International Alliance, RIKEN Center for Genomic Medicine, Tsurumi-ku, Yokohama, Japan.

    Background: Bipolar affective disorder (BAD) is a psychiatric illness characterized by episodes of mania and depression. Although the etiology is not clear, epidemiological studies suggest it is a result of an interaction of genetic and environmental factors. Despite of enormous efforts and abundant studies conducted, none has yet been identified definitively a gene susceptible to bipolar disorder.

    Methods: Ninety-four Bulgarian patients diagnosed with bipolar disorder and 184 Bulgarian healthy individuals, were used for genotyping of 191 single nucleotide polymorphisms (SNPs) by TaqMan and/or Invader assays. Seventeen SNPs that revealed P value less than 0.05 in the first screening were genotyped using an additional independent set of samples, consisting of 78 BAD cases and 372 controls.

    Results: After applying the Bonferonni correction on genotyping results of 172 cases and 556 controls, only one SNP, rs1800883, in the HTR5A gene revealed a significant level of P value (P=0.000097; odds ratio=1.80 (95%CI, 1.27-2.54); corrected P=0.017).

    Conclusions: Our findings suggest that HTR5A gene could play an important role in the pathogenesis of bipolar disorder in our population. However these findings should be viewed with caution and replication studies in other populations are necessary in support of these findings.

    Funded by: Medical Research Council: G0800509

    Journal of affective disorders 2009;117;1-2;87-97

  • 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

  • Association between the G1001C polymorphism in the GRIN1 gene promoter and schizophrenia in the Iranian population.

    Galehdari H, Pooryasin A, Foroughmand A, Daneshmand S and Saadat M

    Shahid Chamran University, Ahvaz, Iran. galehdari@scu.ac.ir

    Schizophrenia is a complex genetic disorder to which genetic variation in the glutamatergic signaling pathways is believed to play a substantial role in the etiology of the disease. Association studies have implicated the N-methyl-D-aspartate receptor subunit gene, GRIN1, as a candidate gene for schizophrenia. In this report, we used a case control study to establish the possible association between the G1001C polymorphism in the GRIN1 gene promoter region and schizophrenia in an Iranian cohort of 200 unrelated patients and 200 controls. The allelic and genotypic frequencies of the polymorphism were determined using polymerase chain reaction restriction fragment length polymorphism. Data analysis using logistic regression and the Mantel-Haenszel chi-square test revealed a strong association between the G1001C polymorphism and schizophrenia (CG genotype: odds ratio (OR) = 2.12, 95% confidence interval (CI) 1.34-3.48, P = 0.001 and CC genotype: OR = 29.10, 95% CI 3.40-565.78, P < 0.001). Furthermore, the C allele is significantly associated with an increasing risk of schizophrenia.

    Journal of molecular neuroscience : MN 2009;38;2;178-81

  • 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

  • A comparison of the molecular bases for N-methyl-D-aspartate-receptor inhibition versus immobilizing activities of volatile aromatic anesthetics.

    Sewell JC, Raines DE, Eger EI, Laster MJ and Sear JW

    Nuffield Department of Anesthetics, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, UK.

    Background: Aromatic anesthetics exhibit a wide range of N-methyl-d-aspartate (NMDA) receptor inhibitory potencies and immobilizing activities. We sought to characterize the molecular basis of NMDA receptor inhibition using comparative molecular field analysis (CoMFA), and compare the results to those from an equivalent model for immobilizing activity.

    Methods: Published potency data for 14 compounds were supplemented with new values for 2 additional agents. The anesthetics were divided into a training set (n = 12) used to formulate the activity models and a test set (n = 4) used to independently assess the models' predictive capability. The anesthetic structures were geometry optimized using ab initio quantum mechanics and aligned by field-fit minimization to provide the best correlation between the steric and electrostatic fields of the molecules and one or more lead structures. Orientations that yielded CoMFA models with the greatest predictive capability (assessed by leave-one-out cross-validation) were retained.

    Results: The final CoMFA model for the inhibition of NR1/NR2B NMDA receptors explained 99.3% of the variance in the observed activities of the 12 training set agents (F(2,)(9) = 661.5, P < 0.0001). The model effectively predicted inhibitory potency for the training set (cross-validated r(2)(CV) = 0.944) and 4 excluded test set compounds (predictive r(2)(Pred) = 0.966). The equivalent model for immobility in response to noxious stimuli explained 98.0% of the variance in the observed activities for the training set (F(2,)(9) = 219.2, P < 0.0001) and exhibited adequate predictive capability for both the training set (r(2)(CV) = 0.872) and test set (r(2)(Pred) = 0.926) agents. Comparison of pharmacophoric maps showed that several key steric and electrostatic regions were common to both activity models, but differences were observed in the relative importance of these key regions with respect to the two aspects of anesthetic activity.

    Conclusions: The similarities in the pharmacophoric maps are consistent with NMDA receptors contributing part of the immobilizing activity of volatile aromatic anesthetics.

    Funded by: NIGMS NIH HHS: 1P01 GM47818, P01 GM047818, P01 GM058448, P01 GM58448, R01 GM061927

    Anesthesia and analgesia 2009;108;1;168-75

  • Developmental regulation of the NMDA receptor subunits, NR3A and NR1, in human prefrontal cortex.

    Henson MA, Roberts AC, Salimi K, Vadlamudi S, Hamer RM, Gilmore JH, Jarskog LF and Philpot BD

    Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27705, USA.

    Subunit composition of N-methyl-D-aspartate-type glutamate receptors (NMDARs) dictates their function, yet the ontogenic profiles of human NMDAR subunits from gestation to adulthood have not been determined. We examined NMDAR mRNA and protein development in human dorsolateral prefrontal cortex (DLPFC), an area in which NMDARs are critical for higher cognitive processing and NMDAR hypofunction is hypothesized in schizophrenia. Using quantitative reverse transcriptase-polymerase chain reaction and western blotting, we found NR1 expression begins low prenatally, peaks in adolescence, yet remains high throughout life, suggesting lifelong importance of NMDAR function. In contrast, NR3A levels are low during gestation, surge soon after birth, and decline progressively through adolescence and into adulthood. Because NR3A subunits uniquely attenuate NMDAR-mediated currents, limit calcium influx, and suppress dendritic spine formation, high levels during early childhood may be important for regulating neuroprotection and activity-dependent sculpting of synapses. We also examined whether subunit changes underlie reduced NMDAR activity in schizophrenia. Our results reveal normal NR1 and NR3A protein levels in DLPFC from schizophrenic patients, indicating that NMDAR hypofunction is unlikel 1f40 y to be maintained by gross changes in NR3A-containing NMDARs or overall NMDAR numbers. These data provide insights into NMDAR functions in the developing CNS and will contribute to designing pharmacotherapies for neurological disorders.

    Funded by: NEI NIH HHS: R01 EY018323, R01 EY018323-02; NIGMS NIH HHS: 2R25GM055336; NIMH NIH HHS: MH064065

    Cerebral cortex (New York, N.Y. : 1991) 2008;18;11;2560-73

  • MMP-7 cleaves the NR1 NMDA receptor subunit and modifies NMDA receptor function.

    Szklarczyk A, Ewaleifoh O, Beique JC, Wang Y, Knorr D, Haughey N, Malpica T, Mattson MP, Huganir R and Conant K

    Johns Hopkins University, Department of Neurology, Pathology Bldg. Rm. 625, 600 N. Wolfe St., Baltimore, MD 21287, USA.

    Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that play a role in the inflammatory response. These enzymes have been well studied in the context of cancer biology and inflammation. Recent studies, however, suggest that these enzymes also play roles in brain development and neurodegenerative disease. Select MMPs can target proteins critical to synaptic structure and neuronal survival, including integrins and cadherins. Here, we show that one member of the MMP family, MMP-7, which may be released from cells, including microglia, can target a protein critical to synaptic function. Through analysis of extracts from murine cortical slice preparations, we show that MMP-7 cleaves the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor to generate an N-terminal fragment of approximately 65 kDa. Moreover, studies with recombinant protein show that MMP-7-mediated cleavage of NR1 occurs at amino acid 517, which is extracellular and just distal to the first transmembrane domain. Data suggest that NR2A, which shares sequence homology with NR1, is also cleaved following treatment of slices with MMP-7, while select AMPA receptor subunits are not. Consistent with a potential effect of MMP-7 on ligand binding, additional experiments demonstrate that NMDA-mediated calcium flux is significantly diminished by MMP-7 pretreatment of cultures. In addition, the AMPA/NMDA ratio is increased by MMP-7 pretreatment. These data suggest that synaptic function may be altered in neurological conditions associated with increased levels of MMP-7.

    Funded by: NIA NIH HHS: R01 AG023471, R21 AG034849; NIDA NIH HHS: DA024447, R21 DA024447; NIMH NIH HHS: R01 MH077542, R21 MH072534; NINDS NIH HHS: NS052580, R21 NS052580

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;11;3757-67

  • 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

  • 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

  • Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors.

    Perroy J, Raynaud F, Homburger V, Rousset MC, Telley L, Bockaert J and Fagni L

    Institut de Génomique Fonctionnelle, CNRS UMR 5203, INSERM U661, Universités de Montpellier 1 and 2, Montpellier 34094, France.

    Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.

    The Journal of biological chemistry 2008;283;11;6799-805

  • Temperature dependence of NR1/NR2B NMDA receptor channels.

    Cais O, Sedlacek M, Horak M, Dittert I and Vyklicky L

    Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic.

    N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.

    Neuroscience 2008;151;2;428-38

  • 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

  • Possible association between genetic variants at the GRIN1 gene and schizophrenia with lifetime history of depressive symptoms in a German sample.

    Georgi A, Jamra RA, Klein K, Villela AW, Schumacher J, Becker T, Paul T, Schmael C, Höfels S, Klopp N, Illig T, Propping P, Cichon S, Nöthen MM, Schulze TG and Rietschel M

    Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, D-68159 Mannheim, Germany.

    Genetic variation in glutamatergic signalling pathways is believed to play a substantial role in the aetiology of schizophrenia. The N-methyl-D-aspartate receptor subunit gene GRIN1 has been proposed as a candidate gene for schizophrenia. We tested for a potential association between schizophrenia and four single nucleotide polymorphisms (rs4880213, rs11146020, rs6293, and rs10747050) and one microsatellite marker at GRIN1 in a German sample of 354 patients and 323 controls. We found significant associations in single-marker and haplotype-based analyses (P<0.05). Significance was more pronounced (P<0.01) in the subset of patients with a lifetime history of major depression, a subgroup of schizophrenia described previously as a promising phenotypic subtype in genetic studies of schizophrenia. Although significances did not withstand correction for multiple testing, the results of our exploratory analysis warrant further studies on GRIN1 and schizophrenia.

    Psychiatric genetics 2007;17;5;308-10

  • Requirement of PSD-95 for dopamine D1 receptor modulating glutamate NR1a/NR2B receptor function.

    WH, Yang S, Shi WX, Jin GZ and Zhen XC

    Department of Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.

    Aim: To elucidate the role of scaffold protein postsynaptic density (PSD)-95 in the dopamine D1 receptor (D1R)-modulated NR1a/NR2B receptor response.

    Methods: The human embryonic kidney 293 cells expressing D1R (tagged with the enhanced yellow fluorescent protein) and NR1a/NR2B with or without co-expression of PSD-95 were used in the experiments. The Ca2+ influx measured by imaging technique was employed to monitor N-methyl-D-aspartic acid receptors (NMDAR) function.

    Results: The application of dopamine (DA, 100 micromol/L) did not alter glutamate/glycine (Glu/Gly)-induced NMDAR-mediated Ca2+ influx in cells only expressing the D1R/NR1a/NR2B receptor. However, DA increased Glu/Gly-induced Ca2+ influx in a concentration-dependent manner while the cells were co-expressed with PSD-95. D1R-stimulated Ca2+ influx was inhibited by a selective D1R antagonist SCH23390. Moreover, pre-incubation with either the protein kinase A (PKA) inhibitor H89, or the protein kinase C (PKC) inhibitor chelerythrine attenuated D1R-enhanced Ca2+ influx induced by the N-methyl-D-aspartic acid (NMDA) agonist. The results clearly indicate that D1R-modulated NR1a/NR2B receptor function depends on PSD-95 and is subjected to the regulation of PKA and PKC.

    Conclusion: The present study provides the first evidence that PSD-95 is essential in D1R-regulated NR1a/NR2B receptor function.

    Acta pharmacologica Sinica 2007;28;6;756-62

  • 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

  • [An association study between GRIN1, BDNF genes and bipolar disorder].

    Liu M, Ling SH, Li WB, Wang CY, Chen DF and Wang G

    Beijing Anding Hospital Affiliated to the Capital University of Medical Sciences, Beijing, China. shuerya@163.com

    To evaluate the role of inherited gene variations in GRIN1 (glutamate receptor, ionotropic NMDA1), BDNF (brain derived neurotrophic factor) genes in human bipolar disorder, we selected 4 single nucleotide polymorphisms in GRIN1, BDNF (2 SNPs in each gene) and made SNPs analysis in 100 unrelated cases and 100 controls by TaqMan. Then we compared genotypes differences between cases and controls. The software SHEsis was also used to make haplotype analysis. The significant results were obtained, showing that the SNPs in GRIN1 gene were related to the BP (P < 0.05). In addition, the combined haplotype T/G had a significant difference in the two groups (P < 0.05). The SNPs in BDNF gene showed no statistical significance. These results confirm that the GRIN1 gene confers susceptibility to bipolar disorder.

    Yi chuan = Hereditas 2007;29;1;41-6

  • Subunit-specific roles of glycine-binding domains in activation of NR1/NR3 N-methyl-D-aspartate receptors.

    Awobuluyi M, Yang J, Ye Y, Chatterton JE, Godzik A, Lipton SA and Zhang D

    Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

    N-Methyl-D-aspartate receptors (NMDARs) composed of NR1 and NR3 subunits differ from other NMDAR subtypes in that they require glycine alone for activation. However, little else is known about the activation mechanism of these receptors. Using NMDAR glycine-site agonists/antagonists in conjunction with functional mutagenesis of the NR1 and NR3 ligand-binding cores, we demonstrate quite surprisingly that agonist binding to NR3 alone is sufficient to activate a significant component of NR1/NR3 receptor currents. Thus, the apo conformation of NR1 in NR1/NR3 receptors is permissive for receptor activation. Agonist-bound NR1 may also contribute to peak NR1/NR3 receptor currents but specifically enables significant NR1/NR3 receptor current decay under the conditions studied here, pre-sumably via a slow component of desensitization. Ligand studies of NR1/NR3 receptors also suggest differential agonist selectivity between NR3 and NR1, as some high-affinity NR1 agonists only minimally activate NR1/NR3 receptors, whereas other NR1 agonists are as potent as glycine. Furthermore, liganded NR3 subunits seem necessary for effective engagement of NR1 in NR1/NR3 receptor activation, suggesting significant interactivity between the two subunits. NR3 subunits thus induce plasticity in NR1 with respect to subunit assembly and ligand binding/channel coupling that is unique among ligand-gated ion channel subunits.

    Funded by: NEI NIH HHS: R01-EY05477, R01-EY09024; NICHD NIH HHS: P01-HD29587; NINDS NIH HHS: R01-NS43434

    Molecular pharmacology 2007;71;1;112-22

  • Altered neuregulin 1-erbB4 signaling contributes to NMDA receptor hypofunction in schizophrenia.

    Hahn CG, Wang HY, Cho DS, Talbot K, Gur RE, Berrettini WH, Bakshi K, Kamins J, Borgmann-Winter KE, Siegel SJ, Gallop RJ and Arnold SE

    Cellular and Molecular Neuropathology Program, Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. hahnc@mail.med.upenn.edu

    Recent molecular genetics studies implicate neuregulin 1 (NRG1) and its receptor erbB in the pathophysiology of schizophrenia. Among NRG1 receptors, erbB4 is of particular interest because of its crucial roles in neurodevelopment and in the modulation of N-methyl-D-aspartate (NMDA) receptor signaling. Here, using a new postmortem tissue-stimulation approach, we show a marked increase in NRG1-induced activation of erbB4 in the prefrontal cortex in schizophrenia. Levels of NRG1 and erbB4, however, did not differ between schizophrenia and control groups. To evaluate possible causes for this hyperactivation of erbB4 signaling, we examined the association of erbB4 with PSD-95 (postsynaptic density protein of 95 kDa), as this association has been shown to facilitate activation of erbB4. Schizophrenia subjects showed substantial increases in erbB4-PSD-95 interactions. We found that NRG1 stimulation suppresses NMDA receptor activation in the human prefrontal cortex, as previously reported in the rodent cortex. NRG1-induced suppression of NMDA receptor activation was more pronounced in schizophrenia subjects than in controls, consistent with enhanced NRG1-erbB4 signaling seen in this illness. Therefore, these findings suggest that enhanced NRG1 signaling may contribute to NMDA hypofunction in schizophrenia.

    Funded by: NIMH NIH HHS: MH63946, MH64045

    Nature medicine 2006;12;7;824-8

  • 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

  • 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

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

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

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

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

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

  • First in vivo evidence of an NMDA receptor deficit in medication-free schizophrenic patients.

    Pilowsky LS, Bressan RA, Stone JM, Erlandsson K, Mulligan RS, Krystal JH and Ell PJ

    Funded by: NIAAA NIH HHS: KO5 AA 014906-02

    Molecular psychiatry 2006;11;2;118-9

  • Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation.

    Miglio G, Varsaldi F and Lombardi G

    DiSCAFF Department, Eastern Piedmont University, Via Bovio 6, 28100 Novara, Italy.

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 microg/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

    Biochemical and biophysical research communications 2005;338;4;1875-83

  • Pharmacogenetics and obesity: common gene variants influence weight loss response of the norepinephrine/dopamine transporter inhibitor GW320659 in obese subjects.

    Spraggs CF, Pillai SG, Dow D, Douglas C, McCarthy L, Manasco PK, Stubbins M and Roses AD

    Genetics Research, GlaxoSmithKline Research and Development, Medicines Research Centre, Stevenage, UK. colin.f.spragg@gsk.com

    Background: GW320659, a highly selective neuronal norepinephrine and dopamine re-uptake inhibitor, has been evaluated for the treatment of obesity. Scrutiny of the weight loss data from a phase II study (GlaxoSmithKline study OBS20001) showed a wide variation in weight loss response following GW320659 treatment and the possibility that the study population might include subgroups with enhanced weight loss response.

    Methods: Pharmacogenetic analysis was performed in 191 subjects prospectively ascertained from a Phase II dose ranging study to evaluate the influence of genotype on weight loss efficacy and safety of GW320659 in obese subjects.

    Results: Common genetic polymorphisms in the drug target (norepinephrine transporter protein 1, SLC6A2) and mechanism pathway (NMDA receptor channel NR1 subunit, GRIN1) were associated with increased weight loss following GW320659 treatment in a proportion (36%) of the study population. In the patient subgroup selected for these genotypes, GW320659 (15 mg/day) produced a significant difference in mean weight loss of 7.84 kg (SD 5.23, n = 14), compared to 2.53 kg (SD 5.17, n = 24) in the subgroup that did not possess the genotypes (P = 0.006). This subgroup also showed a highly significant weight loss response for GW320659 compared to placebo (+0.31 kg, SD 3.32, n = 16) with the same genotypes (P < 0.0001). In addition, there was no difference in placebo response between either subgroup.

    Conclusions: Polymorphisms in SLC6A2 and GRIN1 could be used to maximize effective obesity pharmacotherapy by norepinephrine/dopamine transporter inhibitors by identifying patients that may be predisposed to particularly good treatment weight loss response.

    Pharmacogenetics and genomics 2005;15;12;883-9

  • Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity.

    Kwaśnicka-Crawford DA and Vincent SR

    Graduate Program in Neuroscience, Department of Psychiatry and the Brain Research Centre, The University of British Columbia, Vancouver, Canada BC V6T 1Z3.

    Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions.

    Biochemical and biophysical research communications 2005;336;2;565-71

  • 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

  • An association study of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B) in schizophrenia with universal DNA microarray.

    Qin S, Zhao X, Pan Y, Liu J, Feng G, Fu J, Bao J, Zhang Z and He L

    Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 230030, PR China.

    Dysfunction of the N-methyl-D-aspartate (NMDA) receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of several antagonists and on observation of genetic animal models. To conduct association analysis of the NMDA receptors in the Chinese population, we examined 16 reported SNPs across the NMDA receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B), five of which were identified in the Chinese population. In this study, we combined universal DNA microarray and ligase detection reaction (LDR) for the purposes of association analysis, an approach we considered to be highly specific as well as offering a potentially high throughput of SNP genotyping. The association study was performed using 253 Chinese patients with schizophrenia and 140 Chinese control subjects. No significant frequency differences were found in the analysis of the alleles but some were found in the haplotypes of the GRIN2B gene. The interactions between the GRIN1 and GRIN2B genes were evaluated using the multifactor-dimensionality reduction (MDR) method, which showed a significant genetic interaction between the G1001C in the GRIN1 gene and the T4197C and T5988C polymorphisms in the GRIN2B gene. These findings suggest that the combined effects of the polymorphisms in the GRIN1 and GRIN2B genes might be involved in the etiology of schizophrenia.European Journal of Human Genetics (2005) 13, 807-814. doi:10.1038/sj.ejhg.5201418 Published online 20 April 2005.

    European journal of human genetics : EJHG 2005;13;7;807-14

  • 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

  • The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines.

    Tolias KF, Bikoff JB, Burette A, Paradis S, Harrar D, Tavazoie S, Weinberg RJ and Greenberg ME

    Neurobiology Program, Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. We report that the Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with RNAi and dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines.

    Funded by: NICHD NIH HHS: P30-HD18655; NINDS NIH HHS: NS-04550, NS-07484, NS-39444, NS-44306, R01 NS045500

    Neuron 2005;45;4;525-38

  • Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling.

    Fernández-Monreal M, López-Atalaya JP, Benchenane K, Cacquevel M, Dulin F, Le Caer JP, Rossier J, Jarrige AC, Mackenzie ET, Colloc'h N, Ali C and Vivien D

    CNRS UMR 6185, University of Caen, Centre Cyceron, Bd. Henri Becquerel, BP 5229, 14074, Caen, France.

    Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-D-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59-64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the A 1f40 TD of NR1 and to modulate the NMDA receptor function.

    The Journal of biological chemistry 2004;279;49;50850-6

  • Systematic screening for mutations in the human N-methyl-D-aspartate receptor 1 gene in schizophrenic patients from the German population.

    Paus S, Rietschel M, Schulze TG, Ohlraun S, Diaconu CC, Van Den Bogaert A, Maier W, Propping P, Cichon S and Nöthen MM

    Department of Neurology, Institute of Human Genetics, University of Bonn, Bonn, Germany. spaus@uni-bonn.de

    Evidence for a dysfunction of the N-methyl-D-aspartate (NMDA) type of ionotropic glutamate receptors in schizophrenic patients, comes from neurochemical and clinical pharmacologic data. Therefore, the NMDAR1 gene can be regarded as an interesting candidate gene for schizophrenia. Several groups have tried to identify variants of this gene in schizophrenic patients in different, however not in German, populations. We sought to identify sequence changes of potential functional relevance in genomic DNA from 46 German unrelated schizophrenic patients by means of single-strand conformation analysis. No mutations of likely functional relevance were observed. We identified two synonymous coding Single Nucleotide Polymorphisms (cSNPs) in exons 6 and 7, and two SNPs in exon-flanking intronic sequences. Genotype distribution of these four SNPs was not significantly different between schizophrenic patients and controls. Our results suggest that the NMDAR1 subunit is not frequently involved in the development of schizophrenia in the German population.

    Psychiatric genetics 2004;14;4;233-4

  • The NR1 subunit of the glutamate/NMDA receptor in the superior temporal cortex in schizophrenia and affective disorders.

    Nudmamud-Thanoi S and Reynolds GP

    Division of Psychiatry and Neuroscience, Queen's University Belfast, White Medical Building, Belfast BT97BL, UK.

    The NMDA receptor has been implicated in the pathophysiology of several diseases including schizophrenia and affective disorders. We have investigated the NR1 subunit of the NMDA receptor in a well-defined series of psychiatric cases using radioligand binding and quantitative immunoblotting techniques. Saturable radioligand binding of [(3)H]L-689,560 to the glycine site on this subunit of the NMDA receptor was undertaken in superior temporal cortex of patients with schizophrenia, bipolar disorder, depression and matched control subjects. A tendency towards an increased receptor density was found in schizophrenia. A significant decrease in NMDA receptor density below control value was found in both bipolar and depressive disorders. The immunoblotting technique was used to identify NR1 protein in the same series of cases of which two bands were identified consistent with NR1 splice variants. A tendency to a decrease in the density of the NR1 upper band below control values was found in bipolar and depressed patients, but not schizophrenics. Consistent with this observation, the ratio between the upper and lower NR1-immunoreactive bands showed a significant decrease in bipolar disorder, although the ratio in depression did not reach significance. No significant difference was found in the NR1 lower band in any patient group compared with control. The finding of an increase NMDA receptor density in schizophrenia is consistent with the previous reports, with a possible compensatory response to glutamatergic deficits in superior temporal cortex in schizophrenia. The findings in affective disorders are interesting in respect of reports of cortical NMDA receptor deficits in suicide victims, although antidepressant drug treatment may contribute to these changes.

    Neuroscience letters 2004;372;1-2;173-7

  • Expression of epidermal N-methyl-D-aspartate receptors (NMDAR1) depends on formation of the granular layer--analysis in diseases with parakeratotic cornification.

    Fischer M, William T, Helmbold P, Wohlrab J and Marsch WCh

    Department of Dermatology and Venerology, Martin-Luther-University Halle-Wittenberg, Ernst-Kromayer-Str. 5, 06097 Halle (Saale), Germany. matthias.fischer@medizin.uni-halle.de

    Ionotrope glutamate receptors of the N-methyl-D-aspartate (NMDA) receptor type are expressed on keratinocytes and influence the intracellular calcium concentration. The importance of NMDA receptors in pathophysiological processes in the skin is, however, still unclear. Epidermal distribution patterns of NMDA receptors were investigated in dermatoses with parakeratotic cornification (psoriasis vulgaris and verrucae vulgares) and compared to the expression of filaggrin. The expression of NMDA receptors (R1 component) in paraffin-embedded normal epidermis (n = 22), psoriasis vulgaris (n = 21) and verrucae vulgares (n = 23) was examined and evaluated by means of digital image analysis. For quantitative characterization of the distribution patterns, a quotient was formed of the expression in the stratum granulosum and stratum basale ("NMDA ratio"). The distribution of NMDAR1 was compared to the immunohistochemical expression of filaggrin. Additionally the expression of filaggrin was investigated in HaCaT cells after treatment with the NMDA receptor antagonist MK-801. NMDA receptors were demonstrated in the epidermis of all preparations. In healthy skin, the highest receptor density was found in the stratum granulosum. This distribution pattern was basically also present in the dermatoses examined. Thus, the occurrence of parakeratosis in psoriasis vulgaris, but not in verrucae vulgares, was characterized by a significant reduction in the NMDA ratio (reduced expression of NMDAR1 in the upper epidermis). The immunohistochemical distribution of filaggrin was similar to that of NMDAR1. In HaCaT cells MK-801 suppressed the expression of filaggrin. NMDA receptors are expressed in human epidermis under physiological conditions especially in the stratum granulosum. Their reduced expression within parakeratotic epidermis in psoriasis vulgaris may be evidence of impaired intracellular calcium influx in this disease.

    Archives of dermatological research 2004;296;4;157-62

  • Characterization of the expression and function of N-methyl-D-aspartate receptor in keratinocytes.

    Morhenn VB, Murakami M, O'Grady T, Nordberg J and Gallo RL

    Division of Dermatology, University of California, San Diego, CA, USA. verajonath@aol.com

    The N-methyl-D-aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK-801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.

    Funded by: NIAMS NIH HHS: AR45676

    Experimental dermatology 2004;13;8;505-11

  • NF-kappaB site interacts with Sp factors and up-regulates the NR1 promoter during neuronal differentiation.

    Liu A, Hoffman PW, Lu W and Bai G

    Department of Biomedical Sciences, Dental School, Program in Neuroscience, and Program in Cellular and Molecular Biology, University of Maryland, Baltimore, Maryland 21201, USA.

    The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.

    Funded by: NINDS NIH HHS: NS38077

    The Journal of biological chemistry 2004;279;17;17449-58

  • Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2.

    Gingrich JR, Pelkey KA, Fam SR, Huang Y, Petralia RS, Wenthold RJ and Salter MW

    Brain and Behaviour Program, Hospital for Sick Children, Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5G 1X8.

    Src is the prototypic protein tyrosine kinase and is critical for controlling diverse cellular functions. Regions in Src define structural and functional domains conserved in many cell signaling proteins. Src also contains a region of low sequence conservation termed the unique domain, the function of which has until now remained enigmatic. Here, we show that the unique domain of Src is a protein-protein interaction region and we identify NADH dehydrogenase subunit 2 (ND2) as a Src unique domain-interacting protein. ND2 is a subunit of complex I in mitochondria, but we find that ND2 interacts with Src outside this organelle at excitatory synapses in the brain. ND2 acts as an adapter protein anchoring Src to the N-methyl-d-aspartate (NMDA) receptor complex, and is crucial for Src regulation of synaptic NMDA receptor activity. By showing an extramitochondrial action for a protein encoded in the mitochondrial genome, we identify a previously unsuspected means by which mitochondria regulate cellular function, suggesting a new paradigm that may be of general relevance for control of Src signaling.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;16;6237-42

  • 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

  • Increased numbers of coassembled PSD-95 to NMDA-receptor subunits NR2B and NR1 in human epileptic cortical dysplasia.

    Ying Z, Bingaman W and Najm IM

    Department of Neurology, The Cleveland Clinic Foundation, Cleveland, Ohio, USA. yingz@ccf.org

    Purpose: Glutamatergic transmission between neurons occurs at chemical synapses. The N-methyl-d-aspartate (NMDA)-receptor subclass of ionotropic glutamate receptors has been implicated in the epileptogenic mechanisms in human cortical dysplasia (CD). NMDA receptors are clustered at the postsynaptic membrane by anchoring to the postsynaptic density protein PSD-95, a putative ion channel-clustering protein. In this study, we quantitatively investigated the coassembly of PSD-95 to NR2B and NR1 in human epileptogenic cortex as compared with nonepileptic cortex.

    Methods: We used coimmunoprecipitation and immunoblotting techniques to quantify and compare the numbers of coassembled PSD-95 with NR2B, PSD-95 with NR1, and NR2B with NR1 in the membrane proteins of brain tissues resected from four patients (aged 3.5, 6, 14, and 18 years) with medically intractable neocortical epilepsy associated with CD. The resected cortical tissues were grouped into epileptic and nonepileptic, as determined by prolonged subdural electrode recordings in three patients and direct intraoperative electrocorticographic recording in one patient.

    Results: In all patients, the amounts of immunoprecipitated complexes, which reflect the numbers of coassembled PSD-95 proteins to NR2B subunits, were increased in epileptic cortex as compared with nonepileptic cortex.

    Conclusions: These results suggest that increased coassembly of NR2B and NR1 with PSD-95 may underlie one of the cellular mechanisms that contribute to the in situ increased hyperexcitability, leading to seizure generation in focal CD.

    Funded by: NINDS NIH HHS: K08 NS-02046, R21 NS42354

    Epilepsia 2004;45;4;314-21

  • Selective loss of NMDA receptor NR1 subunit isoforms in Alzheimer's disease.

    Hynd MR, Scott HL and Dodd PR

    Department of Biochemistry, University of Queensland, Australia.

    Previous work had shown that the ratio of NMDA receptor NR1 subunit mRNA transcripts containing an N-terminal splice cassette to those that do not is markedly lower in regions of the Alzheimer's disease (AD) brain that are susceptible to pathological damage, compared with spared regions in the same cases or homotropic regions in controls. To elucidate the origins of this difference in proportionate expression, we measured the absolute levels of each of the eight NR1 transcripts by quantitative internally standardized RT-PCR assay. Expression of transcripts with the cassette was strongly attenuated in susceptible regions of Alzheimer's brain, whereas expression of non-cassette transcripts differed little from that in controls. The expression of other NR1 splice variants was not associated with pathology relevant to disease status, although some combinations of splice cassettes were well maintained in AD cases. The population profile of NR1 transcripts in occipital cortex differed from the profiles in other brain regions studied. Western analysis confirmed that the expression of protein isoforms containing the N-terminal peptide was very low in susceptible areas of the Alzheimer's brain. Cells that express NR1 subunits with the N-terminal cassette may be selectively vulnerable to toxicity in AD.

    Journal of neurochemistry 2004;89;1;240-7

  • Decreased phosphorylation of NMDA receptor type 1 at serine 897 in brains of patients with Schizophrenia.

    Emamian ES, Karayiorgou M and Gogos JA

    The Rockefeller University, Laboratory of Human Neurogenetics, New York, New York 10021, USA.

    NMDA receptor hypofunction in schizophrenia has been inferred by a large number of clinical and preclinical observations; however, whether and how NMDA receptors are exactly involved in the pathogenesis of schizophrenia are still unknown and subject to interpretation. Here we show, in two independent samples of brains from patients with schizophrenia, a significant decrease in the phosphorylation level at serine 897 (S897) of the NMDA receptor type 1 (NR1) subunit. Our finding, together with a previous report that antipsychotics increase phosphorylation of NR1 at S897 in vivo, strongly suggests that insufficient phosphorylation at S897 may contribute to the neuronal pathology underlying schizophrenia.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;7;1561-4

  • 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

  • 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;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

  • Polymorphisms in the N-methyl-D-aspartate receptor 1 and 2B subunits are associated with alcoholism-related traits.

    Wernicke C, Samochowiec J, Schmidt LG, Winterer G, Smolka M, Kucharska-Mazur J, Horodnicki J, Gallinat J and Rommelspacher H

    Department of Clinical Neurobiology, University Hospital Benjamin Franklin, Free University of Berlin, Berlin, Germany.

    Background: This study examined the hypothesis that allelic variants of the ionotropic glutamatergic N-methyl-D-aspartate receptor (NMDAR) are associated with vulnerability to alcoholism and some related traits.

    Methods: We investigated the silent G2108A and C2664T polymorphisms of the NMDAR1 and the NMDAR2B genes, respectively. The case control study included 367 alcoholic and 335 control subjects of German origin. The family-based study comprised 81 Polish alcoholic patients and their parents using the transmission disequilibrium test.

    Results: The genotype frequencies of the NMDAR1 polymorphism differed significantly between control and alcoholic subjects. This difference was also observed in more homogenous subgroups of alcoholic subjects with vegetative withdrawal syndrome and Cloninger type 1. Patients with a history of delirium tremens or seizures during withdrawal showed a significantly increased prevalence of the A allele. Genotyping of the NMDAR2B polymorphism revealed a significantly reduced T allele in Cloninger type 2 alcoholics and in patients reporting an early onset compared with control subjects. Our family-based study for NMDAR2B, revealed a trend to a preferred transmission of the C allele by the fathers, and families with early-onset patients contributed most to this trend.

    Conclusions: These results suggest that variants in NMDAR genes are associated with alcoholism and related traits.

    Biological psychiatry 2003;54;9;922-8

  • 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

  • NMDA receptor 1 expression in the brainstem of human infants and its relevance to the sudden infant death syndrome (SIDS).

    Machaalani R and Waters KA

    Department of Medicine, The University of Sydney, Westmead, Sydney, Australia.

    The N-methyl-D-aspartate (NMDA) glutamatergic receptor is widely expressed in the brain during the early postnatal period and, among other functions is involved in cardiorespiratory control and in cell death by excitotoxic mechanisms. This study examined NMDA receptor-1 (NR1) expression in the human infant brainstem and assessed whether expression differed between non-SIDS and SIDS infants. NRI mRNA was identified using non-radioactive in situ hybridization and quantified by optical density. NRI protein was identified by immunohistochemistry and quantified by cellular counting. Eight nuclei of the mid-medulla and 2 nuclei of the rostral pons were studied. NRI mRNA and protein were expressed in all nuclei studied, confirming that the NMDA receptor is widely distributed in the human infant brainstem. Compared to non-SIDS infants (n = 10). SIDS infants (n = 15) had increased mRNA in 6 nuclei of the mid-medulla (p < 0.05 for all) while protein was increased in the dorsal motor nucleus of the vagus (p = 0.04) and decreased in the nucleus of the spinal trigeminal tract (p = 0.03). No differences were observed in the rostral pons. This preliminary study suggests that abnormalities of the glutamatergic system are present in SIDS victims. Further studies will be required to delineate these abnormalities and to investigate potential underlying mechanisms and sequelae.

    Journal of neuropathology and experimental neurology 2003;62;10;1076-85

  • 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

  • MCP-1 (CCL2) protects human neurons and astrocytes from NMDA or HIV-tat-induced apoptosis.

    Eugenin EA, D'Aversa TG, Lopez L, Calderon TM and Berman JW

    Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

    Acquired immunodeficiency syndrome (AIDS)-associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV-1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA-induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co-treatment with MCP-1 or RANTES but not IP-10. The protective effect of RANTES was blocked by antibodies to MCP-1, indicating that RANTES protection is mediated by the induction of MCP-1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co-treatment with MCP-1 inhibited tat and NMDA-induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP-1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.

    Funded by: NIMH NIH HHS: MH52974; NINDS NIH HHS: NS11920, T32NS07098

    Journal of neurochemistry 2003;85;5;1299-311

  • N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia: TDT and case-control analyses.

    Martucci L, Wong AH, Trakalo J, Cate-Carter T, Wong GW, Macciardi FM and Kennedy JL

    Neurogenetics Section, CAMH, Clarke Division, University of Toronto, Toronto, Ontario, Canada.

    The N-methyl-d-aspartate glutamate receptors (NMDAR) act in the CNS as regulators of the release of neurotransmitters such as dopamine, noradrenaline, acetylcholine, and GABA. It has been suggested that a weakened glutamatergic tone increases the risk of sensory overload and of exaggerated responses in the monoaminergic system, which is consistent with the symptomatology of schizophrenia. We studied two silent polymorphisms in GRIN1. GRIN1/1 is a G/C substitution localized on the 5' untranslated region; GRIN1/10 is an A/G substitution localized in exon 6 of GRIN1. Minor allele frequencies in our sample were calculated to be 0.05 and 0.2 respectively. We genotyped 86 nuclear families and 91 ethnically matched case-control pairs. Both samples were collected from the Toronto area. We tested the hypothesis that GRIN1 polymorphisms were associated with schizophrenia using the transmission disequilibrium test (TDT) and comparing allele frequencies between cases and controls. The results are as follows: GRIN1/1: chi(2) = 2.19, P = 0.14; GRIN1/10: chi(2) = 1.5, P = 0.22. For the case-control sample: GRIN1/1: chi(2) = 0.013, P = 0.908; GRIN1/10: chi(2) = 0.544, P = 0.461. No significant results were obtained. Haplotype analyses showed a borderline significant result for the 2,1 haplotype (chi(2) = 3.86, P-value = 0.049). An analysis of variance (ANOVA) to evaluate the association between genetic makeup and age at onset was performed, with no significant results: GRIN1/1, F[df = 2] = 0.42, P-value = 0.659; GRIN1/10, F[df = 2] = 0.16, P-value = 0.853. We are currently collecting additional samples to increase the power of the analyses.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2003;119B;1;24-7

  • Disruption of the neuronal PAS3 gene in a family affected with schizophrenia.

    Kamnasaran D, Muir WJ, Ferguson-Smith MA and Cox DW

    Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.

    Schizophrenia and its subtypes are part of a complex brain disorder with multiple postulated aetiologies. There is evidence that this common disease is genetically heterogeneous, with many loci involved. In this report, we describe a mother and daughter affected with schizophrenia, who are carriers of a t(9;14)(q34;q13) chromosome. By mapping on flow sorted aberrant chromosomes isolated from lymphoblast cell lines, both subjects were found to have a translocation breakpoint junction between the markers D14S730 and D14S70, a 683 kb interval on chromosome 14q13. This interval was found to contain the neuronal PAS3 gene (NPAS3), by annotating the genomic sequence for ESTs and performing RACE and cDNA library screenings. The NPAS3 gene was characterised with respect to the genomic structure, human expression profile, and protein cellular localisation to gain insight into gene function. The translocation breakpoint junction lies within the third intron of NPAS3, resulting in the disruption of the coding potential. The fact that the bHLH and PAS domains are disrupted from the remaining parts of the encoded protein suggests that the DNA binding and dimerisation functions of this protein are destroyed. The daughter (proband), who is more severely affected, has an additional microdeletion in the second intron of NPAS3. On chromosome 9q34, the translocation breakpoint junction was defined between D9S752 and D9S972 and no genes were found to be disrupted. We propose that haploinsufficiency of NPAS3 contributes to the cause of mental illness in this family.

    Journal of medical genetics 2003;40;5;325-32

  • Glycine binding primes NMDA receptor internalization.

    Nong Y, Huang YQ, Ju W, Kalia LV, Ahmadian G, Wang YT and Salter MW

    Programme in Brain and Behaviour, Hospital for Sick Children, , University of Toronto, Toronto, Ontario M5G 1X8, Canada.

    NMDA (N-methyl-d-aspartate) receptors (NMDARs) are a principal subtype of excitatory ligand-gated ion channel with prominent roles in physiological and disease processes in the central nervous system. Recognition that glycine potentiates NMDAR-mediated currents as well as being a requisite co-agonist of the NMDAR subtype of 'glutamate' receptor profoundly changed our understanding of chemical synaptic communication in the central nervous system. The binding of both glycine and glutamate is necessary to cause opening of the NMDAR conductance pore. Although binding of either agonist alone is insufficient to cause current flow through the channel, we report here that stimulation of the glycine site initiates signalling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis. Glycine binding alone does not cause the receptor to be endocytosed; this requires both glycine and glutamate site activation of NMDARs. The priming effect of glycine is mimicked by the NMDAR glycine site agonist d-serine, and is blocked by competitive glycine site antagonists. Synaptic as well as extrasynaptic NMDARs are primed for internalization by glycine site stimulation. Our results demonstrate transmembrane signal transduction through activating the glycine site of NMDARs, and elucidate a model for modulating cell-cell communication in the central nervous system.

    Nature 2003;422;6929;302-7

  • Evidence that the N-methyl-D-aspartate subunit 1 receptor gene (GRIN1) confers susceptibility to bipolar disorder.

    Mundo E, Tharmalingham S, Neves-Pereira M, Dalton EJ, Macciardi F, Parikh SV, Bolonna A, Kerwin RW, Arranz MJ, Makoff AJ and Kennedy JL

    Neurogenetics Section, Centre for Addiction and Mental Health (CAMH), Department of Psychiatry, University of Toronto, Clarke Site R-31, Toronto, Ontario, Canada M5T 1R8.

    There is evidence for the involvement of glutamatergic transmission in the pathogenesis of major psychoses. The two most commonly used mood stabilizers (ie lithium and valproate) have been found to act via the N-methyl-D-aspartate receptor (NMDAR), suggesting a specific role of NMDAR in the pathogenesis of bipolar disorder (BP). The key subunit of the NMDAR, named NMDA-1 receptor, is coded by a gene located on chromosome 9q34.3 (GRIN1). We tested for the presence of linkage disequilibrium between the GRIN1 (1001-G/C, 1970-A/G, and 6608-G/C polymorphisms) and BP. A total of 288 DSM-IV Bipolar I, Bipolar II, or schizoaffective disorder, manic type, probands with their living parents were studied. In all, 73 triads had heterozygous parents for the 1001-G/C polymorphism, 174 for the 1970-A/G, and 48 for the 6608-G/C. These triads were suitable for the final analyses, that is, the transmission disequilibrium test (TDT) and the haplotype-TDT. For the 1001-G/C and the 6608-G/C polymorphisms, we found a preferential transmission of the G allele to the affected individuals (chi(2)=4.765, df=1, P=0.030 and chi(2)= 8.395, df=1, P=0.004, respectively). The 1001G-1970A-6608A and the 1001G-1970A-6608G haplotypes showed the strongest association with BP (global chi(2)=14.12, df=4, P=0.007). If these results are replicated there could be important implications for the involvement of the GRIN1 in the pathogenesis of BP. The role of the gene variants in predicting the response to mood stabilizers in BP should also be investigated.

    Molecular psychiatry 2003;8;2;241-5

  • Systematic mutation analysis of the human glutamate receptor, ionotropic, N-methyl-D-aspartate 1 gene(GRIN1) in schizophrenic patients.

    Hung CC, Chen HY and Chen CH

    Institute of Human Genetics, Tzu-Chi University, Hualien City, Taiwan. cchen@mail.tcu.edu.tw

    Schizophrenia is a severe, complex mental disorder with unknown etiology. Abnormal glutamate neurotransmission has been proposed as one of the hypotheses of the pathogenesis of schizophrenia. Mohn recently reported that transgenic mice with the reduced glutamate receptor, ionotropic, -methyl-D-aspartate 1 gene (GRIN1) (formerly referred to as NMDAR1) expression display schizophrenia-like behaviors, which can be ameliorated by antipsychotic drug treatment. Their report promoted us to examine whether mutations in the human GRIN1 gene may convey genetic susceptibility to schizophrenia. To test this possibility, we systematically screened mutations in the promoter region and in all the exons of the human GRIN1 gene in a cohort of Chinese schizophrenic patients from Taiwan. Using single-strand conformation polymorphism analysis and autosequencing, we identified two single nucleotide polymorphisms, designated g.-1140G>A and g.-855G>C, respectively, at the 5'-untranslated region of the human GRIN1 gene. Genetic association study, however, revealed no association of these two single nucleotide polymorphisms with schizophrenia in our patients. Besides, no other mutations of the human GRIN1 gene were detected in this study. Our data suggest that the human GRIN1 gene may not contribute substantially to the genetic etiology of schizophrenia in our population.

    Psychiatric genetics 2002;12;4;225-30

  • 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

  • Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor.

    Lee FJ, Xue S, Pei L, Vukusic B, Chéry N, Wang Y, Wang YT, Niznik HB, Yu XM and Liu F

    Department of Neuroscience, Centre for Addiction and Mental Health, Clarke Division, University of Toronto, Toronto, M5T 1R8, Ontario, Canada

    Dopamine D1-like receptors, composed of D1 and D5 receptors, have been documented to modulate glutamate-mediated fast excitatory synaptic neurotransmission. Here, we report that dopamine D1 receptors modulate NMDA glutamate receptor-mediated functions through direct protein-protein interactions. Two regions in the D1 receptor carboxyl tail can directly and selectively couple to NMDA glutamate receptor subunits NR1-1a and NR2A. While one interaction is involved in the inhibition of NMDA receptor-gated currents, the other is implicated in the attenuation of NMDA receptor-mediated excitotoxicity through a PI-3 kinase-dependent pathway.

    2002;111;2;219-30

  • Alteration of branch site consensus sequence and enhanced pre-mRNA splicing of an NMDAR1 intron not associated with schizophrenia.

    Hammond L, Castanotto D, Rice SR, Nimgaonkar VL, Wirshing DA, Rossi JJ, Heston LL and Sobell JL

    Division of Molecular Medicine, City of Hope National Medical Center, Duarte, California 91010-0269, USA.

    Aberrant splicing of pre-mRNA is recognized to account for a significant minority of disease-causing mutations. The N-methyl-D-aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African-American male near a weak putative branch-site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in-frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer-extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African-American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease.

    American journal of medical genetics 2002;114;6;631-6

  • 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

  • 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

  • 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

  • Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity.

    Grunwald IC, Korte M, Wolfer D, Wilkinson GA, Unsicker K, Lipp HP, Bonhoeffer T and Klein R

    Department of Molecular Neurobiology, Max-Planck-Institute of Neurobiology, Am Klopferspitz 18a, D-82152, Martinsried, Germany.

    During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2(-/-) hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.

    Neuron 2001;32;6;1027-40

  • 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

  • An NMDA receptor signaling complex with protein phosphatase 2A.

    Chan SF and Sucher NJ

    Department of Biology and Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Special Administrative Region, China.

    Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked. The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits. We hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS. A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait. The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain. Immunoprecipitation studies indicated that the NR3A subunit formed a stable complex with PP2A in the rat brain in vivo. Association of PP2A with NMDARs led to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1. Stimulation of NMDARs led to the dissociation of PP2A from the complex and the reduction of PP2A activity. A peptide corresponding to the PP2A-NR3A binding domain functioned as a negative regulator of PP2A activity. These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state. The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism in addition to the traditional ionotropic functions of NMDARs.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;20;7985-92

  • Effect of gestational ethanol exposure on the NMDA receptor complex in rat forebrain: from gene transcription to cell surface.

    Hughes PD, Wilson WR and Leslie SW

    Division of Pharmacology and Toxicology, College of Pharmacy and the Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, 78712, USA. ph@uts.cc.utexas.edu

    Effects of gestational ethanol exposure on the trafficking of the NMDA receptor complex were investigated. Studies focused on three distinct processes in NMDA receptor translocation: (1) the level of gene transcription (2) nascent NMDA receptor subunits (NR) associated with the endoplasmic reticulum bound chaperone protein calnexin and (3) NMDA receptors associated with the cell surface anchoring protein PSD-95. Forebrain mRNA and membrane proteins were isolated from postnatal day 1 rat pups from prenatally ethanol exposed, pair-fed and ad libitum experimental groups. Ribonuclease protection assays were carried out to determine the levels of NR2A, NR2B, and NR2C mRNA within the treatment groups determined. Results indicated that gestational ethanol exposure did not affect the gene transcription of the NR2 subunits. Immunoprecipitation experiments were conducted with an anti-calnexin antibody or an anti-PSD-95 antibody and the immunoprecipitates probed for NR1 and NR2 subunits. Within the anti-calnexin immunoprecipitates, no NR2A, NR2B or NR2C subunits were detectable, but a significant pool of NR1 subunits was identified. These findings suggest that NR1 subunits but not NR2 subunits are associated with calnexin within the endoplasmic reticulum. Further, gestational ethanol exposure significantly increased the NR1 polypeptide levels in the anti-calnexin immunoprecipitate. Anti-PSD-95 immunoprecipitates revealed an abundance of NR1 and NR2B subunits, and these complexes were unaffected by gestational ethanol exposure. No NR2A or NR2C subunits were detected. These results suggest that gestational ethanol exposure significantly affects the assembly and transport of NMDA receptors. Gestational ethanol exposure may not alter the composition of the PSD-95 associated NMDA receptor complex.

    Funded by: NIAAA NIH HHS: 1 R01 AA11836, R37 AA05809

    Brain research. Developmental brain research 2001;129;2;135-45

  • 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. 1f40 , 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

  • Assembly with the NR1 subunit is required for surface expression of NR3A-containing NMDA receptors.

    Perez-Otano I, Schulteis CT, Contractor A, Lipton SA, Trimmer JS, Sucher NJ and Heinemann SF

    Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA. otano@salk.edu

    Functional NMDA receptors are heteromultimeric complexes of the NR1 subunit in combination with at least one of the four NR2 subunits (A-D). Coexpression of NR3A, an additional subunit of the NMDA receptor family, modifies NMDA-mediated responses. It is unclear whether NR3A interacts directly with NR1 and/or NR2 subunits and how such association might regulate the intracellular trafficking and membrane expression of NR3A. Here we show that NR3A coassembles with NR1-1a and NR2A to form a receptor complex with distinct single-channel properties and a reduced relative calcium permeability. NR3A associates independently with both NR1-1a and NR2A in the endoplasmic reticulum, but only heteromeric complexes containing the NR1-1a NMDA receptor subunit are targeted to the plasma membrane. Homomeric NR3A complexes or complexes composed of NR2A and NR3A were not detected on the cell surface and are retained in the endoplasmic reticulum. Coexpression of NR1-1a facilitates the surface expression of NR3A-containing receptors, reduces the accumulation of NR3A subunits in the endoplasmic reticulum, and induces the appearance of intracellular clusters where both subunits are colocalized. Our data demonstrate a role for subunit oligomerization and specifically assembly with the NR1 subunit in the trafficking and plasma membrane targeting of the receptor complex.

    Funded by: NEI NIH HHS: R01 EY05477; NICHD NIH HHS: P01 HD29587; NINDS NIH HHS: R01 NS28709, R01 NS34383

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;4;1228-37

  • Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms.

    Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, Bredt DS and Nicoll RA

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

    Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.

    Nature 2000;408;6815;936-43

  • EphB receptors interact with NMDA receptors and regulate excitatory synapse formation.

    Dalva MB, Takasu MA, Lin MZ, Shamah SM, Hu L, Gale NW and Greenberg ME

    Division of Neuroscience, Children's Hospital, and the Department of Neurobiology Harvard Medical School 02115, Boston, MA, USA.

    EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, as perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function.

    Funded by: NCI NIH HHS: CA43855; NICHD NIH HHS: HD18655; NINDS NIH HHS: R01 NS045500

    Cell 2000;103;6;945-56

  • A multivariate analysis of 59 candidate genes in personality traits: the temperament and character inventory.

    Comings DE, Gade-Andavolu R, Gonzalez N, Wu S, Muhleman D, Blake H, Mann MB, Dietz G, Saucier G and MacMurray JP

    Department of Medical Genetics, City of Hope Medical Center, Duarte, CA 91010, USA.

    Cloninger (Cloninger CR. Neurogenetic adaptive mechanisms in alcoholism. Science 1987: 236: 410-416) proposed three basic personality dimensions for temperament: novelty seeking, harm avoidance, and reward dependence. He suggested that novelty seeking primarily utilized dopamine pathways, harm avoidance utilized serotonin pathways, and reward dependence utilized norepinephrine pathways. Subsequently, one additional temperament dimension (persistence) and three character dimensions (cooperativeness, self-directedness, and self-transcendence) were added to form the temperament and character inventory (TCI). We have utilized a previously described multivariate analysis technique (Comings DE, Gade-Andavolu R, Gonzalez N et al. Comparison of the role of dopamine, serotonin, and noradrenergic genes in ADHD, ODD and conduct disorder. Multivariate regression analysis of 20 genes. Clin Genet 2000: 57: 178-196; Comings DD, Gade-Andavolu R, Gonzalez N et al. Multivariate analysis of associations of 42 genes in ADHD, ODD and conduct disorder. Clin Genet 2000: in press) to examine the relative role of 59 candidate genes in the seven TCI traits and test the hypothesis that specific personality traits were associated with specific genes. While there was some tendency for this to be true, a more important trend was the involvement of different ratios of functionally related groups of genes, and of different genotypes of the same genes, for different traits.

    Funded by: NIDA NIH HHS: R01-DA08417

    Clinical genetics 2000;58;5;375-85

  • 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

  • NMDA spikes in basal dendrites of cortical pyramidal neurons.

    Schiller J, Major G, Koester HJ and Schiller Y

    Department of Physiology and Biophysics, Technion Medical School, Bat-Galim, Haifa, Israel. Jackie@tx.technion.ac.il

    Basal dendrites are a major target for synaptic inputs innervating cortical pyramidal neurons. At present little is known about signal processing in these fine dendrites. Here we show that coactivation of clustered neighbouring basal inputs initiated local dendritic spikes, which resulted in a 5.9 +/- 1.5 mV (peak) and 64.4 +/- 19.8 ms (half-width) cable-filtered voltage change at the soma that amplified the somatic voltage response by 226 +/- 46%. These spikes were accompanied by large calcium transients restricted to the activated dendritic segment. In contrast to conventional sodium or calcium spikes, these spikes were mediated mostly by NMDA (N-methyl-D-aspartate) receptor channels, which contributed at least 80% of the total charge. The ionic mechanism of these NMDA spikes may allow 'dynamic spike-initiation zones', set by the spatial distribution of glutamate pre-bound to NMDA receptors, which in turn would depend on recent and ongoing activity in the cortical network. In addition, NMDA spikes may serve as a powerful mechanism for modification of the cortical network by inducing long-term strengthening of co-activated neighbouring inputs.

    Nature 2000;404;6775;285-9

  • 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

  • Evidence relating human verbal memory to hippocampal N-methyl-D-aspartate receptors.

    Grunwald T, Beck H, Lehnertz K, Blümcke I, Pezer N, Kurthen M, Fernández G, Van Roost D, Heinze HJ, Kutas M and Elger CE

    Department of Epileptology, Bonn University Medical Center, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. thomas@jersey.med.uni-bonn.de

    Studies in rodents and nonhuman primates have linked the activity of N-methyl-D-aspartate (NMDA) receptors within the hippocampus to animals' performance on memory-related tasks. However, whether these receptors are similarly essential for human memory is still an open question. Here we present evidence suggesting that hippocampal NMDA receptors, most likely within the CA1 region, do participate in human verbal memory processes. Words elicit a negative event-related potential (ERP) peaking around 400 ms within the anterior mesial temporal lobe (AMTL-N400). Ketamine, an NMDA-receptor antagonist, reduces the amplitude of the AMTL-N400 (in contrast to other hippocampal potentials) on initial presentation, eliminates the typical AMTL-N400 amplitude reduction with repetition, and leads to significant memory impairment. Of the various hippocampal subfields, only the density of CA1 neurons correlates with the word-related ERPs that are reduced by ketamine. Altogether, our behavioral, anatomical, and electrophysiological results indicate that hippocampal NMDA receptors are involved in human memory.

    Funded by: NIA NIH HHS: AG08313, R01 AG008313; NICHD NIH HHS: HD22614, R01 HD022614; NIMH NIH HHS: MH52883

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;21;12085-9

  • 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

  • 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

  • The glutamate receptor ion channels.

    Dingledine R, Borges K, Bowie D and Traynelis SF

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

    Pharmacological reviews 1999;51;1;7-61

  • Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.

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

    Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, USA.

    Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and alpha-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of alpha-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of alpha-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or alpha-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from alpha-actinin by two distinct but converging calcium-dependent processes: competitive displacement of alpha-actinin by calmodulin and reduction in the affinity of alpha-actinin for C0 after binding of calcium to alpha-actinin.

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

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;4;1165-78

  • 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

  • N-methyl-D-aspartate and dopamine receptor involvement in the modulation of locomotor activity and memory processes.

    Adriani W, Felici A, Sargolini F, Roullet P, Usiello A, Oliverio A and Mele A

    Dip. Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy.

    In this study we report on the effects of N-methyl-D-aspartate (NMDA)- and dopamine (DA)-receptor manipulation on the modulation of one-trial inhibitory avoidance response and the encoding of spatial information, as assessed with a non-associative task. Further, a comparison with the well-known effects of the manipulation of these two receptor systems on locomotor activity is outlined. It is well assessed that NMDA-receptor blockage induces a stimulatory action on locomotor activity similar to that exerted by DA agonists. There is evidence showing that the nucleus accumbens is involved in the response induced by both NMDA antagonists and DA agonists. We show results indicating a functional interaction between these two neural systems in modulating locomotor activity, with D2 DA-receptor antagonists (sulpiride and haloperidol) being more effective than the D1 antagonist (SCH 23390) in blocking MK-801-induced locomotion. A different profile is shown in the effects of NMDA antagonists and DA agonists in the modulation of memory processes. In one-trial inhibitory avoidance response, NMDA antagonists (MK-801 and CPP) impair the response on test day, while DA agonists exert a facilitatory effect; furthermore, sub-effective doses of both D1 (SKF 23390) and D2 (quinpirole) are able to attenuate the impairing effect in a way similar to that induced by NMDA antagonists. The effects of NMDA- and DA-acting drugs on the response to spatial novelty, as assessed with a task designed to study the ability of animals to react to discrete spatial changes, are in good accord with the effects observed on passive avoidance. The results show that NMDA as well as DA antagonists, at low doses, selectively impair the reactivity of mice to spatial changes. In a last series of experiments, the possible role of NMDA receptors located in the nucleus accumbens was investigated regarding reactivity to spatial novelty. The experiments gave apparently contrasting results: while showing an impairing effect of focal administrations of NMDA antagonists in the nucleus accumbens on reactivity to spatial novelty, no effect of ibotenic acid lesions of the same structure was observed.

    Experimental brain research 1998;123;1-2;52-9

  • Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors.

    Zhang S, Ehlers MD, Bernhardt JP, Su CT and Huganir RL

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

    Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. We report here that the mutation of a specific calmodulin binding site in the CO region of the NR1 subunit of the NMDA receptor blocks CDI. Moreover, intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. Furthermore, this inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2 known to interact with the CO region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton.

    Neuron 1998;21;2;443-53

  • 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

  • Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin.

    Wechsler A and Teichberg VI

    Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel.

    The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vit 1f40 ro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.

    The EMBO journal 1998;17;14

  • 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

  • Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A.

    Das S, Sasaki YF, Rothe T, Premkumar LS, Takasu M, Crandall JE, Dikkes P, Conner DA, Rayudu PV, Cheung W, Chen HS, Lipton SA and Nakanishi N

    Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The NMDA (N-methyl-D-aspartate) subclass of glutamate receptor is essential for the synaptic plasticity thought to underlie learning and memory and for synaptic refinement during development. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or chi-1), which is expressed primarily during brain development. NR3A co-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.

    Nature 1998;393;6683;377-81

  • Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.

    Allison DW, Gelfand VI, Spector I and Craig AM

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

    We used actin-perturbing agents and detergent extraction of primary hippocampal cultures to test directly the role of the actin cytoskeleton in localizing GABAA receptors, AMPA- and NMDA-type glutamate receptors, and potential anchoring proteins at postsynaptic sites. Excitatory postsynaptic sites on dendritic spines contained a high concentration of F-actin that was resistant to cytochalasin D but could be depolymerized using the novel compound latrunculin A. Depolymerization of F-actin led to a 40% decrease in both the number of synaptic NMDA receptor (NMDAR1) clusters and the number of AMPA receptor (GluR1)-labeled spines. The nonsynaptic NMDA receptors appeared to remain clustered and to coalesce in cell bodies. alpha-Actinin-2, which binds both actin and NMDA receptors, dissociated from the receptor clusters, but PSD-95 remained associated with both the synaptic and nonsynaptic receptor clusters, consistent with a proposed cross-linking function. AMPA receptors behaved differently; on GABAergic neurons, the clusters redistributed to nonsynaptic sites, whereas on pyramidal neurons, many of the clusters appeared to disperse. Furthermore, in control neurons, AMPA receptors were detergent extractable from pyramidal cell spines, whereas AMPA receptors on GABAergic neurons and NMDA receptors were unextractable. GABAA receptors were not dependent on F-actin for the maintenance or synaptic localization of clusters. These results indicate fundamental differences in the mechanisms of receptor anchoring at postsynaptic sites, both regarding the anchoring of a single receptor (the AMPA receptor) in pyramidal cells versus GABAergic interneurons and regarding the anchoring of different receptors (AMPA vs NMDA receptors) at a single class of postsynaptic sites on pyramidal cell dendritic spines.

    Funded by: NIGMS NIH HHS: GM52111-01; NINDS NIH HHS: NS33184

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;7;2423-36

  • Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NR1.

    Lin JW, Wyszynski M, Madhavan R, Sealock R, Kim JU and Sheng M

    Howard Hughes Medical Institute and Department of Neurobiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.

    The molecular machinery underlying neurotransmitter receptor immobilization at postsynaptic sites is poorly understood. The NMDA receptor subunit NR1 can form clusters in heterologous cells via a mechanism dependent on the alternatively spliced C1 exon cassette in its intracellular C-terminal tail, suggesting a functional interaction between NR1 and the cytoskeleton. The yeast two-hybrid screen was used here to identify yotiao, a novel coiled coil protein that interacts with NR1 in a C1 exon-dependent manner. Yotiao mRNA (11 kb) is present modestly in brain and abundantly in skeletal muscle and pancreas. On Western blots, yotiao appears as an approximately 230 kDa band that is present in cerebral cortex, hippocampus, and cerebellum. Biochemical studies reveal that yotiao fractionates with cytoskeleton-associated proteins and with the postsynaptic density. With regard to immunohistochemistry, two anti-yotiao antibodies display a somatodendritic staining pattern similar to each other and to the staining pattern of NR1. Yotiao was colocalized by double-label immunocytochemistry with NR1 in rat brain and could be coimmunoprecipitated with NR1 from heterologous cells. Thus yotiao is an NR1-binding protein potentially involved in cytoskeletal attachment of NMDA receptors. Consistent with a general involvement in postsynaptic structure, yotiao was also found to be specifically concentrated at the neuromuscular junction in skeletal muscle.

    Funded by: NINDS NIH HHS: NS33145, NS35050

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;6;2017-27

  • Splice variant-specific interaction of the NMDA receptor subunit NR1 with neuronal intermediate filaments.

    Ehlers MD, Fung ET, O'Brien RJ and Huganir RL

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

    NMDA receptors are excitatory neurotransmitter receptors critical for synaptic plasticity and neuronal development in the mammalian brain. These receptors are found highly concentrated in the postsynaptic membrane of glutamatergic synapses. To investigate the molecular mechanisms underlying NMDA receptor localization, we used the yeast two-hybrid system to identify proteins expressed in the brain that interact with the NMDA receptor subunit NR1. Here we report that the 68 kDa neurofilament subunit NF-L directly interacts with the NR1 subunit. This interaction occurs between the cytoplasmic C-terminal domain of NR1 and the rod domain of NF-L. However, NR1 splice variants lacking the first C-terminal exon cassette (C1) failed to associate with NF-L. Immunogold electron microscopy revealed a preferential localization of NR1 at the ends of in vitro-assembled neurofilaments. Overexpression of C1 cassette-containing NR1 constructs in fibroblast cells disrupted the assembly of recombinant neurofilaments. In addition, NR1 and NF-L cofractionated in detergent-treated rat brain synaptic plasma membranes. Furthermore, NR1 and NF-L colocalize in the dendrites and growth cones of cultured hippocampal neurons. These results demonstrate the splice variant-specific association of NR1 with neurofilaments and suggest a possible mechanism for anchoring or localizing NMDA receptors in the neuronal plasma membrane.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;2;720-30

  • 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

  • Cloning and localization of exon 5-containing isoforms of the NMDAR1 subunit in human and rat brains.

    Nash NR, Heilman CJ, Rees HD and Levey AI

    Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322, U.S.A.

    Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N-terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1-3b (hNMDA1-1) and NMDAR1-4b (hNMDA1-2), both bearing the insert, is reported here. A monoclonal antibody generated against the N-terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of approximately 115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21-amino acid N-terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5-containing isoforms of the NMDAR1 subunit in both rat and human brain.

    Journal of neurochemistry 1997;69;2;485-93

  • Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies.

    Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT and Huganir RL

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

    Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.

    The Journal of biological chemistry 1997;272;8;5157-66

  • Competitive binding of alpha-actinin and calmodulin to the NMDA receptor.

    Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM and Sheng M

    Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.

    The mechanisms by which neurotransmitter receptors are immobilized at postsynaptic sites in neurons are largely unknown. The activity of NMDA (N-methyl-D-aspartate) receptors is mechanosensitive and dependent on the integrity of actin, suggesting a functionally important interaction between NMDA receptors and the postsynaptic cytoskeleton. alpha-Actinin-2, a member of the spectrin/dystrophin family of actin-binding proteins, is identified here as a brain postsynaptic density protein that colocalizes in dendritic spines with NMDA receptors and the putative NMDA receptor-clustering molecule PSD-95. alpha-Actinin-2 binds by its central rod domain to the cytoplasmic tail of both NR1 and NR2B subunits of the NMDA receptor, and can be immunoprecipitated with NMDA receptors and PSD-95 from rat brain. Intriguingly, NR1-alpha-actinin binding is directly antagonized by Ca2+/calmodulin. Thus alpha-actinin may play a role in both the localization of NMDA receptors and their modulation by Ca2+.

    Nature 1997;385;6615;439-42

  • 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

  • The messenger RNAs for the N-methyl-D-aspartate receptor subunits show region-specific expression of different subunit composition in the human brain.

    M, Le Bourdellès B, Heavens RP, Kelly S, Smith D, Butler A, Hammans R, Hills R, Xuereb JH, Hill RG, Whiting PJ and Sirinathsinghji DJ

    Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, U.K.

    The expression of the messenger RNAs encoding N-methyl-D-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl-D-aspartate R1-1 messenger RNA but much weaker signals for N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4, N-Methyl-D-aspartate R1-2 was not detectable. N-methyl-D-aspartate R2B was the only N-methyl-D-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 were strongly expressed in the dentate gyrus, CA3-CA1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl-D-aspartate R1-2 and N-methyl-D-aspartate R1-3. The messenger RNAs for both N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B, but not N-methyl-D-aspartate R2C, subunits were expressed in the hippocampus. In the temporal cortex all N-methyl-D-aspartate RI isoforms were expressed (N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 being the most abundant) and N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B but not N-methyl-D-aspartate R2C were also moderately expressed. In the brain stem N-methyl-D-aspartate R1-4 was strongly expressed in various nuclei including the locus coeruleus, nucleus centralis superior and deep pontine nuclei. Only weak expression was seen for N-methyl-D-aspartate RI-1 and N-methyl-D-aspartate R1-3 but not N-methyl-D-aspartate RI-2; of the N-methyl-D-aspartate R2 subunits only N-methyl-D-aspartate R2C was found to be expressed in these nuclei. In the cerebellum all the N-methyl-D-aspartate I isoforms were expressed (mostly N-methyl-D-aspartate R1-4) in the Purkinje layer which also expressed N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2C. In the molecular layer cells were found expressing N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2B and cells in the granule layer were found to express N-methyl-D-aspartate R1-1, N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2C only. Preliminary studies indicated that the messenger RNA for the N-methyl-D-aspartate R2D subunit was not expressed in the above areas of brain. These results give the first demonstration of the distribution of N-methyl-D-aspartate receptor subunit messenger RNAs in the human brain. The region-specific expression of subunit combinations suggests a heterogeneity of N-methyl-D-aspartate receptors with diverse physiological/pathophysiological roles and provides a rationale for the development of discriminatory N-methyl-D-aspartate receptor antagonists to target selective neuronal populations.

    Neuroscience 1996;73;2;429-47

  • 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

  • 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

  • Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit.

    Ehlers MD, Zhang S, Bernhadt JP and Huganir RL

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

    NMDA (N-methyl-D-aspartate) receptors are excitatory neurotransmitter receptors in the brain critical for synaptic plasticity and neuronal development. These receptors are Ca2+-permeable glutamate-gated ion channels whose physiological properties are regulated by intracellular Ca2+. We report here the purification of a 20 kDa protein identified as calmodulin that interacts with the NR1 subunit of the NMDA receptor. Calmodulin binding to the NR1 subunit is Ca2+ dependent and occurs with homomeric NR1 complexes, heteromeric NR1/NR2 subunit complexes, and NMDA receptors from brain. Furthermore, calmodulin binding to NR1 causes a 4-fold reduction in NMDA channel open probability. These results demonstrate that NMDA receptor function can be regulated by direct binding of calmodulin to the NR1 subunit, and suggest a possible mechanism for activity-dependent feedback inhibition and Ca2+-dependent inactivation of NMDA receptors.

    Cell 1996;84;5;745-55

  • 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

  • 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

  • Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1).

    Zimmer M, Fink TM, Franke Y, Lichter P and Spiess J

    Max-Planck-Institut für experimentelle Medizin, Abteilung Molekulare Neuroendokrinologie, Göttingen, Germany.

    The complete gene encoding the human N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) has been isolated on a single cosmid clone. The gene is composed of 21 exons distributed over a total length of about 31 kb. More than 24 kb were sequenced. Exons 4, 20 and 21 are identical in their amino-acid sequence to those exons that are subject to alternative splicing in rat, indicating that all eight NMDAR1 isoforms found in rat will also be expressed in the human brain. Computer analysis of the pre-mRNA sequence revealed no secondary structures stable enough to explain alternative splicing. We suggest that cell-specific factors control expression of different isoforms. The promoter region contains two perfect copies of the recognition sequence for the Drosophila even-skipped protein, indicating that the developmentally regulated expression of NMDAR1 is controlled by a homeobox protein. The complete cosmid clone covering NMDAR1 was mapped to chromosome 9q34.3-qter by fluorescent in situ hybridization (FISH). The telomeric location is supported by an imperfect (CA)n repeat homologous to a subtelomeric repeat on chromosome 16p.

    Gene 1995;159;2;219-23

  • 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

  • Cloning and sequence analysis of additional splice variants encoding human N-methyl-D-aspartate receptor (hNR1) subunits.

    Foldes RL, Rampersad V and Kamboj RK

    Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.

    Two cDNA clones representing previously unidentified human N-methyl-D-aspartate receptor (hNR1) subunit polypeptides were isolated and sequenced. Clone hNR1-4 was isolated from a human hippocampus cDNA library and was presumably generated by alternative RNA splicing in the 3' amino acid (aa) coding regions. The hNR1-4 cDNA demonstrated an 85.7% nucleotide (nt) identity to the corresponding rat NR1 (rNR1) cDNA. The nt sequence of hNR1-4 would encode a protein that has a 99.8% identity with the corresponding rNR1 subunit. Clone hNR1N was isolated by polymerase chain reaction (PCR)-mediated amplification of a 0.6-kb DNA fragment from human cerebellum cDNA. The nt sequence of this DNA fragment was identical to previously isolated hNR1 cDNA clones, except for the presence of a 63-bp DNA insertion that would encode an additional 21 aa. This DNA insertion occurs in the 5' aa coding regions of hNR1 and presumably represents an exon that is subject to alternative splicing. The nt and aa sequences of this exon are identical between human and rat.

    Gene 1994;147;2;303-4

  • 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

  • Cloning and sequence analysis of cDNAs encoding human hippocampus N-methyl-D-aspartate receptor subunits: evidence for alternative RNA splicing.

    Foldes RL, Rampersad V and Kamboj RK

    Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.

    Several cDNA clones encoding human N-methyl-D-aspartate receptor (hNR1) subunit polypeptides were isolated from a human hippocampus library. Degenerate oligodeoxyribonucleotide (oligo) primers based on the published rat NR1 (rNR1) amino acid (aa) sequence [K. Moriyoshi et al. Nature 354 (1991) 31-37] amplified a 0.7-kb fragment from a human hippocampus cDNA library, via the polymerase chain reaction (PCR). This fragment was used as a probe for subsequent hybridization screening. DNA sequence analysis of 28 plaque-purified clones indicated three distinct classes, designated hNR1-1, hNR1-2 and hNR1-3, presumably generated by alternative RNA splicing. One of these clones, hNR1-1(5A), was isolated as a full-length cDNA. The hNR1-2 and hNR1-3 cDNAs represented 66.8 and 98.9%, respectively, of the total aa coding information predicted for the polypeptides. The hNR1 cDNAs demonstrated an 84-90.8% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequences of hNR1-1, hNR1-2 and hNR1-3 would encode 885-, 901- and 938-aa proteins, respectively, that have 99.1-99.8% identity with the corresponding rodent NR1 (roNR1) subunits. The changes between the predicted aa sequences of hNR1 and the corresponding roNR1 subunits are confined to the extracellular N-terminal regions. We have also identified two possible allelic variations of the hNR1-3 cDNA that result in aa substitutions in the extracellular N- and C-terminal regions. One of these naturally occurring aa variations is situated within a potential glutamate-binding site.

    Gene 1993;131;2;293-8

  • Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain.

    Tingley WG, Roche KW, Thompson AK and Huganir RL

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

    The NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A-2D). A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function and play a role in many forms of synaptic plasticity such as long-term potentiation. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.

    Nature 1993;364;6432;70-3

  • Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively.

    Collins C, Duff C, Duncan AM, Planells-Cases R, Sun W, Norremolle A, Michaelis E, Montal M, Worton R and Hayden MR

    Department of Medical Genetics, University of British Columbia, Canada.

    A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.3 using in situ hybridization studies and NMDARA1 to human chromosome 8 using a somatic cell hybrid panel. Because the gene causing HD has been localized to chromosome 4p16.3, the chromosome assignments reported here are inconsistent with either of these genes playing a causative role in the molecular pathology of HD. However, it is noteworthy that the gene for torsion dystonia has also been localized by genetic studies to 9q34.3, the same regional map location as NMDAR1.

    Funded by: NIMH NIH HHS: MH00778

    Genomics 1993;17;1;237-9

  • Molecular cloning, functional expression, and pharmacological characterization of an N-methyl-D-aspartate receptor subunit from human brain.

    Planells-Cases R, Sun W, Ferrer-Montiel AV and Montal M

    Department of Biology, University of California, San Diego, La Jolla 92093-0319.

    A cDNA encoding a full-length N-methyl-D-aspartate (NMDA) receptor subunit 1, hNR1, was isolated from a human brain cDNA library. The hNR1 cDNA encodes an open reading frame of approximately 2.7 kb that shares high homology with the rat brain NMDA receptor subunit 1 and the mouse zeta 1 subunit. The hNR1 sequence, however, diverges from the rodent and murine homologs near the C terminus, suggesting that they represent alternatively spliced messages of the same gene. Oocytes injected with cRNA synthesized from the hNR1 cDNA express glutamate and NMDA-activated currents in the presence of glycine. Currents are blocked by the NMDA-receptor-specific antagonists 2-amino-5-phosphovaleric acid and 7-chlorokynurenate, and the open channel blockers MK-801 and phencyclidine, by Mg2+ ions in a voltage-dependent manner, and by Zn2+. Expressed hNR1 homomeric receptor channels exhibit the high Ca2+ permeability characteristic of neuronal NMDA receptors. Therefore, the cDNA clone hNR1 codes for a human brain NMDA receptor subunit cognate to the rodent and murine brain NR1 subunits.

    Funded by: NIGMS NIH HHS: 5T32GM08326; NIMH NIH HHS: MH-00778, MH-44638

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;11;5057-61

  • Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

    Younkin DP, Tang CM, Hardy M, Reddy UR, Shi QY, Pleasure SJ, Lee VM and Pleasure D

    Neurology Research, Children's Hospital of Philadelphia, PA.

    Glutamate receptor (GluR) channels are responsible for a number of fundamental properties of the mammalian central nervous system, including nearly all excitatory synaptic transmission, synaptic plasticity, and excitotoxin-mediated neuronal death. Although many human and rodent neuroblast cell lines are available, none has been directly shown to express GluR channels. We report here that cells from the human teratocarcinoma line NT2 are induced by retinoic acid to express neuronal N-methyl-D-aspartate (NMDA) and non-NMDA GluR channels concomitant with their terminal differentiation into neuron-like cells. The molecular and physiologic characteristics of these human GluR channels are nearly identical to those in central nervous system neurons, as demonstrated by PCR and patch clamp recordings, and the cells demonstrate glutamate-induced neurotoxicity.

    Funded by: NINDS NIH HHS: NS08075, NS25044, NS28158

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;6;2174-8

  • Molecular cloning and chromosomal localization of the key subunit of the human N-methyl-D-aspartate receptor.

    Karp SJ, Masu M, Eki T, Ozawa K and Nakanishi S

    Institute for Immunology, Kyoto University Faculty of Medicine, Japan.

    A complementary DNA encoding the key subunit of the human N-methyl-D-aspartate (NMDA) receptor (NMDAR1) has been cloned using a probe derived from the rat NMDAR1 cDNA. The cDNA encodes a 938-amino acid protein, which 1e33 shows 99% amino acid homology with the rat counterpart. Of the 7 of 938 amino acids which are different, three occur in the region of the signal peptide and the others in the extracellular amino-terminal domain preceding the 4 putative transmembrane segments. Expression in Xenopus oocytes demonstrated that the single protein encoded by the cloned cDNA possesses the electrophysiological and pharmacological properties characteristic of the NMDA receptor, including Ca2+ permeability, voltage-dependent Mg2+ block, and inhibition by selective antagonists such as Zn2+ and channel blockers. The high evolutionary conservation in the structure and properties of NMDAR1 argues strongly for the importance of this receptor in functions of glutamate neurotransmission. RNA blot analysis showed abundant expression of mRNA whose size is about 4.5 and 4.8 kilonucleotides. The human gene encoding the NMDAR1 subunit has been mapped to chromosome 9q34.3 by the analyses of blot hybridization of a DNA panel of human/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes.

    The Journal of biological chemistry 1993;268;5;3728-33

  • 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 (9)

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
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
L00000032 G2C Homo sapiens Pocklington H1 Human orthologues of cluster 1 (mouse) from Pocklington et al (2006) 21
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
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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