G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
glutamate receptor, ionotropic, N-methyl D-aspartate 2D
G00000066 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000070899 (Vega human gene)
ENSG00000105464 (Ensembl human gene)
2906 (Entrez Gene)
294 (G2Cdb plasticity & disease)
GRIN2D (GeneCards)
602717 (OMIM)
Marker Symbol
HGNC:4588 (HGNC)
Protein Sequence
O15399 (UniProt)

Synonyms (2)

  • EB11
  • NR2D

Literature (49)

Pubmed - other

  • 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 analysis of the glutamic acid decarboxylase 2 and the glutamine synthetase genes (GAD2, GLUL) with schizophrenia.

    Arai S, Shibata H, Sakai M, Ninomiya H, Iwata N, Ozaki N and Fukumaki Y

    Division of Human Molecular Genetics, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

    Objective: As dysfunction of glutamatergic neurotransmission is one of the plausible hypotheses for the pathogenesis of schizophrenia, genes involved in the glutamate neurotransmitter system are candidates for schizophrenia susceptibility. The aim of this study is to clarify the contribution of two genes encoding glutamate metabolic enzymes: the glutamic acid decarboxylase 2 gene (GAD2) and the glutamine synthetase gene (GLUL), in schizophrenia.

    Methods: We genotyped 300 Japanese schizophrenia patients and 300 healthy controls for 14 single nucleotide polymorphisms (SNPs) in GAD2 (approximately 91 kb in size) and six SNPs in GLUL (approximately 14 kb in size). We examined 'single-point' association as well as pairwise haplotype association for all SNPs with schizophrenia.

    Results: We observed no significant 'single-point' associations with the disease in any of the 20 SNPs after correction for multiple testing using False Discovery Rate. We also observed no significant haplotype associations with False Discovery Rate. Furthermore, we analyzed gene-gene interactions, including six glutamate receptor genes we have reported previously in the association studies of GRIA4, GRIN2D, GRIK3, GRIK4, GRIK5, and GRM3, using the multifactor dimensionality reduction method. The best interaction model, however, did not show the statistical significance.

    Conclusion: These results suggest that GAD2 and GLUL do not play a major role in schizophrenia pathogenesis and there is no gene-gene interaction between the eight genes in the Japanese population.

    Psychiatric genetics 2009;19;1;6-13

  • Neurotransmission and bipolar disorder: a systematic family-based association study.

    Shi J, Badner JA, Hattori E, Potash JB, Willour VL, McMahon FJ, Gershon ES and Liu C

    Department of Psychiatry, University of Chicago, Chicago, Illinois 60637, USA. kjacobso@bsd.uchicago.edu

    Neurotransmission pathways/systems have been proposed to be involved in the pathophysiology and treatment of bipolar disorder for over 40 years. In order to test the hypothesis that common variants of genes in one or more of five neurotransmission systems confer risk for bipolar disorder, we analyzed 1,005 tag single nucleotide polymorphisms in 90 genes from dopaminergic, serotonergic, noradrenergic, GABAergic, and glutamatergic neurotransmitter systems in 101 trios and 203 quads from Caucasian bipolar families. Our sample has 80% power to detect ORs >or= 1.82 and >or=1.57 for minor allele frequencies of 0.1 and 0.5, respectively. Nominally significant allelic and haplotypic associations were found for genes from each neurotransmission system, with several reaching gene-wide significance (allelic: GRIA1, GRIN2D, and QDPR; haplotypic: GRIN2C, QDPR, and SLC6A3). However, none of these associations survived correction for multiple testing in an individual system, or in all systems considered together. Significant single nucleotide polymorphism associations were not found with sub-phenotypes (alcoholism, psychosis, substance abuse, and suicide attempts) or significant gene-gene interactions. These results suggest that, within the detectable odds ratios of this study, common variants of the selected genes in the five neurotransmission systems do not play major roles in influencing the risk for bipolar disorder or comorbid sub-phenotypes.

    Funded by: NIAAA NIH HHS: AA00231, U10AA0503; NIDA NIH HHS: 5R01DA018662; NIMH NIH HHS: R01 MH061613, R01 MH061613-08, R01 MH065560, R01 MH065560-05, R01 MH080425, R01 MH080425-01

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;7;1270-7

  • Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening.

    Wu C, Ma MH, Brown KR, Geisler M, Li L, Tzeng E, Jia CY, Jurisica I and Li SS

    Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

    Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.

    Proteomics 2007;7;11;1775-85

  • 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

  • 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

  • Cultivated keratinocytes express N-methyl-D-aspartate receptors of the NMDAR2D type.

    Fischer M, Fiedler E, Seidel C, Meiss F, Klapperstück T, Helmbold P and Dammann R

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

    N-methyl-D-aspartate receptors (NMDAR) can regulate the intracellular calcium concentration of keratinocytes (KC) and seem to be important for their growth and differentiation. The objective of this study was to identify the subtype(s) of this receptor expressed by KC in vitro. The mRNA was isolated from primary cultures of KC as well as from a KC cell line (HaCaT) and expression of the NMDAR subtypes determined by using RT-PCR. At the mRNA level, we found expression of only the constant NMDAR1 as well as the subtype NMDAR2D. In contrast to the other subtypes of NMDAR, NMDAR2D is characterized by low influence of magnesium to the receptor function. This characteristic is consistent with previously published functional investigations in KC. The identification of the NMDAR2D subtype in KC may be of value for the development of new therapeutic approaches.

    Archives of dermatological research 2006;297;7;316-8

  • Identification of single-nucleotide polymorphisms in the human N-methyl-D-aspartate receptor subunit NR2D gene, GRIN2D, and association study with schizophrenia.

    Makino C, Shibata H, Ninomiya H, Tashiro N and Fukumaki Y

    Division of Disease Genes, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

    Objectives: The glutamatergic dysfunction is one of the main hypotheses for the pathophysiology of schizophrenia. N-methyl-D-aspartate receptors are of major interest because phencyclidine, a non-competitive antagonist of N-methyl-D-aspartate receptors, produces a schizophrenia-like psychosis. Therefore, the genes encoding N-methyl-D-aspartate receptor subunits are strong candidates for schizophrenia susceptibility genes. We focused on the N-methyl-D-aspartate receptor subunit NR2D gene in the case-control study of schizophrenia.

    Methods: We screened for polymorphisms in exons, exon-intron boundaries and the 5' upstream region of GRIN2D by direct sequencing in 32 Japanese patients. Out of the total 13 single-nucleotide polymorphisms identified, we genotyped 200-201 Japanese patients and 219-221 controls for nine common single-nucleotide polymorphisms (minor allele frequency over 0.05).

    Results: None of the nine single-nucleotide polymorphisms showed significant differences in genotype and allele frequencies between cases and controls. We observed significant associations of pairwise haplotypes in three combinations of four single-nucleotide polymorphisms, INT10SNP-EX13SNP2, EX13SNP2-EX13SNP3 and EX6SNP-EX13SNP2, with the disease even after the Bonferroni correction (P=1.094 x 10(-6), Pcorrected=2.297 x 10(-5), P=2.825 x 10(-6), Pcorrected=5.933 x 10(-5) and P=2.02 x 10(-4), Pcorrected=4.242 x 10(-3), respectively). The same results were also obtained using the false discovery rate (BL) method at the threshold P value, 2.908 x 10(-3).

    Conclusions: We conclude that the GRIN2D locus is a possible genomic region contributing to schizophrenia susceptibility in the Japanese population.

    Psychiatric genetics 2005;15;3;215-21

  • 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 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

  • 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

  • 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

  • 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

  • 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

  • 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

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

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

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

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

    Funded by: Medical Research Council: G9309834, G9810900

    Molecular psychiatry 2002;7;5;508-14

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

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

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

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

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

  • 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

  • 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

  • Interaction of the N-methyl-D-aspartic acid receptor NR2D subunit with the c-Abl tyrosine kinase.

    Glover RT, Angiolieri M, Kelly S, Monaghan DT, Wang JY, Smithgall TE and Buller AL

    Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.

    The COOH-terminal domain of the NR2D subunit of the NMDA receptor contains proline-rich regions that show striking homology to sequences known to bind to Src homology 3 (SH3) domains. To determine whether the proline-rich region of the NR2D subunit interacts with specific SH3 domains, in vitro SH3 domain binding assays were performed. A proline-rich fragment of the NR2D subunit (2D(866-1064)) bound to the Abl SH3 domain but not to the SH3 domains from Src, Fyn, Grb2, GAP, or phospholipase C-gamma (PLCgamma). Co-immunoprecipitation of NR2D with Abl suggests stable association of NR2D and Abl in transfected cells. The SH3 domain plays an important role in the negative regulation of Abl kinase activity. To determine whether the interaction of NR2D with the Abl SH3 domain alters Abl kinase activity, Abl was expressed alone or with NR2D in 293T cells. Autophosphorylation of Abl was readily observed when Abl was expressed alone. However, co-expression of Abl with 2D(866-1064) or full-length NR2D inhibited autophosphorylation. 2D(866-1064) did not inhibit DeltaSH3 Abl, indicating a requirement for the Abl SH3 domain in the inhibitory effect. Similarly, 2D(866-1064) did not inhibit the catalytic activity of Abl-PP, which contains two point mutations in the SH2-kinase linker domain that release the negative kinase regulation by the SH3 domain. In contrast, the full-length NR2D subunit partially inhibited the autokinase activity of both DeltaSH3 Abl and Abl-PP, suggesting that NR2D and Abl may interact at multiple sites. Taken together, the data in this report provide the first evidence for a novel inhibitory interaction between the NR2D subunit of the NMDA receptor and the Abl tyrosine kinase.

    Funded by: NCI NIH HHS: CA 81398, R01 CA043054, R37 CA043054; NIAAA NIH HHS: AA 00153

    The Journal of biological chemistry 2000;275;17;12725-9

  • 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

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

    Kurschner C and Yuzaki M

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

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

    Funded by: NCI NIH HHS: P30 CA21765

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

  • 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

  • Expression of a functional N-methyl-D-aspartate-type glutamate receptor by bone marrow megakaryocytes.

    Genever PG, Wilkinson DJ, Patton AJ, Peet NM, Hong Y, Mathur A, Erusalimsky JD and Skerry TM

    Department of Biology, University of York, York, United Kingdom. pg5@york.ac.uk

    Better understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.

    Funded by: Wellcome Trust

    Blood 1999;93;9;2876-83

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

    Wang P, Barks JD and Silverstein FS

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

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

    Funded by: NINDS NIH HHS: NS 31054

    Neuroscience 1999;88;2;585-97

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

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

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

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

    Neurotoxicology 1998;19;4-5;683-8

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

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

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

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

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

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

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

    Kurschner C, Mermelstein PG, Holden WT and Surmeier DJ

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

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

    Funded by: NCI NIH HHS: P30 CA21765

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

  • Functional characterization of human N-methyl-D-aspartate subtype 1A/2D receptors.

    Hess SD, Daggett LP, Deal C, Lu CC, Johnson EC and Veliçelebi G

    SIBIA Neurosciences, Inc., La Jolla, California 92037, USA.

    The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and D-serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7-dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2 occurred at -40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

    Journal of neurochemistry 1998;70;3;1269-79

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

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

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

    Genomics 1998;47;3;423-5

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

    Wollmuth LP, Kuner T and Sakmann B

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

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

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

  • Isolation of estrogen-responsive genes with a CpG island library.

    Watanabe T, Inoue S, Hiroi H, Orimo A, Kawashima H and Muramatsu M

    Department of Biochemistry, Saitama Medical School, Japan.

    In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochrome c oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5' upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5' upstream region of the chicken beta-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

    Molecular and cellular biology 1998;18;1;442-9

  • Cellular distribution of NMDA glutamate receptor subunit mRNAs in the human cerebellum.

    Scherzer CR, Landwehrmeyer GB, Kerner JA, Standaert DG, Hollingsworth ZR, Daggett LP, Veliçelebi G, Penney JB and Young AB

    Department of Neurology, Massachusetts General Hospital, Boston 02114, USA.

    We have used a quantitative in situ hybridization method with human ribonucleotide probes to examine the regional and cellular distribution of N-methyl-D-aspartate receptor (NMDAR) subunit mRNAs in the human cerebellum. Purkinje cells showed very dense labeling for NMDAR1 mRNA, dense labeling for NMDAR2A mRNA, and moderate labeling for NMDAR2D mRNA, whereas labeling for NMDAR2C mRNA was low. Granule cells showed high hybridization signals for the NMDAR1 and NMDAR2C mRNAs and moderate signals for the NMDAR2A and NMDAR2D mRNAs. In addition intense labeling with the NMDAR2B probe was observed in medium-sized neurons with chromophilic cell bodies in the upper part of the granule cell layer, most likely representing Golgi cells. Neurons in the molecular layer, i.e., basket cells and stellate cells, showed moderate hybridization signals for NMDAR1 and NMDAR2D and low signal for NMDAR2C. Each type of cerebellar neuron analyzed displayed a distinct NMDAR2 subunit profile, suggesting that they are likely to have NMDA receptors with distinct properties.

    Funded by: NIA NIH HHS: AG11337; NIMH NIH HHS: MH/NS 31862; NINDS NIH HHS: NS31579

    Neurobiology of disease 1997;4;1;35-46

  • 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

  • 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

  • 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

  • Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics.

    Akbarian S, Sucher NJ, Bradley D, Tafazzoli A, Trinh D, Hetrick WP, Potkin SG, Sandman CA, Bunney WE and Jones EG

    Department of Anatomy and Neurobiology, University of California, Irvine 92717, USA.

    NMDA receptor antagonists can induce a schizophrenia-like psychosis, but the role of NMDA receptors in the pathophysiology of schizophrenia remains unclear. Expression patterns of mRNAs for five NMDA receptor subunits (NR1/NR2A-D) were determined by in situ hybridization in prefrontal, parieto-temporal, and cerebellar cortex of brains from schizophrenics and from neuroleptic-treated and nonmedicated controls. In the cerebral cortex of both schizophrenics and controls, mRNAs for NR1, NR2A, NR2B, and NR2D subunits were preferentially expressed in layers II/III, Va, and VIa, with much higher levels in the prefrontal than in the parieto-temporal cortex. Levels of mRNA for the NR2C subunit were very low overall. By contrast, the cerebellar cortex of both schizophrenics and controls contained very high levels of NR2C subunit mRNA, whereas levels for the other subunit mRNAs were very low, except NR1, for which levels were moderate. Significant alterations in the schizophrenic cohort were confined to the prefrontal cortex. Here there was a shift in the relative proportions of mRNAs for the NR2 subunit family, with a 53% relative increase in expression of the NR2D subunit mRNA. No comparable changes were found in neuroleptic-treated or untreated controls. These findings indicate regional heterogeneity of NMDA receptor subunit expression in human cerebral and cerebellar cortex. In schizophrenics, the alterations in expression of NR2 subunit mRNA in prefrontal cortex are potential indicators of deficits in NMDA receptor-mediated neurotransmission accompanying functional hypoactivity of the frontal lobes.

    Funded by: NIMH NIH HHS: MH44188

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;1;19-30

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

    Bennett BA, Rusyniak DE and Hollingsworth CK

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

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

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

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

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

    Dreyer EB and Lipton SA

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

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

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

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

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

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

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

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

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

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

    Andersen DL, Tannenberg AE, Burke CJ and Dodd PR

    Royal Brisbane Hospital Research Foundation, Australia.

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

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

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

    Kornau HC, Schenker LT, Kennedy MB and Seeburg PH

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

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

    Funded by: NINDS NIH HHS: NS-28710

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

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

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

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

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

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

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

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

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

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

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

Gene lists (7)

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
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
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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