Biomedical subjects

Robert H Lipsky

Publications and source records attributed to Robert H Lipsky.

At least 19 recordsLinked to original sources

PubMed2006

Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder.

A functional serotonin transporter promoter polymorphism, HTTLPR, alters the risk of disease as well as brain morphometry and function. Here, we show that HTTLPR is functionally triallelic. The L(G) allele, which is the L allele with a common G substitution, creates a functional AP2 transcription-factor binding site. Expression assays in 62 lymphoblastoid cell lines representing the six genotypes and in transfected raphe-derived cells showed codominant allele action and low, nearly equivalent expression for the S and L(G) alleles, accounting for more variation in HTT expression than previously recognized. The gain-of-function L(A)L(A) genotype was approximately twice as common in 169 whites with obsessive-compulsive disorder (OCD) than in 253 ethnically matched controls. We performed a replication study in 175 trios consisting of probands with OCD and their parents. The L(A) allele was twofold overtransmitted to the patients with OCD. The HTTLPR L(A)L(A) genotype exerts a moderate (1.8-fold) effect on risk of OCD, which crystallizes the evidence that the HTT gene has a role in OCD.

Adult↗

PubMed2005

The excitoprotective effect of N-methyl-D-aspartate receptors is mediated by a brain-derived neurotrophic factor autocrine loop in cultured hippocampal neurons.

The neuroprotective effect and molecular mechanisms underlying preconditioning with N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre-incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor-mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time- and concentration-dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain-derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF-dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 microM) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4-specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre-incubation with a BDNF-blocking antibody and TrkB-IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA-mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2005

N-methyl-D-aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal-regulated kinase pathway.

N-Methyl-D-aspartate (NMDA) at a subtoxic concentration (100 microM) promotes neuronal survival against glutamate-mediated excitotoxicity via a brain-derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3-K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration- and time-dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK-801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 microM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3-K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl-2, bcl-X(L), and bax mRNA levels,. NMDA increases Bcl-2 and Bcl-X(L) protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate-mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl-2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.

Animals↗

PubMed2005

Haplotype structure of the beta adrenergic receptor genes in US Caucasians and African Americans.

The beta-adrenergic receptors (beta-AR) are G protein-coupled receptors activated by epinephrine and norepinephrine and are involved in a variety of their physiological functions. Previously, three beta-AR genes (ADRB1, ADRB2 and ADRB3) were resequenced, identifying polymorphisms that were used in genetic association studies of cardiovascular and metabolic disorders. These studies have produced intriguing but inconsistent results, potentially because the known functional variants: ADRB1 Arg389Gly and Gly49Ser, ADRB2 Arg16Gly and Gln27Glu, and ADRB3 Arg64Trp provided an incomplete picture of the total functional diversity at these genes. Therefore, we created marker panels for each beta-AR gene that included the known functional markers and also other markers evenly spaced and with sufficient density to identify haplotype block structure and to maximize haplotype diversity. A total of 27 markers were genotyped in 96 US Caucasians and 96 African Americans. In both populations and for each gene, a single block with little evidence of historical recombination was observed. For each gene, haplotype captured most of the information content of each functional locus, even if that locus was not genotyped, and presumably haplotype would capture the signal from unknown functional loci whose alleles are of moderate abundance. This study demonstrates the utility of using beta-AR gene haplotype maps and marker panels as tools for linkage studies on beta-AR function.

Black or African American↗

PubMed2005

BDNF variation and mood disorders: a novel functional promoter polymorphism and Val66Met are associated with anxiety but have opposing effects.

The brain-derived neurotrophic factor (BDNF) gene is critical for neuronal function and survival, and is likely to be important in psychiatric disorders. In this study, we used single-nucleotide polymorphism (SNP) discovery, functional analyses, and genetic association studies to better understand the potential role of BDNF sequence variation in behavior. Screening 480 unrelated individuals for SNPs and genotyping was performed in US Caucasian, American Indian, and African American populations. Lifetime DSM-III-R psychiatric diagnoses were assigned and the Tridimensional Personality Questionnaire (TPQ) was administered to measure anxious temperament (harm avoidance (HA)) and novelty seeking (NS). A novel SNP (-281 C>A) in promoter 1 was discovered that had decreased DNA binding in vitro and decreased basal reporter gene activity in transfected rat hippocampal neurons. The frequency of the -281 A allele was 0.03 in a Caucasian sample, but was virtually absent in other populations. Association analyses in a community-based sample showed that individuals with the -281 A allele (13 heterozygotes) had lower TPQ HA (F=4.8, p<0.05). In contrast, the Met 66 allele was associated with increased HA (F=4.1, p=0.02) and was most abundant in individuals with both anxiety disorders and major depression (p<0.05). Among the Val66Val homozygotes, individuals who were -281 CA heterozygotes had significantly lower HA than the -281 CC homozygotes (p<0.01). Our results suggest that in this population, the low activity -281 A allele may be protective against anxiety and psychiatric morbidity, whereas Met 66 may be a risk allele.

Adult↗

PubMed2005

Association of COMT Val158Met genotype with executive functioning following traumatic brain injury.

Catechol-O-methyltransferase (COMT) is thought to functionally modulate dopamine neurons, thus likely influencing frontal-executive functioning. High enzyme activity (COMT Val) and low enzyme activity (COMT Met) are functional polymorphisms resulting from a G to A transition in exon 4 (codon 158) of the human COMT gene. Decreased cortical dopamine should result in poorer executive functioning. Therefore, the authors hypothesized that individuals with traumatic brain injury (TBI) and the low enzyme activity polymorphism would perform better on tests of executive functioning than individuals with the high enzyme activity polymorphism. One hundred thirteen individuals referred to the Defense and Veterans Brain Injury Center underwent a comprehensive TBI evaluation and were genotyped for the COMT polymorphism. Comparison of mean differences among the COMT genotype groups for several measures of aspects of executive functioning was conducted using analysis of variance (ANOVA) with adjustment for multiple comparisons. Homozygotes for the higher activity allele made more perseverative responses on the Wisconsin Card Sorting Test, while homozygotes for the lower activity allele had the least number of perseverative responses. While it cannot be determined whether TBI influenced the association of COMT Val158Met to executive functioning, these data extend the known relationship of genotype to executive performance seen in healthy comparison subjects and individuals with schizophrenia to individuals with TBI.

Adult↗

PubMed2005

Delineating and understanding cerebellar neuroprotective pathways: potential implication for protecting the cortex.

The cerebellum is a brain region that is resistant to many of the neurodegenerative disorders such as stroke and Alzheimer's disease. In contrast, the neocortex (cerebrum, cerebral cortex) is vulnerable to these disorders. While there may be many reasons for the differences in vulnerability to acute and chronic neurodegenerative disorders, the cerebellum appears to be equipped with the tools necessary to protect itself against these types of insults. Over the last century, evidence has accumulated to suggest that the cerebellum is also involved in memory and higher cognitive function. We have discovered that intrinsic survival pathways exist in cerebellar granule cells that are regulated by low level stimulation of N-methyl-d-aspartate (NMDA) receptors. Activation of NMDA receptors protects vulnerable neurons against glutamate-mediated excitotoxicity acting on NMDA receptors. This report focuses on how modulation of neuronal survival by NMDA receptors through a brain-derived neurotrophic factor (BDNF)-mediated pathway may be incorporated into a network of cerebellar function, particularly in light of recent findings suggesting that the cerebellum plays a vital role in learning, memory, fear conditioning, and cognitive processing.

Animals↗

PubMed2004

Haplotype-based analysis of alpha 2A, 2B, and 2C adrenergic receptor genes captures information on common functional loci at each gene.

The alpha 2-adrenergic receptors (alpha2-AR) mediate physiological effects of epinephrine and norepinephrine. Three genes encode alpha2-AR subtypes carrying common functional polymorphisms (ADRA2A Asn251Lys, ADRA2B Ins/Del301-303 and ADRA2C Ins/Del322-325). We genotyped these functional markers plus a panel of single nucleotide polymorphisms evenly spaced over the gene regions to identify gene haplotype block structure. A total of 24 markers were genotyped in 96 Caucasians and 96 African Americans. ADRA2A and ADRA2B each had a single haplotype block at least 11 and 16 kb in size, respectively, in both populations. ADRA2C had one haplotype block of 10 kb in Caucasians only. For the three genes, haplotype diversity and the number of common haplotypes were highest in African Americans, but a similar number of markers (3-6) per block was sufficient to capture maximum diversity in either population. For each of the three genes, the haplotype was capable of capturing the information content of the known functional locus even when that locus was not genotyped. The alpha2-AR haplotype maps and marker panels are useful tools for genetic linkage studies to detect effects of known and unknown alpha2-AR functional loci.

Black or African American↗

PubMed2004

Disrupted in schizophrenia 1 (DISC1): association with schizophrenia, schizoaffective disorder, and bipolar disorder.

Schizophrenia, schizoaffective disorder, and bipolar disorder are common psychiatric disorders with high heritabilities and variable phenotypes. The Disrupted in Schizophrenia 1 (DISC1) gene, on chromosome 1q42, was originally discovered and linked to schizophrenia in a Scottish kindred carrying a balanced translocation that disrupts DISC1 and DISC2. More recently, DISC1 was linked to schizophrenia, broadly defined, in the general Finnish population, through the undertransmission to affected women of a common haplotype from the region of intron 1/exon 2. We present data from a case-control study of a North American white population, confirming the underrepresentation of a common haplotype of the intron 1/exon 2 region in individuals with schizoaffective disorder. Multiple haplotypes contained within four haplotype blocks extending between exon 1 and exon 9 are associated with schizophrenia, schizoaffective disorder, and bipolar disorder. We also find overrepresentation of the exon 9 missense allele Phe607 in schizoaffective disorder. These data support the idea that these apparently distinct disorders have at least a partially convergent etiology and that variation at the DISC1 locus predisposes individuals to a variety of psychiatric disorders.

Adult↗

PubMed2004

Differential expression of human COMT alleles in brain and lymphoblasts detected by RT-coupled 5' nuclease assay.

RATIONALE: A common polymorphism, Val158Met, alters catechol- O-methyltransferase (COMT) enzyme activity and has been linked to psychiatric phenotypes. Bray et al. (2003) reported that COMT is subject to differential allele expression in brain, finding modest (13-22%) underexpression of a haplotype containing Val158. However, disparate findings by another group who used the same method, but in lymphoblasts, raise the issues of tissue specificity, magnitude of differential expression, and identity of loci altering expression. OBJECTIVES: We measured COMT allele expression ratios in heterozygous human lymphoblast cell lines and brains. METHODS: Using transcribed single nucleotide polymorphisms as endogenous reporters, we developed an RT-coupled 5' nuclease assay for allele expression ratios and applied it to 63 COMT rs4818(C>G) heterozygotes and 68 Val158Met [rs4680(G>A)] heterozygotes. RESULTS: For rs4818(C>G), the C allele was overexpressed relative to the G allele in 18 of 27 lymphoblast lines and 23 of 36 brains. For Val158Met, Met158 was overexpressed relative to Val158 in all (29 of 29) lymphoblast lines and all (39 of 39) brains. Each of the 22 rs4818 heterozygotes without differential allele expression was a Val158/Val158 homozygote. The Met158 allele was overexpressed by 65-77% when compared with Val158 in lymphoblasts and brain. Haplotype augmented ability to predict expression in brain only. However, the expression of the Val158 allele on the high-expressing haplotype was only 19% higher than Val158 alleles on the other haplotype background. CONCLUSIONS: COMT alleles are differentially expressed. The Met158 allele predicts higher mRNA expression in both brain and lymphoblasts. As exemplified here, the RT-coupled 5' nuclease assay is a reliable method for the quantitative evaluation of cis-acting regulatory effects.

Alleles↗

PubMed2004

Association of specific haplotypes of D2 dopamine receptor gene with vulnerability to heroin dependence in 2 distinct populations.

CONTEXT: Dopamine receptor-mediated pathways play critical roles in the mechanism of addiction. However, associations of the D(2) dopamine receptor gene (DRD2) with substance abuse are controversial. OBJECTIVE: To determine whether susceptibility sites resided at DRD2. DESIGN: Haplotype-based case-control analysis of 2 distinct populations using 10 single nucleotide polymorphisms (SNPs) with heroin dependence. SETTING: Universities of Mainz and Bonn, Germany, and 3 local hospitals in southwestern China. Patients Cases and control subjects recruited from China (486 cases, 313 controls) and Germany (471 cases, 192 controls). INTERVENTIONS: Genotyping for 10 SNPs by 5'-exonuclease fluorescence assays. The D' value of linkage disequilibrium and haplotypes were generated by the expectation-maximization algorithm. MAIN OUTCOME MEASURES: Genotype, allele, and haplotype frequencies were compared between cases and controls by chi(2) tests constructed for each population. An additional 32 SNPs randomly distributed in the genome were genotyped for detecting population admixture in the 2 populations. RESULTS: A haplotype block of 25.8 kilobases (kb) was defined by 8 SNPs extending from SNP3 (TaqIB) at the 5' end to SNP10 site (TaqIA) located 10 kb distal to the 3' end of the gene. Within this block, specific haplotype cluster A (carrying TaqIB1 allele) was associated with a high risk of heroin dependence in Chinese patients (P = 1.425 x 10(-22); odds ratio, 52.80; 95% confidence interval, 7.290-382.5 for 8-SNP analysis). A putative recombination "hot spot" was found near SNP6 (intron 6 ins/del G), creating 2 new daughter haplotypes that were associated with a lower risk of heroin dependence in Germans (P = 1.94 x 10(-11) for 8-SNP analysis). There was no evidence of population stratification in either population. CONCLUSIONS: These results strongly support a role of DRD2 as a susceptibility gene with heroin dependence in Chinese patients and was associated with low risk of heroin dependence in Germans.

Adult↗

PubMed2004

Inhibition of protein kinase C promotes neuronal survival in low potassium through an Akt-dependent pathway.

Cerebellar granule cell neurons undergo apoptotic cell death when subjected to serum-free conditions at physiological concentrations of potassium (5 mM). Protein kinase C (PKC) is known to play a role in preventing neuronal apoptosis under trophic factor deprivation, but its role in protecting cerebellar neurons from cell death under conditions of low potassium is unknown. This study sought to determine the involvement of PKC in neuronal survival and to determine if PKC regulated the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in low physiologic concentrations of potassium. Incubation with a pan-PKC inhibitor, Ro-31-8220 (2 microm), or a specific PKCAlpha inhibitor, Gö6976, protected cerebellar granule cell neurons from low potassium-mediated cell death. In contrast, phorbol ester (TPA, 100 nm), a PKC activator, increased cell death. Incubation with, Ro-31-8220 rescued neurons from cell death induced by the PI 3-K inhibitor, LY294002, suggesting that Ro-31-8220 may affect Akt phosphorylation. Western blot analysis showed that serum-free, low potassium conditions decreased Akt phosphorylation, which was exacerbated by treatment with LY294002. In contrast, PKC inhibitors, Gö6976 or Ro-31-8220, increased Akt phosphorylation approximately two and four-fold, respectively in low potassium conditions. Because Akt activation appears to be critical in promoting neuronal survival under these culture conditions, increased Akt phosphorylation brought about by inhibiting PKC promotes neuronal survival.

Animals↗

PubMed2004

Haplotype architecture of the norepinephrine transporter gene SLC6A2 in four populations.

The norepinephrine transporter (NET) regulates levels of monoamine neurotransmitters integral to a variety of behaviors and autonomic functions. Two SLC6A2 polymorphisms have been used in genetic association studies, generating intriguing but nondefinitive results on traits such as hypertension and mood. One of these SLC6A2 variants is functional but rare. The other is common but not informative over the entire 48 kb SLC6A2 region and is insufficient to capture the functional diversity potentially contained within any SLC6A2 region. To elucidate SLC6A2 haplotype structure and define markers sufficient to capture haplotype diversity within detected haplotype blocks, 26 single-nucleotide polymorphisms (SNPs) were genotyped in 384 individuals evenly divided across Finnish Caucasian, US Caucasian, Plains American Indian, and African American populations. Three conserved blocks, 13.6, 12.5, and 25 kb in size and showing little evidence for historical recombination were observed in all populations. Haplotype diversity in block 1 and numbers of common haplotypes were highest in African Americans, among whom 5-6 optimal markers were sufficient to maximize diversity of each block. For other populations, 2-3 markers/block sufficed, but the optimal markers differed across populations. The SLC6A2 haplotype map and 25-marker panel (excluding the monomorphic one) is a comprehensive tool for genetic linkage studies on phenotypes related to NET function.

Black or African American↗

PubMed2004

Sites in the fourth membrane-associated domain regulate alcohol sensitivity of the NMDA receptor.

N-methyl-D-aspartate (NMDA) receptors are important target sites of alcohol action in the central nervous system. Alcohol inhibits NMDA receptor current by an action on ion channel gating, apparently through a direct action on a region of the NMDA receptor accessible from the extracellular environment. Our previous studies have revealed an important role for a methionine residue (Met823) in membrane-associated domain 4 (M4) of the NR2A subunit in channel gating as well as alcohol sensitivity of the NMDA receptor. The role of sites in M4 of the NMDA receptor NR2A subunit adjacent to Met823 was investigated using tryptophan-scanning mutagenesis and electrophysiological recording. Receptors containing NR1 and NR2A(V820W) or NR2A(M817W) mutant subunits expressed in HEK 293 cells were not functional. The mutation Ala826Trp modified apparent desensitization, and the mutations Ala825Trp and Ala826Trp changed the mean open time of the channel as determined by fluctuation analysis. In addition, the mutations Tyr822Trp and Ala825Trp significantly altered the concentration-response curves for ethanol inhibition. The changes in mean open time did not appear to be able to account for the observed differences in ethanol sensitivity. These results indicate that this region in M4 of the NR2A subunit may be involved in the action of alcohol.

Amino Acid Sequence↗

PubMed2004

AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression.

The signal transduction and molecular mechanisms underlying alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-mediated neuroprotection are unknown. In the present study, we determined a major AMPA receptor-mediated neuroprotective pathway. Exposure of cerebellar granule cells to AMPA (500 microM) + aniracetam (1 microM), a known blocker of AMPA receptor desensitization, evoked an accumulation of brain-derived neurotropic factor (BDNF) in the culture medium and enhanced TrkB-tyrosine phosphorylation following the release of BDNF. AMPA also activated the src-family tyrosine kinase, Lyn, and the downstream target of the phosphatidylinositol 3-kinase (PI3-K) pathway, Akt. Extracellular signal regulated kinase (ERK), a component of the mitogen-activated protein kinase (MAPK) pathway, was also activated. K252a, a selective inhibitor of neurotrophin signaling, blocked the AMPA-mediated neuroprotection. The involvement of BDNF release in protecting neurons by AMPA was confirmed using a BDNF-blocking antibody. AMPA-mediated neuroprotection is blocked by PP1, an inhibitor of src family kinases, LY294002, a PI3-K inhibitor, or U0126, a MAPK kinase (MEK) inhibitor. Neuroprotective concentrations of AMPA increased BDNF mRNA levels that was blocked by the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX). The increase in BDNF gene expression appeared to be the downstream target of the PI3-K-dependent activation of the MAPK cascade since MEK or the PI3-K inhibitor blocked the AMPA receptor-mediated increase in BDNF mRNA. Thus, AMPA receptors protect neurons through a mechanism involving BDNF release, TrkB receptor activation, and a signaling pathway involving a PI3-K dependent activation of MAPK that increases BDNF expression.

Animals↗

PubMed2004

Role of brain-derived neurotrophic factor and NF-kappaB in neuronal plasticity and survival: From genes to phenotype.

PURPOSE: Brain-derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and promotes diverse effects in neurons including development, maintenance of function, synaptic plasticity, and survival in different animal models. We present advances in our understanding of the genomics of the BDNF gene (bdnf) and its regulation by calcium-activated transcription factors, including cAMP response element binding protein (CREB) and more recently, nuclear factor kappaB (NF-kappaB) and discuss these findings in the context of neuronal plasticity and survival. METHODS: We used amplified bdnf complementary DNAs (cDNAs) and genomic DNA templates for direct sequencing and sequence variant discovery, information mining of public databases, and conventional molecular and cellular biology approaches to screen bdnf for novel regulatory elements, alternatively spliced exons, and functional sequence variants. RESULTS: We discovered a candidate NF-kappaB site in promoter 3 of bdnf and showned that activation of N-methyl-D-aspartate (NMDA) inotropic glutamate receptors increased bdnf expression through an NF-kappaB-dependent pathway and extended the finding to show that NF-kappaB was required for NMDA neuroprotection in vitro. In addition, sequence analysis of bdnf cDNAs from different brain regions predicted at least three pre-pro-BDNF protein isoforms, two of which were previously unknown. Each isoform differs at the amino terminus and may have functional importance. CONCLUSIONS: Given the central role that BDNF plays in the developing and adult nervous system, understanding how BDNF is regulated and how it functions will enhance our knowledge of its diverse effects, which may lead to more effective treatments for neurodegenerative disorders and reveal the role of BDNF in complex phenotypes related to behavior.

Animals↗

PubMed2003

Genomics and variation of ionotropic glutamate receptors.

Sequencing of the human, mouse, and rat genomes has enabled a comprehensive informatics approach to gene families. This approach is informative for identification of new members of gene families, for cross-species sequence conservation related to functional conservation, for within-species diversity related to functional variation, and for historical effects of selection. This genome informatics approach also focuses our attention on genes whose genomic locations coincide with linkages to phenotypes. We are identifying ionotropic glutamate receptor (IGR) sequence variation by resequencing technologies, including denaturing high-performance liquid chromoatography (dHPLC), for screening and direct sequencing, and by information mining of public (e.g., dbSNP and ENSEMBL) and private (i.e., Celera Discovery System) sequence databases. Each of the 16 known IGRs is represented in these databases, their positions on a canonical physical map (for example, the Celera map) are established, and comparison to mouse and rat sequences has been performed, revealing substantial conservation of these genes, which are located on different chromosomes but found within syntenic groups of genes. A collection of 38 missense variants were identified by the informatics and resequencing approaches in several of these receptor genes, including GRIN2B, GRIN3B, GRIA2, GRIA3, and GRIK1. This represents only a fraction of the sequence variation across these genes, but, in fact, these may constitute a large fraction of the common polymorphisms at these genes, and these polymorphisms are a starting point for understanding the role of these receptors in neurogenetic variation. Genetically influenced human neurobehavioral phenotypes that are likely to be linked to IGR genetic variants include addictions, anxiety/dysphoria disorders, post-brain injury behavioral disorders, schizophrenia, epilepsy, pain perception, learning, and cognition. Thus, the effects of glutamate receptor variation may be protean, and the task of relating variation to behavior difficult. However, functional variants of (1) catechol-O-methyltransferase, (2) serotonin transporter, and (3) brain-derived neurotrophic factor have recently been linked both to behavioral differences and to intermediate phenotypes, suggesting a pathway by which functional variation at IGRs can be tied to an etiologically complex phenotype.

Animals↗

PubMed2002

A site in the fourth membrane-associated domain of the N-methyl-D-aspartate receptor regulates desensitization and ion channel gating.

The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1-M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met(823)) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC(50) for glycine or the peak NMDA EC(50) but decreased the steady-state NMDA EC(50) and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC(50), and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met(823) in regulation of NMDA receptor function.

Amino Acid Sequence↗