Glutamate Receptor Subunit Gene Research Articles

Aberrant neurogenesis and late onset suppression of synaptic plasticity as well as sustained neuroinflammation in the hippocampal dentate gyrus after developmental exposure to ethanol in rats

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. The present study investigated the effects of maternal oral ethanol (EtOH) exposure (0, 10, or 12.5 % in drinking water) from gestational day 6 until day 21 post-delivery (weaning) on postnatal hippocampal neurogenesis at weaning and in adulthood on postnatal day 77 in rat offspring. At weaning, type-3 neural progenitor cells (NPCs) were decreased in the subgranular zone (SGZ), accompanied by Chrnb2 downregulation and Grin2b upregulation in the dentate gyrus (DG). These results suggested suppression of CHRNB2-mediated cholinergic signaling in γ-aminobutyric acid (GABA)ergic interneurons in the DG hilus and increased glutamatergic signaling through the NR2B subtype of N-methyl-d-aspartate (NMDA) receptors, resulting in NPC reduction. In contrast, upregulation of Chrna7 may increase CHRNA7-mediated cholinergic signaling in immature granule cells, and upregulation of Ntrk2 may cause an increase in somatostatin-immunoreactive (+) GABAergic interneurons, suggesting a compensatory response against NPC reduction. Promotion of SGZ cell proliferation increased type-2a NPCs. Moreover, an increase in calbindin-d-29 K+ interneurons and upregulation of Reln, Drd2, Tgfb2, Il18, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunit genes might participate in this compensatory response. In adulthood, reduction of FOS+ cells and downregulation of Fos and Arc suggested suppression of granule cell synaptic plasticity, reflecting upregulation of Tnf and downregulation of Cntf, Ntrk2, and AMPA-type glutamate receptor genes. In the DG hilus, gliosis and hyper-ramified microglia, accompanying upregulation of C3, appeared at weaning, suggesting contribution to suppressed synaptic plasticity in adulthood. M1 microglia increased throughout adulthood, suggesting sustained neuroinflammation. These results indicate that maternal EtOH exposure temporarily disrupts hippocampal neurogenesis and later suppresses synaptic plasticity. Induction of neuroinflammation might initially ameliorate neurogenesis (as evident by upregulation of Tgfb2 and Il18) but later suppress synaptic plasticity (as evident by upregulation of C3 at weaning and Tnf in adulthood).

NMDARs Drive the Expression of Neuropsychiatric Disorder Risk Genes Within GABAergic Interneuron Subtypes in the Juvenile Brain.

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical, and physiological properties. However, the molecular mechanisms regulating their maturation remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates transcriptional regulation of gene expression in specific subtypes of MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific early developmental Grin1 loss results in a broad downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs in the juvenile brain. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

Expression of neurotransmitter receptors in oral keratinocytes and their response to agonists

This study aimed to investigate whether neurotransmitter receptors in the nervous system were also expressed in oral keratinocytes. Expressions of various neurotransmitter receptor genes in immortalized mouse oral keratinocyte (IMOK) cells were examined by reverse transcriptase polymerase chain reaction. IMOK cells expressed calcitonin gene-related peptide (CGRP) receptor subunit genes Ramp1 and Ramp3 and glutamate receptor subunit genes Grina , Gria3 , Grin1 , Grin2a , and Grin2d . Moreover, IMOK cells expressed Adrb2 and Chrna5 that encode beta 2 adrenergic receptor and cholinergic receptor nicotinic alpha 5 for sympathetic and parasympathetic neurotransmitters, respectively. The expression of Bdkrb1 and Ptger4 , which encode receptors for bradykinin and prostaglandin E2 involved in inflammatory responses, was also observed at low levels. Expressions of Ramp1 and Grina in the mouse gingival epithelium were also confirmed by immunohistochemistry. When the function of neurotransmitter receptors expressed on IMOK cells was tested by intracellular calcium response, CGRP, glutamate, and cholinergic receptors did not respond to their agonists, but the bradykinin receptor responded to bradykinin. Collectively, oral keratinocytes express several neurotransmitter receptors, suggesting the potential regulation of oral epithelial homeostasis by the nervous system.

Pharmacologic Inhibition of ADAM10 Attenuates Brain Tissue Loss, Axonal Injury and Pro-inflammatory Gene Expression Following Traumatic Brain Injury in Mice.

The α-secretase A disintegrin and metalloprotease 10 (ADAM10) regulates various physiological and pathophysiological processes. Despite its broad functional implications during development, plasticity, and disease, no pharmacological approaches to inhibit ADAM10 in acute brain injury have been reported. Here, we examined the effects of the ADAM10 inhibitor GI254023X on the neurological and histopathological outcome after experimental traumatic brain injury (TBI). C57BL/6N mice were subjected to the controlled cortical impact (CCI) model of TBI or sham procedure and received GI254023X or vehicle during the acute phase of injury (n = 40, 100 mg/kg, 25% DMSO, 0.1 M Na2CO3, intraperitoneal, 30 min and 24 h after TBI). GI254023X treatment did not improve neurological deficits from 1 to 7 days post-injury (dpi) but animals treated with GI254023X exhibited smaller brain lesions compared to vehicle treatment. Determination of brain mRNA expression by quantitative PCR showed that TBI-induced up-regulation of Adam10 and Adam17 was not influenced by GI254023X but the up-regulation of the matrix metalloproteinase genes Mmp2 and Mmp9 was attenuated. GI254023X treatment further increased the T cell marker Cd247 but did not affect blood brain barrier integrity, as assessed by Occludin mRNA expression and IgG brain extravasation. However, in agreement with neuroprotective effects of ADAM10 inhibition, GI254023X treatment attenuated axonal injury, as indicated by decreased generation of spectrin breakdown products (SBDPs) and decreased immunostaining using anti-non-phosphorylated neurofilament (SMI-32). Interestingly, reduced axonal injury in GI254023X-treated animals coincided with subtle mRNA dysregulation in the glutamate receptor subunit genes Gria1 and Grin2b. Quantitative PCR also revealed that GI254023X mitigated up-regulation of the pro-inflammatory markers Il6, Tnfa, and Lcn2 but not the up-regulation of the pan-microglia marker Aif1, the M2 microglia marker Arg1 and the reactive astrocyte marker Gfap. Taken together, the ADAM10 inhibitor GI254023X attenuates brain tissue loss, axonal injury and pro-inflammatory gene expression in the CCI model of TBI. These results suggest that ADAM10 may represent a therapeutic target in the acute phase of TBI.

Demonstration of critical role of GRIN3A in nicotine dependence through both genetic association and molecular functional studies.

Nicotine dependence (ND) is a chronic disease with catastrophic effects on individual and public health. The glutamate receptor subunit gene, ionotropic N-methyl-d-aspartate 3A (GRIN3A), encodes a crucial subunit of N-methyl-d-aspartate receptors (NMDARs), which play an essential role in synaptic plasticity in the brain. Although various variants of GRIN3A have been associated with ND in European-American and African-American samples, no study has been reported for the association between GRIN3A and ND in Chinese Han population. We performed an association study of 16 single nucleotide polymorphisms (SNPs) in GRIN3A with ND in 2616 Chinese individuals. SNP-based association analysis indicated that SNP rs1323423 was significantly associated with the Fagerström Test for Nicotine Dependence (FTND) score after correction for multiple testing (P=0.0026). Haplotype-based association analysis revealed that Block 3, formed by rs1323423-rs10989591, was significantly associated with the FTND score after correction for multiple testing (global P=0.0183). Furthermore, luciferase reporter assay demonstrated that the DNA region containing rs1323423 was an enhancer element, the activity of which was significantly impacted by rs1323423 genotype. Considering that rs1323423 is located in a potential enhancer region, we performed GRIN3A editing in HEK293T cells with CRISPR/Cas9 and found that the DNA region around rs1323423 has a regulatory function and the expression of GRIN3A affects the expression of other NMDA subunits. Moreover, we demonstrated that nicotine at a concentration of 100μM decreased expression of GRIN3A in SH-SY5Y and HEK293T cells at the RNA and protein level, respectively. This study provides novel evidence for the involvement of GRIN3A in ND.

Prenatal Bisphenol A Exposure is Linked to Epigenetic Changes in Glutamate Receptor Subunit Gene Grin2b in Female Rats and Humans

Bisphenol A (BPA) exposure has been linked to neurodevelopmental disorders and to effects on epigenetic regulation, such as DNA methylation, at genes involved in brain function. High doses of BPA have been shown to change expression and regulation of one such gene, Grin2b, in mice. Yet, if such changes occur at relevant doses in animals and humans has not been addressed. We investigated if low-dose developmental BPA exposure affects DNA methylation and expression of Grin2b in brains of adult rats. Furthermore, we assessed associations between prenatal BPA exposure and Grin2b methylation in 7-year old children. We found that Grin2b mRNA expression was increased and DNA methylation decreased in female, but not in male rats. In humans, prenatal BPA exposure was associated with increased methylation levels in girls. Additionally, low APGAR scores, a predictor for increased risk for neurodevelopmental diseases, were associated with higher Grin2b methylation levels in girls. Thus, we could link developmental BPA exposure and low APGAR scores to changes in the epigenetic regulation of Grin2b, a gene important for neuronal function, in a sexual dimorphic fashion. Discrepancies in exact locations and directions of the DNA methylation change might reflect differences between species, analysed tissues, exposure level and/or timing.

Study of GRIN2A mutation in epilepsy-aphasia spectrum disorders

To detect potential mutations of the glutamate receptor subunit (GRIN2A) gene and delineate the clinical-genetic characteristics of patients with epilepsy-aphasia spectrum (EAS) disorders. One hundred twenty two patients with Landau-Kleffner syndrome (LKS), epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS), benign childhood epilepsy with centrotemporal spikes (BECT) and BECT variants were recruited. Potential mutations of the GRIN2A gene were screened with Sanger sequencing. And clinical-genetic characteristics for all patients were analyzed. The patients have included 9 LKS, 26 CSWS, 42 BECT variants and 45 BECT. The mean age of onset of seizure or aphasia was 5 years old (10 months to 11 years). Mutation screening has detected 4 possible pathogenic missense mutations including c.2278G>A (p.G760S), c.4153G>T (p.D1385Y), c.1364G>A (p.C455Y) and c.691T>C (p.C231R) in four unrelated probands, which comprised one case with LKS and three with BECT variants. The mutation rate was 11.1% (1/9) for LKS and 7.2% (3/42) for BECT variants. No GRIN2A mutation was found in the 26 patients with CSWS and 45 patients with BECT. Among the 122 probands, 25 (20.5%) patients without a GRIN2A mutation had a positive family history of febrile seizures or epilepsy. GRIN2A mutation do exist in EAS patients, but with a relatively low rate. A proportion of EAS patients without a GRIN2A mutation have a positive family history, which suggested a complex mechanism for EAS.

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression.

RationaleAspartame (L-aspartyl phenylalanine methyl ester) is a non-nutritive sweetener (NNS) approved for use in more than 6000 dietary products and pharmaceuticals consumed by the general public including adults and children, pregnant and nursing mothers. However a recent prospective study reported a doubling of the risk of being overweight amongst 1-year old children whose mothers consumed NNS-sweetened beverages daily during pregnancy. We have previously shown that chronic aspartame (ASP) exposure commencing in utero may detrimentally affect adulthood adiposity status, glucose metabolism and aspects of behavior and spatial cognition, and that this can be modulated by developmental N-methyl-D-aspartate receptor (NMDAR) blockade with the competitive antagonist CGP 39551 (CGP). Since glucose homeostasis and certain aspects of behavior and locomotion are regulated in part by the NMDAR-rich hypothalamus, which is part of the hypothalamic-pituitary-adrenal- (HPA) axis, we have elected to examine changes in hypothalamic and adrenal gene expression in response to ASP exposure in the presence or absence of developmental NMDAR antagonism with CGP, using Affymetrix microarray analysis.ResultsUsing 2-factor ANOVA we identified 189 ASP-responsive differentially expressed genes (DEGs) in the adult male hypothalamus and 2188 in the adrenals, and a further 23 hypothalamic and 232 adrenal genes significantly regulated by developmental treatment with CGP alone. ASP exposure robustly elevated the expression of a network of genes involved in hypothalamic neurosteroidogenesis, together with cell stress and inflammatory genes, consistent with previous reports of aspartame-induced CNS stress and oxidative damage. These genes were not differentially expressed in ASP mice with CGP antagonism. In the adrenal glands of ASP-exposed mice, GABA and Glutamate receptor subunit genes were amongst those most highly upregulated. Developmental NMDAR antagonism alone had less effect on adulthood gene expression and affected mainly hypothalamic neurogenesis and adrenal steroid metabolism. Combined ASP + CGP treatment mainly upregulated genes involved in adrenal drug and cholesterol metabolism.ConclusionASP exposure increased the expression of functional networks of genes involved in hypothalamic neurosteroidogenesis and adrenal catecholamine synthesis, patterns of expression which were not present in ASP-exposed mice with developmental NMDAR antagonism.

THC Treatment Alters Glutamate Receptor Gene Expression in Human Stem Cell-Derived Neurons

Given the cognitive and behavioral effects following in utero Δ9-tetrahydrocannabinol (THC) exposure that have been reported in humans and rodents, it is critical to understand the precise consequences of THC on developing human neurons. Here, we utilize excitatory neurons derived from human-induced pluripotent stem cells (hiPSCs), and report that in vitro THC exposure reduced expression of glutamate receptor subunit genes (GRIA1, GRIA2, GRIN2A, and GRIN2B). By expanding these studies across hiPSC-derived neurons from individuals with a variety of genotypes, we believe that a hiPSC-based model will facilitate studies of the interaction of THC exposure and the genetic risk factors underlying neuropsychiatric disease vulnerability.

Genetic analysis of Ohtahara syndrome and atypical benign partial epilepsy coexisting in a family

Objective To explore the genetic etiologies in 2 siblings with different epileptic encephalopathies (EEs) diagnosed as Ohtahara syndrome(OS) and atypical benign partial epilepsy(ABPE) from one family. Methods The 2 brothers were diagnosed at the Pediatric Neurological Clinic of Peking University First Hospital in September 2013, whose clinical data were collected.The coding region of the syntaxin-binding protein 1 gene (STXBP1) and glutamate receptor subunit gene (GRIN2A) were detected by using Sanger sequencing in the 2 siblings.For the elder brother, targeted next-generation sequencing was further performed to detect the genes associated with epilepsy. Results The younger brother manifested focal motor seizures and tonic spasms in cluster at the age of 1 month.Interictal electroencephalogram (EEG) showed suppression-burst pattern.He was diagnosed as OS.The elder brother had seizure onset at age of 6 years old.Focal motor seizure during sleep was his seizure type.His EEG showed interictal discharges in Rolandic area primarily.Electrical status epilepticus during sleep, epileptic negative myoclonus and intellectual disabilities occurred during the course.He was diagnosed as ABPE.Brain magnetic resonance imagines for both of them were normal.Screening of STXBP1 mutations for the younger brother found a de novo heterozygous mutation: c.1672C>T (p.Q558X). Gene detection for the elder brother and the parents showed negative results. Conclusions Coexistence of distinct EEs was reported in 2 brother siblings: the younger brother had OS associated with a novel nonsense mutation in STXBP1, and the elder brother had ABPE without genetic evidence.This study indicated that different pathological mechanisms might exist underlying the two different EEs in a family. Key words: Epileptic encephalopathy; Ohtahara syndrome; Siblings; Syntaxin-binding protein 1 gene; Glutamate receptor subunit gene

Genetic association analysis of N-methyl-D-aspartate receptor subunit gene GRIN2B and clinical response to clozapine.

Approximately 30% of patients with schizophrenia fail to respond to antipsychotic therapy and are classified as having treatment-resistant schizophrenia. Clozapine is the most efficacious drug for treatment-resistant schizophrenia and may deliver superior therapeutic effects partly by modulating glutamate neurotransmission. Response to clozapine is highly variable and may depend on genetic factors as indicated by twin studies. We investigated eight polymorphisms in the N-methyl-D-aspartate glutamate receptor subunit gene GRIN2B with response to clozapine. GRIN2B variants were genotyped using standard TaqMan procedures in 175 European patients with schizophrenia deemed resistant or intolerant to treatment. Response was assessed using change in Brief Psychiatric Rating Scale scores following six months of clozapine therapy. Categorical and continuous response was assessed using chi-squared test and analysis of covariance, respectively. No associations were observed between the variants and response to clozapine. A-allele carriers of rs1072388 responded marginally better to clozapine therapy than GG-homozygotes; however, the difference was not statistically significant (p = 0.067, uncorrected). Our findings do not support a role for these GRIN2B variants in altering response to clozapine in our sample. Investigation of additional glutamate variants in clozapine response is warranted.

Combined Effects of Simultaneous Exposure to Caffeine and Cocaine in the Mouse Striatum.

Caffeine is the world's most popular psychoactive drug and is also an active adulterant found in many drugs of abuse, including seized cocaine samples. Despite several studies which examine the effects of caffeine or cocaine administered as single agents, little data are available for these agents when given in combination. The purpose of the present study was to determine if combined intake of both psychostimulants can lead to maladaptive changes in striatal function. Mice were injected with abinge regimen (intermittent treatment for 13days) of caffeine (3×5mg/kg), cocaine (3×10mg/kg), or combined administration. We found that chronic caffeine potentiated locomotion induced by cocaine and that both caffeine-treated groups showed sensitization. Striatal tissue was obtained 24h and 7days after last injection (withdrawal) for immunohistochemistry and mRNA expression. Our results show that combined intake of both psychostimulants can increase GFAP immunoreactivity in the striatum at both times post treatment. Gene expression analysis, targeted at dopamine, adenosine, and glutamate receptor subunit genes, revealed significant transcript down-regulation in the dorsal striatum of AMPA, NMDA, D1 and D2 receptor subunit mRNA expression in the group that received combined treatment, but not after individual administration. At withdrawal, we found increased D1 receptor mRNA expression along with increased A1, AMPA, NMDA, and metabotropic subunit expression. A2A mRNA showed decreased expression after both times in all experimental groups. Our study provides evidence that there are striatal alterations mediated by combined caffeine and cocaine administration, and highlights negative outcomes of chronic intake of both psychostimulants.

A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity.

Homeostatic synaptic plasticity (HSP) helps neurons and synapses maintain physiologically appropriate levels of output. The fruit fly Drosophila melanogaster larval neuromuscular junction (NMJ) is a valuable model for studying HSP. Here we introduce a genetic tool that allows fruit fly researchers to examine the lifelong maintenance of HSP with a single cross. The tool is a fruit fly stock that combines the GAL4/UAS expression system with RNA interference (RNAi)-based knock down of a glutamate receptor subunit gene. With this stock, we uncover important new information about the maintenance of HSP. We address an open question about the role that presynaptic CaV2-type Ca2+ channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the CaV2 α1a subunit gene cacophony (cac) can impair homeostatic plasticity at the NMJ. Here we report that reducing cac expression levels by RNAi is not sufficient to impair homeostatic plasticity. The presence of wild-type channels appears to support HSP—even when total CaV2 function is severely reduced. We also conduct an RNAi- and electrophysiology-based screen to identify new factors required for sustained homeostatic signaling throughout development. We uncover novel roles in HSP for Drosophila homologs of Cysteine string protein (CSP) and Phospholipase Cβ (Plc21C). We characterize those roles through follow-up genetic tests. We discuss how CSP, Plc21C, and associated factors could modulate presynaptic CaV2 function, presynaptic Ca2+ handling, or other signaling processes crucial for sustained homeostatic regulation of NMJ function throughout development. Our findings expand the scope of signaling pathways and processes that contribute to the durable strength of the NMJ.

GRIN2A: an aptly named gene for speech dysfunction.

To delineate the specific speech deficits in individuals with epilepsy-aphasia syndromes associated with mutations in the glutamate receptor subunit gene GRIN2A. We analyzed the speech phenotype associated with GRIN2A mutations in 11 individuals, aged 16 to 64 years, from 3 families. Standardized clinical speech assessments and perceptual analyses of conversational samples were conducted. Individuals showed a characteristic phenotype of dysarthria and dyspraxia with lifelong impact on speech intelligibility in some. Speech was typified by imprecise articulation (11/11, 100%), impaired pitch (monopitch 10/11, 91%) and prosody (stress errors 7/11, 64%), and hypernasality (7/11, 64%). Oral motor impairments and poor performance on maximum vowel duration (8/11, 73%) and repetition of monosyllables (10/11, 91%) and trisyllables (7/11, 64%) supported conversational speech findings. The speech phenotype was present in one individual who did not have seizures. Distinctive features of dysarthria and dyspraxia are found in individuals with GRIN2A mutations, often in the setting of epilepsy-aphasia syndromes; dysarthria has not been previously recognized in these disorders. Of note, the speech phenotype may occur in the absence of a seizure disorder, reinforcing an important role for GRIN2A in motor speech function. Our findings highlight the need for precise clinical speech assessment and intervention in this group. By understanding the mechanisms involved in GRIN2A disorders, targeted therapy may be designed to improve chronic lifelong deficits in intelligibility.

Modulation of Behavior and Glutamate Receptor mRNA Expression in Rats after Sub-chronic Administration of Benzo(a)pyrene

Objective The present study aimed to test whether exposure to benzo(a)pyrene [B(a)P] affects spatial learning and short-term memory by modulating the expression of the Gria1 and Grin2a glutamate receptor subunit genes in the hippocampus. Methods Thirty-six 21–24-day-old, male rats were randomly assigned into high-, medium-, and low-dose toxin exposure groups (6.25, 2.5, and 1 mg/kg, respectively) and a control group, each containing nine rats. The behavioral performance of adult rats exposed to sub-chronic administration of B(a)P was monitored by learning and memory tests (Morris water maze). Real-time PCR assays were used to quantify Gria1 and Grin2a gene expression in the hippocampus. Results At medium and high doses, B(a)P impaired spatial learning performance. The crossing-platform-location frequency and the time spent swimming in the platform area, which both relate to short-term memory, were significantly decreased in B(a)P-treated rats compared with controls. The level of Gria1 mRNA increased 2.6–5.9-fold, and the level of Grin2a mRNA increased 10–14.5-fold, with a greater fold increase associated with higher doses of B(a)P. Conclusion We demonstrated that sub-chronic administration of B(a)P inhibits spatial learning and short-term memory, and increases Gria1 and Grin2a expression in the hippocampus. This suggests a relationship of B(a)P exposure levels with Gria1 and Grin2a expression and impairment of short-term and spatial memory.

Comprehensive analysis of candidate genes for photosensitivity using a complementary bioinformatic and experimental approach

Photoparoxysmal response (PPR) is a highly heritable electroencephalographic trait characterized by an increased sensitivity to photic stimulation. It may serve as an endophenotype for idiopathic generalized epilepsy. Family linkage studies identified susceptibility loci for PPR on chromosomes 5q35.3, 8q21.13, and 16p13.3. This study aimed to identify key candidate genes within these loci. We used bioinformatics tools for gene prioritization integrating information on biologic function, sequence data, gene expression, and others. The prime candidate gene from this analysis was sequenced in 48 photopositive probands. Presumed functional implications of identified polymorphisms were investigated using bioinformatics methods. The glutamate receptor subunit gene GRIN2A was identified as a prime candidate gene. Sequence analysis revealed various new polymorphisms. None of the identified variants was predicted to be functionally relevant. We objectified the selection of candidate genes for PPR without an a priori hypothesis. Particularly among the various ion channel genes in the linkage regions, GRIN2A was identified as the prime candidate gene. GRIN2A mutations have recently been identified in various epilepsies. Even though our mutation analysis failed to demonstrate direct involvement of GRIN2A in photosensitivity, in silico gene prioritization may provide a useful tool for the identification of candidate genes within large genomic regions.

Association between a polymorphism in the promoter of a glutamate receptor subunit gene (GRIN2A) and alcoholism

A variable (GT)(n) repeat in the 5'-regulatory region of N-methyl-D-aspartate GRIN2A subtype has recently been identified and associated with psychiatric disorders. In this study, we examined the association of this polymorphism with alcohol dependence. Subject-control analysis included 206 alcohol-dependent and 168 control subjects. Average observed repeat numbers and genotype distributions were significantly different (P-value = 0.001) in alcohol-dependent subjects versus control subjects. Short alleles were significantly less frequent among alcohol-dependent subjects (odds ratio = 0.58, P-value = 7 × 10(-4)). These results could be replicated in an independent sample of 116 alcohol-dependent subjects. For the first time, a significant association was identified between this polymorphism and alcoholism.

P.3.001 NMDA receptor co-determines vulnerability to develop dyskinesia in Huntington's Disease and levodopa-induced dyskinesia but not tardive dyskinesia

Dyskinesia occurs as clinical feature of Huntington's Disease (HD) and as an adverse consequence of chronic exposure to levodopa in Parkinson's disease (levodopa-induced dyskinesia, LID) and to antipsychotic drugs in schizophrenia (tardive dyskinesia, TD). Arning et al. have reported that specific variations in the NR2A glutamate receptor subunit gene (GRIN2A) play a significant role in determining age at onset in HD [1]. These findings have been replicated by Andresen et al. [2]. This subunit of the NMDA glutamate receptor is assumed to play an important role in excitotoxicity of striatal Medium Spiny Neurons (MSN) resulting from over-activation of this receptor. Currently, there are no studies describing the relationship between GRIN2A polymorphisms and the vulnerability to develop drug-induced dyskinesia. Purpose: To investigate the association between TD/LID and 48 tagging SNPs from which 15 of the GRIN2A gene in Russian patients. Methods: After obtaining approval of the study protocol by the ethical committee, suitable participants were recruited from three psychiatric hospitals (Tomsk, Kemerovo, Chita) and the depart-ment of neurology of the Siberian State Medical University of Tomsk, Siberia (Russia). All subjects gave informed consent after proper explanation of the study. DNA extraction and Veracode Assay were conducted according to standard protocols. A total of 48 tagging SNPs were studied of which 15 of GRIN2A. The Abnormal Involuntary Movement Scale was utilized to define the presence of LID/TD. Logistic and logic regression on the binary measurement of dyskinesia (presence, absence) were performed to identify the effect of different variables, respectively SNP-SNP interactions. Results: In total 665 Russian Caucasian patients were included of who 144 were Parkinson's Disease (PD) patients from the neurological department [52 males, 92 females; age 63.1 ± 11.6 years (sample mean ± SD)]. Before analysis participants with more than 5 out of 48 missing SNPs were excluded. Moreover, SNPs with missing values that accounted for more than 10% of the sample were excluded from the analysis, leaving 41 SNPs for statistical analysis. The remaining missing values were imputed with random sampling from the marginal distribution of the proportions of the alleles in the patient population. In PD patients single-SNP logistic regressions identified 2 (rs7192557 and rs8057394) outof a total of 41 SNPs as most influential on the presence of LID on a 5% significance level, after correction for gender and duration of treatment. Logic regression showed that these two SNPs were present in most of the influential SNP combinations. SNP rs7192557 tags to rs1969060 identified by Arning et al. [1] and verified by Andresen et al. [2]. SNP rs8057394 was also identified in the verification study of Arning et al. [3]. The analysis on TD in schizophrenic patients did not identify these two SNPs as being influential. Conclusion: The same SNPs of the GRIN2A gene that play a role in the vulnerability to develop dyskinesias in Huntington Disease seem to be associated with the development of levodopa-induced dyskinesia in Parkinson's Disease but not of TD in schizophrenic patients, probably by determining the activity of the NMDA receptor on striatal MSN.

Association study of N-methyl-D-aspartate glutamate receptor subunit genes and childhood-onset mood disorders

GRIN1 rs4880210 139,143,515 50 G T 0.122 0.878 76 70 0.247 0.619 rs28489906 139,158,220 Intron 2 G A 0.470 0.530 171 170 0.003 0.957 rs6293 139,171,059 Exon 6 A G 0.692 0.303 166 157 0.251 0.616 rs10747050 139,175,697 Intron 11 A G 0.951 0.049 24 33 1.427 0.232 GRIN2A rs1071504 10,187,517 50 C T 0.279 0.721 138 144 0.128 0.721 rs2302711 10,183,199 Intron 2 C T 0.282 0.718 141 140 0.004 0.952 rs1070503 10,172,675 Intron 3 C T 0.866 0.134 67 79 0.987 0.320 rs727605 9,863,065 Intron 5 G A 0.694 0.306 161 133 2.671 0.102 rs8049651 9,851,167 Exon 6 G A 0.726 0.274 153 125 2.825 0.093 rs1014531 9,763,295 Exon 14, 30UTR G A 0.617 0.383 184 163 1.272 0.260 G A 0.797 0.203 117 109 0.283 0.594 rs2284416 13,810,481 Intron 2 T G 0.464 0.536 174 189 0.620 0.431 rs2268115 13,760,992 Intron 3 T G 0.561 0.439 169 170 0.003 0.957 rs2284407 13,733,473 Intron 3 G T 0.582 0.418 186 176 0.276 0.599 rs2268097 13,644,099 Intron 9 A G 0.583 0.417 170 174 0.047 0.829 rs890 13,606,575 Exon 13, 30UTR G T 0.452 0.548 183 172 0.341 0.559

Coupling of Energy Metabolism and Synaptic Transmission at the Transcriptional Level: Role of Nuclear Respiratory Factor 1 in Regulating both CytochromecOxidase and NMDA Glutamate Receptor Subunit Genes

Neuronal activity and energy metabolism are tightly coupled processes. Regions high in neuronal activity, especially of the glutamatergic type, have high levels of cytochrome c oxidase (COX). Perturbations in neuronal activity affect the expressions of COX and glutamatergic NMDA receptor subunit 1 (NR1). The present study sought to test our hypothesis that the coupling extends to the transcriptional level, whereby NR1 and possibly other NR subunits and COX are coregulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), which regulates all COX subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutations, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Grin 1 (NR1), Grin 2b (NR2b) and COX subunit genes, but not of Grin2a and Grin3a genes. These transcripts were upregulated by KCl and downregulated by tetrodotoxin (TTX) in cultured primary neurons. However, silencing of NRF-1 with small interference RNA blocked the upregulation of Grin1, Grin2b, and COX induced by KCl, and overexpression of NRF-1 rescued these transcripts that were suppressed by TTX. NRF-1 binding sites on Grin1 and Grin2b genes are also highly conserved among mice, rats, and humans. Thus, NRF-1 is an essential transcription factor critical in the coregulation of NR1, NR2b, and COX, and coupling exists at the transcriptional level to ensure coordinated expressions of proteins important for synaptic transmission and energy metabolism.