Structure and alternative promoters of the mouse glutamic acid decarboxylase 67 gene.
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ABSTRACT:
SUBMITTER: Yanagawa Y
PROVIDER: S-EPMC1218707 | biostudies-other | 1997 Sep
REPOSITORIES: biostudies-other
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Project description:Reelin and glutamic acid decarboxylase 67 (GAD(67)) expression down-regulation in GABAergic interneurons of mice exposed to protracted treatment with l-methionine (MET) is attributed to RELN and GAD(67) promoter cytosine-5-hypermethylation. This process recruits various transcription repressor proteins [methyl-CpG binding protein (MeCP2) and histone deacetylases (HDACs)] leading to formation of transcriptionally inactive chromatin. Here, we tested the hypothesis that RELN and GAD(67) promoter cytosine-5-hypermethylation induced by a protracted MET treatment is reversible and that repeated administration of HDAC inhibitors influences this process by an activation of DNA-cytosine-5-demethylation. In the frontal cortices of mice receiving MET (5.2 mmol/kg twice a day for 7 days) and killed at 1, 2, 3, 6, and 9 days during MET washout, we measured RELN (base pairs -414 to -242) and GAD(67) (base pairs -1133 to -942) promoter methylation and MeCP2 bound to methylated cytosines of RELN (base pairs -520 to -198) and GAD(67) (base pairs -446 to -760) promoters. Levels of RELN and GAD(67) promoter hypermethylation induced by 7 days of MET treatment declines by approximately 50% after 6 days of MET withdrawal. When valproate (VPA) (2 mmol/kg) or MS-275 (0.015-0.12 mmol/kg), two structurally unrelated HDAC inhibitors, was given after MET treatment termination, VPA and MS-275 dramatically accelerated RELN and GAD(67) promoter demethylation in 48-72 h. At these doses, VPA and MS-275 effectively increased the binding of acetylhistone-3 to RELN and GAD(67) promoters, suggesting that histone-3 covalent modifications modulate DNA demethylation in terminally differentiated neurons, supporting the view that, directly or indirectly, HDAC inhibitors may facilitate DNA demethylation.
| S-EPMC1815468 | biostudies-literature
Project description:GABAergic dysfunction in hippocampus, a key feature of schizophrenia (SZ), may contribute to cognitive impairment in this disorder. In stratum oriens (SO) of sector CA3/2 of the human hippocampus, a network of genes involved in the regulation of glutamic acid decarboxylase GAD67 has been identified. Several of the genes in this network including epigenetic factors histone deacetylase 1 (HDAC1) and death-associated protein 6 (DAXX), the GABAergic enzyme GAD65 as well as the kainate receptor (KAR) subunits GluR6 and 7 show significant changes in expression in this area in SZ. We have tested whether HDAC1 and DAXX regulate GAD67, GAD65, or GluR in the intact rodent hippocampus. Stereotaxic injections of lentiviral vectors bearing shRNAi sequences for HDAC1 and DAXX were delivered into the SO of CA3/2, followed by laser microdissection of individual transduced GABA neurons. Quantitative PCR (QPCR) analyses demonstrated that inhibition of HDAC1 and DAXX increased expression of GAD67, GAD65, and GluR6 mRNA. Inhibition of DAXX, but not HDAC1 resulted in a significant increase in GluR7 mRNA. Our data support the hypothesis that HDAC1 and DAXX play a central role in coordinating the expression of genes in the GAD67 regulatory pathway in the SO of CA3/2.
| S-EPMC6411031 | biostudies-literature
Project description:In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor ?1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis.
| S-EPMC4903066 | biostudies-literature
Project description:Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.
| S-EPMC30678 | biostudies-literature
Project description:BackgroundGABA has important functions in brain plasticity related processes like memory, learning, locomotion and during the development of the nervous system. It is synthesized by the glutamic acid decarboxylase (GAD). There are two isoforms of GAD, GAD1 and GAD2, which are encoded by different genes. During embryonic development the transcription of GAD1 mRNA is regulated by alternative splicing and several alternative transcripts were distinguished in human, mouse and rat. Despite the fact that the structure of GAD1 gene has been extensively studied, knowledge of its exact structural organization, alternative promoter usage and splicing have remained incomplete.ResultsIn the present study we report the identification and characterization of novel GAD1 splicing isoforms (GenBank: KM102984, KM102985) by analyzing genomic and mRNA sequence data using bioinformatics, cloning and sequencing. Ten mRNA isoforms are generated from GAD1 gene locus by the combined actions of utilizing different promoters and alternative splicing of the coding exons. Using RT-PCR we found that GAD1 isoforms share similar pattern of expression in different mouse tissues and are expressed early during development. Quantitative RT-PCR was used to investigate the expression of GAD1 isoforms and GAD2 in olfactory bulb, cortex, medial and lateral striatum, hippocampus and cerebellum of adult mouse. Olfactory bulb showed the highest expression of GAD1 transcripts. Isoforms 1/2 are the most abundant forms. Their expression is significantly higher in the lateral compared to the medial striatum. Isoforms 3/4, 5/6, 7/8 and 9/10 are barely detectable in all investigated regions except of the high expression in olfactory bulb. When comparing GAD1 expression with GAD2 we found that Isoforms 1/2 are the predominant isoforms. In situ hybridization confirmed the predominant expression of Isoforms 7/8 and 9/10 in the olfactory bulb and revealed their weak expression in hippocampus, cerebellum and some other areas known to express GAD1.ConclusionsGeneration of ten splicing isoforms of GAD1 was described including two so far uncharacterized transcripts. GAD1 splicing isoforms producing the shorter, enzymatically inactive GAD25 protein are expressed at very low level in adult mouse brain except in the olfactory bulb that is associated with neurogenesis and synaptic plasticity even during adulthood.
| S-EPMC4295415 | biostudies-literature
Project description:BackgroundConvergent findings indicate that cortical gamma-aminobutyric acid (GABA)ergic circuitry is altered in schizophrenia. Postmortem studies have consistently found lower levels of glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA) in the prefrontal cortex (PFC) of subjects with schizophrenia. At the cellular level, the density of GABA neurons with detectable levels of GAD67 mRNA is ~30% lower across cortical layers. Knowing how this transcript deficit translates to GAD67 protein levels in axonal boutons is important for understanding the impact it might have on GABA synthesis. In addition, because reductions in GAD67 expression before, but not after, the maturation of GABAergic boutons results in a lower density of GABAergic boutons in mouse cortical cultures, knowing if GABAergic bouton density is altered in schizophrenia would provide insight into the timing of the GAD67 deficit.MethodsPFC tissue sections from 20 matched pairs of schizophrenia and comparison subjects were immunolabeled for the vesicular GABA transporter (vGAT) and GAD67.ResultsvGAT+ bouton density did not differ between subject groups, consistent with findings that vGAT mRNA levels are unaltered in the illness and confirming that the number of cortical GABAergic boutons is not lower in schizophrenia. In contrast, in schizophrenia subjects, the proportion of vGAT+ boutons with detectable GAD67 levels (vGAT+/GAD67+ boutons) was 16% lower and mean GAD67 levels were 14% lower in the remaining vGAT+/GAD67+ boutons.ConclusionsOur findings suggest that GABA production is markedly reduced in a subset of boutons in the PFC of schizophrenia subjects and that this reduction likely occurs after the maturation of GABAergic boutons.
| S-EPMC4744808 | biostudies-literature
Project description:Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD(67) expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5-22 micromol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD(67) expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD(67) promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 micromol/kg s.c.), an nAChR blocker that penetrates the blood-brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD(67) expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.
| S-EPMC2570996 | biostudies-literature
Project description: Not available
| S-EPMC2680820 | biostudies-literature
Project description:Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major ?-aminobutyric acid (GABA)-synthesizing enzyme. However, little is known about the relationship of prefrontal GAD67 mRNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function.Quantitative polymerase chain reaction was used to measure GAD67 mRNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no known history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocal immunofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are known to have low levels of GAD67 mRNA in schizophrenia.GAD67 mRNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%).The findings that lower GAD67 mRNA expression is common in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in schizophrenia.
| S-EPMC3273780 | biostudies-literature
Project description:BackgroundThe association of Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) common variants (rs4402960 and rs1470579) with type 2 diabetes (T2D) has been performed in different populations. The aim of this study was to evaluate the association of alternative variants of IGF2BP2; rs6777038, rs16860234 and rs7651090 with glutamic acid decarboxylase antibodies (GADA) negative diabetes in Malaysian Subjects.Methods/principal findingsIGF2BP2; rs6777038, rs16860234 and rs7651090 single nucleotide polymorphisms (SNPs) were genotyped in 1107 GADA negative diabetic patients and 620 control subjects of Asian from Malaysia. The additive genetic model adjusted for age, race, gender and BMI showed that alternative variants; rs6777038, rs16860234 and rs7651090 of IGF2BP2 associated with GADA negative diabetes (OR?=?1.21; 1.36; 1.35, P?=?0.03; 0.0004; 0.0002, respectively). In addition, the CCG haplotype and diplotype CCG-TCG increased the risk of diabetes (OR?=?1.51, P?=?0.01; OR?=?2.36, P?=?0.009, respectively).Conclusions/significanceIGF2BP2 alternative variants were associated with GADA negative diabetes. The IGF2BP2 haplotypes and diplotypes increased the risk of diabetes in Malaysian subject.
| S-EPMC3446917 | biostudies-literature