Association of GAD1 gene polymorphism rs3749034 with the information processing and cognitive event-related potentials in schizophrenia patients and healthy subjects.

OBJECTIVE: Glutamic acid decarboxylase, an enzyme in GABA biosynthesis, is encoded by the GAD1 gene, the transcriptional activity of which is affected by the rs3749034 polymorphism. The aim was to investigate the effects of rs3749034 on cognitive event-related potentials (P300) in healthy subjects and schizophrenic patients. METHODS: Determination of rs3749034 polymorphism was performed in 89 healthy volunteers and 109 schizophrenic patients (males). Two-stimulus oddball task performance and P300 auditory evoked potentials were analyzed and patient symptomatology was assessed using the Positive and Negative Syndrome Scale (PANSS). RESULTS: An increased frequency of C allele carriers was disclosed in patients. In controls, superior task performance was observed in cytosine-thymine carriers, while a greater P300 amplitude and shorter latency were found in C/C carriers. Analogous effects were found in patients with a disease onset before 25 years of age. Higher N5 and lower P3 and G5 PANSS scales were revealed in C/C homozygotes. CONCLUSIONS: The findings substantiate an involvement of GABA-ergic mechanisms in maintaining an optimal excitatory-inhibitory balance and an association of rs3749034 with early-onset disorder and negative symptoms of schizophrenia. SIGNIFICANCE: These results are important for understanding underlying mechanisms and the development of evidence-based methods for assessing the risk of schizophrenia.

Glucose transporter-2 regulation of VMN GABA neuron metabolic sensor and transmitter gene expression.

Glucose transporter-2 (GLUT2) monitors cellular glucose uptake. Astrocyte GLUT2 controls glucose counterregulatory hormone secretion. In vivo gene silencing and laser-catapult-microdissection tools were used here to investigate whether ventromedial hypothalamic nucleus (VMN) GLUT2 may regulate dorsomedial (VMNdm) and/or ventrolateral (VMNvl) γ-aminobutyric acid (GABA) neurotransmission to control this endocrine outflow in female rats. VMN GLUT2 gene knockdown suppressed or stimulated hypoglycemia-associated glutamate decarboxylase (GAD)1 and GAD2 mRNA expression in VMNdm versus VMNvl GABAergic neurons, respectively. GLUT2 siRNA pretreatment also modified co-expressed transmitter marker gene profiles in each cell population. VMNdm GABA neurons exhibited GLUT2 knockdown-sensitive up-regulated 5'-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) transcripts during hypoglycemia. Hypoglycemic augmentation of VMNvl GABA neuron AMPKα2 was refractory to GLUT2 siRNA. GLUT2 siRNA blunted (VMNdm) or exacerbated (VMNvl) hypoglycemic stimulation of GABAergic neuron steroidogenic factor-1 (SF-1) mRNA. Results infer that VMNdm and VMNvl GABA neurons may exhibit divergent, GLUT2-dependent GABA neurotransmission patterns in the hypoglycemic female rat. Data also document differential GLUT2 regulation of VMNdm versus VMNvl GABA nerve cell SF-1 gene expression. Evidence for intensification of hypoglycemic hypercorticosteronemia and -glucagonemia by GLUT2 siRNA infers that VMN GLUT2 function imposes an inhibitory tone on these hormone profiles in this sex.

Age-related Changes of GAD1 mRNA Expression in the Central Inferior Colliculus.

Encoding sounds with a high degree of temporal precision is an essential task for the inferior colliculus (IC) to perform and maintain the accurate processing of sounds and speech. However, the age-related reduction of GABAergic neurotransmission in the IC interrupts temporal precision and likely contributes to presbycusis. As presbycusis often manifests at high or low frequencies specifically, we sought to determine if the expression of mRNA for glutamic decarboxylase 1 (GAD1) is downregulated non-uniformly across the tonotopic axis or cell size range in the aging IC. Using single molecule in situ fluorescent hybridization across young, middle age and old Fisher Brown Norway rats (an aging model that acquires low frequency presbycusis) we quantified individual GAD1 mRNA in small, medium and large GABAergic cells. Our results demonstrate that small GABAergic cells in low frequency regions had ~58% less GAD1 in middle age and continued to decline into old age. In contrast, the amount of GAD1 mRNA in large cells in low frequency regions significantly increased with age. As several studies have shown that downregulation of GAD1 decreases the release of GABA, we interpret our results in two ways. First, the onset of presbycusis may be driven by small GABAergic cells downregulating GAD1. Second, as previous studies demonstrate that GAD67 expression is broadly downregulated in the old IC, perhaps the translation of GAD1 to GAD67 is interrupted in large GABAergic IC cells during aging. These results point to a potential genetic mechanism explaining reduced temporal precision in the aging IC, and in turn, presbycusis.

Risk Stratification by Tissue GAD1 Expression Level in Curatively Resected Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: To improve patient management, new biomarkers are required that stratify prognosis. Here we focused on glutamic acid decarboxylase 1 (GAD1), which is associated with proliferation of lung cancer cells, and investigated its expression and function in esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: We evaluated changes in the proliferative potential of ESCC cell lines using small interfering RNA-mediated GAD1 knockdown techniques. We analyzed GAD1 protein expression using a tissue microarray (TMA) and measured GAD1 mRNA expression to evaluate correlations between the expression level of each tissue and postoperative outcomes of two independent cohorts (the TMA and mRNA cohorts) of patients who underwent radical esophagectomy. RESULTS: GAD1 knockdown reduced cell proliferation. In the TMA cohort, high GAD1 expression significantly correlated with lymph node metastasis and advanced stage. Disease-free survival was significantly shorter in the group with high GAD1 expression, as was overall survival. Multivariate analysis of overall survival showed that positivity for GAD1 was an independent prognostic factor for poor survival. In the mRNA cohort, GAD1 mRNA expression in ESCC tissues was significantly up-regulated compared with that in adjacent noncancerous mucosal tissues. When patients were divided into high- and low-expression groups according to the median GAD1 mRNA expression level in ESCC tissues, overall survival was significantly shortened in the high GAD1 expression group. The incidence of initial hematogenous recurrence was significantly higher in the group with high GAD1 expression. CONCLUSION: GAD1 expression mediates the proliferative potential of ESCC cells, and a high level may serve as a useful prognostic biomarker for patients with ESCC.

GAD1 contributes to the progression and drug resistance in castration resistant prostate cancer.

BACKGROUND: Prostate cancer is currently the second most lethal malignancy in men worldwide due to metastasis and invasion in advanced stages. Studies have revealed that androgen deprivation therapy can induce stable remission in patients with advanced prostate cancer, although most patients will develop castration-resistant prostate cancer (CRPC) in 1-2 years. Docetaxel and enzalutamide improve survival in patients with CRPC, although only for a short time, eventually patients develop primary or secondary resistance, causing disease progression or biochemical relapse. METHODS: The gene expression profiles of docetaxel-sensitive or -resistant prostate cancer cell lines, namely GSE33455, GSE36135, GSE78201, GSE104935, and GSE143408, were sequentially analyzed for differentially expressed genes and progress-free interval significance. Subsequently, the overall survival significance and clinic-pathological features were analyzed by the R package. The implications of hub genes mutations, methylation in prostate cancer and the relationship with the tumor immune cell infiltration microenvironment were assessed with the help of cBioPortal, UALCAN and TISIDB web resources. Finally, effects of the hub genes on the progression and drug resistance in prostate cancer were explored using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, cell phenotype, and drug sensitivity. RESULT: Glutamate decarboxylase 1 (GAD1) was tentatively identified by bioinformatic analysis as an hub gene for the development of drug resistance, including docetaxel and enzalutamide, in prostate cancer. Additionally, GAD1 expression, mutation and methylation were significantly correlated with the clinicopathological features and the tumor immune microenvironment. RT-PCR, immunohistochemistry, cell phenotype and drug sensitivity experiments further demonstrated that GAD1 promoted prostate cancer progression and decreased the therapeutic effect of docetaxel or enzalutamide. CONCLUSION: This research confirmed that GAD1 was a hub gene in the progression and development of drug resistance in prostate cancer. This helped to explain prostate cancer drug resistance and provides new immune-related therapeutic targets and biomarkers for it.

GAD1-mediated GABA elicits aggressive characteristics of human oral cancer cells.

Studies suggest that the expression of glutamate decarboxylase 1 (GAD1), γ-aminobutyric acid (GABA), and GABA receptors are involved in tumor progression. However, the underlying mechanisms of high expression and potential functions of GAD1 and GABA in oral squamous cell carcinoma (OSCC) are not known. In this study, we found that the expressions of GAD1 and GABA were considerably increased in OSCC samples, which were closely associated with clinical stage and lymph node metastasis. The knockdown of GAD1 expression significantly inhibited the proliferation, migration and invasion abilities of OSCC cells by reducing the expression of GABA-mediated GABAB receptors, which could be reversed by exogenous GABA, but did not cause excessive OSCC cell proliferation. And GABA secreted by OSCC cells promoted M2 macrophage polarization for inhibiting anti-tumor immunity by activating GABBR1/ERK/Ca2+. In addition, GABA/GABABR promoted the proliferation and progression of OSCC xenograft tumor. Altogether, our results showed that GAD1 synthetized GABA to promote the malignant progression of OSCC and limits the anti-tumor immunity of macrophages, thereby targeting GABA can be a novel strategy for treating OSCC.

Divergent Transcriptomic Effects of Allopregnanolone in Postpartum Depression.

Brexanolone, a formulation of the neurosteroid allopregnanolone (ALLO), is approved for treating postpartum depression (PPD) and is being investigated for therapeutic efficacy across numerous neuropsychiatric disorders. Given ALLO's beneficial effects on mood in women with PPD compared to healthy control women, we sought to characterize and compare the cellular response to ALLO in women with (n = 9) or without (n = 10, i.e., Controls) past PPD, utilizing our previously established patient-derived lymphoblastoid cell lines (LCLs). To mimic in vivo PPD ALLO-treatment, LCLs were exposed to ALLO or DMSO vehicle for 60 h and RNA-sequenced to detect differentially expressed genes (DEGs, pnominal < 0.05). Between ALLO-treated Control and PPD LCLs, 269 DEGs were identified, including Glutamate Decarboxylase 1 (GAD1), which was decreased 2-fold in PPD. Network analysis of PPD:ALLO DEGs revealed enriched terms related to synaptic activity and cholesterol biosynthesis. Within-diagnosis analyses (i.e., DMSO vs. ALLO) detected 265 ALLO-induced DEGs in Control LCLs compared to only 98 within PPD LCLs, with just 11 DEGs overlapping. Likewise, the gene ontologies underlying ALLO-induced DEGs in PPD and Control LCLs were divergent. These data suggest that ALLO may activate unique and opposing molecular pathways in women with PPD, which may be tied to its antidepressant mechanism.

Developmental and epileptic encephalopathy 89: A novel bi-allelic variant, molecular dynamics simulation, and a comprehensive clinical and molecular profile.

OBJECTIVE: Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult central nervous system, plays an important role during embryonic neural network formation. GAD67 is the rate-limiting enzyme in GABA synthesis, and its deficiency leads to developmental and epileptic encephalopathy 89 (DEE 89). Patients who suffered from this syndrome generally manifested severe to profound neurodevelopmental delay, seizures, and often congenital anomalies such as the cleft palate or/and omphalocele. Up to now, only three papers on this syndrome have been published, and our knowledge about the disease's clinical course and pathophysiology is in its infancy. METHODS: We used whole-exome sequencing (WES) and multiple in-silico tools to detect a potential causative variant in a patient with severe neurodevelopmental delay and refractory epilepsy. Moreover, by molecular docking and molecular dynamics simulation, we investigate the effect of the candidate variant on the GAD67 function and structure. RESULTS: WES data analysis revealed a novel deleterious variant (NM_000817.3: c.850C>T; p.Leu284Phe) in the GAD1 gene, which encodes the GAD67 enzyme. Molecular docking and molecular dynamics simulation showed that this variant has deleterious effects on the structure and function of the GAD67. This study's patient, in addition to typical symptoms of the DEE89, showed microcephaly and clonus in the toe, which were novel clinical findings. SIGNIFICANCE: Our findings expand the mutational and clinical spectrum of DEE 89. Also, by gathering clinical symptoms and genetic findings of previously reported cases, moreover providing a comprehensive clinical picture of the disease, we found that there was no common drug therapy among patients whose epilepsy was controlled. Furthermore, the comparison of clinical symptoms between patients with missense and truncating mutations did not show any significant clinical difference, except that patients with missense mutations did not show cleft palates or omphaloceles.

TorC1 and nitrogen catabolite repression control of integrated GABA shunt and retrograde pathway gene expression.

Despite our detailed understanding of how the lower GABA shunt and retrograde genes are regulated, there is a paucity of validated information concerning control of GAD1, the glutamate decarboxylase gene which catalyzes the first reaction of the GABA shunt. Further, integration of glutamate degradation via the GABA shunt has not been investigated. Here, we show that while GAD1 shares a response to rapamycin-inhibition of the TorC1 kinase, it does so independently of the Gln3 and Gat1 NCR-sensitive transcriptional activators that mediate transcription of the lower GABA shunt genes. We also show that GABA shunt gene expression increases dramatically in response to nickel ions. The α-ketoglutarate needed for the GABA shunt to cycle, thereby producing reduced pyridine nucleotides, derives from the retrograde pathway as shown by a similar high increase in the retrograde reporter, CIT2 when nickel is present in the medium. These observations demonstrate high integration of the GABA shunt, retrograde, peroxisomal glyoxylate cycle, and ß-oxidation pathways.

In silico prediction and in vivo testing of promoters targeting GABAergic inhibitory neurons.

Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. Recombinant adeno-associated virus (rAAV)-based gene therapy targeting GABAergic neurons is a promising treatment for GABA-associated disorders. However, there is a need to develop rAAV-compatible gene-regulatory elements capable of selectively driving expression in GABAergic neurons throughout the brain. Here, we designed several novel GABAergic gene promoters. In silico analyses, including evolutionarily conserved DNA sequence alignments and transcription factor binding site searches among GABAergic neuronal genes, were carried out to reveal novel sequences for use as rAAV-compatible promoters. rAAVs (serotype 9) were injected into the CSF of neonatal mice and into the brain parenchyma of adult mice to assess promoter specificity. In mice injected neonatally, transgene expression was detected in multiple brain regions with very high neuronal specificity and moderate-to-high GABAergic neuronal selectivity. The GABA promoters differed greatly in their levels of expression and, in some brain regions, showed strikingly different patterns of GABAergic neuron transduction. This study is the first report of rAAV vectors that are functional in multiple brain regions using promoters designed by in silico analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.

Predator odor (TMT) exposure potentiates interoceptive sensitivity to alcohol and increases GABAergic gene expression in the anterior insular cortex and nucleus accumbens in male rats.

Post-traumatic stress disorder (PTSD) confers enhanced vulnerability to developing comorbid alcohol use disorder (AUD). Exposure to the scent of a predator, such as the fox odor TMT, has been used to model a traumatic stressor with relevance to PTSD symptomatology. Alcohol produces distinct interoceptive (subjective) effects that may influence vulnerability to problem drinking and AUD. As such, understanding the lasting impact of stressors on sensitivity to the interoceptive effects of alcohol is clinically relevant. The present study used a 2-lever, operant drug discrimination procedure to train male Long-Evans rats to discriminate the interoceptive effects of alcohol (2 g/kg, i.g. [intragastrically]) from water. Upon stable performance, rats underwent a 15-min exposure to TMT. Two weeks later, an alcohol dose-response curve was conducted to evaluate the lasting effects of the TMT stressor on the interoceptive effects of alcohol. The TMT group showed a leftward shift in the effective dose (ED50) of the dose-response curve compared to controls, reflecting potentiated interoceptive sensitivity to alcohol. TMT exposure did not affect response rate. GABAergic signaling in both the anterior insular cortex (aIC) and the nucleus accumbens (Acb) is involved in the interoceptive effects of alcohol and stressor-induced adaptations. As such, follow-up experiments in alcohol-naïve rats examined neuronal activation (as measured by c-Fos immunoreactivity) following TMT and showed that TMT exposure increased c-Fos expression in the aIC and the nucleus accumbens core (AcbC). Two weeks after TMT exposure, Gad-1 gene expression was elevated in the aIC and Gat-1 was increased in the Acb, compared to controls. Lastly, the alcohol discrimination and alcohol-naïve groups displayed dramatic differences in stress reactive behaviors during the TMT exposure, suggesting that alcohol exposure may alter the behavioral response to predator odor. Together, these data suggest that predator odor stressor results in potentiated sensitivity to alcohol, possibly through GABAergic adaptations in the aIC and Acb, which may be relevant to understanding PTSD-AUD comorbidity.

GAD1 alleviates injury-induced optic neurodegeneration by inhibiting retinal ganglion cell apoptosis.

The degeneration of the optic nerve narrows the visual field, eventually causing overall vision loss. This study aimed to identify global protein changes in the retina of optic nerve crushing (ONC) mice and to identify key regulators and pathways involved in injury-induced cell death during the progression of optic neurodegeneration. Label-free quantitative proteomics combined with bioinformatic analysis was performed on retinal protein extracts from ONC and sham-operated mice. Among the 1433 proteins detected, 121 proteins were differentially expressed in the retina of ONC mice. Further bioinformatic analysis showed that various metabolic pathways, including glutamate metabolism and γ-aminobutyric acid (GABA) synthesis, were significantly dysregulated in the injured mouse retinas. Glutamate decarboxylase 1 (GAD1) is the enzyme that converts glutamate into GABA, which was significantly up-regulated during ONC injury. Exogenous GAD1 treatment increased retinal ganglion cell (RGC) survival in the ONC-injured retina. In addition, changes in GAD1 expression were also observed in several other ophthalmic diseases. Vascular endothelial growth factor B (VEGF-B) has previously been reported to protect RGCs from apoptosis and positively regulated the expression of GAD1 in the retina. Notably, combination treatment with GAD1 and VEGF-B also provided strong protection against injury-induced RGC apoptosis. These results suggest that GAD1 expression may serve as an intrinsic protective mechanism that is commonly activated during retinal injury. Targeting GAD1 may serve as a potential strategy to treat optic neurodegenerative diseases.

DNA Methylation Profiles of GAD1 in Human Cerebral Organoids of Autism Indicate Disrupted Epigenetic Regulation during Early Development.

DNA methylation profiling has become a promising approach towards identifying biomarkers of neuropsychiatric disorders including autism spectrum disorder (ASD). Epigenetic markers capture genetic risk factors and diverse exogenous and endogenous factors, including environmental risk factors and complex disease pathologies. We analysed the differential methylation profile of a regulatory region of the GAD1 gene using cerebral organoids generated from induced pluripotent stem cells (iPSCs) from adults with a diagnosis of ASD and from age- and gender-matched healthy individuals. Both groups showed high levels of methylation across the majority of CpG sites within the profiled GAD1 region of interest. The ASD group exhibited a higher number of unique DNA methylation patterns compared to controls and an increased CpG-wise variance. We detected six differentially methylated CpG sites in ASD, three of which reside within a methylation-dependent transcription factor binding site. In ASD, GAD1 is subject to differential methylation patterns that may not only influence its expression, but may also indicate variable epigenetic regulation among cells.

Profile of miR-23 Expression and Possible Role in Regulation of Glutamic Acid Decarboxylase during Postnatal Retinal Development.

As neurotransmitter, GABA is fundamental for physiological processes in the developing retina. Its synthesis enzymes are present during retinal development, although the molecular regulatory mechanisms behind the changes in expression are not entirely understood. In this study, we revealed the expression patterns of glutamic acid decarboxylase 67(GAD67) and its coding gene (GAD1) and its potential miRNA-dependent regulation during the first three postnatal weeks in rat retina. To gain insight into the molecular mechanisms, miRNA-sequencing supported by RT-qPCR and in situ hybridization were carried out. GAD1 expression shows an increasing tendency, peaking at P15. From the in silico-predicted GAD1 targeting miRNAs, only miR-23 showed similar expression patterns, which is a known regulator of GAD1 expression. For further investigation, we made an in situ hybridization investigation where both GAD67 and miR-23 also showed lower expression before P7, with the intensity of expression gradually increasing until P21. Horizontal cells at P7, amacrine cells at P15 and P21, and some cells in the ganglion cell layer at several time points were double labelled with miR-23 and GAD67. Our results highlight the complexity of these regulatory networks and the possible role of miR-23 in the regulation of GABA synthesizing enzyme expression during postnatal retina development.

Weak Association Between the Glutamate Decarboxylase 1 Gene (GAD1) and Schizophrenia in Han Chinese Population.

OBJECTIVES: Schizophrenia (SZ) is a complex psychiatric disorder with high heritability, and genetic components are thought to be pivotal risk factors for this illness. The glutamate decarboxylase 1 gene (GAD1) was hypothesized to be a candidate risk locus for SZ given its crucial role in the GABAergic neurotransmission system, and previous studies have examined the associations of single nucleotide polymorphisms (SNPs) spanning the GAD1 gene with SZ. However, inconsistent results were obtained. We hence examined the associations between GAD1 SNPs and SZ in two independent case-control samples of Han Chinese ancestry. MATERIALS AND METHODS: Two Han Chinese SZ case-control samples, referred as the discovery sample and the replication sample, respectively, were recruited for the current study. The discovery sample comprised of 528 paranoid SZ cases (with age of first onset ≥ 18) and 528 healthy controls; the independent replication sample contained 1,256 early onset SZ cases (with age of first onset < 18) and 2,661 healthy controls. Logistic regression analysis was performed to examine the associations between GAD1 SNPs and SZ. RESULTS: Ten SNPs covering GAD1 gene were analyzed in the discovery sample, and two SNPs showed nominal associations with SZ (rs2241165, P = 0.0181, OR = 1.261; rs2241164, P = 0.0225, OR = 1.219). SNP rs2241164 was also nominally significant in the independent replication sample (P = 0.0462, OR = 1.110), and the significance became stronger in a subsequent meta-analysis combining both discovery and replication samples (P = 0.00398, OR = 1.138). Nevertheless, such association could not survive multiple corrections, although the effect size of rs2241164 was comparable with other SZ risk loci identified in genome-wide association studies (GWAS) in Han Chinese population. We also examined the associations between GAD1 SNPs and SZ in published datasets of SZ GWAS in East Asians and Europeans, and no significant associations were observed. CONCLUSION: We observed weak associations between GAD1 SNPs and risk of SZ in Han Chinese populations. Further analyses in larger Han Chinese samples with more detailed phenotyping are necessary to elucidate the genetic correlation between GAD1 SNPs and SZ.

Reductions in midbrain GABAergic and dopamine neuron markers are linked in schizophrenia.

Reductions in the GABAergic neurotransmitter system exist across multiple brain regions in schizophrenia and encompass both pre- and postsynaptic components. While reduced midbrain GABAergic inhibitory neurotransmission may contribute to the hyperdopaminergia thought to underpin psychosis in schizophrenia, molecular changes consistent with this have not been reported. We hypothesised that reduced GABA-related molecular markers would be found in the midbrain of people with schizophrenia and that these would correlate with dopaminergic molecular changes. We hypothesised that downregulation of inhibitory neuron markers would be exacerbated in schizophrenia cases with high levels of neuroinflammation. Eight GABAergic-related transcripts were measured with quantitative PCR, and glutamate decarboxylase (GAD) 65/67 and GABAA alpha 3 (α3) (GABRA3) protein were measured with immunoblotting, in post-mortem midbrain (28/28 and 28/26 control/schizophrenia cases for mRNA and protein, respectively), and analysed by both diagnosis and inflammatory subgroups (as previously defined by higher levels of four pro-inflammatory cytokine transcripts). We found reductions (21 - 44%) in mRNA encoding both presynaptic and postsynaptic proteins, vesicular GABA transporter (VGAT), GAD1, and parvalbumin (PV) mRNAs and four alpha subunits (α1, α2, α3, α5) of the GABAA receptor in people with schizophrenia compared to controls (p < 0.05). Gene expression of somatostatin (SST) was unchanged (p = 0.485). We confirmed the reduction in GAD at the protein level (34%, p < 0.05). When stratifying by inflammation, only GABRA3 mRNA exhibited more pronounced changes in high compared to low inflammatory subgroups in schizophrenia. GABRA3 protein was expressed by 98% of tyrosine hydroxylase-positive neurons and was 23% lower in schizophrenia, though this did not reach statistical significance (p > 0.05). Expression of transcripts for GABAA receptor alpha subunits 2 and 3 (GABRA2, GABRA3) were positively correlated with tyrosine hydroxylase (TH) and dopamine transporter (DAT) transcripts in schizophrenia cases (GABRA2; r > 0.630, GABRA3; r > 0.762, all p < 0.001) but not controls (GABRA2; r < - 0.200, GABRA3; r < 0.310, all p > 0.05). Taken together, our results support a profound disruption to inhibitory neurotransmission in the substantia nigra regardless of inflammatory status, which provides a potential mechanism for disinhibition of nigrostriatal dopamine neurotransmission.

GAD1 expression and its methylation as indicators of malignant behavior in thymic epithelial tumors.

Thymic epithelial tumors (TETs) comprise thymomas and thymic carcinoma (TC). TC has more aggressive features and a poorer prognosis than thymomas. Genetic and epigenetic alterations in thymomas and TC have been investigated in an attempt to identify novel target molecules for TC. In the present study, genome-wide screening was performed on aberrantly methylated CpG islands in thymomas and TC, and the glutamate decarboxylase 1 gene (GAD1) was identified as the 4th significantly hypermethylated CpG island in TC compared with thymomas. GAD1 catalyzes the production of γ-aminobutyric acid from L-glutamic acid. GAD1 expression is abundant in the brain but rare in other tissues, including the thymus. A total of 73 thymomas and 17 TC tissues were obtained from 90 patients who underwent surgery or biopsy at Tokushima University Hospital between 1990 and 2017. DNA methylation was examined by bisulfite pyrosequencing, and the mRNA and protein expression levels of GAD1 were analyzed using reverse transcription-quantitative PCR and immunohistochemistry, respectively. The DNA methylation levels of GAD1 were significantly higher in TC tissues than in the normal thymus and thymoma tissues, and GAD1 methylation exhibited high sensitivity and specificity for discriminating between TC and thymoma. The mRNA and protein expression levels of GAD1 were significantly higher in TC tissues than in thymomas. Patients with TET with high GAD1 DNA hypermethylation and high mRNA and protein expression levels had significantly shorter relapse-free survival rates than those with low levels. In conclusion, significantly more epigenetic alterations were observed in TC tissues compared with in thymomas, which may contribute to the clinical features and prognosis of patients.

Epigenetic Regulation of Neural Transmission after Cerebellar Fastigial Nucleus Lesions in Juvenile Rats.

Structural and functional abnormalities in the cerebellar midline region, including the fastigial nucleus, have been reported in neuropsychiatric disorders, also comprising the cerebellar cognitive affecting syndrome. In rats, early fastigial lesions reduce social interaction during development and lead to cognitive and emotional deficits in adults, accompanied by compromised neuronal network activity. Since epigenetic mechanisms are implicated in the etiology of neuropsychiatric disorders, we investigated whether fastigial nucleus lesions in juvenile rats would impact epigenetic regulation of neural transmission. The fastigial nucleus was lesioned bilaterally in 23-day-old male rats. Sham-lesion and naïve rats served as controls. DNA methylation was investigated for target genes of the GABAergic, dopaminergic, glutamatergic and oxytocinergic systems in brain regions with anatomic connections to the fastigial nucleus, i.e., medial prefrontal cortex, nucleus accumbens, striatum, thalamus, and sensorimotor cortex. Protein expression was examined for the respective target genes in case of altered DNA methylation between lesion and control groups. Lesioning of the fastigial nucleus led to significant differences in the epigenetic regulation of glutamate decarboxylase 1 and the oxytocin receptor in the nucleus accumbens and the prefrontal cortex. No differences were found for the other target genes and brain regions. Our findings indicate that epigenetic dysregulation after lesioning of the fastigial nucleus may influence long-term recovery and the emergence of behavioral changes. Together with previous behavioral and electrophysiological investigations of this rat model, these observations can play a role in the cerebellar cognitive affective syndrome and other neuropsychiatric disorders.

ACC Glu/GABA ratio is decreased in euthymic bipolar disorder I patients: possible in vivo neurometabolite explanation for mood stabilization.

Bipolar disorder (BD) is characterized by unstable mood states ranging from mania to depression. Although there is some evidence that mood instability may result from an imbalance between excitatory glutamatergic and inhibitory GABA-ergic neurotransmission, few proton magnetic resonance spectroscopy (1H-MRS) studies have measured these two neurometabolites simultaneously in BD. The enzyme glutamic acid decarboxylase (GAD1) catalyzes the decarboxylation of glutamate (Glu) to GABA, and its single nucleotide polymorphisms (SNPs) might influence Glu/GABA ratio. Thus, we investigated Glu/GABA ratio in the dorsal anterior cingulate cortex (dACC) of euthymic BD type I patients and healthy controls (HC), and assessed the influence of both mood stabilizers and GAD1 SNPs on this ratio. Eighty-eight subjects (50 euthymic BD type I patients and 38 HC) underwent 3T 1H-MRS in the dACC (2 × 2 × 4.5 cm3) using a two-dimensional JPRESS sequence and all subjects were genotyped for 4 SNPs in the GAD1 gene. BD patients had lower dACC Glu/GABA ratio compared to HC, where this was influenced by anticonvulsant and antipsychotic medications, but not lithium. The presence of GAD1 rs1978340 allele A was associated with higher Glu/GABA ratio in BD, while patients without this allele taking mood stabilizers had a lower Glu/GABA ratio. The lowering of dACC Glu/GABA could be one explanation for the mood stabilizing action of anticonvulsants and antipsychotics in BD type I euthymia. Therefore, this putative role of Glu/GABA ratio and the influence of GAD1 genotype interacting with mood stabilization medication should be confirmed by further studies involving larger samples and other mood states.ClincalTrials.gov registration: NCT01237158.

Early-infantile onset epilepsy and developmental delay caused by bi-allelic GAD1 variants.

Gamma-aminobutyric acid (GABA) and glutamate are the most abundant amino acid neurotransmitters in the brain. GABA, an inhibitory neurotransmitter, is synthesized by glutamic acid decarboxylase (GAD). Its predominant isoform GAD67, contributes up to ∼90% of base-level GABA in the CNS, and is encoded by the GAD1 gene. Disruption of GAD1 results in an imbalance of inhibitory and excitatory neurotransmitters, and as Gad1-/- mice die neonatally of severe cleft palate, it has not been possible to determine any potential neurological dysfunction. Furthermore, little is known about the consequence of GAD1 disruption in humans. Here we present six affected individuals from six unrelated families, carrying bi-allelic GAD1 variants, presenting with developmental and epileptic encephalopathy, characterized by early-infantile onset epilepsy and hypotonia with additional variable non-CNS manifestations such as skeletal abnormalities, dysmorphic features and cleft palate. Our findings highlight an important role for GAD1 in seizure induction, neuronal and extraneuronal development, and introduce GAD1 as a new gene associated with developmental and epileptic encephalopathy.