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Abstract 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.

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. Diseasefree 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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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). 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 0.05). 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.

Abstract Background and Objectives In a previous genome‐wide screening, we identified hypermethylated CpG islands around glutamate decarboxylase 1 (GAD1) in lung adenocarcinoma (LADC). In this study, we aimed to investigate the methylation and expression status of GAD1 and its prognostic value in patients with LADC. Methods GAD1 methylation and mRNA expression status were analyzed using 33 tumorous and paired non‐tumorous LADC samples and publicly available datasets. The prognostic value of GAD1 overexpression was investigated using publicly available datasets of mRNA levels and 162 cases of LADC by immunohistochemistry. Results The methylation and mRNA expression levels of GAD1, each having a positive correlation, were significantly higher in LADC tumors than in paired non‐tumorous tissues. LADC patients with higher GAD1 mRNA expression showed significantly poorer prognosis for overall survival in publicly available datasets. Higher immunoreactivity of GAD1 was significantly associated with the pathological stage, pleural invasion, lymph vessel invasion, and poorer prognosis for cancer‐specific and disease‐free survival. Multivariate analysis revealed that GAD1 protein overexpression is an independent prognosticator for disease‐free survival. Conclusions GAD1 mRNA and protein expression levels were significant prognostic factors in LADC, suggesting that they might be useful biomarkers to stratify patients with worse clinical outcomes after resection.

ObjectivesSchizophrenia (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 MethodsTwo 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.ResultsTen 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.ConclusionWe 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 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Developmental and epileptic encephalopathies are a heterogeneous group of early-onset epilepsy syndromes dramatically impairing neurodevelopment. Modern genomic technologies have revealed a number of monogenic origins and opened the door to therapeutic hopes. Here we describe a new syndromic developmental and epileptic encephalopathy caused by bi-allelic loss-of-function variants in GAD1, as presented by 11 patients from six independent consanguineous families. Seizure onset occurred in the first 2 months of life in all patients. All 10 patients, from whom early disease history was available, presented with seizure onset in the first month of life, mainly consisting of epileptic spasms or myoclonic seizures. Early EEG showed suppression-burst or pattern of burst attenuation or hypsarrhythmia if only recorded in the post-neonatal period. Eight patients had joint contractures and/or pes equinovarus. Seven patients presented a cleft palate and two also had an omphalocele, reproducing the phenotype of the knockout Gad1-/- mouse model. Four patients died before 4 years of age. GAD1 encodes the glutamate decarboxylase enzyme GAD67, a critical actor of the γ-aminobutyric acid (GABA) metabolism as it catalyses the decarboxylation of glutamic acid to form GABA. Our findings evoke a novel syndrome related to GAD67 deficiency, characterized by the unique association of developmental and epileptic encephalopathies, cleft palate, joint contractures and/or omphalocele. [ABSTRACT FROM AUTHOR]

The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington's and Alzheimer's diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders. [ABSTRACT FROM AUTHOR]

Background and Objectives: In a previous genome‐wide screening, we identified hypermethylated CpG islands around glutamate decarboxylase 1 (GAD1) in lung adenocarcinoma (LADC). In this study, we aimed to investigate the methylation and expression status of GAD1 and its prognostic value in patients with LADC. Methods: GAD1 methylation and mRNA expression status were analyzed using 33 tumorous and paired non‐tumorous LADC samples and publicly available datasets. The prognostic value of GAD1 overexpression was investigated using publicly available datasets of mRNA levels and 162 cases of LADC by immunohistochemistry. Results: The methylation and mRNA expression levels of GAD1, each having a positive correlation, were significantly higher in LADC tumors than in paired non‐tumorous tissues. LADC patients with higher GAD1 mRNA expression showed significantly poorer prognosis for overall survival in publicly available datasets. Higher immunoreactivity of GAD1 was significantly associated with the pathological stage, pleural invasion, lymph vessel invasion, and poorer prognosis for cancer‐specific and disease‐free survival. Multivariate analysis revealed that GAD1 protein overexpression is an independent prognosticator for disease‐free survival. Conclusions: GAD1 mRNA and protein expression levels were significant prognostic factors in LADC, suggesting that they might be useful biomarkers to stratify patients with worse clinical outcomes after resection. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Schizophrenia is a devastating neurodevelopmental disorder that affects approximately 1% of the population. Reduced expression of the 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of the disease and is encoded by the GAD1 gene. In schizophrenia, GAD67 downregulation occurs in multiple interneuronal subpopulations, including the cannabinoid receptor type 1 positive (CNR1+) cells, but the functional consequences of these disturbances are not well understood. To investigate the role of the CNR1-positive GABA-ergic interneurons in behavioral and molecular processes, we employed a novel, miRNA-mediated transgenic mouse approach. We silenced the Gad1 transcript using a miRNA engineered to specifically target Gad1 mRNA under the control of Cnr1 bacterial artificial chromosome. Behavioral characterization of our transgenic mice showed elevated and persistent conditioned fear associated with an auditory cue and a significantly altered response to an amphetamine challenge. These deficits could not be attributed to sensory deficits or changes in baseline learning and memory. Furthermore, HPLC analyses revealed that Cnr1/Gad1 mice have enhanced serotonin levels, but not dopamine levels in response to amphetamine. Our findings demonstrate that dysfunction of a small subset of interneurons can have a profound effect on behavior and that the GABA-ergic, monoamine, and cannabinoid systems are functionally interconnected. The results also suggest that understanding the function of various interneuronal subclasses might be essential to develop knowledge-based treatment strategies for various mental disorders including schizophrenia and substance abuse.

The type of neuronal activity determines the outcome of gene expression. Hence, the characterization of underlying mechanisms in transcriptome alterations may serve as a biomarker and provide new intervention methods for the treatment of pathologic conditions. Parrish et al. (Parrish RR, Codadu NK, Racca C, Trevelyan AJ. J Neurophysiol 120: 2358-2367, 2018) show that the changes in interneuronal gene transcription are correlated with the type of the activated neuronal population and that the initiation route of Ras/ERK MAPK pathway determines the polarity of the gene expression. [ABSTRACT FROM AUTHOR]

The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula, and other brain regions, in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptors mRNA were not detected, using the same method. Water-deprivation for 24 hrs, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze test, and this effect was mimicked by bilateral microinfusion of VP into the CeA. Anxious behavior induced by either water deprivation or VP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of central amygdala inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.

The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula and other brain regions in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala. [ABSTRACT FROM AUTHOR]

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., Competing Interests: J.B.S. and J.T.B. declare that they own stock in and are employees of REGENXBIO, Inc., (© 2023 The Authors.)

Background: GABA 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. Results: In 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. Conclusions: Generation 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. [ABSTRACT FROM AUTHOR]