Добірка наукової літератури з теми "GAD67 gene"

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in β-cell destruction and immune regulation. A major target of β-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of β-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1β (1 ng/ml, 24 h), tumour necrosis factor α (TNF-α; 200 units/ml, 24 h) or interferon γ (IFN-γ; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-α (1000 units/ml), TNF-β (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor β2 (TGF-β2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1β, TNF-α or IFN-γ was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1β and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1β, TNF-α or IFN-γ by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of β-cell destruction in IDDM.

γ-Aminobutyric acid is synthesized by glutamic acid decarboxylase (GAD), which has two forms, GAD65 and GAD67. Genomic clones coding mouse GAD67 (mGAD67) have been isolated. The restriction map of the overlapping clones covers a region of more than 45 kb of genomic DNA. The mGAD67 gene contains 16 translated exons in addition to an exon which is preferentially expressed in foetal brain. The rapid amplification of 5′-cDNA ends showed that mGAD67 gene transcripts have two different 5′-untranslated regions. Analysis of the genomic clones encompassing the 5′-exons revealed that the two transcripts arose from a single gene by alternative splicing using two different donor sites and a common acceptor. The exons were found 1.5 and 0.6 kb upstream of exon 1. The corresponding promoter regions of these exons have a number of putative regulatory elements, including Sp1- and Krox-24-binding sites. Analysis of mGAD67 transcripts demonstrated that each of the 5′-untranslated exons was expressed in mouse brain. In contrast, exon 0A, but not exon 0B, was expressed in mouse testis and pancreas. These results suggest that these transcripts may be regulated under the control of independent promoters.

Epigenetics is a gene–environment interaction mechanism, manifested mostly through changes in regulatory gene expression. Stress is an established environmental factor known to induce epigenetic changes. This study aimed to assess the long-term effect of stress as juveniles, or juvenile and adult stress, on alterations in glutamic acid decarboxylase genes (GAD65, GAD67). We assessed DNA methylation and RNA expression in four rat groups: (1) control group, (2) juvenile stress group sacrificed two days following stress exposure (JSe) (RNA only), (3) juvenile stress group sacrificed as adults (JS), and (4) juvenile and adult stress group (JS + AS). Three different areas of the brain were examined in each group: the dorsal dentate gyrus (dDG), the dorsal CA1 (dCA1), and the basolateral amygdala (BLA). A significantly low methylation level of GAD65 in the BLA was observed among the JS group, followed by almost complete recovery among the JS + AS group. However, in dDG, an opposite trend was captured, and higher GAD65 methylation was found in JS. In addition, RNA levels were found to be decreased in JS compared to JSe and JS + AS. These findings can point to a possible mechanism: while juvenile stress may enhance a better coping strategy with life challenges, additional stress in adulthood may trigger a contradictory response, either beneficial or harmful.

Synaptic contacts onto motoneurons were studied in mice in which the gene for the trkB neurotrophin receptor was knocked out selectively in a subset of spinal motoneurons. The extent of contacts by structures immunoreactive for either of two different vesicular glutamate transporters (VGLUT1 and VGLUT2), the vesicular GABA transporter, or glutamic acid decarboxylase 67 (GAD67) with the somata of motoneurons, was studied in wild type and trkB knockout cells in tamoxifen treated male and female SLICK-trkB−/−mice. Selective knockout of the trkB gene resulted in a marked reduction in contacts made by VGLUT2- and GAD67-immunoreactive structures in both sexes and a significant reduction in contacts containing only glycine in male mice. No reduction was found for glycinergic contacts in female mice or for VGLUT1 immunoreactive contacts in either sex. Signaling through postsynaptic trkB receptors is considered to be an essential part of a cellular mechanism for maintaining the contacts of some, but not all, synaptic contacts onto motoneurons.

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.

Introduction Alteration of GABergic neurotransmission is accused to be sharing in the cognitive impairment in schizophrenia. Exploring the relation between the neuronal expression of GABergic genes and cognitive impairment in living patients through modeling of schizophrenia is an important step to know more about the core of the pathophysiology of this disorder Objectives Altered genetic expression of GAD 67 may have an important role in the pathophysiology of cognitive impairment in schizophrenia Methods . Reprogramming of human fibroblasts into human induced pluripotent stem cells (hIPSc) then neuronal differentiation was performed in 20 patients presenting with schizophrenia and 20 matched controls. Real time Polymerase chain reaction was done for measurement of genetic expression of GAD 65, GAD 67 and GABA beta 2. The Digit Symbol task, block design, block design task and similarities tasks from the Wechsler Adult Intelligence Scale., Trail A and Trail B making tests in addition to Rey-Osterrieth Complex Figure Test (ROCF) were applied to measure cognitive functions . Results There were lower means of GAD65, GAD67 and GABA beta2genetic expression in the patients group with significant statistical difference between the 2 groups. The down regulation of GAD 67 in patients presenting with schizophrenia is positively correlated with impairment in executive functions. Conclusions GAD 67 gene expression had the most significant correlations with the cognitive assessment in both patients and controls. The presence of those statistically significant correlations in both groups points to the possible role of GAD 67 gene functioning in the pathophysiology of cognitive impairment in schizophrenia Disclosure No significant relationships.

IntroductionThe glutamate system is implicated both in mood disorders and schizophrenia. Mice lacking metabotropic mGlu5 receptors (mGluR5 KO) display schizophrenia-like abnormalities. Additionally, mGluR5 antagonists represent promising alternative anxiolytics/antidepressants. However, the underlying age-specific molecular/cellular mechanisms are only partially understood.ObjectivesWe aimed at identifying molecular alterations associated with a genetically induced mGluR5 deletion, which results in a schizophrenia-like phenotype. Additionally, we investigated age-specific effects of mGluR5 antagonists on emotional behaviour and c-fos activation.MethodsFor analysis of mRNA and protein levels we performed Real-time RT-PCR and Western blot investigations of brains from mGluR5 KO and wild-type mice. Additionally we used classical behavioral tests for determining anxiety- and depression-like changes triggered by the mGluR5 antagonist 2-Methyl-6-(phenylethynyl)pyridine (MPEP). Finally, we used profiling of c-Fos expression, as marker of neuronal activity, induced by MPEP from postnatal day 16 (P16) to adulthood (P90).ResultsWe found reduced expression levels of reelin, GAD65, GAD67, parvalbumin, as well as NMDA and AMPA receptor subunits in mGluR5 KO mice, especially in the prefrontal cortex (PFC). We measured age-specific alterations in emotional behaviour of mGluR5 KO mice, with marked increase of anxiety during aging. There was a remarkably conserved activation of the paraventricular nucleus of the hypothalamus, implicated in stress regulation, by MPEP at all investigated ages, whereas the extended amygdala was specifically activated in adulthood only.ConclusionsOur animal data provide new insights into the potential role of mGluR5 in neurochemical and behavioural changes associated with schizophrenia and mood disorders during the lifespan.Disclosure of interestThe authors have not supplied their declaration of competing interest.

L’alcolismo è uno tra i maggiori problemi di natura sociale con forti ripercussioni sulla salute pubblica. I disturbi alcol correlati osservati più frequentemente sono rappresentati dalla dipendenza e dall’abuso. Secondo alcuni tra i criteri diagnostici del DSM IV per la caratterizzazione della dipendenza, vi sono il desiderio incoercibile di assumere la sostanza e di provarne gli effetti (carving) e la perdita di controllo della frequenza e/o quantita’ di sostanza somministrata e coazione a ripetere l’assunzione. La dipendenza da alcol rappresenta una patologia comportamentale complessa che riflette interazioni tra fattori di rischio genetici ed ambientali. Infatti, studi di natura familiare, su gemelli e su fratelli adottati indicano che i fattori genetici contribuiscono per il 40-60% nel rischio di sviluppare alcol dipendenza. L’alcol esercita il suo effetto principale nel cervello dove agisce a livello delle membrane cellulari e a livello intracellulare dove è coinvolto nella trasduzione del segnale. Il sistema dell’acido gamma-amminobutirrico (GABA), il principale neurotrasmettitore inibitore del sistema nervoso centrale, è uno dei potenziali bersagli dell’alcol. In particolare, si ritiene che la GAD67, enzima limitante la sintesi del GABA, possa contribuire in modo determinante all’azione dell’alcol. Questo lavoro di tesi ha previsto l’analisi di polimorfismi a singolo nucleotide (SNPs) del gene GAD67 in possibile associazione alla condizione di alcol dipendenza. 2 La ricerca, strutturata come studio associazionale di tipo caso-controllo, ha avuto come oggetto l’analisi di 283 individui di sesso maschile, di cui 107 soggetti alcol dipendenti e 176 controlli. Specificatamente, sono stati analizzati 26 SNPs localizzati nelle regioni promotrici, introniche ed esoniche del gene GAD67 mediante metodica SNPStream (Beckman Coulter). I risultati ottenuti hanno evidenziato una differenza statisticamente significativa (p=0.0030) nella distribuzione genotipica dello SNP esonico rs 11542313 responsabile di una mutazione silente (His-His).
Alcoholism is one of the major social, economic and public health problems facing the world today. Social drinking can be associated with development of alcohol tolerance, leading in turn to abuse and dependence. Primary features of alcoholism include loss of control over consumption, obsessional thoughts about the next drink, and continuation of abuse despite negative health effects and social consequences. Epidemiologically alcoholism is defined as a heterogeneous, complex disorder with both genetic and environmental influences. A meta-analysis of twin studies has shown that the genetic influence of all addictive substances ranges from 40–70% and the heritability of alcoholism, derived from nearly 10000 twin pairs, is found to be 50%, showing that genetic and environmental risk factors for alcoholism are almost equally important although they may differ in the different populations. Additionally alcoholism is a disease with an underlying complex of biochemical pathophysiology. Unlike other addictive drugs, alcohol has widespread effects throughout the brain; it acts at a variety of targets within cell membranes and in the intracellular signal transduction, inducing effects on neurotransmitter and neurohormone membrane receptors and receptor-gated and voltage-activated ion channels. In the central nervous system, ethanol alters the activity of several neurotransmitters including monoamines and gamma-aminobutyric acid 2 (GABA). Genetic vulnerability to alcoholism is therefore likely due to a number of genes of small varying degrees in many neurotransmitter systems and signal transduction pathways. In particular, several studies suggest that GABA may be involved in alcohol withdrawal and tolerance; the genes encoding for glutamate decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis, are of potential interest for their association to ethanol consumption and alcohol dependence. In our project we evaluated, in a case-control association study, the relationship between the GAD gene and alcohol use disorder. Specifically, the total cohort analyzed was 283 male individuals, 107 of which were alcohol dependent subjects and 176 controls. As a whole, in all experiments carried out a total of 26 single nucleotide polymorphisms (SNPs) localized in the promoter, exonic and intronic regions of the GAD 67 gene by using GenomeLab SNPStream Genotyping System technology were analyzed. Our results showed a significant difference in genotype distribution of one SNP (rs 11542313) localized in the exon 3 of the GAD 67 gene that is responsible for a silent mutation (His-His).

Parkinson’s disease (PD) and autism are prevalent diseases in two disparate age groups. The neuropathology underlying these diseases involves the major neurotransmitters, dopamine and GABA, and/ or their receptors. The current study investigated mRNA gene expressions of the GAD67 in autistic striatum and the DRD1 in the Parkinsonian dorsolateral prefrontal cortex. In situ hybridization histochemistry for GAD67 mRNA levels in postmortem striatal specimens from autistic individuals was compared to those of normal controls. Similarly, a nonradioactive in situ hybridization newly emerging method, RNAscope, was used to assess the D1 receptor mRNA gene expression in postmortem specimens of the dorsolateral prefrontal cortex of PD and control brains. The GAD67 mRNA labeling intensity that was measured on X-ray films and on emulsion radioautograph sections did not vary significantly between the autistic samples and the normal control samples. On the other hand, DRD1 mRNA levels showed a significant increase in the Parkinsonian dorsolateral prefrontal cortex specimens as compared to their normal counterparts. The GAD65 mRNA labeling results corresponded with the GAD67 mRNA levels. The similar GAD67 and GAD65 mRNA patterns in the autism group and the control group may suggest that the hyper-excitability hypothesis can be accounted for by an increase in the glutamatergic activity rather than a decrease in the GABAergic system. The increase in the DRD1 mRNA in the Parkinson’s disease dorsolateral prefrontal cortex may be interpreted in light of the expected upregulation of the D1 receptor in cases of dopamine depletion as the treatment-status was unknown. In conclusion, research investigating the neurotransmitters’ gene expression in Parkinson’s disease and in autism spectrum disorder needs more neurobiological studies in order to establish some knowledge regarding the temporality, and the genetic profile mapping of the diseases. Likewise, more research is encouraged to relate the symptoms and behaviors associated with disease to their anatomical origins.