GAD67 Promoter Research Articles

DNMT1 Mediates Chronic Pain–Related Depression by Inhibiting GABAergic Neuronal Activation in the Central Amygdala

BackgroundChronic pain can induce depressive emotion. DNA methyltransferases (DNMTs) have been shown to be involved in the development of chronic pain and depression. However, the role and mechanism of DNMTs in chronic pain–induced depression are not well understood. MethodsIn well-established spared nerve injury (SNI)–induced chronic pain–related depression models, the expression of DNMTs and the functional roles and underlying mechanisms of DNMT1 in central amygdala (CeA) GABAergic (gamma-aminobutyric acidergic) neurons were investigated using molecular, pharmacological, electrophysiological, optogenetic, and chemogenetic techniques and behavioral tests. ResultsDNMT1, but not DNMT3a or DNMT3b, was upregulated in the CeA of rats with SNI-induced chronic pain–depression. Inhibition of DNMT1 by 5-Aza or viral knockdown of DNMT1 in GABAergic neurons in the CeA effectively ameliorated the depression-like behaviors induced by chronic pain. The DNMT1 action was associated with methylation at the CpG-rich Gad1 promoter and GAD67 downregulation, leading to a decrease of GABAergic neuronal activity. Optogenetic activation of GABAergic neurons in the CeA improved SNI-induced depression-like behaviors. Moreover, optogenetic or chemogenetic inhibition of GABAergic neurons in the CeA reversed DNMT1 knockdown–induced improvement of depression-like behaviors in SNI mice. ConclusionsOur findings suggest that DNMT1 is involved in the development of chronic pain–related depression by epigenetic repression of GAD67, leading to the inhibition of GABAergic neuronal activation. This study indicates that DNMT1 could be a potential target for the treatment of chronic pain–related depression.

Glutamic Acid Decarboxylase 1 Gene Methylation and Panic Disorder Severity: Making the Connection by Brain Gray Matter Volume.

ObjectiveThis study aimed to test the hypothesis that the relationship between glutamic acid decarboxylase (GAD) 1 gene methylation and severity of clinical symptoms of panic disorder (PD) is mediated by the effect of GAD1 gene methylation on gray matter volume (GMV) and the effect of GMV on PD.MethodsPanic disorder (n = 24) patients were recruited consecutively from the Affiliated Brain Hospital of Nanjing Medical University through outpatient and public advertising, eligible healthy controls (HCs) (n = 22) were recruited from public advertising. We compared GMV and GAD1 gene methylation in PD and HCs to estimate the differences, and on the basis of the relationship between gray matter volumes and GAD1 gene methylation in PD patients was evaluated, the role of GMV as a mediator of GAD1 gene methylation and PD clinical symptoms was analyzed.ResultsPanic disorder patients had significantly lower methylation in the GAD1 promoter region on Cytosine-phosphate-guanine (CPG) 7 than HCs (t = 2.380, p = 0.021). Pearson correlation analysis found a significant negative association between cg171674146 (cg12) site and clinical severity (n = 24, r = −0.456, p = 0.025). Compared to HCs, patients with PD had decreased gray matter volumes in several brain regions, which were also associated with PD severity. Left postcentral gyrus (PoCG) GMV mediated the association between cg12 methylation and PD severity, and there was a significant mediation effect of right angular gyrus (ANG) gray matter volumes on the relationship between cg12 methylation and PD severity.LimitationNo direct results can be derived for methylation patterns in different brain regions; the study is cross-sectional; relatively small size.

Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina.

The amacrine neurons in the mammalian retina comprise a large variety of cell types with distinct properties and functions that serve to integrate and modulate signals presented to output neurons. The majority of them use either glycine or GABA as inhibitory neurotransmitters and express the glycine transporter 1 (GlyT1) or glutamic acid decarboxylase (GAD67) and GABA transporters (GAT1 and GAT3), as a glycinergic or GABAergic marker respectively. We report here a novel subpopulation of amacrine neurons expressing both, GABAergic and glycinergic markers, in retinas from wild-type C57BL/6J mice and two transgenic lines. In retinal sections from the transgenic line expressing eGFP under the control of the glycine transporter 2, eGFP expression was exclusively found in cell bodies and dendrites of inhibitory amacrine neurons, identified for their immunoreactivity to syntaxin 1A. All of the glycinergic and a large portion of the GABAergic amacrine neurons contained eGFP; of these, 8-10% of GlyT1 positive neurons were also labeled either with GAD67, GAT1 or GAT3. These findings were confirmed in retinas from a wild-type and a mouse line expressing eGFP under the GAD67 promoter and two different anti-GlyT1 antibodies, showing the presence of a subpopulation with a dual phenotype. Moreover, eGFP-positive dendrites on both mouse lines were found juxtaposed to GlyR subunits and the scaffold protein gephyrin in several areas of the inner plexiform layer, demonstrating the glycinergic character of these neurons. This dual phenotype was also demonstrated in primary retina cultures, in which isolated neurons were positive for GlyT1 and GAD67 or GAT1/3. Altogether, these data provide compelling evidence of a subpopulation of dual inhibitory, glycinergic/GABAergic amacrine neurons. The co-release of both neurotransmitters may serve to strengthen the inhibition on ganglion cells under synaptic hyperexcitability.

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

Programming for increased expression of hippocampal GAD67 mediated the hypersensitivity of the hypothalamic\u2013pituitary\u2013adrenal axis in male offspring rats with prenatal ethanol exposure

An imbalance of excitatory and inhibitory signals in the brain has been proposed to be one of the main pathological features of various diseases related to hypothalamic–pituitary–adrenal axis (HPAA) dysfunction. Excessive glutamate release induces neuronal excitotoxicity, while glutamic acid decarboxylase (GAD) 67 promotes the transformation of excessive glutamate to γ-aminobutyric acid (GABA). Our previous studies demonstrated that prenatal ethanol exposure (PEE) causes foetal over-exposure to maternal corticosterone and hypersensitivity of the HPAA after birth, but its intrauterine programming mechanism is unknown. In this study, PEE was shown to lead to an enhanced potential excitatory ability of the hypothalamus and hypersensitivity of the HPAA, as well as mild abnormal hippocampal morphology, demethylation of the -1019 to -691-bp region in the hippocampal GAD67 promoter and upregulation of GAD67 expression accompanied by a reduction in glutamatergic neurons and increase in GABAergic neurons in PEE male offspring. Similar changes were also found in PEE male foetal rats. Furthermore, corticosterone increased the expression of the glucocorticoid receptor (GR) and GAD67 in foetal hippocampal H19-7 cells in a concentration-dependent manner, accompanied by demethylation of the GAD67 promoter, a decrease in glutamatergic neurons and increase in GABAergic neurons. The GR inhibitor, mifepristone, reversed the effects of corticosterone on H19-7 cells. These results suggested that PEE-induced excessive corticosterone can lead to upregulation of GAD67 through epigenetic modification mediated by the GR in the male foetal hippocampus, thereby weakening the negative regulation of the HPAA by the hippocampus and increasing the potential excitatory ability of the hypothalamus. These changes persisted until after birth, resulting in hypersensitivity of the HPAA. However, gender differences were observed in the hippocampal development, morphology and GAD67 expression associated with PEE. Programming for the increased expression of hippocampal GAD67 is a potential mechanism responsible for the hypersensitivity of the HPAA in PEE male rats.

GAD1 alternative transcripts and DNA methylation in human prefrontal cortex and hippocampus in brain development, schizophrenia.

Genetic variations and adverse environmental events in utero or shortly after birth can lead to abnormal brain development and increased risk of schizophrenia. γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, plays a vital role in normal brain development. GABA synthesis is controlled by enzymes derived from two glutamic acid decarboxylase (GAD) genes, GAD1 and GAD2, both of which produce transcript isoforms. While the full-length GAD1 transcript (GAD67) has been implicated in the neuropathology of schizophrenia, the transcript structure of GAD1 in the human brain has not been fully characterized. In this study, with the use of RNA sequencing and PCR technologies, we report the discovery of 10 novel transcripts of GAD1 in the human brain. Expression levels of four novel GAD1 transcripts (8A, 8B, I80 and I86) showed a lifespan trajectory expression pattern that is anticorrelated with the expression of the full-length GAD1 transcript. In addition, methylation levels of two CpG loci within the putative GAD1 promoter were significantly associated with the schizophrenia-risk SNP rs3749034 and with the expression of GAD25 in dorsolateral prefrontal cortex (DLPFC). Moreover, schizophrenia patients who had completed suicide and/or were positive for nicotine exposure had significantly higher full-length GAD1 expression in the DLPFC. Alternative splicing of GAD1 and epigenetic state appear to play roles in the developmental profile of GAD1 expression and may contribute to GABA dysfunction in the PFC and hippocampus of patients with schizophrenia.

Epigenetic regulation of RELN and GAD1 in the frontal cortex (FC) of autism spectrum disorder (ASD) subjects

Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains.We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP).We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments.Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged amounts of 5hmC present in these regions, suggests the possibility that DNMT1 interacts with and alters MECP2 binding properties to selected promoters. Comparisons between data obtained from the FC with CB studies showed some common themes between brain regions which are discussed.

GABAergic Neurons and Their Modulatory Effects on GnRH3 in Zebrafish.

γ-Aminobutyric acid (GABA) is a major amino acid neurotransmitter in the vertebrate brain. To provide detailed information on the distribution of the GABA in zebrafish (Danio rerio), neurons were labeled with mCherry driven by the glutamic acid decarboxylase 67 (gad67) promoter. In the transgenic line Tg(gad67:mCherry), mCherry-positive gad67 cell bodies were predominantly localized to the olfactory bulb, pallial zones, subpallium zones, parvocellular preoptic nucleus, periventricular gray zone of optic tectum, torus semicircularis, posterior tuberculum, medial longitudinal fascicle, caudal zone of periventricular hypothalamus, and oculomotor nucleus. mCherry-positive fibers were widely distributed in the olfactory bulbs, subpallium, thalamus, ventral hypothalamic zone, tectum opticum, mesencephalon, and rhombencephalon. mCherry-positive neurons were also observed in the retina and the spinal cord. The anatomical relationships between GABAergic and gonadotrophin-releasing hormone 3 (GnRH3) neurons were investigated by crossing Tg(gad67:mCherry) fish with the previously established Tg(gnrh3:EGFP) transgenic line. GnRH3 cell bodies and fibers were contacted by GABAergic fibers directly in the ventral telencephalon and anterior tuberal nucleus. A subpopulation of GnRH3 neurons in the ventral telencephalic area was also labeled with mCherry, so some GnRH3 neurons are also GABAergic. GABAB receptor agonist (baclofen) and antagonist (CGP55845) treatments indicated that GABAB receptor signaling inhibited gnrh3 expression in larval fish but was stimulatory in adult fish. The expression of pituitary lhβ and fshβ was stimulated by intraperitoneal injection of baclofen in adult fish. We conclude that GABA via GABAB receptors regulates GnRH3 neurons in a developmentally dependent manner in zebrafish.

Behavioral and molecular neuroepigenetic alterations in prenatally stressed mice: relevance for the study of chromatin remodeling properties of antipsychotic drugs.

We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg−1 twice a day for 5 days) but not by haloperidol (1 mg kg−1 twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention.

Lentiviral expression of GAD67 and CCK promoter-driven opsins to target interneurons in vitro and in vivo.

The ability to manipulate the activity of interneurons with optogenetic tools offers the possibility of interfering with diseases caused by altered neuronal inhibition and synchrony, including epilepsy and schizophrenia. To develop vectors for therapeutic approaches, targeting optogenetic constructs to interneurons is therefore a key requirement. We investigated whether the interneuron-specific promoters glutamic acid decarboxylase (GAD)67 and cholecystokinin (CCK) allowed targeted lentiviral delivery of opsins to interneurons as a whole, or specifically CCK+ interneurons. We generated lentiviral (LV) plasmids encoding channelrhodopsin (ChR2) and halorhodopsin (NpHR) tagged with fluorophores and driven by GAD67 or CCK promoters. Adeno-associated virus (AAV) and LV vectors carrying opsins driven by pyramidal cell promoters were used as controls. We transduced neuronal cultures and rodent brain in vivo, immunostained specimens 6-8 weeks after in vivo injection and 7-14 days after in vitro transduction, and evaluated volume and specificity of expression by confocal microscopy. In vitro, 90% (19/21) of LV-CCK-NpHR2.0-EYFP expressing neurons were CCK+. In vivo, LV-GAD67-ChR2-mCherry was expressed in 2.6% (5/193), LV-GAD67-NpHR2.0-EYFP in approximately 15% (43/279) and LV-CCK-NpHR2.0-EYFP in 47% (9/19) of hippocampal GABA+ interneurons. GAD67 vectors expressed in larger volumes than CCK-driven constructs. AAV vector controls achieved the largest expression volumes. LV-CCK-NpHR2.0-EYFP may be useful for targeting CCK+ interneurons in culture. GAD67/CCK-driven lentiviral constructs are expressed in vivo, although expression is not specific for interneurons. Overall, expression levels are low compared to opsins driven by pyramidal cell promoters. A better understanding of GAD67 and CCK promoter structure or alternative techniques is required to reliably target opsins to interneurons using viral vectors.

Toward dissecting the etiology of schizophrenia: HDAC1 and DAXX regulate GAD67 expression in an in vitro hippocampal GABA neuron model.

Schizophrenia (SZ) is associated with GABA neuron dysfunction in the hippocampus, particularly the stratum oriens of sector CA3/2. A gene expression profile analysis of human postmortem hippocampal tissue followed by a network association analysis had shown a number of genes differentially regulated in SZ, including the epigenetic factors HDAC1 and DAXX. To characterize the contribution of these factors to the developmental perturbation hypothesized to underlie SZ, lentiviral vectors carrying short hairpin RNA interference (shRNAi) for HDAC1 and DAXX were used. In the hippocampal GABA neuron culture model, HiB5, transduction with HDAC1 shRNAi showed a 40% inhibition of HDAC1 mRNA and a 60% inhibition of HDAC1 protein. GAD67, a enzyme associated with GABA synthesis, was increased twofold (mRNA); the protein showed a 35% increase. The expression of DAXX, a co-repressor of HDAC1, was not influenced by HDAC1 inhibition. Transduction of HiB5 cells with DAXX shRNAi resulted in a 30% inhibition of DAXX mRNA that translated into a 90% inhibition of DAXX protein. GAD1 mRNA was upregulated fourfold, while its protein increased by ~30%. HDAC1 expression was not altered by inhibition of DAXX. However, a physical interaction between HDAC1 and DAXX was demonstrated by co-immunoprecipitation. Inhibition of HDAC1 or DAXX increased expression of egr-1, transcription factor that had previously been shown to regulate the GAD67 promoter. Our in vitro results point to a key role of both HDAC1 and DAXX in the regulation of GAD67 in GABAergic HiB5 cells, strongly suggesting that these epigenetic/transcription factors contribute to mechanisms underlying GABA cell dysfunction in SZ.

Maternal immune activation induces GAD1 and GAD2 promoter remodeling in the offspring prefrontal cortex

Maternal infection during pregnancy increases the risk of neurodevelopmental disorders in the offspring. In addition to its influence on other neuronal systems, this early-life environmental adversity has been shown to negatively affect cortical γ-aminobutyric acid (GABA) functions in adult life, including impaired prefrontal expression of enzymes required for GABA synthesis. The underlying molecular processes, however, remain largely unknown. In the present study, we explored whether epigenetic modifications represent a mechanism whereby maternal infection during pregnancy can induce such GABAergic impairments in the offspring. We used an established mouse model of prenatal immune challenge that is based on maternal treatment with the viral mimetic poly(I:C). We found that prenatal immune activation increased prefrontal levels of 5-methylated cytosines (5mC) and 5-hydroxymethylated cytosines (5hmC) in the promoter region of GAD1, which encodes the 67-kDa isoform of the GABA-synthesising enzyme glutamic acid decarboxylase (GAD67). The early-life challenge also increased 5mC levels at the promoter region of GAD2, which encodes the 65-kDa GAD isoform (GAD65). These effects were accompanied by elevated GAD1 and GAD2 promoter binding of methyl CpG-binding protein 2 (MeCP2) and by reduced GAD67 and GAD65 mRNA expression. Moreover, the epigenetic modifications at the GAD1 promoter correlated with prenatal infection-induced impairments in working memory and social interaction. Our study thus highlights that hypermethylation of GAD1 and GAD2 promoters may be an important molecular mechanism linking prenatal infection to presynaptic GABAergic impairments and associated behavioral and cognitive abnormalities in the offspring.

DNA-methyltransferase1 (DNMT1) binding to CpG rich GABAergic and BDNF promoters is increased in the brain of schizophrenia and bipolar disorder patients

The down regulation of glutamic acid decarboxylase67 (GAD1), reelin (RELN), and BDNF expression in brain of schizophrenia (SZ) and bipolar (BP) disorder patients is associated with overexpression of DNA methyltransferase1 (DNMT1) and ten–eleven translocase methylcytosine dioxygenase1 (TET1). DNMT1 and TET1 belong to families of enzymes that methylate and hydroxymethylate cytosines located proximal to and within cytosine phosphodiester guanine (CpG) islands of many gene promoters, respectively. Altered promoter methylation may be one mechanism underlying the down-regulation of GABAergic and glutamatergic gene expression. However, recent reports suggest that both DNMT1 and TET1 directly bind to unmethylated CpG rich promoters through their respective Zinc Finger (ZF-CXXC) domains. We report here, that the binding of DNMT1 to GABAergic (GAD1, RELN) and glutamatergic (BDNF-IX) promoters is increased in SZ and BP disorder patients and this increase does not necessarily correlate with enrichment in promoter methylation. The increased DNMT1 binding to these promoter regions is detected in the cortex but not in the cerebellum of SZ and BP disorder patients, suggesting a brain region and neuron specific dependent mechanism. Increased binding of DNMT1 positively correlates with increased expression of DNMT1 and with increased binding of MBD2. In contrast, the binding of TET1 to RELN, GAD1 and BDNF-IX promoters failed to change. These data are consistent with the hypothesis that the down-regulation of specific GABAergic and glutamatergic genes in SZ and BP disorder patients may be mediated, at least in part, by a brain region specific and neuronal-activity dependent DNMT1 action that is likely independent of its DNA methylation activity.

Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii.

The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotine-sensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments.

Lower Expression of Glutamic Acid Decarboxylase 67 in the Prefrontal Cortex in Schizophrenia: Contribution of Altered Regulation by Zif268

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

Acute ethanol alters multiple histone modifications at model gene promoters in the cerebral cortex.

Ethanol (EtOH) exposure alters gene expression in the cerebral cortex (CCx); however, mechanisms of EtOH-induced gene regulation are not well understood. We hypothesized that EtOH regulates gene expression by differentially altering histone modifications at gene promoters that are up- and down-regulated by EtOH. Such epigenetic mechanisms may ultimately contribute to EtOH-induced neuro-adaptations that underlie tolerance, dependence, and EtOH-use disorders. Eight-week-old, male C57BL/6J mice were treated with 3 g/kg EtOH (intraperitoneally) or saline and sacrificed 6 hours after injection; the CCx and hippocampus (HC) were immediately removed and flash frozen. Chromatin immunoprecipitation was used to study the association of model gene promoters with histone modifications. Western blot was used to detect global changes in the histone modifications studied. We also used a polymerase chain reaction (PCR) array to identify changes in expression of chromatin-modifying enzymes. In CCx, acute EtOH decreased expression of Gad1, Hdac2, and Hdac11, which was associated with decreased histone acetylation at the Gad1 and Hdac2 promoters; we also identified increased expression of Mt1, Mt2, Egr1, which was associated with increased H3K4me3 levels at the Mt2 promoter and decreased H3K27me3 levels at the Mt1 promoter. We identified an increase in global levels of H3K4me3 in CCx as well as a global increase in H3K9ac and H3K14ac in HC. The PCR array identified decreased expression of Csrp2 bp, Hdac2, and Hdac11 as well as increased expression of Kat2b in CCx. Acute EtOH induces chromatin remodeling at model up- and down-regulated genes in CCx. Different patterns of histone modifications at these gene promoters indicate that EtOH may be acting through multiple histone-modifying enzymes to alter gene expression; in particular, differential expression of Kat2b, Hdac2, Hdac11, and Csrp2 bp in CCx may mediate EtOH-induced chromatin remodeling. Additional studies are necessary to determine the relationship between EtOH-induced changes in histone-modifying enzymes, specific EtOH-induced histone modifications, and gene expression.

Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms related to altered social interactions/communication and restricted and repetitive behaviors. In addition to genetic risk, epigenetic mechanisms (which include DNA methylation/demethylation) are thought to be important in the etiopathogenesis of ASD. We studied epigenetic mechanisms underlying the transcriptional regulation of candidate genes in cerebella of ASD patients, including the binding of MeCP2 (methyl CpG binding protein-2) to the glutamic acid decarboxylase 67 (GAD1), glutamic acid decarboxylase 65 (GAD2), and Reelin (RELN) promoters and gene bodies. Moreover, we performed methyl DNA immunoprecipitation (MeDIP) and hydroxymethyl DNA immunoprecipitation (hMeDIP) to measure total 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the same regions of these genes. The enrichment of 5-hmC and decrease in 5-mC at the GAD1 or RELN promoters detected by 5-hmC and 5-mC antibodies was confirmed by Tet-assisted bisulfite (TAB) pyrosequencing. The results showed a marked and significant increase in MeCP2 binding to the promoter regions of GAD1 and RELN, but not to the corresponding gene body regions in cerebellar cortex of ASD patients. Moreover, we detected a significant increase in TET1 expression and an enrichment in the level of 5-hmC, but not 5-mC, at the promoters of GAD1 and RELN in ASD when compared with CON. Moreover, there was increased TET1 binding to these promoter regions. These data are consistent with the hypothesis that an increase of 5-hmC (relative to 5-mC) at specific gene domains enhances the binding of MeCP2 to 5-hmC and reduces expression of the corresponding target genes in ASD cerebella.

Nicotine Restores Wt-Like Levels of Reelin and GAD67 Gene Expression in Brain of Heterozygous Reeler Mice

Important reduction of reelin, a neural development- and plasticity-associated protein, and glutamic acid decarboxylase (GAD67) are reported in brains of schizophrenic patients. These individuals are consistently engaged in tobacco smoking and nicotine is thought to alleviate negative behavioral symptoms or cognitive alterations. In mouse brain, nicotine has been shown to reduce GAD67 promoter methylation and increase its transcription. We assessed the effects of administration of nicotine (1mg/kgs.c.) for 6days, in male mice heterozygous for reelin (HRM), a putative model for symptoms related to schizophrenia. Expression of reelin, GAD67 and brain-derived neurotrophic factor (BDNF) was measured in different brain areas. RNA expression analysis evidenced genotype-related changes, with a marked reduction in reelin and GAD67 gene expression in prefrontal cortex, hippocampus, cerebellum, and striatum from HRM. Nicotine treatment selectively reversed the HRM-related phenotype in most brain areas and increased BDNF gene expression in cortex and hippocampus of both genotypes. Locomotor performance in their home cage revealed that HRM subjects were characterized by general hyperactivity; with nicotine administration restoring WT-like levels of locomotion. These findings are interpreted within the hypothesis of pre-existing vulnerability (based on haploinsufficiency of reelin) to brain and behavioral disorders and regulative effects associated with nicotine exposure.

Expression of GABAergic and glutamatergic phenotypic markers in hypothalamic proopiomelanocortin neurons

Hypothalamic proopiomelanocortin (POMC) neurons have traditionally been defined by their peptide transmitters, which are important regulators of energy balance and reward. Recent work shows that POMC neurons can also release the amino acid transmitters γ-aminobutyric acid (GABA) and glutamate, although studying GABAergic and glutamatergic populations of POMC neurons has been hindered by the difficulty in reliably identifying amino acid (AA) transmitter phenotypes. In the present study, fluorescent in situ hybridization and immunohistochemistry were used to identify POMC neurons and to detect the presence of mRNA for the transporters responsible for packaging either GABA (vesicular GABA transporter [vGAT]) or glutamate (vesicular glutamate transporter [vGLUT]) into vesicles, as well as the enzymes responsible for GABA synthesis, glutamic acid decarboxylase (GAD)65 and GAD67. Approximately 7% of POMC neurons expressed vGlut2 and the highest percentage of vGlut2-positive POMC cells were located in the rostral arcuate nucleus. Despite the reports of GABA release from POMC neurons, vGat was not detected in POMC neurons, although Gad65 and Gad67 were present in ~40% of POMC neurons. Approximately half of the vGlut2-expressing POMC cells also expressed Gad65. Markers of neurotransmitter phenotype were better detected by using in situ hybridization techniques rather than transgenic expression of fluorophores under the control of the vGat or Gad67 promoters. It is now clear that the expression of markers of AA phenotype provides a useful means to identify distinct subpopulations of POMC neurons. Additionally, the method described will be useful to explore the possibility that plasticity of AA phenotype is an important aspect of POMC neuron function.

A Steep Learning Curve: Decoding Epigenetic Influence on Behavior and Mental Health

Research on epigenetics has surged in the past two decades as it has become apparent that changes in gene function aside from those related to DNA mutations or natural variations may be integral factors in numerous perplexing health disorders. But much remains unknown about this relatively new field. Of the thousands of epigenetics studies published,1 a few hundred have addressed behavioral and mental health outcomes, but only a fraction of those have dealt with fetal or childhood exposures or outcomes. However, early results in the niche field of behavioral epigenetics suggest such studies could provide insights into behavioral and mental health conditions such as autism spectrum disorders (ASDs), attention deficit/hyperactivity disorder, schizophrenia, bipolar disorder, depression, and anxiety.