Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABAA receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5␣-reductase (5␣-R) type I and 3␣-hydroxysteroid dehydrogenase (3␣-HSD). This study evaluates 5␣-R type I and 3␣-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67͞65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100␤ immunohistochemistry. We demonstrate that 5␣-R type I and 3␣-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5␣-R type I nor 3␣-HSD mRNAs are expressed in S100␤-or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67͞65 antibodies to mark GABAergic neurons, we failed to detect 5␣-R type I and 3␣-HSD in cortical and hippocampal GABAergic interneurons. However, 5␣-R type I and 3␣-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABAA receptors, either with an autocrine or a paracrine mechanism or by reaching GABAA receptor intracellular sites through lateral membrane diffusion.3␣-hydroxysteroid dehydrogenase ͉ 5␣-reductase (type I) ͉ GABAergic neurons ͉ glutamatergic neurons T he neurosteroids 3␣-hydroxy-5␣-pregnan-20-one [allopregnanolone (ALLO)] and 3␣,21-dihydroxy-5␣-pregnan-20-one [tetrahydrodeoxycorticosterone (THDOC)] are potent positive allosteric modulators of GABA action at GABA A receptors (1-6). These neurosteroids can be synthesized in the brain from progesterone (7) or deoxycorticosterone (8, 9), respectively, by the sequential action of two enzymes, 5␣-reductase (5␣-R) type I and 3␣-hydroxysteroid dehydrogenase (3␣-HSD) (10).Two types (I and II) of 5␣-Rs, which convert progesterone into 5␣-dihydroprogesterone (5␣-DHP) or convert deoxycorticosterone into 5␣-dihydrodeoxycorticosterone (5␣-DHDOC), have been identified in tissues of rodents and humans (11). Whereas 5␣-R type I and II are abundantly expressed in several peripheral tissues, 5␣-R type I is the most abundant 5␣-R molecular form detected in the adult brains of rats, mice, and humans (11-17). The human brain expresses four types of 3␣-HSD, which, under different optimal conditions, either catalyze the reduction of 5␣-DHP into ALLO or reverse this reaction (18). So far, only one 3␣-HSD isoform has been identified in the rat or mouse brain (19)(20)(21)(22). The mRNA sequences of 5␣-R type I (Ϸ88%) and 3␣-HSD (Ϸ89%) are highly homologous in mouse (5␣-R type I GeneBank access...

Among the most consistent results of studies of post-mortem brain tissue from schizophrenia patients (SZP) is the finding that in this disease, several genes expressed by GABAergic neurons are downregulated. This downregulation may be caused by hypermethylation of the relevant promoters in affected neurons. Indeed, increased numbers of GABAergic interneurons expressing DNA methyltransferase 1 (DNMT1) mRNA have been demonstrated in the prefrontal cortex (PFC) of SZP using in situ hybridization. The present study expands upon these findings using nested competitive reverse transcription-polymerase chain reaction combined with laser-assisted microdissection to quantitate the extent of DNMT1 mRNA overexpression in distinct populations of GABAergic neurons obtained from either layer I or layer V of the PFC of SZP. In a cohort of eight SZP and eight non-psychiatric subject (NPS) post-mortem BA9 tissue samples, DNMT1 mRNA was found to be selectively expressed in GABAergic interneurons and virtually absent in pyramidal neurons. DNMT1 mRNA expression was approximately threefold higher in GABAergic interneurons microdissected from layer I of SZP relative to the same neurons microdissected from NPS. GABAergic interneurons obtained from layer V of the same samples displayed no difference in DNMT1 mRNA expression between groups. In the same samples, the GABAergic neuron-specific glutamic acid-decarboxylase 67 (GAD 67 ) and reelin mRNAs were underexpressed twofold in GABAergic interneurons isolated from layer I of SZP relative to GABAergic interneurons microdissected from layer I of NPS, and unaltered in GABAergic interneurons of layer V. These findings implicate an epigenetically mediated layer I GABAergic dysfunction in the pathogenesis of schizophrenia, and suggest novel strategies for treatment of the disease.

Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD 67 expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5-22 mol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD 67 expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD67 promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 mol/kg s.c.), an nAChR blocker that penetrates the blood-brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD67 expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.antagonists ͉ epigenetic mechanisms ͉ nicotinic acetylcholine receptor agonists ͉ schizophrenia T obacco smoking is frequently abused by schizophrenia patients (SZP) (for reviews see refs. 1 and 2). Because nicotine is a potent cholinergic receptor agonist that is inhaled with tobacco smoking and both the expression and function of nicotinic acetylcholine receptors (nAChRs) are down-regulated in the brain of SZP, one may conclude that the high level of tobacco smoking in these patients represents an attempt to self-medicate; i.e., correction of some disease-associated abnormalities of cholinergic (nicotinic) neurotransmission (3, 4), possibly related to the decrease of GABAergic function occurring in the brain of SZP (5-9).Typically, plasma nicotine levels in heavy smokers (Ϸ20-30 cigarettes a day) oscillate between 0.3 and 0.6 M. Because in humans nicotine half-life is Ϸ2 h, the nicotine plasma levels in heavy smokers progressively increase during the day but fluctuate in a ''peak and trough'' fashion after each cigarette (10, 11). These submicromolar concentrations of nicotine, which act at heteroolig...

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.

Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann’s area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layers I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in and PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.