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...

A down-regulation of reelin and glutamic acid decarboxylase (GAD) 67 mRNAs was detected in ␥-aminobutyric acid (GABA)ergic cortical interneurons of schizophrenia (SZ) postmortem brains (10), suggesting that the availability of GABA and reelin may be decreased in SZ cortex. In situ hybridization of the mRNA encoding for DNA-methyltransferase 1, which catalyzes the methylation of promoter CpG islands, shows that the expression of this mRNA is increased in cortical GABAergic interneurons but not in pyramidal neurons of SZ brains. Counts of reelin mRNA-positive neurons in Brodmann's area 10 of either nonpsychiatric subjects or SZ patients show that the expression of reelin mRNA is decreased in layer-I, -II, and -IV GABAergic interneurons of SZ patients. These findings are consistent with the hypothesis that the increase of DNA-methyltransferase 1 expression in telencephalic GABAergic interneurons of SZ patients causes a promoter hypermethylation of reelin and GAD 67 and perhaps of other genes expressed in these interneurons. It is difficult to decide whether this dysfunction of GABAergic neurons detected in SZ is responsible for this disease or is a consequence of this disorder. Although at present we cannot differentiate between these two alternatives, it is important to consider that so far a molecular pathology of cortical GABAergic neurons appears to be the most consistent finding associated with SZ morbidity.

Cortical DNA-methyltransferase 1 (DNMT1) is preferentially expressed in interneurons secreting GABA where it very likely contributes to promoter CpG island hypermethylation, thus causing a down-regulation of promoter functions. To consolidate and expand on previous findings that, in the cortex of schizophrenia (SZ) brains, glutamic acid decarboxylase 67 (GAD67) expression is downregulated whereas that of DNMT1 is up-regulated, we studied both parameters in Brodmann's area (BA) 9 from the McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center, Belmont, MA). In BA9 of SZ and bipolar disorder patients with psychosis, DNMT1 mRNA and protein expression preferentially increases in layer I, II, and IV interneurons, and this increase is paralleled by a decreased number of GAD67 mRNA-positive neurons. The increase in DNMT1 and the decrease in GAD67-expressing neurons were unrelated to postmortem interval, pH, RNA quality, or to the presence, dose, or duration of antipsychotic (APS) medication, with the exception of a subgroup of SZ patients treated with a combination of valproate and APS in which the expression of DNMT1 failed to change. The DNMT1 increase and the GAD67 decrease in BA9 interneurons are significant features of SZ and bipolar disorder with psychosis. Interestingly, the DNMT1 increase failed to occur when patients with psychosis received a combination of valproate and APS treatment but not APS monotherapy. bipolar disorder ͉ glutamic acid decarboxylase ͉ schizophrenia ͉ valproate ͉ antipsychotics A lthough monozygotic twins carry identical genomic sequences, they can exhibit a number of differences. In fact, in the absence of nucleotide sequence mutations, DNA can be modified by methylation of the cytosine ring. Such structural DNA modifications can be termed ''epigenetic,'' to indicate a modification that occurred after the operation of conventional genetic mechanisms (1).In neurons, epigenetic hypermethylation in the 5Ј position of the cytosine ring expressed in promoter CpG islands of various genes including that of glutamic acid decarboxylase 67 (GAD 67 ) and reelin is catalyzed by DNA-methyltransferases (2, 3). One of them, the DNA-methyltransferase 1 (DNMT1), is preferentially expressed in cortical GABA-secreting neurons of Brodmann's area (BA) 10, and BA17, and in the medium spiny neurons of the caudate nucleus (4).GABAergic interneurons of schizophrenia (SZ) patient (SZP) brains (BA10) exhibit a decrease of reelin and GAD 67 expression (4-6) and an increased expression of DNMT 1 (4).In a recent study (7), it was found that the selective expression of the human reelin gene in GABAergic neurons is regulated by an epigenetic cytosine hypermethylation of promoter CpG islands. In fact, when DNMT1 function is inhibited by the addition of aza-2Ј-deoxycytidine or down-regulated by the induction of demethylation elicited by histone deacetylase inhibitors, the in vitro expression of reelin increases severalfold (7). Moreover, in the hippocampus and frontal cortex of mice receiving methionine (6.6 m...

The association of the histone deacetylase (HDAC) inhibitor valproate (VPA) with atypical antipsychotics has become a frequent treatment strategy for schizophrenia and bipolar disorder. Because the VPA doses administered are elevated, one cannot assume that the benefits of the VPA plus antipsychotic treatment are exclusively related to the covalent modifications of nucleosomal histone tails. We compared the actions of N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide derivative (MS-275), which is a potent HDAC inhibitor in vitro, with the actions of VPA for their ability to (i) increase the acetylated status of brain nucleosomal histone tail domains and (ii) to regulate brain histone-RELN and histone-GAD67 promoter interactions. MS-275 increases the content of acetylhistone 3 (Ac-H3) in the frontal cortex. Whereas this response peaks after a s.c. injection of 15 mol͞kg, the increase in Ac-H3 content in the hippocampus becomes significant only after an injection of 60 mol͞kg, suggesting that MS-275 is 30-to 100-fold more potent than VPA in increasing Ac-H3 in these brain regions. In contrast to VPA, MS-275, in doses up to 120 mol͞kg, fails to increase Ac-H3 content in the striatum. Chromatin immunoprecipitation shows that MS-275 increases Ac-H3-RELN and Ac-H3-GAD67 promoter interaction in the frontal cortex. These results suggest that MS-275 is a potent brain region-selective HDAC inhibitor. It is likely that, in addition to MS-275, other benzamide derivatives, such as sulpiride, are brain-region selective inhibitors of HDACs. Hence, some benzamide derivatives may express a greater efficacy than VPA as an adjunctive to antipsychotics in the treatment of epigentically induced psychiatric disorders. bipolar disorder ͉ reelin ͉ schizophrenia ͉ histone code ͉ chromatin remodeling A prefrontal cortex GABAergic neuron dysfunction, which is characterized by a reduction of the 67-kDa form of glutamic acid decarboxylase (GAD 67 ) and reelin expression, is one of the most consistent neuropathological findings in postmortem brain studies of schizophrenia (SZ) and bipolar (BP) disorder (1-9). These expression deficits cannot be explained by reelin or GAD 67 gene haploinsufficiency (10, 11). Converging epidemiological (12), histological, and biochemical (10, 13-17) evidence suggests that the pathogenesis of this dysfunction may be related to a disruption of epigenetic signaling, resulting in the selective hypermethylation of several GABAergic gene promoters that characterize SZ as a selective defect of gene transcription in GABAergic cortical neurons (13,14). Such hypermethylation is very likely mediated by the overexpression of DNA methyltransferase 1 (DNMT1) (15, 18), which has been found to be operative in cortical and subcortical GABAergic interneurons of SZ and BP patients.The long-term objective of this line of research is to identify drugs that selectively correct a basic defect of SZ, which is an epigenetic GABAergic neuron dysfunction, by directly or indirectly reducing the RELN and GAD 67 pr...

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.