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.

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.

Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection.

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.

An epigenetic mouse model for molecular and behavioral neuropathologies related to schizophrenia vulnerability.

Reelin and glutamic acid decarboxylase (GAD)67 expressed by cortical gamma-aminobutyric acid-ergic interneurons are down-regulated in schizophrenia. Because epidemiological studies of schizophrenia fail to support candidate gene haploinsufficiency of Mendelian origin, we hypothesize that epigenetic mechanisms (i.e., cytosine hypermethylation of CpG islands present in the promoter of these genes) may be responsible for this down-regulation. Protracted l-methionine (6.6 mmolkg for 15 days, twice a day) treatment in mice elicited in brain an increase of S-adenosyl-homocysteine, the processing product of the methyl donor S-adenosyl-methionine, and a marked decrease of reelin and GAD67 mRNAs in both WT and heterozygous reeler mice. This effect of l-methionine was associated with an increase in the number of methylated cytosines in the CpG island of the reelin promoter region. This effect was not observed for GAD65 or neuronal-specific enolase and was not replicated by glycine doses 2-fold greater than those of l-methionine. Prepulse inhibition of startle declined at a faster rate as the prepulsestartle interval increased in mice receiving l-methionine. Valproic acid (2 mmolkg for 15 days, twice a day) reverted l-methionine-induced down-regulation of reelin and GAD67 in both WT and heterozygous reeler mice, suggesting an epigenetic action through the inhibition of histone deacetylases. The same dose of valproate increased acetylation of histone H3 in mouse brain nearly 4-fold. This epigenetic mouse model may be useful in evaluating drug efficacy on schizophrenia vulnerability. Hence the inhibition of histone deacetylases could represent a pharmacological intervention mitigating epigenetically induced vulnerability to schizophrenia in individuals at risk.