The complex neurodevelopmental disorder schizophrenia is thought to be induced by an interaction between predisposing genes and environmental stressors. In order to get a better insight into the aetiology of this complex disorder, animal models have been developed. In this review, we summarize mRNA expression profiling studies on neurodevelopmental, pharmacological and genetic animal models for schizophrenia. We discuss parallels and contradictions among these studies, and propose strategies for future research.

Reelin plays an important role in the development and function of the brain and has been linked to different neuropsychiatric diseases. To further clarify the connection between reelin and psychiatric disorders, we studied the factors that influence the expression of reelin gene (RELN) and its different isoforms. We examined the total expression of RELN, allelic expression, and two alternative RELN isoforms in postmortem brain samples from patients with schizophrenia and bipolar disorder, as well as unaffected controls. We did not find a significant reduction in the total expression of RELN in schizophrenia or bipolar disorder. However, we did find a significant reduction of the proportion of the short RELN isoform, missing the C-terminal region in bipolar disorder, and imbalance in the allelic expression of RELN in schizophrenia. In addition, we tested the association between variation in RELN expression and rs7341475, an intronic SNP that was found to be associated with schizophrenia in women. We did not find an association between rs7341474 and the total expression of RELN either in women or in the entire sample. However, we observed a nominally significant effect of genotype-by-sex interaction on the variation in microexon skipping. Women with the risk genotype of rs7341475 (GG) had a higher proportion of microexon skipping, which is the isoform predominant in tissues outside the brain, while men had the opposite trend. Finally, we tested 83 SNPs in the gene region for association with expression variation of RELN, but none were significant. Our study further supports the connection between RELN dysfunction and psychiatric disorders, and provides a possible functional role for a schizophrenia associated SNP. Nevertheless, the positive associations observed in this study needs further replication as it may have implications for understanding the biological causes of schizophrenia and bipolar disorder.

BACKGROUND Epigenetic changes may play a role in the etiology of psychotic diseases. It has been demonstrated that the serotonin receptor, 5HTR1A, is implicated in schizophrenia (SCZ) and bipolar disorder (BPD). The aim of this study was to investigate the methylation status of a promoter region of the 5HTR1A gene in BPD and SCZ patients. METHODS Our study included 58 BPD and 40 SCZ (DSM-IV criteria) as well as 67 control subjects. DNA was extracted from blood leukocytes and high-resolution melt (HRM) method was used for analysis. RESULTS Non-parametric analysis of variance (Kruskal-Wallis) within groups was significant: H=67.6; p<0.0001. The Mann-Whitney U-test showed increased methylation level in both BPD (Z=-7.4; p<0.0001) and SCZ (Z=4.2; p<0.0001) compared to controls. No effect either of age or gender by own, was observed. ANCOVA revealed a modest effect of age/gender covariance (F=3.99; p<0.048). LIMITATION We used a peripheral tissue. The relationship between methylation of blood and brain DNA is not well known. Data need to be replicated in a brain tissue. CONCLUSION We observed increased DNA methylation in the promoter region of the 5HTR1A gene of SCZ and BPD. This could explain the reported decrease of the receptor expression. The current study supports the growing interest of DNA methylation in psychopathology.

Previous work suggests that the methylenetetrahydrofolate reductase gene (MTHFR) functional polymorphism A1298C may be a risk factor for schizophrenia. In this study, the genetic association between the MTHFR A1298C polymorphism and schizophrenia was investigated in 379 patients with schizophrenia and 380 age- and sex-matched controls subjects. The results showed an association between the 1298C allele and the disorder (OR 1.39, 95% confidence interval 1.08-1.79). This provides further evidence that the MTHFR A1298C polymorphism may play a role in conferring risk for schizophrenia in the Chinese Han population.

Genome-wide association studies (GWAS) with small sample size have had limited statistical power in identifying schizophrenia susceptibility genes. This is exemplified by the fact that one of the most convincing associations was detected only after meta-analyses of three different GWAS. Here we used meta-analysis to study the association of two single-nucleotide polymorphisms (SNPs)(rs7341475 and rs17746501) previously indicated to be associated with schizophrenia by a GWAS of Ashkenazi Jews (AJ). In the initial report, rs7341475 was associated only in women, while rs17746501 was associated in both men and women. We collected genotyping results of samples published in four GWAS for the two SNPs, additional to results from AJ. We used the Mantel-Haenszel method to combine the data of the different samples. For both SNPs, the results of the meta-analysis of all samples, including the initial report, did not reach a genome-wide significance level. However, the association between rs7341475 and schizophrenia in women, after excluding the data from AJ, was significant (P = 9.0 x 10(-3)), with a calculated odds ratio (OR) of 1.11, much smaller than the original result. Association between rs17746501 and schizophrenia was significant in four of the new samples, showing evidence for heterogeneity and very small effect when tested across all samples (P = 0.016, OR = 1.06). These findings suggest that the two SNPs might have a small effect on schizophrenia risk and suggest that meta-analyses of very large samples are needed to adequately study the contribution of common variants to schizophrenia susceptibility.

Epigenetics commonly refers to the developmental process by which cellular traits are established and inherited without a change in DNA sequence. These mechanisms of cellular memory also orchestrate gene expression in the adult brain and recent evidence suggests that the "epigenome" represents a critical interface between environmental signals, activation, repression and maintenance of genomic responses, and persistent behavior. We here review the current state of knowledge regarding the contribution of the epigenome toward the development of psychiatric disorders.(c) 2010 Wiley Periodicals, Inc. Dev. Psychobiol.

Epigenetics is a rapidly growing field and holds great promise for a range of human diseases, including brain disorders such as Rett syndrome, anxiety and depressive disorders, schizophrenia, Alzheimer disease and Huntington disease. This review is concerned with the pharmacology of epigenetics to treat disorders of the epigenome whether induced developmentally or manifested/acquired later in life. In particular, we will focus on brain disorders and their treatment by drugs that modify the epigenome. While the use of DNA methyl transferase inhibitors and histone deacetylase inhibitors in in vitro and in vivo models have demonstrated improvements in disease-related deficits, clinical trials in humans have been less promising. We will address recent advances in our understanding of the complexity of the epigenome with its many molecular players, and discuss evidence for a compromised epigenome in the context of an ageing or diseased brain. We will also draw on examples of species differences that may exist between humans and model systems, emphasizing the need for more robust pre-clinical testing. Finally, we will discuss fundamental issues to be considered in study design when targeting the epigenome.

Neurobiological disorders have diverse manifestations and symptomology. Neurodegenerative disorders, such as Alzheimer's disease, manifest late in life and are characterized by, among other symptoms, progressive loss of synaptic markers. Developmental disorders, such as autism spectrum, appear in childhood. Neuropsychiatric and affective disorders, such as schizophrenia and major depressive disorder, respectively, have broad ranges of age of onset and symptoms. However, all share uncertain etiologies, with opaque relationships between genes and environment. We propose a 'Latent Early-life Associated Regulation'(LEARn) model, positing latent changes in expression of specific genes initially primed at the developmental stage of life. In this model, environmental agents epigenetically disturb gene regulation in a long-term manner, beginning at early developmental stages, but these perturbations might not have pathological results until significantly later in life. The LEARn model operates through the regulatory region (promoter) of the gene, specifically through changes in methylation and oxidation status within the promoter of specific genes. The LEARn model combines genetic and environmental risk factors in an epigenetic pathway to explain the etiology of the most common, that is, sporadic, forms of neurobiological disorders.

Gamma-aminobutyric acid A (GABA(A)) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the expression of four GABA(A) receptor subunits and observed significant reductions in GABRA1, GABRA2, GABRA3, and GABRB3 in parietal cortex (Brodmann's Area 40 (BA40)), while GABRA1 and GABRB3 were significantly altered in cerebellum, and GABRA1 was significantly altered in superior frontal cortex (BA9). The presence of seizure disorder did not have a significant impact on GABA(A) receptor subunit expression in the three brain areas. Our results demonstrate that GABA(A) receptors are reduced in three brain regions that have previously been implicated in the pathogenesis of autism, suggesting widespread GABAergic dysfunction in the brains of subjects with autism.

I review and evaluate genetic and genomic evidence salient to the hypothesis that the development and evolution of psychotic spectrum conditions have been mediated in part by alterations of imprinted genes expressed in the brain. Evidence from the genetics and genomics of schizophrenia, bipolar disorder, major depression, Prader-Willi syndrome, Klinefelter syndrome, and other neurogenetic conditions support the hypothesis that the etiologies of psychotic spectrum conditions commonly involve genetic and epigenetic imbalances in the effects of imprinted genes, with a bias towards increased relative effects from imprinted genes with maternal expression or other genes favouring maternal interests. By contrast, autistic spectrum conditions, including Kanner autism, Asperger syndrome, Rett syndrome, Turner syndrome, Angelman syndrome, and Beckwith-Wiedemann syndrome, commonly engender increased relative effects from paternally expressed imprinted genes, or reduced effects from genes favouring maternal interests. Imprinted-gene effects on the etiologies of autistic and psychotic spectrum conditions parallel the diametric effects of imprinted genes in placental and foetal development, in that psychotic spectrum conditions tend to be associated with undergrowth and relatively-slow brain development, whereas some autistic spectrum conditions involve brain and body overgrowth, especially in foetal development and early childhood. An important role for imprinted genes in the etiologies of psychotic and autistic spectrum conditions is consistent with neurodevelopmental models of these disorders, and with predictions from the conflict theory of genomic imprinting.

The neuronal GABAergic mechanisms that mediate the symptomatic beneficial effects elicited by a combination of antipsychotics with valproate (a histone deacetylase inhibitor) in the treatment of psychosis (expressed by schizophrenia or bipolar disorder patients) are unknown. This prompted us to investigate whether the beneficial action of this combination results from a modification of histone tail covalent esterification or is secondary to specific chromatin remodeling. The results suggest that clozapine, or sulpiride associated with valproate, by increasing DNA demethylation with an unknown mechanism, causes a chromatin remodeling that brings about a beneficial change in the epigenetic GABAergic dysfunction typical of schizophrenia and bipolar disorder patients.

Recent advances in schizophrenia (SZ) research indicate that the telencephalic gamma-aminobutyric acid (GABA)ergic neurotransmission deficit associated with this psychiatric disorder probably is mediated by the hypermethylation of the glutamic acid decarboxylase 67 (GAD(67)), reelin and other GABAergic promoters. A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to induce a DNA-cytosine demethylation by altering the chromatin remodeling with valproate (VPA). When co-administered with VPA, the clinical efficacy of atypical antipsychotics is enhanced. This prompted us to investigate whether this increase in drug efficacy is related to a modification of GABAergic-promoter methylation via chromatin remodeling. Our previous and present results strongly indicate that VPA facilitates chromatin remodeling when it is associated with clozapine or sulpiride but not with haloperidol or olanzapine. This remodeling might contribute to reelin- and GAD(67)-promoter demethylation and might reverse the GABAergic-gene-expression downregulation associated with SZ morbidity.

Reelin mRNA and protein levels are reduced by approximately 50% in various cortical structures of postmortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. In addition, the mRNA encoding the methylating enzyme, DNA methyltransferase 1, is up-regulated in the same neurons that coexpress reelin and glutamic acid decarboxylase 67. We have analyzed the extent and pattern of methylation within the CpG island of the reelin promoter in genomic DNA isolated from cortices of schizophrenia patients and nonpsychiatric subjects. Ten (The Stanley Foundation Neuropathology Consortium) and five (Harvard Brain Collection) schizophrenia patients and an equal number of nonpsychiatric subjects were selected from each brain collection. Genomic DNA was isolated, amplified (from base pair -527 to base pair +322) after bisulphite treatment, and sequenced. The results show that within the promoter region there were interesting regional variations. There was increased methylation at positions -134 and 139, which is particularly important for regulation, because this portion of the promoter is functionally competent based on transient transfection assays. This promoter region binds a protein present in neuronal precursor nuclear extracts that express very low levels of reelin mRNA; i.e., an oligonucleotide corresponding to this region and that contains methylated cytosines binds more tightly to extracts from nonexpressing cells than the nonmethylated counterpart. Collectively, the data show that this promoter region has positive and negative properties and that the function of this complex cis element relates to its methylation status.

The polygenic nature of complex psychiatric disorders suggests a common pathway that may be involved in the down-regulation of multiple genes through an epigenetic mechanism. To investigate the role of methylation in down-regulating the expression of mRNAs that may be associated with the schizophrenia phenotype, we have adopted a cell-culture model amenable to this line of investigation. We have administered methionine (2 mM) to primary cultures of cortical neurons prepared from embryonic day 16 mice and show that this treatment down-regulated reelin and glutamic acid decarboxylase 67 (GAD67) mRNA expression but not that corresponding to neuron-specific enolase mRNA. Moreover, methionine increased methylation of the reelin promoter, suggesting a possible mechanism for the observed change. These cultures contain a mixed population of neurons and glia. Approximately 83% of the neurons are GABAergic based on GAD immunoreactivity, and these neurons coexpress high levels of reelin and DNA methyltransferase (Dnmt) 1 immunoreactivity. To examine whether Dnmt1 regulates reelin gene expression, we used an antisense approach to reduce (knock down) Dnmt1 expression. The reduced Dnmt1 mRNA and protein were accompanied by increased reelin mRNA expression. More importantly, the Dnmt1 knockdown blocked the methionine-induced reelin and GAD67 mRNA down-regulation. These data support the hypothesis that the reduced amounts of reelin and GAD67 mRNAs documented in postmortem schizophrenia brain may be the consequence of a Dnmt1-mediated hypermethylation of the corresponding promoters.

The methylation and demethylation of CpG dinucleotides that are embedded in promoters play an important role in controlling gene transcription. In the mammalian brain, CpG promoter methylation is a postreplicative process mediated by a group of DNA methyltransferases (DNMT), such as DNMT1 and DNMT3a, DNMT3b. Several studies demonstrate that in addition to DNMTs, promoter methylation in the brain can be regulated by a putative DNA demethylation process that specifically removes the methyl group from the carbon-5 of cytosines. To test the existence of a possible active DNA demethylation activity in postmitotic neuronal or glial cells, we incubated an SssI methylated mouse reelin (Reln) promoter fragment (-720 to +140) with nuclear extracts from the mouse frontal cortex (FC). We observed the presence of DNA demethylation activity, which was increased in FC nuclear extracts from mice treated with valproate (VPA, 2.2 mmol/kg, twice a day for 3 days). VPA not only reduces anxiety, and cognitive deficits, and other symptoms in bipolar disorder (BP) disorder and schizophrenia (SZ) patients but also upregulates Reln and glutamic acid decarboxylase 67 (Gad67) mRNA/protein expression by reducing the methylation of their promoters. We believe that the identification of an enzyme in brain that facilitates DNA-demethylation and an understanding of how drugs induce DNA demethylation are crucial to progress in a new line of pharmacological interventions to treat neurodevelopment, neuropsychiatric, and neurodegenerative diseases.

The role of methylation in the history of psychiatry has traversed a storied path. The original trans-methylation hypothesis was proposed at a time when chlorpromazine had been synthesized but not yet marketed as an antipyschotic (Thorazine). The premise was that abnormal metabolism led to the methylation of biogenic amines in the brains of schizophrenia patients and that these hallucinogenic compounds produced positive symptoms of the disease. At the time, psychiatry was very interested in drugs such as mescaline and lysergic acid diethyl amide that replicated clinical symptoms and understood that these compounds might provide a biological basis for psychosis. The amino acid methionine (MET) was given to patients in the hopes of confiriming the transmethylation hypothesis. However with time, many realized that the hunt for an endogenous psychotropic compound would remain elusive. We now believe that the MET studies may have produced a toxic reaction in susceptible patients by disrupting epigenetic regulation in the brain. The focus of the current review is on the coordinate regulation of multiple promoters expressed in neurons that may be modulated through methylation. While certainly the identification of genes and promoters regulated epigenetically has been steadily increasing over the years, there have been few studies that examine methylation changes as a consequence of increased levels of a dietary amino acid such as methionine (MET). We suggest that the MET mouse model may provide information regarding the indentification of genes that are regulated by epigenetic perturbations. In addition to our studies with the reelin and GAD67 promoters, we also have evidence that additional promoters expressed in select neurons of the brain are similarly affected by MET administration. We suggest that to expand our knowledge of epigenetically-responsive promoters using MET might allow for a better appreciation of global methylation changes occurring in selected brain regions.

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNMT-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, HDAC inhibitors, activate these two genes with a comparable dose- and time-dependence. In parallel, both groups of drugs decrease DNMT1, DNMT3A and DNMT3B protein levels, and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes, containing all three DNMTs, MeCP2 and HDAC1, from the corresponding promoters and increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

Reelin and GAD67 mRNAs and protein levels are substantially reduced in post-mortem brains of schizophrenia patients. Increasing evidence suggests that the observed down-regulation of reelin and GAD67 gene expression may be caused by the dysfunction of epigenetic regulatory pathways operative in cortical GABAergic interneurons. To explore whether human reelin and GAD67 mRNAs are coordinately regulated through DNA methylation-dependent mechanisms, we studied the effects of DNA methyltransferase inhibitors on reelin and GAD67 expression in NT-2 neuronal precursor cells. Competitive RT-PCR with internal standards was used to quantitate mRNA levels. The data showed that reelin and GAD67 mRNAs are induced in the same dose- and time-dependent manners. We further demonstrated that the activation of these two genes correlated with a reduction in DNA methyltransferase activity and DNMT1 protein levels. Time-course Western blot analysis showed that DNMT1 protein down-regulation occurs temporally prior to the reelin and GAD67 mRNA increase. In addition, ChIP assays demonstrated that the activation of the reelin gene correlates with dissociation of DNMT1 and MeCP2 from the promoter, and an increased acetylation of histones H3 in the region. Collectively, our data strongly imply that human reelin and GAD67 genes are coordinately regulated through epigenetic mechanisms that include the action of DNMT1. Our study also suggests that negative regulation of the reelin gene involves methylation-dependent recruitment of DNMT1, MeCP2, and certain HDACs, which most likely reduce the activity of the promoter by shifting the surrounding chromatin into a more compact state.

Levels of acetylated Histone 3 and 4 proteins are strongly predictive of a chromatin structure that is conducive to gene expression. In cell and animal studies, valproic acid is a potent inhibitor of histone deactylating enzymes, and consequently results in increased levels of acetylated Histone 3 (acH3) and acetylated Histone 4 proteins (acH4). To examine this effect in a clinical setting, 14 schizophrenic and bipolar patients were treated with valproic acid (Depakote ER(R)), either as monotherapy or in combination with antipsychotics, over a period of 4 weeks. AcH3 and acH4 levels from lymphocyte nuclear protein extracts were measured by Western Blot. Treatment with Depakote ER resulted in a significant increase of acH3 and a trend-level increase of acH4. Levels of valproic acid were positively and significantly correlated with percent increase in acH3 but not acH4. Schizophrenia patients were significantly less likely to increase their acH3 and acH4 levels after 4 weeks on Depakote ER. The authors consider these results in the context of future application of HDAC inhibitors to the treatment of psychiatric disorders.

The accessibility of cognate binding sites within a gene promoter can be modified by the condensation or relaxation of local chromatin structure. Local chromatin structure is in turn programmed by covalent modifications of cytosine bases in DNA and amino acid residues in histone protein tails. These chemical and physical adaptations around gene promoters can significantly change levels of mRNA expression. Furthermore, linear patterns of covalent modification of histone protein tails are emerging as a distinct regulatory code--another form of cellular memory. Because chromatin structure can be modified by conventional pharmacologic therapy, a novel approach to the regulation of neuronal gene expression in clinical populations is possible.

High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.

The methylation and demethylation of CpG dinucleotides that are embedded in promoters play an important role in controlling gene transcription. In the mammalian brain, CpG promoter methylation is a postreplicative process mediated by a group of DNA methyltransferases (DNMT), such as DNMT1 and DNMT3a, DNMT3b. Several studies demonstrate that in addition to DNMTs, promoter methylation in the brain can be regulated by a putative DNA demethylation process that specifically removes the methyl group from the carbon-5 of cytosines. To test the existence of a possible active DNA demethylation activity in postmitotic neuronal or glial cells, we incubated an SssI methylated mouse reelin (Reln) promoter fragment (-720 to +140) with nuclear extracts from the mouse frontal cortex (FC). We observed the presence of DNA demethylation activity, which was increased in FC nuclear extracts from mice treated with valproate (VPA, 2.2 mmol/kg, twice a day for 3 days). VPA not only reduces anxiety, and cognitive deficits, and other symptoms in bipolar disorder (BP) disorder and schizophrenia (SZ) patients but also upregulates Reln and glutamic acid decarboxylase 67 (Gad67) mRNA/protein expression by reducing the methylation of their promoters. We believe that the identification of an enzyme in brain that facilitates DNA-demethylation and an understanding of how drugs induce DNA demethylation are crucial to progress in a new line of pharmacological interventions to treat neurodevelopment, neuropsychiatric, and neurodegenerative diseases.

In the kidney, expression of 25-hydroxyvitamin D-1-alpha-hydroxylase (CYP27B1) is primarily under negative transcriptional control by the vitamin D receptor (VDR)(Takeyama et al., 1997), while PTH is a positive regulator. We have previously demonstrated that a bHLH transcription factor, VDIR (also called TCF3), directly binds to a newly identified negative vitamin D response element in the promoter. Association of VDR with VDIR results in recruitment of an HDAC2 co-repressor complex leading to transrepression that is also dependent on the 1alpha,25-dihydroxyvitamin D ligand (abbreviated VD or 1alpha,25(OH)2D)(Murayama et al., 2004; Fujiki et al., 2005). However, TSA (an HDAC inhibitor) could not fully abrogate 1a,25(OH)(2)D(3)-induced transrepression. To explore the unknown mechanism of TSA-insensitive transrepression by VD, we further characterized the VDIR-VDR co-repressor complex. Using a biochemical purification approach, we found DNA methyltransferases (Dnmts) in the VDTR-VDR complex, and showed that transrepression is mediated by these Dnmts. DNA methylation was induced by VD at CpG sites in the promoter as well as exon regions of the human CYP27B1 gene. PTH was unexpectedly found to induce demethylation of the methylated CpG sites in the CYP27B1 promoter with derepression of transcription, and this DNA demethylation step in response to PTH required phosphorylation of MBD4. Thus, the study of the CYP27B1 gene promoter has uncovered a novel molecular mechanism of hormonal control of gene regulation at two epigenetic levels, namely histone acetylation and DNA methylation.

The expression levels of caspase-3, a major contributor to the execution of neuronal apoptosis, markedly decrease in the process of brain maturation. We have previously cloned the rat caspase-3 gene promoter and identified its essential regulatory elements. In the present study, we extended previous findings by examining transcriptional regulation of caspase-3 expression in the rat brain of two different ages, corresponding to the immature and mature brain. In particular, we determined that the rate of transcription initiation substantially declines during brain maturation. Furthermore, we established that mRNA levels of Ets1, Ets2 and Sp1 do not change in the brain with maturation, suggesting that these transcription factors do not contribute to age-dependent caspase-3 down-regulation. Hence, we examined a role of DNA methylation and histone modification in this process. Utilizing bisulfite DNA sequencing, we determined the presence of age-dependent differentially-methylated fragments within the caspase-3 promoter region. Strikingly, differentially methylated CpG sites correspond to the predicted binding sites for a number of transcription factors that have been previously shown to be involved in neuronal development and differentiation. Moreover, using chromatin immunoprecipitation, we found that mature brains displayed significantly lower levels of histone 3 acetylated Lys14 and histone 4 acetylated Lys5, 8, 12, and 16. This observation is consistent with the decreased level of expression of caspase-3 in the mature brain. Together with our observation that histone deacetylase inhibitor, trichostatin A, increased the level of caspase-3 mRNA in cortical neurons in vitro, these results further indicate an important role of epigenetic factors in the regulation of caspase-3 gene expression.

Epigenetic modifications at the histone level affect gene regulation in response to extracellular signals. However, regulated epigenetic modifications at the DNA level, especially active DNA demethylation, in gene activation are not well understood. Here we report that DNA methylation/demethylation is hormonally switched to control transcription of the cytochrome p450 27B1 (CYP27B1) gene. Reflecting vitamin-D-mediated transrepression of the CYP27B1 gene by the negative vitamin D response element (nVDRE), methylation of CpG sites ((5m)CpG) is induced by vitamin D in this gene promoter. Conversely, treatment with parathyroid hormone, a hormone known to activate the CYP27B1 gene, induces active demethylation of the (5m)CpG sites in this promoter. Biochemical purification of a complex associated with the nVDRE-binding protein (VDIR, also known as TCF3) identified two DNA methyltransferases, DNMT1 and DNMT3B, for methylation of CpG sites, as well as a DNA glycosylase, MBD4 (ref. 10). Protein-kinase-C-phosphorylated MBD4 by parathyroid hormone stimulation promotes incision of methylated DNA through glycosylase activity, and a base-excision repair process seems to complete DNA demethylation in the MBD4-bound promoter. Such parathyroid-hormone-induced DNA demethylation and subsequent transcriptional derepression are impaired in Mbd4(-/-) mice. Thus, the present findings suggest that methylation switching at the DNA level contributes to the hormonal control of transcription.

The role of methylation in the history of psychiatry has traversed a storied path. The original trans-methylation hypothesis was proposed at a time when chlorpromazine had been synthesized but not yet marketed as an antipyschotic (Thorazine). The premise was that abnormal metabolism led to the methylation of biogenic amines in the brains of schizophrenia patients and that these hallucinogenic compounds produced positive symptoms of the disease. At the time, psychiatry was very interested in drugs such as mescaline and lysergic acid diethyl amide that replicated clinical symptoms and understood that these compounds might provide a biological basis for psychosis. The amino acid methionine (MET) was given to patients in the hopes of confiriming the transmethylation hypothesis. However with time, many realized that the hunt for an endogenous psychotropic compound would remain elusive. We now believe that the MET studies may have produced a toxic reaction in susceptible patients by disrupting epigenetic regulation in the brain. The focus of the current review is on the coordinate regulation of multiple promoters expressed in neurons that may be modulated through methylation. While certainly the identification of genes and promoters regulated epigenetically has been steadily increasing over the years, there have been few studies that examine methylation changes as a consequence of increased levels of a dietary amino acid such as methionine (MET). We suggest that the MET mouse model may provide information regarding the indentification of genes that are regulated by epigenetic perturbations. In addition to our studies with the reelin and GAD67 promoters, we also have evidence that additional promoters expressed in select neurons of the brain are similarly affected by MET administration. We suggest that to expand our knowledge of epigenetically-responsive promoters using MET might allow for a better appreciation of global methylation changes occurring in selected brain regions.

Reduced levels of glutamic acid decarboxylase(67)(GAD(67)), an essential enzyme for GABA synthesis, is one of the most consistent gene expression changes found in the frontal cortex of patients with schizophrenia. Recently this reduction has been shown to extend to other areas including primary sensory, primary motor and anterior cingulate (ACC) cortices. To determine the extent to which additional cortical and subcortical regions may be affected in schizophrenia, we measured the level of GAD(67) mRNA in previously unexplored areas including the orbitofrontal (OFC) and superior temporal (STG) cortices as well as the caudate, putamen, nucleus accumbens, medial dorsal thalamus and anterior thalamus using in situ hybridization. We also examined GAD(67) mRNA levels in all these regions in individuals with bipolar disorder and major depression. ANCOVA comparing GAD(67) mRNA levels in all four diagnostic groups revealed a significant reduction ( approximately 30%) in layers III and IV of the OFC of patients with schizophrenia and bipolar disorder. A priori t-tests comparing GAD(67) mRNA levels between the schizophrenia and control groups revealed significant reductions in the ACC, STG, striatum and thalamus. These findings suggest that there may be a widespread reduction in GABA neurotransmission due to a decrease in the synthesis of GAD(67) in subjects with psychiatric disorders. The resulting decrease in inhibitory tone across multiple brain areas may contribute to the psychotic behavior observed in patients with schizophrenia and bipolar disorder.

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNMT-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, HDAC inhibitors, activate these two genes with a comparable dose- and time-dependence. In parallel, both groups of drugs decrease DNMT1, DNMT3A and DNMT3B protein levels, and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes, containing all three DNMTs, MeCP2 and HDAC1, from the corresponding promoters and increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

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 mumol/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 GAD(67) promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 mumol/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 GAD(67) expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.

Stromal cell-derived factor alpha (SDF1alpha) and its cognate receptor CXCR4 play an important role in neuronal development in the hippocampus, but the genes directly regulated by SDF1alpha/CXCR4 signaling are unknown. To study the role of CXCR4 targeted genes in neuronal development, we used neuronal cultures established from embryonic day 18 rats. Hippocampal neurons express CXCR4 receptor proteins, and are stimulated by SDF1alpha resulting in activation of extracellular signal-regulated kinase (ERK)1/2 and the transcription factor CREB. SDF1alpha rapidly induces the expression of early growth response gene Egr1, a transcription factor involved in activity-dependent neuronal responses, in a concentration-dependent manner. Gel-shift analysis showed that SDF1alpha enhances DNA binding activity to the Egr1-containing promoter for GAD67. Chromatin immunoprecipitation analysis using an Egr1 antibody indicated that SDF1alpha stimulation increases binding of Egr1 to a GAD67 promoter DNA sequence. SDF1alpha stimulation increases the expression of GAD67 at both the mRNA and protein levels, and increases the amount and neurite localization of GABA in neurons already expressing GABA. SDF1alpha-induced Egr1/GAD67 expression is mediated by the G protein-coupled CXCR4 receptor and activation of the ERK pathway. Reduction of Egr1 gene expression using siRNA technology lowers the level of GAD67 transcripts and inhibits SDF1alpha-induced GABA production. Inhibitin of CXCR4 in the developing mouse brain in utero greatly reduced Egr1 and GAD67 mRNA levels and GAD67 protein levels, suggesting a pivotal role for CXCR4 signaling in the development of GABAergic neurons in vivo. Our data suggest that SDF1alpha/CXCR4/G protein/ERK signaling induces the expression of GAD67 system via Egr1 activation, a mechanism that may promote the maturation of GABAergic neurons during development.