Recent evidence suggests that epigenetic mechanisms play a role in psychiatric diseases. In this study, we considered rats with neonatal ventral hippocampal lesions (NVHL) that are currently used for modeling neurodevelopmental aspects of schizophrenia. Contribution of epigenetic regulation to the effects of the lesion was investigated, using a histone deacetylase (HDAC) inhibitor. Lesioned or sham-operated rats were treated with the general HDAC inhibitor phenylbutyrate, which was injected daily from the day after surgery until adulthood. Changes in the volume of the lesion were monitored by magnetic resonance imaging (MRI). Anxiety was analyzed in the Plus Maze Test. Hypersensitivity of the dopaminergic system was evaluated by measuring the locomotor response to apomorphine. An associative conditioning test rewarded with food was used to evaluate learning abilities. The volume of the lesions expanded long after surgery, independently of the treatment, as assessed by MRI. Removal of the ventral hippocampus reduced anxiety, and this remained unchanged when animals were treated with phenylbutyrate. In contrast, NVHL rats' hypersensitivity to apomorphine and deterioration of the associative learning were reduced by the treatment. Global HDAC activity, which was increased in the prefrontal cortex of lesioned non-treated rats, was found to be reversed by HDAC inhibition. The study provides evidence that chromatin remodeling may be useful for limiting behavioral consequences due to lesioning of the ventral hippocampus at an early age. This represents a novel approach for treating disorders resulting from insults occurring during brain development.

Cell-free circulating DNA carries not only tumor-specific changes in its sequence but also distinctive epigenetic marks, namely DNA methylation, in certain GC-rich fragments. These fragments are usually located within the promoters and first exons of many genes, comprising CpG islands. Analysis of DNA methylation using cell-free circulating DNA can facilitate development of very accurate biomarkers for detection, diagnosis, prediction of response to therapy and prognosis of outcomes. Recent data suggest that benign and inflammatory diseases have very specific methylation patterns within cell-free circulating DNA, which are different from the pattern of a malignant tumor of the same organ. In addition, specific methylation patterns have been detected for cancers of different organs, so a differential diagnosis of site-specific cancer appears feasible. Currently, cancer-related applications dominate the field, although methylation-based biomarkers may also be possible for other diseases, including neurodegenerative and psychiatric disorders.

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.

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.

A recent report suggests that the down-regulation of reelin and glutamic acid decarboxylase (GAD(67)) mRNAs represents 2 of the more consistent findings thus far described in post-mortem material from schizophrenia (SZ) patients [reviewed in. Neurochemical markers for schizophrenia, bipolar disorder amd major depression in postmortem brains. Biol Psychiatry 57, 252-260]. To study mechanisms responsible for this down-regulation, we have analyzed the promoter of the human reelin gene. Collectively, our studies suggest that SZ is characterized by a gamma-amino butyric acid (GABA)-ergic neuron pathology presumably mediated by promoter hypermethylation facilitated by the over-expression of the methylating enzyme DNA methyltransferase (Dnmt) 1. Using transient expression assays, promoter deletions and co-transfection assays with various transcription factors, we have shown a clear synergistic action that is a critical component of the mechanism of the trans-activation process. Equally important is the observation that the reelin promoter is more heavily methylated in brain regions in patients diagnosed with SZ as compared to non-psychiatric control subjects [Grayson, D. R., Jia, X., Chen, Y., Sharma, R. P., Mitchell, C. P.,& Guidotti, A., et al.(2005). Reelin promoter hypermethylation in schizophrenia. Proc Natl Acad Sci U S A 102, 9341-9346]. The combination of studies in cell lines and in animal models of SZ, coupled with data obtained from post-mortem human material provides compelling evidence that aberrant methylation may be part of a core dysfunction in this psychiatric disease. More interestingly, the hypermethylation concept provides a coherent mechanism that establishes a plausible link between the epigenetic misregulation of multiple genes that are affected in SZ and that collectively contribute to the associated symptomatology.

Nicotine improves cognitive performance and attention in both experimental animals and in human subjects, including patients affected by neuropsychiatric disorders. However, the specific molecular mechanisms underlying nicotine-induced behavioral changes remain unclear. We have recently shown in mice that repeated injections of nicotine, which achieve plasma concentrations comparable to those reported in high cigarette smokers, result in an epigenetically induced increase of glutamic acid decarboxylase 67 (GAD(67)) expression. Here we explored the impact of synthetic α(4)β(2) and α(7) nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α(4)β(2) and a lower affinity full agonist at α(7) neuronal nAChR, injected in doses of 1-5 mg/kg/s.c. twice daily for 5 days, elicited a 30-40% decrease of cortical DNA methyltransferase (DNMT)1 mRNA and an increased expression of GAD(67) mRNA and protein. This upregulation of GAD(67) was abolished by the nAChR antagonist mecamylamine. Furthermore, the level of MeCP(2) binding to GAD(67) promoters was significantly reduced following VAR administration. This effect was abolished when VAR was administered with mecamylamine. Similar effects on cortical DNMT1 and GAD(67) expression were obtained after administration of A-85380, an agonist that binds to α(4)β(2) but has negligible affinity for α(3)β(4) or α(7) subtypes containing nAChR. In contrast, PNU-282987, an agonist of the homomeric α(7) nAChR, failed to decrease cortical DNMT1 mRNA or to induce GAD(67) expression. The present study suggests that the α(4)β(2) nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α(7) nAChR agonists.

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: 5alpha-reductase (5alpha-R) type I and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). This study evaluates 5alpha-R type I and 3alpha-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100beta immunohistochemistry. We demonstrate that 5alpha-R type I and 3alpha-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5alpha-R type I nor 3alpha-HSD mRNAs are expressed in S100beta- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5alpha-R type I and 3alpha-HSD in cortical and hippocampal GABAergic interneurons. However, 5alpha-R type I and 3alpha-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.

RATIONALE: Cortical gamma-aminobutyric acid (GABA)ergic neurons contribute to the orchestration of pyramidal neuron population firing as follows:(1) by releasing GABA on GABA(A) and GABA(B) receptors,(2) by releasing reelin in the proximity of integrin receptors located on cortical pyramidal neuron dendritic spines, and (3) through reelin contributing to the regulation of dendritic spine plasticity by modulating dendritic resident mRNA translation. In schizophrenia (SZ) and bipolar (BP) postmortem brains, the downregulation of mRNAs encoding glutamic acid decarboxylase 67 (GAD(67)) and reelin decreases the cognate proteins coexpressed in prefrontal cortex (PFC) GABAergic neurons. This finding has been replicated in several laboratories. Such downregulation suggests that the neuropil hypoplasticity found in the PFC of SZ and BP disorder patients may depend on a downregulation of GABAergic function, which is associated with a decrease in reelin secretion from GABAergic neuron axon terminals on dendrites, somata, or axon initial segments of pyramidal neurons. Indirectly, this GABAergic neuron downregulation may play a key role in the expression of positive and negative symptoms of SZ and BP disorders. OBJECTIVES: The above described GABAergic dysfunction may be addressed by pharmacological interventions to treat SZ and BP disorders using specific benzodiazepines (BZs), which are devoid of intrinsic activity at GABA(A) receptors including alpha(1) subunits but that act as full positive allosteric modulators of GABA action at GABA(A) receptors containing alpha(2), alpha(3), or alpha(5) subunits. These drugs are expected to enhance GABAergic signal transduction without eliciting sedation, amnesia, and tolerance or dependence liabilities. RESULTS AND CONCLUSIONS: BZs, such as diazepam, although they are efficient in equilibrating GABA(A) receptor signal transduction in a manner beneficial in the treatment of positive and negative symptoms of SZ, may not be ideal drugs, because by mediating a full positive allosteric modulation of GABA(A) receptors containing the alpha(1) subunit, they contribute to sedation and to the development of tolerance after even a brief period of treatment. In contrast, other BZ-binding site ligands, such as 6-(2bromophenyl)-8-fluoro-4H-imidazo [1,5-a][1,4] benzodiazepine-3-carboxamide (imidazenil), which fail to allosterically and positively modulate the action of GABA at GABA(A) receptors with alpha(1) subunits but that selectively allosterically modulate cortical GABA(A) receptors containing alpha(5) subunits, contribute to the anxiolytic, antipanic, and anticonvulsant actions of these ligands without producing sedation, amnesia, or tolerance. Strong support for the use of imidazenil in psychosis emerges from experiments with reeler mice or with methionine-treated mice, which express a pronounced reelin and GAD(67) downregulation that is also operative in SZ and BP disorders. In mice that model SZ symptoms, imidazenil increases signal transduction at GABA(A) receptors containing alpha(5) subunits and contributes to the reduction of behavioral deficits without producing sedation or tolerance liability. Hence, we suggest that imidazenil may be considered a prototype for a new generation of positive allosteric modulators of GABA(A) receptors, which, either alone or in combination with neuroleptics, should be evaluated in GABAergic dysfunction operative in the treatment of SZ and BP disorders with psychosis.

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.