SUMMARY Schizophrenia (SCZ) is among the most disabling of mental disorders. Several neurobiological hypotheses have been postulated as responsible for SCZ pathogenesis: polygenic/multifactorial genomic defects, intrauterine and perinatal environment-genome interactions, neurodevelopmental defects, dopaminergic, cholinergic, serotonergic, gamma-aminobutiric acid (GABAergic), neuropeptidergic and glutamatergic/N-Methyl-D-Aspartate (NMDA) dysfunctions, seasonal infection, neuroimmune dysfunction, and epigenetic dysregulation. SCZ has a heritability estimated at 60-90%. Genetic studies in SCZ have revealed the presence of chromosome anomalies, copy number variants, multiple single-nucleotide polymorphisms of susceptibility distributed across the human genome, aberrant single nucleotide polymorphisms (SNPs) in microRNA genes, mitochondrial DNA mutations, and epigenetic phenomena. Pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variation in specific candidate genes and the positive and adverse effects of drug treatment. Approximately, 18% of neuroleptics are major substrates of CYP1A2 enzymes, 40% of CYP2D6, and 23% of CYP3A4; 24% of antidepressants are major substrates of CYP1A2 enzymes, 5% of CYP2B6, 38% of CYP2C19, 85% of CYP2D6, and 38% of CYP3A4; 7% of benzodiazepines are major substrates of CYP2C19 enzymes, 20% of CYP2D6, and 95% of CYP3A4. About 10-20% of Western populations are defective in genes of the CYP superfamily. Only 26% of Southern Europeans are pure extensive metabolizers for the trigenic cluster integrated by the CYP2D6+CYP2C19+CYP2C9 genes. The pharmacogenomic response of SCZ patients to conventional psychotropic drugs also depends on genetic variants associated with SCZ-related genes. Consequently, the incorporation of pharmacogenomic procedures both to drugs in development and drugs on the market would help to optimize therapeutics in SCZ and other central nervous system (CNS) disorders.

Parental effects are a major source of phenotypic plasticity and may influence offspring phenotype in concert with environmental demands. Studies of "environmental epigenetics" suggest that (1) DNA methylation states are variable and that both demethylation and remethylation occur in post-mitotic cells, and (2) that remodeling of DNA methylation can occur in response to environmentally driven intracellular signaling pathways. Studies of mother-offspring interactions in rodents suggest that parental signals influence the DNA methylation, leading to stable changes in gene expression. If parental effects do indeed enhance the "match" between prevailing environmental demands and offspring phenotype, then the potential for variation in environmental conditions over time would suggest a mechanism that requires active maintenance across generations through parental signaling. We suggest that parental regulation of DNA methylation states is thus an ideal candidate mechanism for parental effects on phenotypic variation.

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.

DNA methylation is an epigenetic mechanism in which the methyl group is covalently coupled to the C5 position of the cytosine residue of CpG dinucleotides. DNA methylation generally leads to gene silencing and is catalyzed by a group of enzymes known as DNA methyltransferases (Dnmt). During development, the epigenome undergoes waves of demethylation and methylation changes. As a result, there are cell type/tissue-specific DNA methylation patterns. Since DNA methylation changes only happen during DNA replication to maintain methylation patterns on hemimethylated DNA or establish new methylation, Dnmt expression generally decreases greatly after cell division. However, significant levels of Dnmts were noticed specifically in postmitotic neurons, suggesting a functional importance of Dnmt in the nervous system. Accumulating evidence showed that DNA methylation correlates with certain neuropsychiatric disorders such as schizophrenia, Rett syndrome, and ICF syndrome. Studies of methyl-CpG-binding proteins, Dnmt inhibitors, and Dnmt knockout mice also explored the key role of DNA methylation in neural development, plasticity, learning, and memory. Though an enzyme exhibiting DNA demethylation capability in vertebrates still remains to be identified, DNA methylation status in the CNS appeared to be reversible at certain genomic loci. This supports a maintenance role of Dnmt to prevent active demethylation in postmitotic neurons. Taken together, DNA methylation provides an epigenetic mechanism of gene regulation in neural development, function, and disorders.

BACKGROUND: A restrictive chromatin state has been thought to be operant in the pathophysiology of schizophrenia. Our objective was to ascertain whether differences exist between baseline levels of a repressive chromatin mark such as dimethylated lysine 9 of histone 3 (H3K9me2) in patients with schizophrenia and healthy controls and whether a histone deacetylase (HDAC) inhibitor in an in vitro assay would differentially affect chromatin structure based on diagnosis. METHODS: We obtained blood samples from 19 healthy controls and 25 patients with schizophrenia and isolated their lymphocytes. We measured baseline H3K9me2 levels (normalized to total histone 1) in the lymphocytes from all participants via Western blot analysis. To examine the effects of an HDAC inhibitor on H3K9me2, we cultured the lymphocytes from participants with trichostatin A (TSA) for 24 hours and then measured changes in H3K9me2 relative to the control condition (dimethyl sulfoxide). RESULTS: Patients with schizophrenia had significantly higher mean baseline levels of H3K9me2 than healthy controls (6.52 v. 2.78, p = 0.028). Moreover, there was a significant negative correlation between age at onset of illness and levels of H3K9me2 (Spearman's rho =-0.588, p = 0.008). In the lymphocyte cultures, TSA induced divergent responses in terms of H3K9me2 levels from patients with schizophrenia compared with healthy controls (F(1,14)= 5.082, p = 0.041). LIMITATIONS: The use of lymphocytes to study schizophrenia has its limitations because they may not be appropriate models of synaptic activity or other brain-specific activities. CONCLUSION: Our results provide further evidence that schizophrenia is associated with a restrictive chromatin state that is also less modifiable using HDAC inhibitors.

The cost of psychiatric illness to the UK economy was recently estimated at pound77 billion annually. Despite years of research no firm aetiological explanation exists, and with no physiological or biochemical markers diagnosis is made entirely on a behavioural basis. All current pharmacological therapies are associated with serious long-term side effects. Substantial evidence supports the involvement of one-carbon cycle dysregulation in psychiatric illness, but this is not currently used as a basis for diagnosis or treatment. The present paper reviews the evidence for one-carbon cycle dysregulation in schizophrenic, bipolar, depressed and autistic patients. Also presented are novel findings from the field of epigenetics, which demonstrate how the one-carbon cycle-derived methyl donor S-adenosylmethionine influences the expression of key genes in the brain affecting memory, learning, cognition and behaviour, genes whose expression is reduced to varying degrees in these patient groups. Clinical evidence that nutritional supplements can rectify one-carbon cycle activity, and restore normal gene expression, suggests a novel approach to the development of biochemical tests and simple, non-harmful treatments for some psychiatric patients. Conversely, evidence from animal studies highlights the dangers of exposing the unborn fetus to very high dietary levels of folic acid, a one-carbon cycle cofactor. Fetal adaptations to a high-folate environment may interfere with folate metabolism postnatally, with serious consequences for the epigenetic regulation of gene expression. The public health implications of these diverse scenarios indicate an urgent need for further research in this field.

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.

An emerging theme in the field of neuroscience is that processes critical for neurodevelopment have been co-opted by the adult nervous system to subserve synaptic plasticity and cognition. In this review, we highlight a surprising intersection of two developmental processes that together play a critical role in synaptic plasticity, memory formation and cognition. Reelin, a large glycoprotein associated with the extracellular matrix, is crucial for cortical and cerebellar development. Recent data from several groups indicate that reelin plays a unique modulatory role in the induction of synaptic plasticity in the hippocampus, and that normal levels of reelin in the adult brain are essential for successful formation of certain forms of long-term memory. Given that both increases and decreases in reelin expression have significant effects on plasticity and memory, regulation of reelin expression is predicted to have significant effects on neural function. Epigenetic regulation of transcription is critical for differentiation of cellular phenotype in metazoans. Dozens of reports in the last few years have demonstrated that epigenetics is involved in modulating gene expression in the adult nervous system and subserves plasticity and memory formation. We review a series of studies that demonstrate that the reelin promoter is subject to differential DNA methylation in the adult nervous system, and that perturbations in reelin promoter methylation correlate with alterations in memory formation and cognition. Thus, two distinct developmental processes, reelin-mediated signaling and epigenetic-based transcriptional regulation, appear to have synergized in the adult nervous system to create a sensitive and robust system for modulation of synaptic plasticity, and ultimately provide a powerful set of tools to probe the molecular basis of cognition.

DNA methylation is an epigenetic mechanism that plays a critical role in the repression of gene expression. Here, we show that DNA methyltransferase (DNMT) inhibition in hippocampal neurons results in activity-dependent demethylation of genomic DNA and a parallel decrease in the frequency of miniature EPSCs (mEPSCs), which in turn impacts neuronal excitability and network activity. Treatment with DNMT inhibitors reveals an activity-driven demethylation of brain-derived neurotrophic factor promoter I, which is mediated by synaptic activation of NMDA receptors, because it is susceptible to AP-5, a blocker of NMDA receptors. The specific effect of DNMT inhibition on spontaneous excitatory neurotransmission requires gene transcription and is occluded in the absence of the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2). Interestingly, enhancing excitatory activity, in the absence of DNMT inhibitors, also produces similar decreases in DNA methylation and mEPSC frequency, suggesting a role for DNA methylation in the control of homeostatic synaptic plasticity. Furthermore, adding excess substrate for DNA methylation (S-adenosyl-l-methionine) rescues the suppression of mEPSCs by DNMT inhibitors in wild-type neurons, as well as the defect seen in MeCP2-deficient neurons. These results uncover a means by which NMDA receptor-mediated synaptic activity drives DNA demethylation within mature neurons and suppresses basal synaptic function.

Histone deactylase enzymes are responsible for the deacetylation of histone tails, and consequently influence gene regulation through their ability to modify chromatin structure surrounding promoter regions. We analyzed the microarray collection of the National Brain Databank to investigate differential expression of these enzymes in the prefrontal cortices of control, schizophrenia and bipolar subjects. HDAC1 expression levels were significantly higher in schizophrenia versus normal subjects. The mRNA expression level of an epigenetically regulated schizophrenia candidate gene GAD67 was strongly and negatively correlated with the mRNA expression levels of HDAC1, HDAC3 and HDAC4 levels. These findings provide additional support for the proposal that epigenetic factors are operative in the brain pathology of patients with schizophrenia.

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 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.

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.

Prefrontal cortex (Brodmann's area 9) levels of the methyl donor S-adenosyl methionine were increased by about two-fold in schizophrenia and bipolar disorder patients, but not in unipolar depressed patients compared with nonpsychiatric subjects from the Stanley Foundation Neuropathology Consortium (Bethesda, Maryland, USA). Neither age, brain weight and pH, hemisphere, post-mortem interval, disease onset/duration, nor cumulative dose of fluphenazine affected S-adenosyl methionine content. In schizophrenia and bipolar disorder patients, the increase of S-adenosyl methionine is associated with an overexpression of DNA methyltransferase-1 mRNA in Brodmann's area 9 GABAergic neurons. Hence, the increased expression of S-adenosyl methionine and DNA methyltransferase-1 may contribute to promoter cytosine 5-methylation and to downregulation of the expression of mRNAs encoding for reelin and GAD67 in cortical GABAergic neurons of schizhophrenia and bipolar disorder patients.

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.

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.

Inhibitory GABAergic interneurons of prefrontal cortex (PFC) appear to play an important role in the regulation of intermittent pyramidal neuron columnary firing and in the neuronal plasticity that mediate cognitive functions. In schizophrenia (SZ), cognitive defects and dysfunctions in pyramidal neuronal columnary firing appear to depend on abnormalities of GABAergic neurons. These abnormalities include a decrease of GAD67 and reelin expression, which result in a reduction of cortical inhibitory input to spine postsynaptic densities as a result of the decrease of GABA concentration at the synaptic cleft, and of neurotrophic stimuli as a result of the decrease of reelin secreted into the extracellular matrix. Our studies show that alterations in chromatin remodeling related to a selective upregulation of DNA-5-cytosine methyltransferase (DNMT) expression in GABAergic neurons of SZ PFC may induce a hypermethylation of reelin and GAD67 promoter CpG islands, which downregulates their expression. In addition, we report preliminary evidence suggesting that by targeting this chromatin-remodeling deficit with inhibitors of histone deacetylases (HDAC), it may be possible to reduce the DNMT upregulation via a covalent modification of nucleosomal histone tails, underscoring the possibility that by addressing a chromatin remodeling deficit, one may treat psychiatric disorders.

Reln mRNA and protein levels are reduced by approximately 50% in various cortical structures of post-mortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. To study mechanisms responsible for this down-regulation, we have analyzed the promoter of the human reelin gene. We show that the reelin promoter directs expression of a reporter construct in multiple human cell types: neuroblastoma cells (SHSY5Y), neuronal precursor cells (NT2), differentiated neurons (hNT) and hepatoma cells (HepG2). Deletion constructs confirmed the presence of multiple elements regulating Reln expression, although the promoter activity is promiscuous, i.e. activity did not correlate with expression of the endogenous gene as reflected in terms of reelin mRNA levels. Co-transfection of the -514 bp human reelin promoter with either Sp1 or Tbr1 demonstrated that these transcription factors activate reporter expression by 6- and 8.5-fold, respectively. Within 400 bp of the RNA start site there are 100 potential CpG targets for DNA methylation. Retinoic acid (RA)-induced differentiation of NT2 cells to hNT neurons was accompanied by increased reelin expression and by the appearance of three DNase I hypersensitive sites 5' to the RNA start site. RA-induced differentiation was also associated with demethylation of the reelin promoter. To test if methylation silenced reelin expression, we methylated the promoter in vitro prior to transfection. In addition, we treated NT2 cells with the methylation inhibitor aza-2'-deoxycytidine and observed a 60-fold increase in reelin mRNA levels. The histone deacetylase inhibitors trichostatin A (TSA) and valproic acid also induced expression of the endogenous reelin promoter, although TSA was considerably more potent. These findings indicate that one determinant responsible for regulating reelin expression is the methylation status of the promoter. Our data also raise the interesting possibility that the down-regulation of reelin expression documented in psychiatric patients might be the consequence of inappropriate promoter hypermethylation.

DNA methylation is an epigenetic regulatory mechanism commonly associated with transcriptional silencing. DNA methyltransferases (DNMTs) are a family of distantly related proteins that both catalyze the de novo formation of 5-methylcytosine and maintain these methylation marks in cell-specific patterns in virtually all mitotic cells of the body. In the adult brain, methylation occurs in progenitor cells of the neurogenic zones and in post-mitotic neurons. Of the DNMTs, DNMT1 and DNMT3a are most highly expressed in post-mitotic neurons. While it has been commonly thought all post-mitotic neurons and glia express DNMTs at comparable levels, the co-expression of selected DNMTs with markers of distinct neurotransmitter phenotypes has not been previously examined in detail in the mouse. To this end, we analyzed the expression of DNMT1 and DNMT3a along with GAD67 in the brains of the GAD67-GFP knockin mice. After first confirming that GFP immunopositive neurons were also GAD67-positive, we showed that in the motor cortex, piriform cortex, striatum, CA1 region of the hippocampus, dentate gyrus, and basolateral amygdala (BLA), GFP immunofluorescence coincided with the signal corresponding to DNMT1 and DNMT3a. A detailed examination of cortical neurons, particularly in cortical layers III to V, showed that ∼30% of NeuN immunopositive neurons were also DNMT1 positive. These data do not exclude the expression of DNMT1 or DNMT3a in glutamatergic neurons and glia. However, they suggest that their expression is very low compared with the levels present in GABAergic neurons.

DNA methylation is an epigenetic regulatory mechanism commonly associated with transcriptional silencing. DNA methyltransferases (DNMTs) are a family of distantly related proteins that both catalyze the de novo formation of 5-methylcytosine and maintain these methylation marks in cell-specific patterns in virtually all mitotic cells of the body. In the adult brain, methylation occurs in progenitor cells of the neurogenic zones and in post-mitotic neurons. Of the DNMTs, DNMT1 and DNMT3a are most highly expressed in post-mitotic neurons. While it has been commonly thought all post-mitotic neurons and glia express DNMTs at comparable levels, the co-expression of selected DNMTs with markers of distinct neurotransmitter phenotypes has not been previously examined in detail in the mouse. To this end, we analyzed the expression of DNMT1 and DNMT3a along with GAD67 in the brains of the GAD67-GFP knockin mice. After first confirming that GFP immunopositive neurons were also GAD67-positive, we showed that in the motor cortex, piriform cortex, striatum, CA1 region of the hippocampus, dentate gyrus, and basolateral amygdala (BLA), GFP immunofluorescence coincided with the signal corresponding to DNMT1 and DNMT3a. A detailed examination of cortical neurons, particularly in cortical layers III to V, showed that ∼30% of NeuN immunopositive neurons were also DNMT1 positive. These data do not exclude the expression of DNMT1 or DNMT3a in glutamatergic neurons and glia. However, they suggest that their expression is very low compared with the levels present in GABAergic neurons.

P11 (S100A10) has been associated with the pathophysiology of depression both in human and rodent models. Different types of antidepressants have been shown to increase P11 levels in distinct brain regions and P11 gene therapy was recently proven effective in reversing depressive-like behaviours in mice. However, the molecular mechanisms that govern P11 gene expression in response to antidepressants still remain elusive. In this study we report decreased levels of P11, associated with higher DNA methylation in the promoter region, in the prefrontal cortex of the Flinders Sensitive Line (FSL) genetic rodent model of depression. This hypermethylated pattern was reversed to normal, as indicated by the control line, after chronic administration of escitalopram (a selective serotonin reuptake inhibitor; SSRI). The escitalopram-induced hypomethylation was associated with both an increase in P11 gene expression and a reduction in mRNA levels of two DNA methyltransferases that have been shown to maintain DNA methylation in adult forebrain neurons (Dnmt1 and Dnmt3a). In conclusion, our data further support a role for P11 in depression-like states and suggest that this gene is controlled by epigenetic mechanisms that can be affected by antidepressant treatment.

Aberrant transcriptional regulation may be one of the key components of the pathophysiology of mood disorders. DNA methylation generally acts as an epigenetic gene silencing mechanism and is catalyzed by a group of enzymes known as DNA methyltransferases (DNMTs). Several lines of evidence have suggested aberrant DNA methylation in patients with neuropsychiatric disorders and in animal models for psychiatric disorders. However, the involvement of DNMTs in the pathophysiology of mood disorders is not completely understood. In this study, we aimed to determine whether there are alterations in the expression of DNMTs mRNA in mood disorder patients. We used quantitative real-time PCR to measure the mRNA expression of four DNMT isoforms in the peripheral white blood cells of major depressive disorder (MDD) and bipolar disorder (BPD) patients during a depressive and a remissive episode. We found that the levels of DNMT1 mRNA were significantly decreased in a depressive but not in a remissive state of MDD and BPD. In addition, the levels of DNMT3B mRNA in MDD were significantly increased in a depressive but not in a remissive state. Thus, our data suggest that the altered expression of DNMTs is state dependent and that the aberrant epigenetic gene regulations caused by the altered expression of DNMT1 and DNMT3B may be associated with the pathophysiology of mood disorders.

Activation of group II metabotropic glutamate receptors (mGlu2 and -3 receptors) has shown a potential antipsychotic activity, yet the underlying mechanism is only partially known. Altered epigenetic mechanisms contribute to the pathogenesis of schizophrenia and currently used medications exert chromatin remodeling effects. Here, we show that systemic injection of the brain-permeant mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268; 0.3-1 mg/kg i.p.) increased the mRNA and protein levels of growth arrest and DNA damage 45-β (Gadd45-β), a molecular player of DNA demethylation, in the mouse frontal cortex and hippocampus. Induction of Gadd45-β by LY379268 was abrogated by the mGlu2/3 receptor antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495; 1 mg/kg i.p.). Treatment with LY379268 also increased the amount of Gadd45-β bound to specific promoter regions of reelin, brain-derived neurotrophic factor (BDNF), and glutamate decarboxylase-67 (GAD67). We directly assessed gene promoter methylation in control mice and in mice pretreated for 7 days with the methylating agent methionine (750 mg/kg i.p.). Both single and repeated injections with LY379268 reduce cytosine methylation in the promoters of the three genes, although the effect on the GAD67 was significant only in response to repeated injections. Single and repeated treatment with LY379268 could also reverse the defect in social interaction seen in mice pretreated with methionine. The action of LY379268 on Gadd45-β was mimicked by valproate and clozapine but not haloperidol. These findings show that pharmacological activation of mGlu2/3 receptors has a strong impact on the epigenetic regulation of genes that have been linked to the pathophysiology of schizophrenia.

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.

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.

Parenting and the early environment influence the risk for various psychopathologies. Studies in the rat suggest that variations in maternal care stably influence DNA methylation, gene expression, and neural function in the offspring. Maternal care affects neural development, including the GABAergic system, the function of which is linked to the pathophysiology of diseases including schizophrenia and depression. Postmortem studies of human schizophrenic brains have revealed decreased forebrain expression of glutamic acid decarboxylase 1 (GAD1) accompanied by increased methylation of a GAD1 promoter. We examined whether maternal care affects GAD1 promoter methylation in the hippocampus of adult male offspring of high and low pup licking/grooming (high-LG and low-LG) mothers. Compared with the offspring of low-LG mothers, those reared by high-LG dams showed enhanced hippocampal GAD1 mRNA expression, decreased cytosine methylation, and increased histone 3-lysine 9 acetylation (H3K9ac) of the GAD1 promoter. DNA methyltransferase 1 expression was significantly higher in the offspring of low- compared with high-LG mothers. Pup LG increases hippocampal serotonin (5-HT) and nerve growth factor-inducible factor A (NGFI-A) expression. Chromatin immunoprecipitation assays revealed enhanced NGFI-A association with and H3K9ac of the GAD1 promoter in the hippocampus of high-LG pups after a nursing bout. Treatment of hippocampal neuronal cultures with either 5-HT or an NGFI-A expression plasmid significantly increased GAD1 mRNA levels. The effect of 5-HT was blocked by a short interfering RNA targeting NGFI-A. These results suggest that maternal care influences the development of the GABA system by altering GAD1 promoter methylation levels through the maternally induced activation of NGFI-A and its association with the GAD1 promoter.

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 defense of deleterious retrotransposition of intracisternal A particle (IAP) elements, IAP loci are heavily methylated and silenced in mouse somatic cells. To determine whether IAP is also repressed in pluripotent stem cells by DNA methylation, we examined IAP expression in demethylated mouse embryonic stem cells (mESCs) and epiblast-derived stem cells. Surprisingly, in demethylated ESC cultures carrying mutations of DNA methyltransferase I (Dnmt1), no IAP transcripts and proteins are detectable in undifferentiated Oct4(+) ESCs. In contrast, approximately 3.6% of IAP-positive cells are detected in Oct4(-) Dnmt1(-/-) cells, suggesting that the previously observed increase in IAP transcripts in the population of Dnmt1(-/-) ESCs could be accounted for by this subset of Oct4(-) Dnmt1(-/-) ESCs undergoing spontaneous differentiation. Consistent with this possibility, a dramatic increase of IAP mRNA (>100-fold) and protein expression was observed in Dnmt1(-/-) ESC cultures upon induction of differentiation through the withdrawal of leukemia-inhibitory factor for 6 or more days. Interestingly, both mRNAs and proteins of IAP can be readily detected in demethylated Oct4(+) epiblast-derived stem cells as well as differentiated mouse embryo fibroblasts, neurons, and glia upon conditional Dnmt1 gene deletion. These data suggest that mESCs are a unique stem cell type possessing a DNA methylation-independent IAP repression mechanism. This methylation-independent mechanism does not involve Dicer-mediated action of microRNAs or RNA interference because IAP expression remains repressed in Dnmt1(-/-); Dicer(-/-) double mutant ESCs. We suggest that mESCs possess a unique DNA methylation-independent mechanism to silence retrotransposons to safeguard genome stability while undergoing rapid cell proliferation for self-renewal.

Docetaxel is an effective chemotherapy drug to treat breast cancer but the underlying molecular mechanisms of drug resistance are not fully understood. DNA methylation is an epigenetic event, involved in the control of gene expression, which is known to play an important role in cancer and chemotherapy drug resistance. To investigate the role of DNA methylation in docetaxel resistance in breast cancer we used two human breast cancer cell lines (MCF-7 and MDA-MB-231) that were made resistant to docetaxel. Docetaxel-resistant sub-lines were treated with different concentrations of decitabine. Global methylation and DNA methyltransferase (DNMT) activity was measured using an ELISA-based assay. Quantitative real-time PCR was used to study DNMT gene expression. Cell viability was studied by MTT assay. Global methylation was increased in MCF-7 but not significantly changed in MDA-MB-231 docetaxel-resistant cells. Decreased DNMT activity and decreased DNMT1 and DNMT3b mRNA expression was associated with docetaxel resistance in both cell lines. To investigate how the components of the DNA methylation machinery may contribute towards docetaxel resistance, decitabine (5-aza-2'-deoxycytidine), an inhibitor of DNA methylation, was used. Decitabine treatment decreased global methylation, DNMT activity and DNMT1, DNMT3a and DNMT3b mRNA expression in MDA-MB-231 docetaxel-resistant cells. In contrast, decitabine-treated MCF-7 docetaxel-resistant cells showed increased DNMT1, DNMT3a and DNMT3b mRNA expression indicating a cell line specific effect of decitabine. Decitabine treatment increased resistance in MCF-7 docetaxel-resistant cells and in the parental MCF-7 and MDA-MB231 docetaxel-sensitive cell lines, however, it did not alter response to docetaxel in MDA-MB-231 docetaxel-resistant cells. This study demonstrates that changes in the DNA methylation machinery are associated with resistance to docetaxel in breast cancer cells. The use of epigenetic therapies, as a strategy to overcome drug resistance, needs to be investigated more fully to determine their effectiveness in different cancers and for different chemotherapy drugs.