Evidence is emerging that several diseases and behavioral pathologies result from defects in gene function. The best-studied example is cancer, but other diseases such as autoimmune disease, asthma, type 2 diabetes, metabolic disorders, and autism display aberrant gene expression. Gene function may be altered by either a change in the sequence of the DNA or a change in epigenetic programming of a gene in the absence of a sequence change.With epigenetic drugs, it is possible to reverse aberrant gene expression profiles associated with different disease states. Several epigenetic drugs targeting the DNA methylation and histone deacetylation enzymes have been tested in clinical trials. Understanding the epigenetic machinery and the differential roles of its components in specific disease states is essential for developing targeted epigenetic therapy. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 49 is January 06, 2009. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

The genome is programmed by the epigenome. Two of the fundamental components of the epigenome are chromatin structure and covalent modification of the DNA molecule itself by methylation. DNA methylation patterns are sculpted during development and it has been a long held belief that they remain stable after birth in somatic tissues. Recent data suggest that DNA methylation is dynamic later in life in postmitotic cells such as neurons and thus potentially responsive to different environmental stimuli throughout life. We hypothesize a mechanism linking the social environment early in life and long-term epigenetic programming of behavior and responsiveness to stress and health status later in life. We will also discuss the prospect that the epigenetic equilibrium remains responsive throughout life and that therefore environmental triggers could play a role in generating interindividual differences in human behavior later in life. We speculate that exposures to different environmental toxins alters long-established epigenetic programs in the brain as well as other tissues leading to late-onset disease. Environ. Mol. Mutagen., 2008.(c) 2007 Wiley-Liss, Inc.

GABAergic dysfunction is present in the hippocampus in schizophrenia (SZ) and bipolar disorder (BD). The trisynaptic pathway was "deconstructed" into various layers of sectors CA3/2 and CA1 and gene expression profiling performed. Network association analysis was used to uncover genes that may be related to regulation of glutamate decarboxylase 67 (GAD67), a marker for this system that has been found by many studies to show decreased expression in SZs and BDs. The most striking change was a down-regulation of GAD67 in the stratum oriens (SO) of CA2/3 in both groups; CA1 only showed changes in the SO of schizophrenics. The network generated for GAD67 contained 25 genes involved in the regulation of kainate receptors, TGF-beta and Wnt signaling, as well as transcription factors involved in cell growth and differentiation. In SZs, IL-1beta,(GRIK2/3), TGF-beta2, TGF-betaR1, histone deacetylase 1 (HDAC1), death associated protein (DAXX), and cyclin D2 (CCND2) were all significantly up-regulated, whereas in BDs, PAX5, Runx2, LEF1, TLE1, and CCND2 were significantly down-regulated. In the SO of CA1 of BDs, where GAD67 showed no expression change, TGF-beta and Wnt signaling genes were all up-regulated, but other transcription factors showed no change in expression. In other layers/sectors, BDs showed no expression changes in these GAD67 network genes. Overall, these results are consistent with the hypothesis that decreased expression of GAD67 may be associated with an epigenetic mechanism in SZ. In BD, however, a suppression of transcription factors involved in cell differentiation may contribute to GABA dysfunction.

In the cerebral prefrontal cortex (PFC), DNA-methyltransferase 1 (DNMT1), the enzyme that catalyzes the methylation of cytosine at carbon atoms in position 5 in CpG dinucleotides, is expressed selectively in GABAergic neurons and is upregulated in layers I and II of schizophrenia (SZ) and bipolar disorder patients with psychosis (BDP). To replicate these earlier findings and to verify whether overexpression of DNMT1 and the consequent epigenetic decrease of reelin and glutamic acid decarboxylase (GAD) 67 mRNA expression also occur in GABAergic medium spiny neurons of the caudate nucleus (CN) and putamen (PT) of SZ and BDP, we studied the entire McLean 66 Cohort (Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA) including SZ and BDP, which were matched with nonpsychiatric subjects. The data demonstrate that in GABAergic medium spiny neurons of CN and PT, unlike in GABAergic neurons of layer I and II PFC, the increased expression of DNMT1 and the decrease of reelin and GAD67 occur in SZ but not in BDP. This suggests that different epigenetic mechanisms must exist in the pathogenesis underlying SZ and BDP and implies that these disorders might involve two separate entities that are characterized by a well-defined neuropathology.

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.

We investigated the effects of agents that induce reelin mRNA expression in vitro on the methylation status of the human reelin promoter in neural progenitor cells (NT2). NT2 cells were treated with the histone deacetylase inhibitors, trichostatin A (TSA) and valproic acid (VPA), and the methylation inhibitor aza-2'-deoxycytidine (AZA) for various times. All three drugs reduced the methylation profile of the reelin promoter relative to untreated cells. The acetylation status of histones H3 and H4 increased following treatment with VPA and TSA at times as short as 15 min following treatment; a result consistent with the reported mode of action of these drugs. Chromatin immunoprecipitation experiments showed that these changes were accompanied by changes occurring at the level of the reelin promoter as well. Interestingly, AZA decreased reelin promoter methylation without concomittantly increasing histone acetylation. In fact, after prolonged treatments with AZA, the acetylation status of histones H3 and H4 decreased relative to untreated cells. We also observed a trend towards reduced methylated H3 after 18 h treatment with TSA and VPA. Our data indicate that while TSA and VPA act to increase histone acetylation and reduce promoter methylation, AZA acts only to decrease the amount of reelin promoter methylation.

Cortical DNA-methyltransferase 1 (DNMT1) is preferentially expressed in interneurons secreting GABA where it very likely contributes to promoter CpG island hypermethylation, thus causing a down-regulation of promoter functions. To consolidate and expand on previous findings that, in the cortex of schizophrenia (SZ) brains, glutamic acid decarboxylase 67 (GAD67) expression is down-regulated whereas that of DNMT1 is up-regulated, we studied both parameters in Brodmann's area (BA) 9 from the McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center, Belmont, MA). In BA9 of SZ and bipolar disorder patients with psychosis, DNMT1 mRNA and protein expression preferentially increases in layer I, II, and IV interneurons, and this increase is paralleled by a decreased number of GAD67 mRNA-positive neurons. The increase in DNMT1 and the decrease in GAD67-expressing neurons were unrelated to postmortem interval, pH, RNA quality, or to the presence, dose, or duration of antipsychotic (APS) medication, with the exception of a subgroup of SZ patients treated with a combination of valproate and APS in which the expression of DNMT1 failed to change. The DNMT1 increase and the GAD67 decrease in BA9 interneurons are significant features of SZ and bipolar disorder with psychosis. Interestingly, the DNMT1 increase failed to occur when patients with psychosis received a combination of valproate and APS treatment but not APS monotherapy.

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.

Reelin glycoprotein is a secretory serine protease with dual roles in mammalian brain: embryologically, it guides neurons and radial glial cells to their corrected positions in the developing brain; in adult brain, Reelin is involved in a signaling pathway which underlies neurotransmission, memory formation and synaptic plasticity. Disruption of Reelin signaling pathway by mutations and selective hypermethylation of the Reln gene promoter or following various pre- or postnatal insults may lead to cognitive deficits present in neuropsychiatric disorders like autism or schizophrenia.

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.

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.

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.

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.

BACKGROUND: Reelin and GAD(67) expression is downregulated in cortical interneurons of schizophrenia (SZ) patients. This downregulation is probably mediated by epigenetic hypermethylation of the respective promoters caused by the selective increase of DNA-methyltransferase 1 in GABAergic neurons. Mice receiving methionine (MET) provide an epigenetic model for neuropathologies related to SZ. We studied whether MET-induced epigenetic reelin promoter hypermethylation and the associated behavioral alterations can be reduced by valproate in doses that inhibit histone deacetylases (HDACs). METHODS: Mice treated with either methionine (MET)(5.2 mmol/kg/SC/twice daily) or valproate (1.5 mmol/kg/SC/twice daily) or MET+ valproate combination were tested for prepulse inhibition of startle (PPI) and social interaction (SI). S-adenosylmethionine, acetylated histone 3, reelin promoter methylation, and reelin mRNA were assayed in the frontal cortex. RESULTS: Valproate enhances acetylated histone 3 content, and prevents MET-induced reelin promoter hypermethylation, reelin mRNA downregulation, and PPI and SI deficits. Imidazenil, a positive allosteric modulator at GABA(A) receptors containing alpha(5) subunits but inactive at receptors including alpha(1) subunits, normalizes MET-induced behavioral changes. CONCLUSION: This MET-induced epigenetic mouse models the neurochemical and behavioral aspects of SZ that can be corrected by positively modulating the action of GABA at alpha(5)-containing GABA(A) receptors with imidazenil or by inhibiting HDACs with valproate, thus opening exciting new avenues for treatment of epigenetically modified chromatin in SZ morbidity.

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.

Schizophrenia postmortem brain is characterized by gamma aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.

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.

In this review, we discuss changes in the regulation of gene expression in the central nervous system (CNS) associated with DNA (cytosine-5) methylation, chromatin remodeling and post-translational covalent modifications of histones. During brain development, abnormal intrinsic or extrinsic cues may compromise epigenetic processes regulating neural stem cell proliferation and differentiation and thus directly or indirectly could contribute to altered epiphenotypes leading to psychiatric disorders. These mechanisms, that include chromatin remodeling and reversible changes in promoter methylation patterns, are largely expressed by terminally differentiated cortical GABAergic neurons. These neurons are unique among various brain cell subtypes because they express high levels of DNA-methyltransferase-1 (DNMT1). Moreover, DNMT1 expression is further increased in schizophrenia (SZ) and bipolar (BP) disorder brains. To unravel how this pathological DNMT1 overexpression induces GABAergic neuronal dysfunction in SZ and in other psychoses, we report on how alterations in methylation modify the expression of susceptible vulnerability genes such as reelin or GAD67 in these neurons. The results encourage the view that promoter hypermethylation in GABAergic neurons that occurs in SZ represents a testable target for novel therapeutic strategies to treat this disorder.

In the cerebral prefrontal cortex (PFC), DNA-methyltransferase 1 (DNMT1), the enzyme that catalyzes the methylation of cytosine at carbon atoms in position 5 in CpG dinucleotides, is expressed selectively in GABAergic neurons and is upregulated in layers I and II of schizophrenia (SZ) and bipolar disorder patients with psychosis (BDP). To replicate these earlier findings and to verify whether overexpression of DNMT1 and the consequent epigenetic decrease of reelin and glutamic acid decarboxylase (GAD) 67 mRNA expression also occur in GABAergic medium spiny neurons of the caudate nucleus (CN) and putamen (PT) of SZ and BDP, we studied the entire McLean 66 Cohort (Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, MA) including SZ and BDP, which were matched with nonpsychiatric subjects. The data demonstrate that in GABAergic medium spiny neurons of CN and PT, unlike in GABAergic neurons of layer I and II PFC, the increased expression of DNMT1 and the decrease of reelin and GAD67 occur in SZ but not in BDP. This suggests that different epigenetic mechanisms must exist in the pathogenesis underlying SZ and BDP and implies that these disorders might involve two separate entities that are characterized by a well-defined neuropathology.

Human studies suggest that a variety of prenatal stressors are related to high risk for cognitive and behavioral abnormalities associated with psychiatric illness (Markham and Koenig, 2011). Recently, a downregulation in the expression of GABAergic genes (i.e., glutamic acid decarboxylase 67 and reelin) associated with DNA methyltransferase (DNMT) overexpression in GABAergic neurons has been regarded as a characteristic phenotypic component of the neuropathology of psychotic disorders (Guidotti et al., 2011). Here, we characterized mice exposed to prenatal restraint stress (PRS) in order to study neurochemical and behavioral abnormalities related to development of schizophrenia in the adult. Offspring born from non-stressed mothers (control mice) showed high levels of DNMT1 and 3a mRNA expression in the frontal cortex at birth, but these levels progressively decreased at post-natal days (PND) 7, 14, and 60. Offspring born from stressed mothers (PRS mice) showed increased levels of DNMTs compared to controls at all time-points studied including at birth and at PND 60. Using GAD67-GFP transgenic mice, we established that, in both control and PRS mice, high levels of DNMT1 and 3a were preferentially expressed in GABAergic neurons of frontal cortex and hippocampus. Importantly, the overexpression of DNMT in GABAergic neurons was associated with a decrease in reelin and GAD67 expression in PRS mice in early and adult life. PRS mice also showed an increased binding of DNMT1 and MeCP2, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine in specific CpG-rich regions of the reelin and GAD67 promoters. Thus, the epigenetic changes in PRS mice are similar to changes observed in the post-mortem brains of psychiatric patients. Behaviorally, adult PRS mice showed hyperactivity and deficits in social interaction, prepulse inhibition, and fear-conditioning that were corrected by administration of valproic acid (a histone deacetylase inhibitor) or clozapine (an atypical antipsychotic with DNA-demethylation activity). Taken together, these data show that prenatal stress in mice induces abnormalities in the DNA methylation network and in behaviors indicative of a schizophrenia-like phenotype. Thus, PRS mice may be a valid model for the investigation of new drugs for schizophrenia treatment targeting DNA methylation. This article is part of a Special Issue entitled 'Neurodevelopment Disorder'.

Translational studies are becoming more common in schizophrenia research. The past couple of decades witnessed the emergence of novel ideas regarding schizophrenia pathophysiology that originated from both human and animal studies. The findings that glutamate and gamma-aminobutyric acid transmission are affected in the disease led to the hypothesis of altered inhibitory neurotransmission as critical for cognitive deficits and to an exploration of novel therapeutic approaches aimed at restoring excitation-inhibition balance. Much is to be done yet to elucidate the ultimate mechanisms by which excitation and inhibition are affected in this disorder; a comprehensive translational effort is necessary to address what may cause altered GABA function, for example. Here, we present an overview of the excitation-inhibition imbalance hypothesis in schizophrenia and discuss ongoing efforts aimed at determining whether cortical inhibitory interneurons are affected by oxidative stress during development.

Deficits in cognitive control, a core disturbance of schizophrenia, appear to emerge from impaired prefrontal gamma oscillations. Cortical gamma oscillations require strong inhibitory inputs to pyramidal neurons from the parvalbumin basket cell (PVBC) class of GABAergic neurons. Recent findings indicate that schizophrenia is associated with multiple pre- and postsynaptic abnormalities in PVBCs, each of which weakens their inhibitory control of pyramidal cells. These findings suggest a new model of cortical dysfunction in schizophrenia in which PVBC inhibition is decreased to compensate for an upstream deficit in pyramidal cell excitation. This compensation is thought to rebalance cortical excitation and inhibition, but at a level insufficient to generate the gamma oscillation power required for high levels of cognitive control.

Deficits of cognitive control in schizophrenia are associated with altered gamma oscillations in the prefrontal cortex. Paralbumin basket interneurons, which innervate the perisomatic region of pyramidal neurons, appear to play a key role in generating cortical gamma oscillations. In the prefrontal cortex of subjects with schizophrenia, alterations are present in both pre- and post-synaptic markers of the strength of GABA inputs from parvalbumin basket neurons to pyramidal neurons. These alterations may contribute to the neural substrate for impaired gamma oscillations in 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.

Transcriptional regulation of gene expression is thought to play a pivotal role in activity-dependent neuronal differentiation and circuit formation. Here, we investigated the role of histone deacetylase 9 (HDAC9), which regulates transcription by histone modification, in the development of neocortical neurons. The translocation of HDAC9 from nucleus to cytoplasm was induced by an increase of spontaneous firing activity in cultured mouse cortical neurons. This nucleocytoplasmic translocation was also observed in postnatal development in vivo. The translocation-induced gene expression and cellular morphology was further examined by introducing an HDAC9 mutant that disrupts the nucleocytoplasmic translocation. Expression of c-fos, an immediately-early gene, was suppressed in the mutant-transfected cells regardless of neural activity. Moreover, the introduction of the mutant decreased the total length of dendritic branches, whereas knockdown of HDAC9 promoted dendritic growth. These findings indicate that chromatin remodeling with nucleocytoplasmic translocation of HDAC9 regulates activity-dependent gene expression and dendritic growth in developing cortical neurons.

Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour. Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component. Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown. Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (gamma-aminobutyric acid)-containing interneurons. In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons. In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input. Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1. In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells. By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons. Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.

Dysregulated glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. In particular, hypofunction of the NMDA glutamate receptor has been proposed to play an important role in mediating cognitive deficits in patients. The two NMDA receptor subunits, NR2A and NR2B, are distinctly regulated during development and are associated with different intracellular pathways and functions, which suggest that these receptors play separate roles in the control of higher cognitive functions such as learning and memory. Trafficking of the NR2B subunit-containing receptor is regulated by a microtubule-associated trafficking complex consisting of the KIF17, APBA1, CASK, and mLin7 proteins. Several studies have demonstrated an integrated functional regulation of this trafficking complex with NR2B receptor subunit expression, which in turn has been linked to higher cognitive functions. In the present work, we investigated whether expression of this NR2B-associated trafficking complex might be abnormal in schizophrenia. We analyzed the expression of KIF17, APBA1, CASK, mLin7A and mLin7C in postmortem brain from patients with schizophrenia a comparison group. Analysis of transcripts for all of these proteins revealed particularly prominent expression in cortical layer III and layer IV, which overlapped with NR2B but not NR2A transcripts. We found altered expression of transcripts for the CASK, ABPA1, and mLin7 molecules and the CASK, mLin7 proteins, suggesting that NR2B-containing NMDA receptor transport could be selectively compromised in schizophrenia, and that these changes likely involve altered NR2B function in a subset of cortical neurons.

Dopamine is a critical modulator of prefrontal cortical function, and it is known to be dysfunctional in schizophrenia. Current hypotheses on schizophrenia highlight developmental aspects and genetic predisposition for the disease; yet, symptom onset typically occurs during adolescence. Several aspects of prefrontal cortical circuits and their modulation by dopamine mature postnatally, as late as during adolescence. Here we review studies assessing the postnatal trajectory of dopamine control of GABA interneurons, a neuronal population that has been long suspected to be critical for schizophrenia pathophysiology. Dopamine modulation of fast-spiking interneurons changes dramatically during adolescence (postnatal day 45-50 in rats) with D2 agonists switching from being mildly inhibitory in prepubertal rats to strongly excitatory in young adult rats. In vivo recordings in adult rats reveal that deep-layer pyramidal neurons respond to endogenous DA release with suppression of firing while interneurons are activated. In adult rats with a neonatal ventral hippocampal lesion (NVHL), an extensively studied developmental model of schizophrenia, the maturation in the D2 modulation of interneuron physiology fails to occur, rendering a disinhibited prefrontal cortex. Abnormal interneuron maturation may therefore impair cognitive function in the adult animal.

Dysbindin has been implicated in the pathogenesis of schizophrenia, but little is known about how dysbindin affects neuronal function in the circuitry underlying psychosis and related behaviors. Using a dysbindin knockout line (dys(-/-)) derived from the natural dysbindin mutant Sandy mice, we have explored the role of dysbindin in dopamine signaling and neuronal function in the prefrontal cortex (PFC). Combined cell imaging and biochemical experiments revealed a robust increase in the dopamine receptor D2, but not D1, on cell surface of neurons from dys(-/-) cortex. This was due to an enhanced recycling and insertion, rather than reduced endocytosis, of D2. Disruption of dysbindin gene resulted in a marked decrease in the excitability of fast-spiking (FS) GABAergic interneurons in both PFC and striatum. Dys(-/-) mice also exhibited a decreased inhibitory input to pyramidal neurons in layer V of PFC. The increased D2 signaling in dys(-/-) FS interneurons was associated with a more pronounced increase in neuronal firing in response to D2 agonist, compared to that in wild-type interneurons. Taken together, these results suggest that dysbindin regulates PFC function by facilitating D2-mediated modulation of GABAergic function.