Prenatal stress alters transcription of NMDA-type glutamate receptors in the hippocampus.

Prenatal stress increases the risk of neurodevelopmental disorders. NMDA-type glutamate receptor (NMDAR) activity plays an important pathophysiological role in the cortico-hippocampal circuit in these disorders. We tested the hypothesis that transcription of NMDAR subunits is modified in the frontal cortex (FCx) and hippocampus after exposure to prenatal restraint stress (PRS) in mice. At 10 weeks of age, male PRS offspring (n = 20) and non-stressed controls (NS, n = 20) were treated with haloperidol (1 mg/kg), clozapine (5 mg/kg) or saline twice daily for 5 days, before measuring social approach (SOC). Saline-treated and haloperidol-treated PRS mice had reduced SOC relative to NS (P < 0.01), but clozapine-treated PRS mice had similar SOC to NS mice. These effects of PRS were associated with increased transcription of NMDAR subunits encoded by GRIN2A and GRIN2B genes in the hippocampus but not FCx. GRIN transcription in FCx correlated positively with SOC, but hippocampal GRIN transcription had negative correlation with SOC. The ratio of GRIN2A/GRIN2B transcription is known to increase during development but was lower in PRS mice. These results suggest that GRIN2A and GRIN2B transcript levels are modified in the hippocampus by PRS, leading to life-long deficits in social behavior. These data have some overlap with the molecular pathophysiology of schizophrenia. Similar to PRS in mice, schizophrenia, has been associated with social withdrawal, with increased GRIN2 expression in the hippocampus, and reduced GRIN2A/GRIN2B expression ratios in the hippocampus. These findings suggest that PRS in mice may have construct validity as a preclinical model for antipsychotic drug development.

Acute and Protracted Prenatal Stress Produce Mood Disorder-Like and Ethanol Drinking Behaviors in Male and Female Adult Offspring.

Background: Alcohol use disorder (AUD) is a complex and chronic relapsing brain disease, which is often co-morbid with psychiatric disorders such as anxiety and depression. AUD phenotypes differ in men and women. Although genetic factors play an important role in its pathophysiology, epidemiologic evidence suggests that during prenatal development, individuals are more vulnerable to the negative effects of environmental factors that may predispose them to AUD later in life. We explored the effects of prenatal stress on the development of AUD phenotypes as well as anxiety- and depression-like behaviors using rat model. Methods: In this study, timed-pregnant Sprague Dawley dams were used. Dams in the control group were left undisturbed throughout gestation, whereas dams in stress groups were either subjected to protracted or acute restraint stress under bright light. At adulthood, the anxiety-like, ethanol drinking, and sucrose drinking behaviors were measured using the Light/Dark Box test and two-bottle free-choice procedure. Results: Compared to the control group, both the male and female offspring in the stress groups exhibited anxiety-like behavior and consumed significantly higher amounts of ethanol in which the acute stress group demonstrated the higher ethanol preference. Moreover, male but not female offspring from the stress groups had decreased sucrose preferences. Conclusion: These findings suggest that protracted and acute prenatal stress in late pregnancy can induce in anxiety-, depressive-like behaviors, and excessive ethanol intake in adult offspring.

Altered Expression and In Vivo Activity of mGlu5 Variant a Receptors in the Striatum of BTBR Mice: Novel Insights Into the Pathophysiology of Adult Idiopathic Forms of Autism Spectrum Disorders.

BACKGROUND: mGlu5 metabotropic glutamate receptors are considered as candidate drug targets in the treatment of "monogenic" forms of autism spectrum disorders (ASD), such as Fragile- X syndrome (FXS). However, despite promising preclinical data, clinical trials using mGlu5 receptor antagonists to treat FXS showed no beneficial effects. OBJECTIVE: Here, we studied the expression and function of mGlu5 receptors in the striatum of adult BTBR mice, which model idiopathic forms of ASD, and behavioral phenotype. METHODS: Behavioral tests were associated with biochemistry analysis including qPCR and western blot for mRNA and protein expression. In vivo analysis of polyphosphoinositides hydrolysis was performed to study the mGlu5-mediated intracellular signaling in the striatum of adult BTBR mice under basal conditions and after MTEP exposure. RESULTS: Expression of mGlu5 receptors and mGlu5 receptor-mediated polyphosphoinositides hydrolysis were considerably high in the striatum of BTBR mice, sensitive to MTEP treatment. Changes in the expression of genes encoding for proteins involved in excitatory and inhibitory neurotransmission and synaptic plasticity, including Fmr1, Dlg4, Shank3, Brd4, bdnf-exon IX, Mef2c, and Arc, GriA2, Glun1, Nr2A, and Grm1, Grm2, GriA1, and Gad1 were also found. Behaviorally, BTBR mice showed high repetitive stereotypical behaviors, including self-grooming and deficits in social interactions. Acute or repeated injections with MTEP reversed the stereotyped behavior and the social interaction deficit. Similar effects were observed with the NMDA receptor blockers MK-801 or ketamine. CONCLUSION: These findings support a pivotal role of mGlu5 receptor abnormal expression and function in idiopathic ASD adult forms and unveil novel potential targets for therapy.

Prenatal stress induced chromatin remodeling and risk of psychopathology in adulthood.

New insights into the pathophysiology of psychiatric disorders suggest the existence of a complex interplay between genetics and environment. This notion is supported by evidence suggesting that exposure to stress during pregnancy exerts profound effects on the neurodevelopment and behavior of the offspring and predisposes them to psychiatric disorders later in life. Accumulated evidence suggests that vulnerability to psychiatric disorders may result from permanent negative effects of long-term changes in synaptic plasticity due to altered epigenetic mechanisms (histone modifications and DNA methylation) that lead to condensed chromatin architecture, thereby decreasing the expression of candidate genes during early brain development. In this chapter, we have summarized the literature of clinical studies on psychiatric disorders induced by maternal stress during pregnancy. We also discussed the epigenetic alterations of gene regulations induced by prenatal stress. Because the clinical manifestations of psychiatric disorders are complex, it is obvious that the biological progression of these diseases cannot be studied only in postmortem brains of patients and the use of animal models is required. Therefore, in this chapter, we have introduced a well-established mouse model of prenatal stress (PRS) generated in restrained pregnant dams. The behavioral phenotypes of the offspring (PRS mice) born to the stressed dam and underlying epigenetic changes in key molecules related to synaptic activity were described and highlighted. PRS mice may serve as a useful model for investigating the pathogenesis of psychiatric disorders and may be a useful tool for screening for the potential compounds that may normalize aberrant epigenetic mechanisms induced by prenatal stress.

Causal roles of stress kinase JNK2 in DNA methylation and binge alcohol withdrawal-evoked behavioral deficits.

Excessive binge alcohol intake is a common drinking pattern in humans, especially during holidays. Cessation of the binge drinking often leads to aberrant withdrawal behaviors, as well as serious heart rhythm abnormalities (clinically diagnosed as Holiday Heart Syndrome (HHS)). In our HHS mouse model with well-characterized binge alcohol withdrawal (BAW)-induced heart phenotypes, BAW leads to anxiety-like behaviors and cognitive impairment. We have previously reported that stress-activated c-Jun NH(2)-terminal kinase (JNK) plays a causal role in BAW-induced heart phenotypes. In the HHS brain, we found that activation of JNK2 (but not JNK1 and JNK3) in the prefrontal cortex (PFC), but not hippocampus and amygdala, led to anxiety-like behaviors and impaired cognition. DNA methylation mediated by a crucial DNA methylation enzyme, DNA methyltransferase1 (DNMT1), is known to be critical in alcohol-associated behavioral deficits. In HHS mice, JNK2 in the PFC (but not hippocampus and amygdala) causally enhanced total genomic DNA methylation via increased DNMT1 expression, which was regulated by enhanced binding of JNK downstream transcriptional factor c-JUN to the DNMT1 promoter. JNK2-specific inhibition either by an inhibitor JNK2I or JNK2 knockout completely offset c-JUN-regulated DNMT1 upregulation and restored the level of DNA methylation in HHS PFC to the baseline levels seen in sham controls. Strikingly, either JNK2-specific inhibition or genetic JNK2 depletion or DNMT1 inhibition (by an inhibitor 5-Azacytidine) completely abolished BAW-evoked behavioral deficits. In conclusion, our studies revealed a novel mechanism by which JNK2 drives BAW-evoked behavioral deficits through a DNMT1-regulated DNA hypermethylation. JNK2 could be a novel therapeutic target for alcohol withdrawal treatment and/or prevention.

Genome-wide methylation in alcohol use disorder subjects: implications for an epigenetic regulation of the cortico-limbic glucocorticoid receptors (NR3C1).

Environmental factors, including substance abuse and stress, cause long-lasting changes in the regulation of gene expression in the brain via epigenetic mechanisms, such as DNA methylation. We examined genome-wide DNA methylation patterns in the prefrontal cortex (PFC, BA10) of 25 pairs of control and individuals with alcohol use disorder (AUD), using the Infinium® MethylationEPIC BeadChip. We identified 5254 differentially methylated CpGs (pnominal < 0.005). Bioinformatic analyses highlighted biological processes containing genes related to stress adaptation, including the glucocorticoid receptor (encoded by NR3C1). Considering that alcohol is a stressor, we focused our attention on differentially methylated regions of the NR3C1 gene and validated the differential methylation of several genes in the NR3C1 network. Chronic alcohol drinking results in a significant increased methylation of the NR3C1 exon variant 1H, with a particular increase in the levels of 5-hydroxymethylcytosine over 5-methylcytosine. These changes in DNA methylation were associated with reduced NR3C1 mRNA and protein expression levels in PFC, as well as other cortico-limbic regions of AUD subjects when compared with controls. Furthermore, we show that the expression of several stress-responsive genes (e.g., CRF, POMC, and FKBP5) is altered in the PFC of AUD subjects. These stress-response genes were also changed in the hippocampus, a region that is highly susceptible to stress. These data suggest that alcohol-dependent aberrant DNA methylation of NR3C1 and consequent changes in other stress-related genes might be fundamental in the pathophysiology of AUD and lay the groundwork for treatments targeting the epigenetic mechanisms regulating NR3C1 in AUD.

Kami-shoyo-san ameliorates sociability deficits in ovariectomized mice, a putative female model of autism spectrum disorder, via facilitating dopamine D1 and GABAA receptor functions.

ETHNOPHARMACOLOGICAL RELEVANCE: Kami-shoyo-san (KSS) is a Kampo formula used clinically for menopause-related symptoms in Japan. However, the effect of KSS on autism spectrum disorder (ASD), a developmental disorder with a higher prevalence in males than in females, has not been reported yet. AIM OF THE STUDY: It is accepted generally that dysfunction in the GABAergic system is associated with pathogenesis of ASD. In our previous study, a decrease in brain allopregnanolone (ALLO), a positive allosteric GABAA receptor modulator, induced ASD-like symptoms such as impaired sociability-related performance and increased repetitive self-grooming behavior in male mice, and that KSS ameliorated these behavioral abnormalities via GABAA receptor- and dopamine D1 receptor-mediated mechanisms. In this study, to better understand a gender difference in the prevalence of ASD, we examined whether dissection of ovary (OVX), a major organ secreting progesterone in females, causes ASD-like behaviors in a manner dependent on brain ALLO levels, and if so, how KSS affects the behaviors. MATERIALS AND METHODS: Six-week-old ICR female mice received ovariectomy, and KSS (74 mg/kg and 222 mg/kg, p.o.) were treated before 1 h starting each behavioral test. The sociability, social anxiety-like behavior, and self-grooming behavior were analyzed by the resident-intruder test, mirror chamber test, and open field test, respectively. After finishing the behavioral experiment, the ALLO content in the brain was measured by ELISA. Furthermore, we examined the effects of OVX on the neuro-signaling pathways in the prefrontal cortex and striatum by Western blotting. RESULTS: The results revealed that OVX induced sociability deficits and social anxiety-related behaviors, but not repetitive self-grooming behavior, and that these behavioral changes were accompanied not only by a decrease of brain ALLO levels, but also by impairment of CREB- and CaMKIIα-mediated neuro-signaling in the prefrontal cortex. Moreover, the administration of KSS had no effect on the brain ALLO level, but significantly ameliorated the OVX-induced behavioral and neurochemical changes via facilitation of GABAA receptor and dopamine D1 receptor-mediated neurotransmission. CONCLUSIONS: These findings suggest that a decrease in gonadal hormone-derived ALLO plays a major role in ASD-like behaviors in female mice and that KSS is beneficial for the treatment of ASD in females.

N-Phthalyl-l-Tryptophan (RG108), like Clozapine (CLO), Induces Chromatin Remodeling in Brains of Prenatally Stressed Mice.

Schizophrenia (SZ), schizoaffective (SZA), and bipolar (BP) disorder are neurodevelopmental psychopathological conditions related, in part, to genetic load and, in part, to environmentally induced epigenetic dysregulation of chromatin structure and function in neocortical GABAergic, glutamatergic, and monoaminergic neurons. To test the above hypothesis, we targeted our scientific efforts on identifying whether the molecular epigenetic signature of postmortem brains of patients with SZ, SZA, and BP disorder are also present in the brains of adult mice born from dams prenatally restraint stressed (PRS) during gestation. The brains of PRS mice, which are similar to the brains of patients with SZ and BP disorder, show an ∼2-fold increased binding of DNMT1 to psychiatric candidate promoters (glutamic acid decarboxylase 67, Reelin, and brain-derived neurotrophic factor), leading to their hypermethylation, reduced expression, as well as the behavioral endophenotypes reminiscent of those observed in the above psychiatric disorders. To establish whether clozapine (CLO) produces its behavioral and molecular action through a causal involvement of DNA methylation/demethylation processes, we compared the epigenetic action of CLO with that of the DNMT1 competitive inhibitor N-phthalyl-l-tryptophan (RG108). The intracerebroventricular injection of RG108 (20 nmol/day per 5 days), similar to the systemic administration of CLO, corrects the altered behavioral and molecular endophenotypes that are typical of PRS mice. These results are consistent with an epigenetic etiology underlying the behavioral endophenotypic profile in PRS mice. Further, it suggests that PRS mice may be useful in the preclinical screening of antipsychotic drugs acting to correct altered epigenetic mechanisms.

Epigenetic Alterations in Prenatal Stress Mice as an Endophenotype Model for Schizophrenia: Role of Metabotropic Glutamate 2/3 Receptors.

Mice subjected to prenatal restraint stress (PRS mice) showed biochemical and behavioral abnormalities consistent with a schizophrenia-like phenotype (Matrisciano et al., 2016). PRS mice are characterized by increased DNA-methyltransferase 1 (DNMT1) and ten-eleven methylcytosine dioxygenase 1 (TET1) expression levels and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Activation of group II metabotropic glutamate receptors (mGlu2 and-3 receptors) showed a potential epigenetically-induced antipsychotic activity by reversing the molecular and behavioral changes observed in PRS mice. This effect was most likely caused by the increase in the expression of growth arrest and DNA damage 45-ß (Gadd45-ß) protein, a molecular player of DNA demethylation, induced by the activation of mGlu2/3 receptors. This effect was mimicked by clozapine and valproate but not by haloperidol. Treatment with the selective mGlu2/3 receptors agonist LY379268 also increased the amount of Gadd45-ß bound to specific promoter regions of reelin, BDNF, and GAD67. A meta-analysis of several clinical trials showed that treatment with an orthosteric mGlu2/3 receptor agonist improved both positive and negative symptoms of schizophrenia, but only in patients who were early-in-disease and had not been treated with atypical antipsychotic drugs (Kinon et al., 2015). Our findings show that PRS mice are valuable model for the study of epigenetic mechanisms involved in the pathogenesis of schizophrenia and support the hypothesis that pharmacological modulation of mGlu2/3 receptors could impact the early phase of schizophrenia and related neurodevelopmental disorders by regulating epigenetic processes that lie at the core of the disorders.

Prenatal stress leads to chromatin and synaptic remodeling and excessive alcohol intake comorbid with anxiety-like behaviors in adult offspring.

Epidemiologic evidence suggests that individuals during their prenatal development may be especially vulnerable to the effects of environmental factors such as stress that predisposes them to psychiatric disorders including alcohol use disorder (AUD) later in life. Currently, the epigenetic mechanisms of anxiety comorbid with AUD induced by prenatal stress (PRS) remain to be elucidated. Here, we examined anxiety-like and alcohol drinking behaviors in adult offspring of prenatally stressed dam (PRS-mice) using elevated plus maze, light/dark box and two-bottle free-choice paradigm. It was found that PRS-mice exhibit heightened anxiety-like behaviors and increased alcohol intake in adulthood and these behavioral deficits were associated with a significant decrease in dendritic spine density (DSD) in medial prefrontal cortex (mPFC) relative to non-stressed mice (NS mice). To determine the mechanisms by which PRS reduces DSD, we examined the expressions of key genes associated with synaptic plasticity, including activity regulated cytoskeleton associated protein (Arc), spinophilin (Spn), postsynaptic density 95 (Psd95), tropomyosin receptor kinase B (TrkB), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and period 2 (Per2) in mPFC of PRS and NS mice. The mRNA levels of these genes were significantly decreased in PRS mice. Methylated DNA and chromatin immunoprecipitation studies revealed hyper DNA methylation or reduced histone H3K14 acetylation on promoters of above genes suggesting that epigenetic dysregulation may be responsible for the deficits in their expression. Findings from this study suggest that prenatal stress induced abnormal epigenetic mechanisms and synaptic plasticity-related events may be associated with anxiety-like and alcohol drinking behaviors in adulthood.

Genipin normalizes depression-like behavior induced by prenatal stress through inhibiting DNMT1.

Synthetic antidepressants in current use for the complex etiopathogeneses of depression have slow response and remission as well as various unpleasant side effects. As a result, it is imperative to develop new antidepressants with more effectiveness and less severe side effects. Recent studies demonstrated that genipin, the aglycon of geniposide, extracted from Gardenia jasminoides Ellis has antidepressive effects. However, knowledge regarding the molecular mechanisms of its antidepressant effects remains limited. Employing a depression-like mouse model, we confirmed that genipin is capable of correcting depressions-like behaviors induced by prenatal stress in offspring from prenatally stressed dams (defined as PRS mice). In further experiments, we found that the effect of genipin on PRS mice occurs through DNA demethylation by inhibiting DNA methyltransferase 1 (DNMT1), normalizing the expression of reduced brain-derived neurotrophic factor (BDNF) in the hippocampus.

Gestational stress induces depressive-like and anxiety-like phenotypes through epigenetic regulation of BDNF expression in offspring hippocampus.

Exposure to stressful life events during pregnancy exerts profound effects on neurodevelopment and increases the risk for several neurodevelopmental disorders including major depression. The mechanisms underlying the consequences of gestational stress are complex and remain to be elucidated. This study investigated the effects of gestational stress on depressive-like behavior and epigenetic modifications in young adult offspring. Gestational stress was induced by a combination of restraint and 24-hour light disturbance to pregnant dams throughout gestation. Depressive-like and anxiety-like behaviors of young adult offspring were examined. The expression and promoter methylation of brain derived neurotrophic factor (BDNF) were measured using RT-qPCR, Western blot, methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP). In addition, the expressions of histone deacetylases (HDACs) and acetylated histone H3 lysine 14 (AcH3K14) were also analyzed. Our results show that offspring from gestational stress dams exhibited depressive-like and anxiety-like behaviors. Biochemically, stress-offspring showed decreased expression of BDNF, increased expression of DNMT1, HDAC1, and HDAC2, and decreased expression of AcH3K14 in the hippocampus as compared to non-stress offspring. Data from MeDIP and ChIP assays revealed an increased methylation as well as decreased binding of AcH3K14 on specific BDNF promoters. Pearson analyses indicated that epigenetic changes induced by gestational stress were correlated with depressive-like and anxiety-like behaviors. These data suggest that gestational stress may be a suitable model for understanding the behavioral and molecular epigenetic changes observed in patients with depression.

Maternal immune activation induces GAD1 and GAD2 promoter remodeling in the offspring prefrontal cortex.

Maternal infection during pregnancy increases the risk of neurodevelopmental disorders in the offspring. In addition to its influence on other neuronal systems, this early-life environmental adversity has been shown to negatively affect cortical γ-aminobutyric acid (GABA) functions in adult life, including impaired prefrontal expression of enzymes required for GABA synthesis. The underlying molecular processes, however, remain largely unknown. In the present study, we explored whether epigenetic modifications represent a mechanism whereby maternal infection during pregnancy can induce such GABAergic impairments in the offspring. We used an established mouse model of prenatal immune challenge that is based on maternal treatment with the viral mimetic poly(I:C). We found that prenatal immune activation increased prefrontal levels of 5-methylated cytosines (5mC) and 5-hydroxymethylated cytosines (5hmC) in the promoter region of GAD1, which encodes the 67-kDa isoform of the GABA-synthesising enzyme glutamic acid decarboxylase (GAD67). The early-life challenge also increased 5mC levels at the promoter region of GAD2, which encodes the 65-kDa GAD isoform (GAD65). These effects were accompanied by elevated GAD1 and GAD2 promoter binding of methyl CpG-binding protein 2 (MeCP2) and by reduced GAD67 and GAD65 mRNA expression. Moreover, the epigenetic modifications at the GAD1 promoter correlated with prenatal infection-induced impairments in working memory and social interaction. Our study thus highlights that hypermethylation of GAD1 and GAD2 promoters may be an important molecular mechanism linking prenatal infection to presynaptic GABAergic impairments and associated behavioral and cognitive abnormalities in the offspring.

Brain-derived neurotrophic factor epigenetic modifications associated with schizophrenia-like phenotype induced by prenatal stress in mice.

BACKGROUND: Prenatal stress (PRS) is considered a risk factor for several neurodevelopmental disorders including schizophrenia (SZ). An animal model involving restraint stress of pregnant mice suggests that PRS induces epigenetic changes in specific GABAergic and glutamatergic genes likely to be implicated in SZ, including the gene for brain-derived neurotrophic factor (BDNF). METHODS: Studying adult offspring of pregnant mice subjected to PRS, we explored the long-term effects of PRS on behavior and on the expression of key chromatin remodeling factors including DNA methyltransferase 1, ten-eleven-translocation hydroxylases, methyl CpG binding protein 2, histone deacetylases, and histone methyltransferases and demethylase in the frontal cortex and hippocampus. We also measured the expression of BDNF. RESULTS: Adult PRS offspring demonstrate behavioral abnormalities suggestive of SZ and molecular changes similar to changes seen in postmortem brains of patients with SZ. This includes a significant increase in DNA methyltransferase 1 and ten-eleven-translocation hydroxylase 1 in the frontal cortex and hippocampus but not in cerebellum; no changes in histone deacetylases, histone methyltransferases and demethylases, or methyl CpG binding protein 2, and a significant decrease in Bdnf messenger RNA variants. The decrease of the corresponding Bdnf transcript level was accompanied by an enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at Bdnf gene regulatory regions. In addition, the expression of Bdnf transcripts (IV and IX) correlated positively with social approach in both PRS mice and nonstressed mice. CONCLUSIONS: Because patients with psychosis and PRS mice show similar epigenetic signature, PRS mice may be a suitable model for understanding the behavioral and molecular epigenetic changes observed in patients with SZ.

Modeling the molecular epigenetic profile of psychosis in prenatally stressed mice.

Based on postmortem brain studies, our overarching epigenetic hypothesis is that chronic schizophrenia (SZ) is a psychopathological condition involving dysregulation of the dynamic equilibrium among DNA methylation/demethylation network components and the expression of SZ target genes, including GABAergic and glutamatergic genes. SZ has a natural course, starting with a prodromal phase, a first episode that occurs in adolescents or in young adults, and later deterioration over the adult years. Hence, the epigenetic status at each neurodevelopmental stage of the disease cannot be studied just in postmortem brain of chronic SZ patients, but requires the use of neurodevelopmental animal models. We have directed the focus of our research toward studying the epigenetic signature of the SZ brain in the offspring of dams stressed during pregnancy (PRS mice). Adult PRS mice have behavioral deficits reminiscent of behaviors observed in psychotic patients. The adult PRS brain, like that of postmortem chronic SZ patients, is characterized by a significant increase in DNA methyltransferase 1, Tet methylcytosine dioxygenase 1 (TET1), 5-methylcytosine, and 5-hydroxymethylcytosine at SZ candidate gene promoters and a reduction in the expression of glutamatergic and GABAergic genes. In PRS mice, measurements of epigenetic biomarkers for SZ can be assessed at different stages of development with the goal of further elucidating the pathophysiology of this disease and predicting treatment responses at specific stages of the illness, with particular attention to early detection and possibly early intervention.

DNA methylation/demethylation network expression in psychotic patients with a history of alcohol abuse.

BACKGROUND: Recent studies suggest that protracted and excessive alcohol use induces an epigenetic dysregulation in human and rodent brains. We recently reported that DNA methylation dynamics are altered in brains of psychotic (PS) patients, including schizophrenia and bipolar disorder patients. Because PS patients are often comorbid with chronic alcohol abuse, we examined whether the altered expression of multiple members of the DNA methylation/demethylation network observed in postmortem brains of PS patients was modified in PS patients with a history of chronic alcohol abuse. METHODS: DNA-methyltransferase-1 (DNMT1) mRNA-positive neurons were counted in situ in prefrontal cortex samples obtained from the Harvard Brain Tissue Resource Center, Belmont, MA. 10-11-translocation (TETs 1, 2, 3), apolipoprotein B editing complex enzyme (APOBEC-3C), growth and DNA-damage-inducible protein 45ß (GADD45ß), and methyl-binding domain protein-4 (MBD4) mRNAs were measured by quantitative real-time polymerase chain reaction in inferior parietal cortical lobule samples obtained from the Stanley Foundation Neuropathology Consortium, Bethesda, MD. RESULTS: We observed an increase in DNMT1 mRNA-positive neurons in PS patients compared with non-PS subjects. In addition, there was a pronounced decrease in APOBEC-3C and a pronounced increase in GADD45ß and TET1 mRNAs in PS patients with no history of alcohol abuse. In PS patients with a history of chronic alcohol abuse, the numbers of DNMT1-positive neurons were not increased significantly. Furthermore, the decrease in APOBEC-3C mRNA was less pronounced, while the increase in TET1 mRNA had a tendency to be potentiated in those PS patients that were chronic alcohol abusers. GADD45ß and MBD4 mRNAs were not influenced by alcohol abuse. The effect of chronic alcohol abuse on DNA methylation/demethylation network enzymes cannot be attributed to confounding demographic variables or to the type and dose of medication used. CONCLUSIONS: Based on these results, we hypothesize that PS patients may abuse alcohol as a potential attempt at self-medication to normalize altered DNA methylation/demethylation network pathways. However, before accepting this conclusion, we need to study alterations in the DNA methylation/demethylation pathways and the DNA methylation dynamics in a substantial number of alcoholic PS and non-PS patients. Additional investigation may also be necessary to determine whether the altered DNA methylation dynamics are direct or the consequence of an indirect interaction of alcohol with the neuropathogenetic mechanisms underlying psychosis.

Growth arrest and DNA-damage-inducible, beta (GADD45b)-mediated DNA demethylation in major psychosis.

Aberrant neocortical DNA methylation has been suggested to be a pathophysiological contributor to psychotic disorders. Recently, a growth arrest and DNA-damage-inducible, beta (GADD45b) protein-coordinated DNA demethylation pathway, utilizing cytidine deaminases and thymidine glycosylases, has been identified in the brain. We measured expression of several members of this pathway in parietal cortical samples from the Stanley Foundation Neuropathology Consortium (SFNC) cohort. We find an increase in GADD45b mRNA and protein in patients with psychosis. In immunohistochemistry experiments using samples from the Harvard Brain Tissue Resource Center, we report an increased number of GADD45b-stained cells in prefrontal cortical layers II, III, and V in psychotic patients. Brain-derived neurotrophic factor IX (BDNF IXabcd) was selected as a readout gene to determine the effects of GADD45b expression and promoter binding. We find that there is less GADD45b binding to the BDNF IXabcd promoter in psychotic subjects. Further, there is reduced BDNF IXabcd mRNA expression, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine at its promoter. On the basis of these results, we conclude that GADD45b may be increased in psychosis compensatory to its inability to access gene promoter regions.

Activation of group II metabotropic glutamate receptors promotes DNA demethylation in the mouse brain.

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-ß (Gadd45-ß), a molecular player of DNA demethylation, in the mouse frontal cortex and hippocampus. Induction of Gadd45-ß 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-ß 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-ß 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.

Selective α4ß2 nicotinic acetylcholine receptor agonists target epigenetic mechanisms in cortical GABAergic neurons.

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)ß(2) and α(7) nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α(4)ß(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)ß(2) but has negligible affinity for α(3)ß(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)ß(2) nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α(7) nAChR agonists.

Analysis of the GAD1 promoter: trans-acting factors and DNA methylation converge on the 5' untranslated region.

GAD67 corresponds to one of two enzymes that decarboxylates glutamate to produce γ-aminobutyric acid, the main inhibitory neurotransmitter in the mammalian central nervous system, hence defining the cellular phenotype of a diverse set of inhibitory interneurons of the brain. Reduced cortical GAD67 mRNA levels have consistently been reported in schizophrenia and bipolar disorder with psychosis. The human gene encoding GAD67, GAD1, is located on chromosome 2q31.1 and the transcriptional start site resides within a large CpG island that spans a region extending from upstream through the first exon. We have analyzed the GAD1 promoter using transient transfection analysis of upstream and downstream sequences in NT2 cells, a human neuroprogenitor cell line. Interestingly, results from these studies show that cis-acting regulatory elements are located downstream of the RNA start site and are in the region corresponding to the first exon. Trans-acting factors such as Pitx2 and the Dlx family of transcription factors are active in promoting downstream reporter expression even when all of the 5' flanking sequences are removed. However, those constructs that contain an internal deletion from +66 to +173 bp fail to support expression even when these factors are provided in trans. We have previously shown that the Class I histone deacetylase inhibitor MS-275 potently activates GAD1 mRNA expression in NT2 cells suggesting the possibility that the promoter is sensitive to drugs that induce chromatin remodeling. Using methyl DNA immuneprecipitation of MS-275-treated NT2 cells, we provide data showing that Class I HDAC inhibition mediated an increase in GAD1 expression and that this was accompanied by decreased GAD1 promoter methylation. Moreover, the reduced levels of GAD1 DNA methylation are highest in those regions proximal to the location of the in vitro defined cis-acting regulatory elements. Our data suggest that changes in promoter methylation associated with gene regulation are not random but overlap the locations of proximal cis-acting elements. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.