David P. Gavin

Histone deactylase 1 expression is increased in the prefrontal cortex of schizophrenia subjects: analysis of the National Brain Databank microarray collection.

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.

Valproate induces DNA demethylation in nuclear extracts from adult mouse brain.

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.

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.

Upregulation of TET1 and downregulation of APOBEC3A and APOBEC3C in the parietal cortex of psychotic patients.

Increasing evidence suggests that epigenetic dysfunction may account for the alteration of gene transcription present in neuropsychiatric disorders such as schizophrenia (SZ), bipolar disorder (BP) and autism. Here, we studied the expression of the ten-eleven translocation (TET) gene family and activation-induced deaminase/apolipoprotein B mRNA-editing enzymes (AID/APOBEC) in the inferior parietal lobule (IPL) (BA39-40) and the cerebellum of psychotic (PSY) patients, depressed (DEP) patients and nonpsychiatric (CTR) subjects obtained from the Stanley Foundation Neuropathology Consortium Medical Research Institute. These two sets of enzymes have a critical role in the active DNA demethylation pathway. The results show that TET1, but not TET2 and TET3, mRNA and protein expression was increased (two- to threefold) in the IPL of the PSY patients compared with the CTR subjects. TET1 mRNA showed no change in the cerebellum. Consistent with the increase of TET1, the level of 5-hydroxymethylcytosine (5hmC) was elevated in the IPL of PSY patients but not in the other groups. Moreover, higher 5hmC levels were detected at the glutamic acid decarboxylase67 (GAD67) promoter only in the PSY group. This increase was inversely related to the decrease of GAD67 mRNA expression. Of 11 DNA deaminases measured, APOBEC3A mRNA was significantly decreased in the PSY and DEP patients, while APOBEC3C was decreased only in PSY patients. The other APOBEC mRNA studied failed to change. Increased TET1 and decreased APOBEC3A and APOBEC3C found in this study highlight the possible role of altered DNA demethylation mechanisms in the pathophysiology of psychosis.

Histone deacetylase inhibitors and candidate gene expression: An in vivo and in vitro approach to studying chromatin remodeling in a clinical population

OBJECTIVE The emerging field of psychiatric epigenetics is constrained by the dearth of research methods feasible in living patients. With this focus, we report on two separate approaches, one in vitro and one in vivo, developed in our laboratory. METHOD In the first approach, we isolated lymphocytes from 12 subjects and cultured their cells with either 0.7 mM valproic acid (VPA), 100 nM Trichostatin A (TSA), or DMSO (control) for 24h based upon previous dose response experiments. We then measured GAD67 mRNA expression using realtime RT-PCR, total acetylated histone 3 (H3K9,K14ac) levels using Western blot analysis, and attachment of H3K9,K14ac to the GAD67 promoter using ChIP. In the second approach, we measured GAD67 mRNA and total H3K9,K14ac levels in lymphocytes from 11 schizophrenia and 7 bipolar patients before and after 4 weeks of clinical treatment with Depakote ER (VPA). RESULTS In the first approach, VPA induced a 383% increase in GAD67 mRNA, an 89% increase in total H3K9,K14ac levels, and a 482% increase in H3K9,K14ac attachment to the GAD67 promoter. TSA induced comparable changes on all measures. In the second approach, bipolar subjects had significantly higher baseline levels of H3K9,K14ac compared to subjects with schizophrenia. Subjects with clinically relevant serum levels of VPA (> or = 65 microg/mL) showed a significant increase in GAD67 mRNA expression. CONCLUSIONS Our results utilizing two separate approaches for examining chromatin remodeling in real clinical time provide possible means to investigate epigenetic events in living patients.

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-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.

Active DNA Demethylation in Post-Mitotic Neurons: A Reason for Optimism

Over the last several years proteins involved in base excision repair (BER) have been implicated in active DNA demethylation. We review the literature supporting BER as a means of active DNA demethylation, and explain how the various components function and cooperate to remove the potentially most enduring means of epigenetic gene regulation. Recent evidence indicates that the same pathways implicated during periods of widespread DNA demethylation, such as the erasure of methyl marks in the paternal pronucleus soon after fertilization, are operational in post-mitotic neurons. Neuronal functional identities, defined here as the result of a combination of neuronal subtype, location, and synaptic connections are largely maintained through DNA methylation. Chronic mental illnesses, such as schizophrenia, may be the result of both altered neurotransmitter levels and neurons that have assumed dysfunctional neuronal identities. A limitation of most current psychopharmacological agents is their focus on the former, while not addressing the more profound latter pathophysiological process. Previously, it was believed that active DNA demethylation in post-mitotic neurons was rare if not impossible. If this were the case, then reversing the factors that maintain neuronal identity, would be highly unlikely. The emergence of an active DNA demethylation pathway in the brain is a reason for great optimism in psychiatry as it provides a means by which previously pathological neurons may be reprogrammed to serve a more favorable role. Agents targeting epigenetic processes have shown much promise in this regard, and may lead to substantial gains over traditional pharmacological approaches.

Reduced baseline acetylated histone 3 levels, and a blunted response to HDAC inhibition in lymphocyte cultures from schizophrenia subjects

Dear Editors: Epigenetic modifications resulting in decreased gene expression is a proposed cause of schizophrenia (Petronis, 2004; Costa et al., 2002). The deacetylation of histone 3, catalyzed by the histone deacetylase (HDAC) family of enzymes, is an example of a change in chromatin structure that leads to a restrictive chromatin state and a consequent reduction in gene transcription both globally as well as at individual gene promoters (Berger, 2002). We recently reported a clinical study in which we treated schizophrenia and bipolar subjects with the HDAC inhibitor Depakote ER® (VPA) (Gottlicher et al., 2001) for four weeks. We found that after four weeks of VPA treatment schizophrenia patients had significantly smaller increases in acetylated lysine 9 and 14 of histone 3 (H3K9,K14ac), compared with bipolar subjects, implying that schizophrenia is associated with more ‘rigid’ chromatin (Sharma et al., 2006). In the current study we sought to further these results by testing an in vitro assay using cultured lymphocytes from a clinical population. Based on our previous findings our hypotheses were that H3K9, K14ac levels at baseline would be lower, and that the highly potent HDAC inhibitor, Trichostatin A (TSA), would induce smaller increases in H3K9, K14ac levels in lymphocytes from schizophrenia subjects compared to normal controls. We also expected TSA to induce smaller increases in the expression of the epigenetically regulated schizophrenia candidate gene, GAD1, among schizophrenia subjects. Patients of the University of Illinois Medical Center who fulfilled DSM-IV criteria for schizophrenia or schizoaffective disorder and had not used valproic acid, carbamazepine, or oxcarbazepine in the past 30 days were referred by the screening clinical psychiatrist for the study. Healthy comparison subjects who volunteered for the study, were recruited from among hospital staff and their associates, and were excluded if they reported a history of major mental illness or treatment with valproic acid. The study was approved by the Institutional Review Board at the University Of Illinois College Of Medicine. Cells were incubated with either DMSO (control) or 100nM TSA in media for 24 hours based on previous dose response experiments. Extraction of basic nuclear histone proteins and Western blot analysis procedures of H3K9, K14ac (Millipore #06-599) and total histone 1 (H1) (Millipore #05-457) were performed with H3K9,K14 being normalized to H1, using published procedures, (Sharma et al., 2006). GAD1 was measured using qRT-PCR, analyzed using a Stratagene Mx3005P™ QPCR System, and normalized to the housekeeping gene GAPDH. Schizophrenia subjects had significantly lower baseline H3K9, K14ac levels than normal controls (mean=0.78 [SD=0.47] vs. mean=1.44 [SD=1.34]; p<0.04) (Fig 1A and 1C). In addition, after 24-hours of incubation with TSA, the percent change in H3K9,K14ac levels significantly differed between schizophrenia and normal control subjects in cultured lymphocytes, (mean=11% [SD=16] vs. mean=60% [SD=54]; p<0.01) (Figure 1B and 1C). Fig. 1 Differences in acetylated histone 3 (H3K9, K14) between schizophrenia and normal control subjects. H3K9, K14ac was normalized to total histone 1 (H1). Panel A: Baseline levels of H3K9, K14ac are lower in schizophrenia compared with normal control subjects. ... There was a substantial though not significant difference between the ability of TSA to induce GAD1 expression in schizophrenia and normal subjects (mean percent change=−18% [SD=38] vs. mean percent change=134% [SD=339]; p<0.08). There was a significant correlation between TSA-induced increases in GAD1 expression and H3K9, K14ac among normal subjects (Spearman’s ρ=0.692; n=12; p<0.01), but not among schizophrenia subjects (Spearman’s ρ=−0.476; n=8; p<0.2). We found no significant differences within groups based on race, age, gender, medication use, duration of illness, or number of previous hospitalizations. We did find female subjects to have non-significantly higher baseline H3K9, K14ac levels and greater increases in GAD1 expression following TSA treatment. There exist some limitations to the current study. Presented here are global levels of H3K9, K14ac measured using Western blot analysis. We did not use a chromatin immunoprecipitation (ChIP) analysis, which would have been informative as to the H3K9, K14 levels specifically at the GAD1 promoter. We have recently performed a ChIP assay, and have been able to show that TSA treatment does increase H3K9, K14ac levels at the GAD1 promoter in cultured lymphocytes (unpublished data). Additionally, the current study utilized lymphocytes, which has limitations when used as a proxy to brain tissue. However, supporting its use for the study of epigenetic gene regulation is the fact that epigenetic parameters have been shown to be similar and reliably measurable in a variety of tissues including lymphocytes (Fraga et al., 2005), lymphocytes are exposed to much the same environment as neurons in terms of neurohormones, neuropeptides, chemo/cytokines, metabolites, and medication blood levels, GAD1 expression is similarly repressed in both tissues (Sullivan et al., 2006), and using lymphocytes allows one to measure changes in real clinical time. In the future it may be possible to isolate those schizophrenia patients characterized by ‘rigid’ chromatin, then using HDAC inhibitors to release the chromatin restraints on a global-basis on gene expression thereby allowing for more efficient gene regulation. Once the genome has been ‘relaxed’ patients may be more likely to benefit from conventional pharmacological treatment (Sharma et al., 2005). Alternatively, it may be possible to design chromatin remodeling agents which directly target disease candidate gene expression.

The melatonin receptor MT1 is required for the differential regulatory actions of melatonin on neuronal 'clock' gene expression in striatal neurons in vitro.

Abstract:  Through inhibitory G protein‐coupled melatonin receptors, melatonin regulates intracellular signaling systems and also the transcriptional activity of certain genes. Clock genes are proposed as regulatory factors in forming dopamine‐related behaviors and mood and melatonin has the ability to regulate these processes. Melatonin‐mediated changes in clock gene expression have been reported in brain regions, including the striatum, that are crucial for the development of dopaminergic behaviors and mood. However, it is not known whether melatonin receptors present in striatum mediate these effects. Therefore, we investigated the role of the melatonin/melatonin receptor system on clock gene expression using a model of primary neuronal cultures prepared from striatum. We found that melatonin at the receptor affinity range (i.e., nm) affects the expression of the clock genes mPer1, mClock, mBmal1 and mNPAS2 (neuronal PAS domain protein 2) differentially in a pertussis toxin‐sensitive manner: a decrease in Per1 and Clock, an increase in NPAS2 and no change in Bmal1 expression. Furthermore, mutating MT1 melatonin receptor (i.e., MT1 knockouts, MT1−/−) reversed melatonin‐induced changes, indicating the involvement of MT1 receptor in the regulatory action of melatonin on neuronal clock gene expression. Therefore, by controlling clock gene expression we propose melatonin receptors (i.e., MT1) as novel therapeutic targets for the pathobiologies of dopamine‐related behaviors and mood.

Histone methylation at H3K9: evidence for a restrictive epigenome in schizophrenia.

OBJECTIVE Epigenetic changes are stable and long-lasting chromatin modifications that regulate genomewide and local gene activity. The addition of two methyl groups to the 9th lysine of histone 3 (H3K9me2) by histone methyltransferases (HMT) leads to a restrictive chromatin state, and thus reduced levels of gene transcription. Given the numerous reports of transcriptional down-regulation of candidate genes in schizophrenia, we tested the hypothesis that this illness can be characterized by a restrictive epigenome. METHODS We obtained parietal cortical samples from the Stanley Foundation Neuropathology Consortium and lymphocyte samples from the University of Illinois at Chicago (UIC). In both tissues we measured mRNA expression of HMTs GLP, SETDB1 and G9a via real-time RT-PCR and H3K9me2 levels via western blot. Clinical rating scales were obtained from the UIC cohort. RESULTS A diagnosis of schizophrenia is a significant predictor for increased GLP, SETDB1 mRNA expression and H3K9me2 levels in both postmortem brain and lymphocyte samples. G9a mRNA is significantly increased in the UIC lymphocyte samples as well. Increased HMT mRNA expression is associated with worsening of specific symptoms, longer durations of illness and a family history of schizophrenia. CONCLUSIONS These data support the hypothesis of a restrictive epigenome in schizophrenia, and may associate with symptoms that are notoriously treatment resistant. The histone methyltransferases measured here are potential future therapeutic targets for small molecule pharmacology, and better patient prognosis.