Kayla A. Chase

Valproic acid and chromatin remodeling in schizophrenia and bipolar disorder: Preliminary results from a clinical population

Levels of acetylated Histone 3 and 4 proteins are strongly predictive of a chromatin structure that is conducive to gene expression. In cell and animal studies, valproic acid is a potent inhibitor of histone deactylating enzymes, and consequently results in increased levels of acetylated Histone 3 (acH3) and acetylated Histone 4 proteins (acH4). To examine this effect in a clinical setting, 14 schizophrenic and bipolar patients were treated with valproic acid (Depakote ER), either as monotherapy or in combination with antipsychotics, over a period of 4 weeks. AcH3 and acH4 levels from lymphocyte nuclear protein extracts were measured by Western Blot. Treatment with Depakote ER resulted in a significant increase of acH3 and a trend-level increase of acH4. Levels of valproic acid were positively and significantly correlated with percent increase in acH3 but not acH4. Schizophrenia patients were significantly less likely to increase their acH3 and acH4 levels after 4 weeks on Depakote ER. The authors consider these results in the context of future application of HDAC inhibitors to the treatment of psychiatric disorders.

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.

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.

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.

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.

Nicotine induces chromatin remodelling through decreases in the methyltransferases GLP, G9a, Setdb1 and levels of H3K9me2

Studies examining the epigenetic effects of nicotine are limited, but indicate that nicotine can promote a transcriptionally permissive chromatin environment by increasing acetylation of histone H3 and H4. To further explore nicotine-induced histone modifications, we measured histone methyltransferase (HMT) mRNA expression as well as total and promoter-specific H3K9me2 levels. Following administration of nicotine, HMT mRNA and H3K9me2 levels were examined in mouse primary cortical neuronal culture and cortex extracted from mice injected intraperitoneally, as well as in human lymphocyte culture. Furthermore, Bdnf/BDNF mRNA levels were examined as an epigenetically regulated read-out of gene expression. There was a significant decrease of the HMT GLP, G9a and Setdb1 mRNA expression in the nicotine-treated tissue examined, with significant decreases seen in both total and promoter-specific H3K9me2 levels. Increasing doses of nicotine resulted in significant decreases in Bdnf/BDNF promoter specific H3K9me2 binding, leading to enhanced Bdnf/BDNF transcription. Taken together, our data suggest that nicotine reduces markers of a restrictive epigenomic state, thereby leading to a more permissive epigenomic environment.

Metabolic and Inflammatory Genes in Schizophrenia

Energy metabolism and immunity are characterized as abnormal in schizophrenia. Because these two systems are highly coordinated, we measured expression of prototypic obesogenic and immunogenic genes in freshly harvested PBMC from controls and participants with schizophrenia. We report significant increases in PPARγ, SREBP1, IL-6 and TNFα, and decreases in PPARα and C/EPBα and mRNA levels from patients with schizophrenia, with additional BMI interactions, characterizing dysregulation of genes relating to metabolic-inflammation in schizophrenia.

The value of interleukin 6 as a peripheral diagnostic marker in schizophrenia

BackgroundAssociations between a pro-inflammatory state and schizophrenia have been one of the more enduring findings of psychiatry, with various lines of evidence suggesting a compelling role for IL-6 in the underlying pathogenesis of schizophrenia.MethodsIn this study, we examined IL-6 mRNA levels by real-time RT-PCR from fresh extracted peripheral blood mononuclear cells (PBMC) in normal controls and participants with schizophrenia.ResultsWe found that peripheral PBMC IL-6 mRNA levels, in the absence of any other information, reliably discriminated between a diagnosis of schizophrenia and normal controls. Furthermore, in participants with schizophrenia, we also found elevated levels of IL-6 mRNA with earlier ages of illness onset and worse positive symptom presentation, as measured by the Positive and Negative Syndrome Scale.ConclusionsThese findings provide important and continued support for a pathophysiological role of inflammation in patients with schizophrenia. Future utilization of peripheral IL-6 mRNA levels could be clinically useful during an initial diagnosis and help tailor individualized treatment plans for patients with schizophrenia.

Heterochromatin as an incubator for pathology and treatment non-response: implication for neuropsychiatric illness

Heterochromatin is a higher order assembly that is characterized by a genome-wide distribution, gene-repression, durability and potential to spread. In this light, it is an appealing mechanism to interpret the neurobiology of complex brain disorders such as schizophrenia where downregulation of expression appears to be the norm. H3K9 methylation (H3K9me) can initiate the seeding of a heterochromatin assembly on an inactive or poorly coordinated promoter as a consequence of a decline in transactivators either from disuse or from misuse. H3K9me can extend its influence by spatial spreading through the mechanism of recursively recruiting adapters, such as heterochromatin protein 1 (HP1) homodimers. HP1 itself serves as a platform for other repressive proteins such as DNA methyltransferases. In full color, heterochromatin can occupy genome-wide gene networks, tissue specific ontologies and even rearrange the nuclear architecture. Heterochromatin in the brain is modified by small molecule pharmacology and serves a physiological role in the functioning of dopamine neurons and the construction of memory. From a therapeutic perspective, the durable nature of heterochromatin implies that it may require disassembly before the full genomic-potential of standard pharmacotherapies is achieved, especially in treatment resistant patients.