Epigenetic GABAergic targets in schizophrenia and bipolar disorder

Abstract

It is becoming increasingly clear that a dysfunction of the GABAergic/glutamatergic network in telencephalic brain structures may be the pathogenetic mechanism underlying psychotic symptoms in schizophrenia (SZ) and bipolar (BP) disorder patients. Data obtained in Costa's laboratory (1996-2009) suggest that this dysfunction may be mediated primarily by a downregulation in the expression of GABAergic genes (e.g., glutamic acid decarboxylase₆₇[GAD₆₇] and reelin) associated with DNA methyltransferase (DNMT)-dependent hypermethylation of their promoters. A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to administer drugs, such as the histone deacetylase (HDAC) inhibitor valproate (VPA), that induce DNA-demethylation when administered at doses that facilitate chromatin remodeling. The benefits elicited by combining VPA with antipsychotics in the treatment of BP disorder suggest that an investigation of the epigenetic interaction of these drugs is warranted. Our studies in mice suggest that when associated with VPA, clinically relevant doses of clozapine elicit a synergistic potentiation of VPA-induced GABAergic promoter demethylation. Olanzapine and quetiapine (two clozapine congeners) also facilitate chromatin remodeling but at doses higher than used clinically, whereas haloperidol and risperidone are inactive. Hence, the synergistic potentiation of VPA's action on chromatin remodeling by clozapine appears to be a unique property of the dibenzepines and is independent of their action on catecholamine or serotonin receptors. By activating DNA-demethylation, the association of clozapine or its derivatives with VPA or other more potent and selective HDAC inhibitors may be considered a promising treatment strategy for normalizing GABAergic promoter hypermethylation and the GABAergic gene expression downregulation detected in the postmortem brain of SZ and BP disorder patients. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

FIG 1. Valproate (VPA) and clozapine (CLZ) accelerate the demethylation of methionine (MET*)-induced hypermethylation of reelin promoter

Met*: mice were pretreated with L-methionine (5.2 mmol/kg s.c. twice a day) for 7 days to hypermethylate the reelin promoter. At 7 days MET was withdrawn and mice received vehicle (VEH) or VPA or CLZ as indicated. Reelin promoter methylation was measured using MeDIP and quantitative PCR assays. Similar results were obtained for the GAD₆₇ promoter. (Dong et al., 2007, 2008).

FIG 2. Cytosine methylation in a CpG island-enriched promoter region of reelin comparing various methods

FIG 3. Valproate (VPA) increases reelin and GAD₆₇ mRNAs and reverses L-methionine (MET)-induced downregulation of these mRNAs

MET 5.2 mmol/kg; GLY (glycine) 5.2 mmol/kg; VPA 2.2 mmol/kg s.c. twice daily for 15 days. *p< 0.05 vs VEH; **p< 0.05 vs MET. Tremolizzo et al., 2005.

FIG 4. Clozapine or olanzapine alone or in combination with valproate (VPA) but not haloperidol induce reelin promoter demethylation in the mouse frontal cortex

VPA (70 mg/kg) and antipsychotics were given s.c. twice a day for three days after MET withdrawal. Open circles denote MET-pretreated mice that did not receive VPA. Filled circles denote MET-pretreated mice that received VPA. Open squares denote mice never treated with MET. *p< 0.05 when clozapine or olanzapine in absence of VPA were compared with the respective VEH-treated mice. **p<0.01 when clozapine + VPA or olanzapine + VPA-treated mice were compared with VEH + VPA-treated mice. # p< 0.05 when VEH + VPA-treated mice were compared with the respective VEH-treated mice.

FIG 5. Clozapine increases acetylated H3-lysine9 frontal cortex levels at the reelin promoter

Open bars denote mice that did not receive valproate. Closed bars denote mice that received valproate (70 mg/kg s.c. twice a day for three days) *p< 0.05; **p< 0.01 when compared with the respective controls. Controls are mice that did not receive valproate or clozapine. For details see Dong et al., 2008.

FIG 6. Proposed mechanism of activity-dependent CpG-rich promoter demethylation

A. Following depolarization GADD45 α, β protein levels increase and GADD45 α, β bind to a methylated promoter region proximal to an acetylated histone (green). B. GADD45 recruits a deaminase (DA), which converts 5-methylcytosine to thymine leading to a T:G mismatch. C. GADD45 recruits a DNA glycosylase (GLY), which removes thymine from the T:G mismatch. Thymine is later replaced with an unmethylated cytosine.

FIG 7. Clozapine (CLZ) alone or in combination with valproate (VPA) increases GADD45 β mRNA expression in FC of mice

CLZ (5 mg/kg s.c./3 days/twice a day); VPA (70 mg/kg s.c./3 days/twice a day); VEH, vehicle. GADD45 β mRNA expression was measured two hours after the last drug injection. *p< 0.05, **p< 0.01 vs VEH group. ANOVA followed by Bonferroni comparison; n=4–5 mice per group.

FIG 8. GADD45 β protein levels increase in FC neurons and coalesce in nuclei of mice treated with valproate

Mice were treated with either vehicle or 70 mg/kg s.c. valproate twice a day for three days. Samples were analyzed two hours after the last drug injection. GADD45 β antibody (Santa Cruz) recognized a major band (~ 18 kDa) of immune-reactive material in western blot of FC extracts.I and II denote layers I and II of cortex.

FIG 9. Structures of the antipsychotics used in the study