Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia - PubMed (original) (raw)

Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia

E Dong et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Reduction of prefrontal cortex glutamic acid decarboxylase (GAD67) and reelin (mRNAs and proteins) expression is the most consistent finding reported by several studies of postmortem schizophrenia (SZ) brains. Converging evidence suggests that the reduced GAD67 and reelin expression in cortical GABAergic interneurons of SZ brains is the consequence of an epigenetic hypermethylation of RELN and GAD67 promoters very likely mediated by the overexpression of DNA methyltransferase 1 in cortical GABAergic interneurons. Studies of the molecular mechanisms (DNA methylation plus related chromatin remodeling factors) that cause the down-regulation of reelin and GAD67 in SZ brains have important implications not only to understand the disease pathogenesis but also to improve present pharmacological interventions to treat SZ. The mouse treated with l-methionine models some of the molecular neuropathologies detected in SZ, including the hypermethylation of RELN promoter CpG islands and the down-regulation of reelin and GAD67 expression. We now report that in these mice, RELN and GAD67 promoters express an increased recruitment of methyl-CpG binding domain proteins. In these mice the histone deacetylase inhibitor valproate, which increases acetylated histone content in cortical GABAergic interneurons, also prevents MET-induced RELN promoter hypermethylation and reduces the methyl-CpG binding domain protein binding to RELN and GAD67 promoters. These findings suggest that DNA hypermethylation and the associated chromatin remodeling may be critically important in mediating the epigenetic down-regulation of reelin and GAD67 expression detected in cortical GABAergic interneurons of SZ patients.

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Figures

Fig. 1.

Fig. 1.

GAD67 but not GAD65 protein expression is down-regulated in FC of MET-treated mice. GAD67/β-actin and GAD65/β-actin OD ratios were obtained after Western blot of GAD67 and GAD65 on a 10-20% SDS/PAGE gradient. Each value is the mean ± SE of VEH and MET (5.2 mmol/kg/s.c. twice a day for 15 days)-treated mice (n = 3). *, P < 0.01 VEH vs. MET (Student's t test).

Fig. 2.

Fig. 2.

MET treatment induces a time-related increase in the amount of FC RELN and GAD67 promoters immunoprecipitated with MeCP2 or MBD2 antibodies. RELN (-520 to -225 bp) and GAD67 (-760 to -329 bp) promoter fragments were quantified by using competitive PCR with internal standards (see Materials and Methods). (A1 and A2) The ratios are depicted between the amount of RELN (A1) and GAD67 (A2) promoters immunoprecipitated with MeCP2 antibody (MeCP2-ChIP) and the amount of RELN and GAD67 promoter fragments in the initial nonimmunoprecipitated extract (Input). (B1 and B2) The ratios are depicted between the amount of RELN (B1) and GAD67 (B2) promoters immunoprecipitated with MBD2 antibody (MBD2-ChIP) and the amount of RELN and GAD67 promoter fragments in the initial nonimmunoprecipitated extract (Input). VEH, vehicle; MET, methionine (5.2 mmol/kg s.c. twice a day; the last injection of MET was administered 2 h before killing). The data represent mean ± SE of three mice. *, P < 0.05 vs. vehicle treated group. ANOVA is followed by Dunnett's test.

Fig. 3.

Fig. 3.

VPA prevents the MET-induced increase in the amount of FC RELN (Left) and GAD67 (Right) promoters immunoprecipitated with MeCP2 antibody. In ordinates depicted are the ratios between the amount of RELN and GAD67 promoters immunoprecipitated with MeCP2 antibody (MeCP2-ChIP) and the amount of RELN and GAD67 promoter fragments in the initial nonimmunoprecipitated extract (Input). Treatments: VEH, vehicle; MET, methionine 5.2 mmol/kg s.c. twice a day for 15 days; VPA, valproate 2 mmol/kg s.c. twice a day for 15 days. The data represent mean ± SE of three mice. *, P < 0.05 vs. VPA plus MET-treated group; ANOVA is followed by Dunnett's test.

Fig. 4.

Fig. 4.

VPA enhances the amount of FC RELN and GAD67 promoters immunoprecipitated with acetyl histone (H3) antibody. Depicted are the ratios between the amount of RELN (Left) and GAD67 (Right) promoters immunoprecipitated with acetyl-histone3 (H3) antibody (Ac_H3-ChIP_) and the amount of RELN and GAD67 promoter fragments in the initial nonimmunoprecipitated extract (Input). Mice were injected with VEH or VPA (2 mmol/kg s.c.) 2 hr before killing. The data represent mean ± SE of three experiments *, P < 0.05, Student's t test.

Fig. 5.

Fig. 5.

Proposed mechanism by which mouse RELN promoter hypermethylation and recruitment of chromatin remodeling complexes (MeCP2, HDACs, and corepressors) regulate reelin gene expression. The mouse reelin (RELN) promoter region depicted here is that reported by Tremolizzo et al. (39). Vertical bars represent CpG dinucleotides present in this region. Pink dots denote 5mC present in the sequence. Note the increase of 5mC in MET (methionine)-treated mice and the prevention of this increase in MET plus VPA (valproate)-treated mice. MeCP2 recruits corepressor complexes including HDACs and induces a state of gene repression. VPA induces loss of MeCP2 binding to the promoter and enhances transcription.

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