Clozapine and sulpiride but not haloperidol or olanzapine activate brain DNA demethylation

Abstract

Cortical GABAergic dysfunction, a hallmark of both schizophrenia (SZ) and bipolar (BP) disorder pathophysiologies may relate to the hypermethylation of GABAergic gene promoters (i.e., reelin and GAD67). Benefits elicited by a combination of atypical antipsychotics with valproate (VPA) (a histone deacetylase inhibitor that may also activate brain DNA demethylation) in SZ or BP disorder treatment prompted us to investigate whether the beneficial action of this association depends on induction of a putative DNA demethylase activity. To monitor this activity, we measured the ratio of 5-methyl cytosine to unmethylated cytosine in reelin and GAD67 promoters in the mouse frontal cortex and striatum. We compared normal mice with mice pretreated with l-methionine (5.2 mmol/kg s.c. twice a day for 7 days) to hypermethylate promoters, including reelin and GAD67. Clinically relevant doses of clozapine (CLZ) (3.8 to 15 micromol/kg twice a day s.c. for 3 days) and sulpiride (SULP) (12.5 to 50 micromol/kg twice a day for 3 days) but not clinically relevant doses of haloperidol (HAL) (1.3 to 4 micromol/kg twice a day s.c. for 3 days) or olanzapine (OLZ) (4 to 15 micromol/kg twice a day for 3 days) exhibited dose-related increases in the cortical and striatal demethylation of hypermethylated reelin and GAD67 promoters. These effects of CLZ and SULP were dramatically potentiated by a clinically relevant VPA dose (0.5 mmol/kg twice a day for 3 days). By activating a DNA demethylase, the association of CLZ or SULP with VPA may facilitate a chromatin remodeling that normalizes the GABAergic gene expression down-regulation detected in the telencephalic regions of SZ and BP patients.

Fig. 1.

VPA facilitates reelin promoter demethylation. (A) Mice were pretreated for 7 days with vehicle (open bar) or MET (5.2 mmol/kg s. c. twice a day) (filled bar) to induce hypermethylation of the reelin promoter. (B) After MET treatment termination, VPA (0 to 2.0 mmol/kg s. c. twice a day for 3 days) induces a dose-related decrease of reelin promoter methylation (shaded bars). Reelin promoter methylation was measured 2 h after the last injection of VPA. ¥, depicted are the ratios between the amount of reelin promoter fragment immunoprecipitated with MeCP2 antibodies (MeCP2-ChIP) and the amount of the corresponding promoter fragment in the initial non immunoprecipitated extract (input). The data represent the mean ± SE of 3 to 5 mice. *, P < 0.01 when the effect of VPA is compared with the group with no VPA. (One-way ANOVA followed by Bonferroni comparison).

Fig. 2.

Clozapine and sulpiride alone or in combination with valproate (VPA) but not haloperidol or olanzapine induce reelin promoter demethylation in mouse FC. Mice were pretreated for 7 days with MET (5.2 mmol/kg s. c. twice a day for 7 days). After termination of MET treatment, various doses of clozapine, sulpiride, haloperidol, olanzapine, or vehicle alone or combined with VPA (0.5 mmol/kg s.c.) were administered twice a day for 3 days. Reelin promoter methylation was measured 2 h after the last injection. 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. In these mice, VPA (0.5 mmol/kg) failed to elicit a significant decrease of reelin promoter methylation. The data represent the mean ± SE of three animals per group. *, P < 0.05 when CLZ and SULP in absence of VPA were compared with the respective VEH-treated mice. **, P < 0.01 when sulpiride + VPA- and clozapine + 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. One-way ANOVA followed by Bonferroni comparison. ¥, Cytosine methylation at reelin promoter was measured as described in Fig. 1.

Fig. 3.

Clozapine (CLZ) in combination with valproate (VPA) induces reelin and GAD67 promoter demethylation in mouse striatum. Mice were pretreated for 7 days with MET (5.2 mmol/kg s.c. twice a day for 7 days). After termination of MET treatment, vehicle (open bars), VPA (0.5 mmol/kg; filled bars), CLZ (15 μmol/kg, shaded bars), or a combination of CLZ and VPA (striped bars) were administered s.c. twice a day for 3 days. Reelin or GAD67 promoter methylation was measured 2 h after the last injection. The data represent the mean ± SE of 3 animals per group. *, P < 0.05 when VPA treated mice were compared with VEH treated mice; **, P < 0.01 when CLZ + VPA treataed mice were compared with VEH + VPA treated mice. One-way ANOVA followed by Bonferroni comparison. ¥, cytosine methylation of reelin and GAD67 promoters was determined as described in Fig. 1.

Fig. 4.

Clozapine alone or in combination with valproate (VPA) induces reelin promoter demethylation in mouse FC but not in liver. Mice were pretreated for 7 days with vehicle (open bars) or MET (5.2 mmol/kg twice a day) (filled bars) followed by 3 days with vehicle. After termination of MET treatment, VPA (0.5 mmol/kg, shaded bar), CLZ (15 μmol/kg, checkered bars), or a combination of CLZ with VPA (strip bars) were administered s.c. twice a day for 3 days. Reelin promoter methylation was measured 2 h after the last injection. The data represent the mean ± SE of three animals per group. *, P < 0.05 when CLZ treated mice were compared with the respective VEH-treated mice. **, P < 0.01 when CLZ + VPA-treated mice were compared with either VEH- or VEH+ VPA-treated mice. One-way ANOVA followed by Bonferroni comparison. ¥, Cytosine methylation of reelin promoter was determined as described in Fig. 1.

Fig. 5.

Clozapine and sulpiride increase acetylated H3K9,14 FC levels at reelin promoter in VPA treated mice. The FCs of the same animals used in Fig. 2 were analyzed in this experiment. Open circles denote mice that did not receive VPA. Filled circles denote mice that received VPA. The data represent the mean ± SE of three animals per group. *, P < 0.05 when SULP and CLZ without VPA were compared with the respective VEH-treated mice. **, P < 0.0 2 when SULP + VPA- and CLZ + VPA-treated mice were compared with VEH + VPA-treated mice. #, P < 0.05 when VEH +VPA-treated mice were compared with VEH treated-mice that did not receive VPA. Filled circles, one-way ANOVA followed by Bonferroni comparison.