Fluoxetine epigenetically alters the CaMKIIα promoter in nucleus accumbens to regulate ΔFosB binding and antidepressant effects

Abstract

Chronic social defeat stress in mice produces a susceptible phenotype characterized by several behavioral abnormalities consistent with human depression that are reversed by chronic but not acute exposure to antidepressant medications. Recent work in addiction models demonstrates that the transcription factor ΔFosB and protein kinase calmodulin-dependent protein kinase II (CaMKII) are co-regulated in nucleus accumbens (NAc), a brain reward region implicated in both addiction and depression models including social defeat. Previous work has also demonstrated that ΔFosB is induced in NAc after chronic social defeat stress or after chronic antidepressant treatment, wherein it mediates a pro-resilience or antidepressant-like phenotype. Here, using chromatin immunoprecipitation assays, we found that ΔFosB binds the CaMKIIα gene promoter in NAc and that this binding increases after mice are exposed to chronic social defeat stress. Paradoxically, chronic exposure to the antidepressant fluoxetine reduces binding of ΔFosB to the CaMKIIα promoter and reduces CaMKII expression in NAc, despite the fact that ΔFosB is induced under these conditions. These data suggest a novel epigenetic mechanism of antidepressant action, whereby fluoxetine induces some chromatin change at the CaMKIIα promoter, which blocks the ΔFosB binding. Indeed, chronic fluoxetine reduces acetylation and increases lysine-9 dimethylation of histone H3 at the CaMKIIα promoter in NAc, effects also seen in depressed humans exposed to antidepressants. Overexpression of CaMKII in NAc blocks fluoxetine's antidepressant effects in the chronic social defeat paradigm, whereas inhibition of CaMKII activity in NAc mimics fluoxetine exposure. These findings suggest that epigenetic suppression of CaMKIIα expression in NAc is behaviorally relevant and offer a novel pathway for possible therapeutic intervention in depression and related syndromes.

Figure 1

(a) Western blotting of mouse NAc after 14 daily fluoxetine injections (20 mg/kg IP), analyzed 24 h after the last dose. (b) Quantitation of (a) reveals decreased total CaMKIIα, no change in total Thr²⁸⁶ autophosphorylation but an inferred increase in relative phosphorylation, and an increase in total ΔFosB (n=7–9 mice). (c) Immunohistochemical analysis of CaMKIIα expression in brains of chronic fluoxetine- or vehicle-treated mice. Quantitation of immunohistochemical signal in both NAc shell (d) and core (e) demonstrates fluoxetine-induced decrease in CaMKIIα expression in both control and socially defeated mice (Def), but no change resulting from several forms of chronic stress alone (Def.; C.U.S.: chronic unpredictable stress; or Soc. Iso.: adult social isolation; n=7–9 mice).

Figure 2

(a) qPCR reveals a significant decrease in NAc CaMKIIα, but not in CaMKIIβ, mRNA in response to chronic fluoxetine. (b) qChIP of mouse NAc reveals increased ΔFosB binding to the CaMKIIα promoter after chronic social defeat (Def), but a paradoxical decrease in binding in response to chronic fluoxetine (n=5–6 groups of mice). (c) qChIP shows reduced H3 pan-acetylation and increased H3 lysine 9 dimethylation (2MeK9) at the CaMKIIα promoter in NAc after chronic fluoxetine, with no change in 3MeK27 or 3MeK4 (n=10 groups of mice). (d) A similar analysis reveals increased ΔFosB binding to the CaMKIIβ promoter after Def, but no change in response to chronic fluoxetine (n=5–6 groups of mice). (e) qChIP of mouse NAc reveals increased ΔFosB binding to the Sparc-like 1 (SC-1) promoter after chronic fluoxetine (n=5–6 groups of mice) (*p⩽0.05, two-tailed t-test; ^#p⩽0.05, one-tailed t-test).

Figure 3

(a) Coronal slice of human brain highlighting NAc (red circle; courtesy MSU Human Brain Atlas). (b) qChIP for H3 pan-acetylation across the human CaMKIIα promoter in NAc from control and medicated-depressed patients (n=13–16). (c) Similar analysis of H3 lysine 9 dimethylation (n=13–16). (d) qPCR from two separate human cohorts reveals reduced CaMKIIα mRNA expression in the NAc of medicated-depressed patients (n=13–16). (e) Correlation between CaMKIIα mRNA and H3 acetylation at the CaMKIIα promoter (r²=0.1509; p=0.04 slope deviation from 0) (*p⩽0.05, two-tailed t-test).

Figure 4

Social interaction time (a), time spent in the corner zones (b), and sucrose preference (c) from socially defeated mice before and after surgery for NAc expression of GFP alone or GFP-AC3I indicate that local inhibition of CaMKII activity reverses stress-induced behavioral abnormalities (n=9). (d) Social interaction time and (e) time spent in the corner zones in defeated animals before and after chronic fluoxetine in mice with NAc expression of GFP alone or GFP and CaMKIIα demonstrate that CaMKII overexpression blocks the behavioral effects of fluoxetine (n=8–9) (*p⩽0.05, two-tailed t-test).