Class I HDAC inhibition blocks cocaine-induced plasticity by targeted changes in histone methylation

Abstract

Induction of histone acetylation in the nucleus accumbens (NAc), a key brain reward region, promotes cocaine-induced alterations in gene expression. Histone deacetylases (HDACs) tightly regulate the acetylation of histone tails, but little is known about the functional specificity of different HDAC isoforms in the development and maintenance of cocaine-induced plasticity, and previous studies of HDAC inhibitors report conflicting effects on cocaine-elicited behavioral adaptations. Here we demonstrate that specific and prolonged blockade of HDAC1 in NAc of mice increased global levels of histone acetylation, but also induced repressive histone methylation and antagonized cocaine-induced changes in behavior, an effect mediated in part through a chromatin-mediated suppression of GABAA receptor subunit expression and inhibitory tone on NAc neurons. Our findings suggest a new mechanism by which prolonged and selective HDAC inhibition can alter behavioral and molecular adaptations to cocaine and inform the development of therapeutics for cocaine addiction.

Figure 1

HDAC1 in NAc regulates locomotor responses to cocaine. (a, c, e) Cocaine (coc, 10 mg/kg) locomotor sensitization in floxed- (a) HDAC1 and (c) HDAC2 mice injected with HSV-CreGFP or HSV-GFP, and (e) HDAC3 mice injected with AAV-CreGFP or AAV-GFP. All data are presented as mean ± s.e.m. (a, c, e) A significant main effect (three-way ANOVA on square-root transformed data) of day (a, F_3,90 = 11.257, P < 0.0005; c, F_3,36 = 26.040, P < 0.0001; e, F_3,57 = 19.479, P < 0.001) and drug (c, F_1,12 = 24.238, P < 0.0001) and significant interactions between day and virus (a, F_3,90 = 3.007, P = 0.034) and day and drug (a, F_3,90 = 11.741, P < 0.0005; e, F_3,57 = 11.976, P < 0.001) as well as a trend towards a day by drug by virus interaction (a, F_3,90 = 2.173, P = 0.097) were observed. *P < 0.04 and **P < 0.001, Bonferroni post hoc tests. (b, d, f) qPCR validation of (b) HDAC1, (d) HDAC2, and (f) HDAC3 knockdown and mRNA expression of other HDACs in the NAc of HDAC1^fl/fl, HDAC2^fl/fl and HDAC3^fl/fl mice. *P < 0.05, **P < 0.01 and ***P < 0.0001, student’s t test. (For behavioral experiments: N = 4–6/group (saline condition); N = 6–11/group (cocaine condition). For qPCR validation: N = 6–8/group).

Figure 2

Chronic MS-275 infusion into NAc blocks locomotor responses to cocaine and alters repressive histone methylation. (a) Cocaine (10 mg/kg) locomotor sensitization in animals receiving continuous intra-NAc infusion of MS-275 or vehicle (Veh) (100 μM). A significant main effect (three-way ANOVA on square-root transformed data followed by Bonferroni post hoc tests) of day (F_3,54 = 3.004, P = 0.037) and significant interactions between day and drug (F_3,54 = 4.578, P = 0.006) and day and treatment (F_3,54 = 3.101, P = 0.034) were observed. Day by drug by treatment interaction (F_3,54= 1.607, P = 0.19). (N = 5 or 6/group (vehicle treatment); N = 6 or 7/group (MS-275 treatment). (b–d) Global levels of H3 acetylation and methylation in NAc after 12 days of continuous treatment with MS-275 (100 μM) and 24 hour after repeated cocaine (20 mg/kg, seven daily doses). All data represented as normalized values to GAPDH (b) A significant main effect (two-way ANOVA followed by Bonferroni post hoc tests) of treatment (H3K14ac, F_1,22 = 23.20, ***P < 0.001), (c) a main effect of drug (H3K9ac, F_1,24 = 6.252, **P < 0.02), and (d) a significant interaction between drug and treatment (H3K9me2, F_1,18 = 15.23, P = 0.001) were observed (N = 5–8/group). Full-length blots are presented in Supplemental Figure 6. (e and f) mRNA expression of H3K9 KMTs and quantitative H3K9ac ChIP in NAc after 12 days of continuous treatment with MS-275 (100 μM) and 24 hour after repeated cocaine (20 mg/kg). (e) A significant interaction (two-way ANOVA followed by Bonferroni post hoc and planned student’s t tests) between treatment and drug (G9a, F_1,27 = 11.11, P = 0.0025; SUV39H1- F_1,27 = 12.82, P = 0.0013) was observed (N = 7 or 8/group). (f) A significant effect (one-way ANOVA followed by Bonferroni post hoc tests) of MS-275 with repeated cocaine on H3K9ac binding to H3K9 KMT promoters (G9a, F_2,16 = 8.749, P = 0.0034; GLP, F_2,16 = 21.01, P < 0.0001; SUV39H1, F_2,16 = 9.108, P = 0.0029 was observed (N = 5 or 6/group). ^#P = 0.14 (trend), *P < 0.05 and **P < 0.02, ***P < 0.001. All data are represented as mean ± s.e.m.

Figure 3

HDAC binding to KMT gene promoters after repeated cocaine. (a–c) Quantitative ChIP for HDAC1, 2, and 3 in NAc from animals treated with repeated cocaine at 4 hours after the last injection. Significance determined using Student’s t tests. (a) HDAC1 (GAPDH, t₁₃ = 1.520, P = 0.1524; G9a, t₁₄ = 2.227, *P≤ 0.05; GLP, t₁₄ = 2.090, *P≤ 0.05; SUV39H1, t₁₂ = 1.735, P = 0.108) (N = 7 or 8/group). (b) HDAC2 (GAPDH, t₁₄ = 0.9857, P = 0.341), (G9a, t₁₂ =0.2604, P = 0.799), (GLP, t₁₁ = 0.3611, P = 0.7249), (SUV39H1, t₁₄ = 1.343, P = 0.2006) (N = 5–8/group). (c) HDAC3 (GAPDH, t₁₂ = 1.061, P = 0.3098), (G9a, t₁₂ = 1.624, P = 0.1303), (GLP, t₁₂ = 0.4761, P = 0.6245), (SUV39H1, t₁₂ = .1775, P = 0.8621) (N = 7/group). All data are presented as percent input enrichment.

Figure 4

Chronic MS-275 infusion prevents cocaine-induced changes in GABA_A receptor subunit expression and GABAergic tone in NAc. (a) mRNA expression of GABA_A receptor subunits (N = 5–8/group) in NAc after 12 days of continuous treatment with MS-275 (100 μM) and 24 hours after repeated cocaine (20 mg/kg). A significant interaction (two-way ANOVA followed by Bonferonni post hoc tests) between treatment and drug for many subunits was observed (GABRA1, F_1,27 = 16.62, P = 0.004; GABRA2, F_1,27 = 8.658, P = 0.0066; GABRA3, F_1,26 = 21.83, P < 0.0001; GABRB2, F_1,22 = 21.84, P < 0.0001; GABRG2, F_1,20 = 6.458, P = 0.0194). (b, c) Quantitative H3K9ac and H3K9me2 ChIP in NAc from mice treated with repeated cocaine or chronic MS-275 infusion into NAc with repeated cocaine at 24 hours. Data are presented as percent input enrichment (N = 5 or 6/group). (b) A significant effect (one-way ANOVA followed by Bonferonni post hoc tests) of MS-275 with repeated cocaine on H3K9ac binding at GABA_A receptor subunit promoters (GABRA1, F_2,16 = 26.02, P < 0.0001; GABRA2, F_2,16 = 11.37, P = 0.0012; GABRA3, F_2,14 = 5.841, P = 0.0169; GABRB3, F_2,16 = 14.39, P = 0.0004; Oct-4, F_2,12 = 3.038, P = 0.0932) was observed. (c) A significant difference (one-way ANOVA followed by Bonferonni post hoc tests) in H3K9me2 binding at GABA_A receptor subunit promoters (GABRA1, F_2,16 = 4.902, P = 0.0243; GABRA2, F_2,15 = 3.910, P = 0.0468; GABRA3, F_2,16 = 10.77, P = 0.0015; GABRB3, F_2,15 = 14.83, P = 0.0004; Oct-4, F_2,13 = 1.365, P = 0.2954) was observed. (d) IPSCs from NAc medium spiny neurons from mice treated with chronic saline or cocaine, and exposed to intra-NAc infusion of vehicle or MS-275 (N = 5–8/group). A significant interaction (two-way ANOVA followed by Bonferonni post hoc tests) between treatment and drug was observed for the frequency of spontaneous IPSCs (F_1,23 = 14.43, P = 0.0009). *P < 0.05 and **P < 0.01. All data presented as mean ± s.e.m.