A role for repressive histone methylation in cocaine-induced vulnerability to stress

Abstract

Substance abuse increases an individual's vulnerability to stress-related illnesses, which is presumably mediated by drug-induced neural adaptations that alter subsequent responses to stress. Here, we identify repressive histone methylation in nucleus accumbens (NAc), an important brain reward region, as a key mechanism linking cocaine exposure to increased stress vulnerability. Repeated cocaine administration prior to subchronic social defeat stress potentiated depressive-like behaviors in mice through decreased levels of histone H3 lysine 9 dimethylation in NAc. Cre-mediated reduction of the histone methyltransferase, G9a, in NAc promoted increased susceptibility to social stress, similar to that observed with repeated cocaine. Conversely, G9a overexpression in NAc after repeated cocaine protected mice from the consequences of subsequent stress. This resilience was mediated, in part, through repression of BDNF-TrkB-CREB signaling, which was induced after repeated cocaine or stress. Identifying such common regulatory mechanisms may aid in the development of new therapies for addiction and depression.

Figure 1. Cocaine Enhances Vulnerability to Social Stress

(A) Time-course of saline or cocaine (20 mg/kg/injection) treatment prior to control handling or sub-maximal (8 days) social defeat stress. (B) Repeated cocaine prior to sub-maximal social stress increases social avoidance during a social interaction test. (C) Repeated cocaine treatment decreases an animal's latency to interact with a target under non-stressed conditions, whereas prior drug administration increases an animal's latency to interact after social stress. (D) Repeated cocaine prior to sub-maximal social stress decreases sucrose preference. (E) Neither repeated cocaine nor social defeat affects locomotor activity in an open field. #, ## indicates significant differences from saline injections prior to defeat stress, p<0.05, or 0.01, respectively. * indicates significant differences from non-stressed controls injected with saline, p<0.05. Data are presented as average ± SEM, n = 8–10/group.

Figure 2. Cocaine, Social Stress, and Clinical Depression Similarly Regulate Repressive Histone Methylation in NAc

Repeated cocaine decreases (A) G9a and (B) H3K9me2 protein levels in NAc. (C) Mice subjected to chronic (10 days) social stress can be grouped into susceptible and unsusceptible subpopulations. (D) G9a shows a trend for reduced levels in susceptible mice compared to controls, but is significantly reduced in susceptible animals compared to unsusceptible mice. (E) H3K9me2 expression is reduced in susceptible animals, an effect not seen in unsusceptible animals. For all experiments, n=6–7/group. (F) G9a and (G) H3K9me2 protein are reduced in NAc of human postmortem depressed subjects, n = 8–10/group. *, ** and *** indicates significant differences from control condition, p<0.05, p<0.01 and p<0.001, respectively. #, ##, and ### indicate significant differences from susceptible mice, p<0.05, p<0.01 and p<0.001, respectively. Data are presented as average ± SEM. (H) mRNA expression levels for chromatin modifying enzymes were quantified in the NAc of control, susceptible, and unsusceptible mice after 10 days of social stress, and compared between clinically depressed and control individuals using postmortem tissue. Significant changes are noted by an arrow, and a p value. Increased expression of several genes was observed in unsusceptible mice, as compared to susceptible animals. Likewise, many of these genes were significantly decreased in clinical depression. For mouse work, n = 8/group, and for human analysis, n = 8–10/group.

Figure 3. G9a Overexpression in NAc Reverses Social Defeat-induced Social Deficits

(A) Time-course of chronic (10 days) social defeat stress and HSV-GFP/HSV-G9a overexpression in NAc. (B) Immunohistochemical analysis of HSV-G9a overexpression in NAc. (C) Western blot analysis of G9a protein in NAc four days after HSV infection. (D) Chronic social defeat stress reduces time spent in the social interaction zone during a social interaction test, an effect that can be reversed by viral overexpression of G9a in NAc. (E) Neither social stress nor G9a overexpression affects locomotor activity in an open field. * indicates a significant difference from control condition (non-defeated mice infected with GFP), p<0.05, and # indicates a significant difference from defeated mice infected with GFP, p<0.05. Data are presented as average ± SEM, n = 6 group.

Figure 4. G9a Repression in NAc Promotes Susceptibility to Stress

(A) Time-course of Cre-mediated G9a knockdown in NAc and subsequent sub-maximal (8 days) social defeat stress. (B) Immunohistochemical and (C) western blot validation of G9a knockdown in NAc using HSV-Cre-GFP viruses in G9a^fl/fl mice. (D) G9a repression in NAc enhances social avoidance in a social interaction test. (E) G9a depletion in NAc promotes anhedonia as measured by sucrose preference. * indicates a significant difference from control condition (defeated mice infected with GFP), p<0.05. Data are presented as average ± SEM, n = 6–7/group. (E) G9a knockdown does not affect locomotor activity in an open field.

Figure 5. G9a Overexpression in NAc Prevents Cocaine-induced Vulnerability to Stress

(A) Time-course of saline or cocaine (20 mg/kg/injection) prior to control handling or compressed sub-maximal (8 defeats over 4 days) social defeat stress. (B) Repeated cocaine prior to compressed social stress increases social avoidance during a social interaction test. (C) Repeated cocaine does not affect locomotor activity in an open field following compressed social stress. * indicates significant differences from mice injected with saline, p<0.05, n = 5–6/group. (D) Time-course of saline or cocaine (20 mg/kg/injection) prior to G9a overexpression, followed by compressed social defeat stress. (E) Overexpression of G9a in NAc following repeated cocaine significantly reduces cocaine-induced social avoidance during a social interaction test. (F) Neither prior cocaine, social stress, nor G9a overexpression affects locomotor activity in an open field. * indicates a significant difference from control condition (non-defeated mice infected with GFP), p<0.05, and # indicates a significant difference from defeated mice infected with GFP, p<0.05, n = 10 mice/group. Data are presented as average ± SEM.

Figure 6. Repeated Cocaine and Chronic Social Stress Induce Ras and Downstream BDNFTrkB Signaling in NAc

(A) Cartoon depicting stimulus-dependent regulation of the BDNF-TrkB signaling cascade. (B) Time-course of saline or cocaine (20 mg/kg/injection) prior to G9a overexpression, followed by control handling or compressed sub-maximal (8 defeats over 4 days) social defeat stress and NAc tissue collection. N = 10 mice/group (C) Compressed social stress following repeated cocaine promotes increased expression of Ras, a member of the BDNF-TrkB signaling cascade, thereby increasing the phosphorylation state of downstream members of the pathway. G9a overexpression, following prior cocaine exposure, prevents stress-induced increases in Ras signaling, and decreases the activity (phospho levels) of downstream signaling molecules, including Raf, MEK1/2, ERK1/2 and CREB. The Ras activator, Ras-GRF1, is unaffected by social stress and G9a overexpression (inset). * indicates a significant difference from control condition (non-defeated mice infected with GFP), p<0.05, and # indicates a significant difference from defeated mice infected with GFP, p<0.05, n = 8–9/group. (D) Repeated, but not acute, cocaine treatment increases H-Ras1 mRNA expression in NAc both at 1 and 24 hrs. # indicates a significant difference from an acute cocaine, p<0.05. (E) Repeated cocaine reduces H3K9me2 association and increases acH3K9 binding to the H-Ras1 promoter, n = 4/group (5 mice pooled/n). (F) Like repeated cocaine, chronic (10 days) social stress increases H-Ras1 mRNA expression in NAc of susceptible mice only, n = 7/group. # indicates a significant difference from susceptible mice, p<0.05. (E) Susceptibility to social stress reduces H3K9me2 binding to the H-Ras1 promoter, whereas this repressive mark is increased at the H-Ras1 promoter in unsusceptible mice (no changes were observed in acH3K9 binding after social defeat stress), n = 4/group (5 mice pooled/n). * indicates a significant difference from saline injected or control mice, p<0.05,. Data are presented as average ± SEM.

Figure 7. Local Knockdown of Endogenous CREB in NAc Promotes Antidepressant-like Behavioral Phenotypes

(A) Time-course of CREB knockdown in Creb^fl/fl mice and subsequent analyses of depression associated behaviors. (B) Immunohistochemical validation of CREB knockdown in Creb^fl/fl mice using AAV-Cre-GFP viruses. (C) Quantification of double labeled GFP and CREB positive cells in NAc following knockdown in Creb^fl/fl mice using AAV-Cre-GFP viruses. *** indicates a significant difference from AAV-GFP, p<0.001, n = 5/group. (D) Viral-mediated knockdown of CREB in NAc reduces social avoidance behavior following chronic (10 days) social stress. (E) Knockdown of CREB in NAc decreases the duration of immobility in the forced swim test. (F) Knockdown of CREB in NAc increases sucrose preference without affecting total levels of fluid (sucrose plus water) intake. * indicates a significant difference from control condition (defeated mice infected with GFP), p<0.05, n = 6–8/group. Data are presented as average ± SEM.

Figure 8. Enhanced Vulnerability to Stress Via Cocaine-Induced Priming of BDNF Signaling in NAc

Repeated cocaine increases vulnerability to the depressive-like effects of social defeat stress via priming BDNF signaling through Ras induction in NAc. Under control conditions, BDNF activation of TrkB signaling is limited. However, after repeated cocaine, BDNF-TrkB signaling is elevated in NAc, causing enhanced phosphorylation and activity of downstream signaling mediators including CREB. This cocaine-initiated adaptive response occurs not only through increased BDNF signaling in NAc, but also through increased Ras expression as a result of decreased G9a binding at the H-Ras1 gene promoter. Chronic stress is associated with similar adaptations in this brain region. Ras also appears to be a target for CREB, creating a positive feed-forward loop, promoting CREB activation and Ras expression as well as depressive-like behavior.