Essential role of poly(ADP-ribosyl)ation in cocaine action

Abstract

Many of the long-term effects of cocaine on the brain's reward circuitry have been shown to be mediated by alterations in gene expression. Several chromatin modifications, including histone acetylation and methylation, have been implicated in this regulation, but the effect of other histone modifications remains poorly understood. Poly(ADP-ribose) polymerase-1 (PARP-1), a ubiquitous and abundant nuclear protein, catalyzes the synthesis of a negatively charged polymer called poly(ADP-ribose) or PAR on histones and other substrate proteins and forms transcriptional regulatory complexes with several other chromatin proteins. Here, we identify an essential role for PARP-1 in cocaine-induced molecular, neural, and behavioral plasticity. Repeated cocaine administration, including self-administration, increased global levels of PARP-1 and its mark PAR in mouse nucleus accumbens (NAc), a key brain reward region. Using PARP-1 inhibitors and viral-mediated gene transfer, we established that PARP-1 induction in NAc mediates enhanced behavioral responses to cocaine, including increased self-administration of the drug. Using chromatin immunoprecipitation sequencing, we demonstrated a global, genome-wide enrichment of PARP-1 in NAc of cocaine-exposed mice and identified several PARP-1 target genes that could contribute to the lasting effects of cocaine. Specifically, we identified sidekick-1--important for synaptic connections during development--as a critical PARP-1 target gene involved in cocaine's behavioral effects as well as in its ability to induce dendritic spines on NAc neurons. These findings establish the involvement of PARP-1 and PARylation in the long-term actions of cocaine.

Keywords: drug addiction; histone PARylation; medium spiny neurons.

Fig. 1.

PARP-1 expression and activity are up-regulated in NAc by cocaine. (A) PARP-1 mRNA, protein, and activity, as well as PAR levels, were analyzed in mouse NAc 30 min after repeated (7 d) cocaine (20 mg/kg i.p.) administration. (B) PARP-1 mRNA and protein levels were measured in rat NAc 24 h after 10 d of cocaine self-administration or after 7 d of withdrawal with animals given a challenge dose of cocaine 24 h before analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Data are presented as mean ± SEM (n = 7–11). *P < 0.05, **P < 0.01.

Fig. 2.

Chronic cocaine induces changes in PARP-1 complexes, consistent with a permissive transcriptional environment. (A) Immunoprecipitation (IP) of PARP-1 from NAc extracts of control and chronic cocaine-treated mice followed by Western blots for Brg1, HDAC2, HDAC3, or NF-κB p65 subunit. All data were normalized to PARP-1 and expressed as mean ± SEM (n = 5–7 independent samples/group with each sample representing tissue of three animals). (B) Levels of PAR present on histones H1, H2A, H2B, H3, and H4 after chronic cocaine. Each histone subunit was IPed and Western blotted for PAR. Data are normalized to total amount of each histone subunit pulled down (images shown in Fig. S1) and expressed as mean ± SEM (n = 4–7 independent samples/group as above). *P < 0.05.

Fig. 3.

PARP-1 controls behavioral responses to cocaine. (A) Locomotor response to cocaine (5 mg/kg i.p.) in mice injected intra-NAc with HSV-GFP or HSV-PARP-1. (B) Conditioned place preference for cocaine in mice injected intra-NAc with HSV-GFP or HSV-PARP-1. (C) Number of self-administered infusions of cocaine by rats, trained to self-administer cocaine and then injected intra-NAc with HSV-GFP or HSV-PARP-1, and tested 2–5 d later for self-administering elevating intravenous cocaine doses. (D) Locomotor responses to cocaine in mice with intra-NAc infusion of TIQ-A or vehicle. (E) Conditioned place preference for cocaine in mice injected intra-NAc with HSV-GFP or HSV-PARG. Data are presented as mean ± SEM (n = 7–10). *P < 0.05.

Fig. 4.

ChIP-seq identification of PARP-1 targets in NAc after chronic cocaine administration. (A) Next-generation sequencing plot (https://code.google.com/p/ngsplot/) of PARP-1 binding to genes genome-wide in NAc under saline and chronic cocaine conditions with a higher-magnification view of the TSS (B). (C) Cocaine-induced differential PARP-1 peak distribution. Arrows show the number of genes at which PARP-1 binding is significantly increased or decreased after cocaine. (D) Correlation of PARP-1 peaks with gene expression, the latter based on RNA-seq data. (E) Validation of mRNA expression of PARP-1 target genes after chronic cocaine (n = 8 per group). (F) Validation of ChIP-seq findings by qChIP for PARP-1 binding at key target genes after chronic cocaine (n = 5–7 per group). ^#P < 0.08, *P < 0.05, **P < 0.01.

Fig. 5.

SDK1 is a PARP-1 target that regulates the behavioral response to cocaine through altered spine dynamics. (A) Locomotor response to cocaine in mice injected intra-NAc with HSV-GFP or HSV-SDK1 at indicated cocaine dose (n = 7–10). (B) Conditioned place preference for cocaine in mice injected intra-NAc with HSV-GFP or HSV-SDK1 at indicated cocaine dose (n = 9–11). (C) Dendritic spine analysis of animals injected intra-NAc with HSV-GFP or HSV-SDK1 and treated chronically with saline or cocaine (20 mg/kg i.p.; n = 4–5 mice per group, n = 3–4 neurons per mouse). ^#P < 0.08, *P < 0.05, **P < 0.01.