Arginine Methyltransferase 1 in the Nucleus Accumbens Regulates Behavioral Effects of Cocaine

Abstract

Recent evidence suggests that histone modifications play a role in the behavioral effects of cocaine in rodent models. Histone arginine is known to be methylated by protein arginine N-methyltransferases (PRMTs). Evidence shows that PRMT1 contributes to >90% of cellular PRMT activity, which regulates histone H4 arginine 3 asymmetric dimethylation (H4R3me2a). Though histone arginine methylation represents a chemical modification that is relatively stable compared with other histone alterations, it is less well studied in the setting of addiction. Here, we demonstrate that repeated noncontingent cocaine injections increase PRMT1 activity in the nucleus accumbens (NAc) of C57BL/6 mice. We, subsequently, identify a selective inhibitor of PRMT1, SKLB-639, and show that systemic injections of the drug decrease cocaine-induced conditioned place preference to levels observed with genetic knockdown of PRMT1. NAc-specific downregulation of PRMT1 leads to hypomethylation of H4R3me2a, and hypoacetylation of histone H3 lysine 9 and 14. We also found that H4R3me2a is upregulated in NAc after repeated cocaine administration, and that H4R3me2a upregulation in turn controls the expression of Cdk5 and CaMKII. Additionally, the suppression of PRMT1 in NAc with lentiviral-short hairpin PMRT1 decreases levels of CaMKII and Cdk5 in the cocaine-treated group, demonstrating that PRMT1 affects the ability of cocaine to induce CaMKII and Cdk5 in NAc. Notably, increased H4R3me2a by repeated cocaine injections is relatively long-lived, as increased expression was observed for up to 7 d after the last cocaine injection. These results show the role of PRMT1 in the behavioral effects of cocaine.

Significance statement: This work demonstrated that repeated cocaine injections led to an increase of protein arginine N-methyltransferase (PRMT1) in nucleus accumbens (NAc). We then identified a selective inhibitor of PRMT1 (SKLB-639), which inhibited cocaine-induced conditioned place preference (CPP). Additionally, genetic downregulation of PRMT1 in NAc also attenuated cocaine-caused CPP and locomotion activity, which was associated with decreased expression of histone H4 arginine 3 asymmetric demethylation (H4R3me2a) and hypoacetylation of histone H3 lysine 9 and 14 (acH3K9/K14). This study also showed that H4R3me2a controlled transcriptions of Cdk5 and CaMKII, and that PRMT1 negatively affected the ability of cocaine to induce CaMKII and Cdk5 in NAc. Notably, increased H4R3me2a by repeated cocaine injection was relatively long-lived as increased expression was observed up to 7 d after withdrawal from cocaine. Together, this study suggests that PRMT1 inhibition may serve as a potential therapeutic strategy for cocaine addiction.

Keywords: PRMT inhibitor; behavioral plasticity; cocaine; histone arginine methylation; protein arginine N-methyltransferase 1.

Figure 1.

Regulation of PRMTs by cocaine administration (admin) in NAc. A, Basal expression levels of PRMT mRNA within the NAc expressed as the fold difference from PRMT1 from five mice. B, C57BL/6 mice were injected repeatedly (7 d, 20 mg/kg, i.p., once daily) with cocaine. NAc samples were analyzed 24 h after the last injection. Student's t tests, *p < 0.05, n = 5 saline, n = 5 repeated cocaine. C, Repeated noncontingent cocaine injections upregulated PRMT1 protein expression in NAc (Student's t tests, **p < 0.01, n = 4 saline, n = 4 repeated cocaine). D, Mice were injected over the short term with cocaine (6 d, saline; 1 d, 20 mg/kg, i.p.). NAc samples were analyzed 1 h after the last injection (Student's t tests, *p < 0.05, n = 4 saline and n = 4 acute cocaine). E, The number of the “active” and “inactive” pokes for each daily session of cocaine self-administration, whereby active pokes resulted in cocaine infusion (0.75 g/kg/infusion) and the inactive pokes in both circumstances were without consequence. n = 9 saline, n = 9 cocaine. F, Cocaine self-administration caused elevated expression of PRMT1 mRNA in NAc. Student's t tests, *p < 0.05, n = 5 saline, n = 5 cocaine self-administration. G, Cocaine self-administration increased PRMT1 protein level. Actin was used as the loading control. Student's t tests, *p < 0.05, n = 4 saline, n = 4 cocaine self-administration. Data are presented as the mean ± SEM.

Figure 2.

PRMT1 is increased following cocaine-induced conditioned place preference. A, Timeline of cocaine CPP experiment. B, Cocaine CPP. After conditioning, mice developed a significant preference for the cocaine-paired side. Student's t tests, ***p < 0.001, n = 12 saline, n = 12 cocaine. C, Cocaine CPP caused significant elevation of PRMT1 expression in mRNA level. Student's t tests, ***p < 0.001, n = 5 saline, n = 5 cocaine. D, Representative immunoblot demonstrating elevated PRMT1 expression in NAc following cocaine CPP. Actin was used as the loading control. Student's t tests, *p < 0.05, n = 3 saline, n = 3 cocaine. E, PRMT1 protein expression in NAc measured by immunohistochemistry, 24 h following cocaine CPP (40× objective). F, Ratio of merged positive cells and DAPI-positive cells from E were counted and averaged, respectively. There were a total of 2348 DAPI-positive cells after saline CPP, which were counted from 8 photos in three mice, whereas 2696 cells were counted from 11 photos in four mice challenged by cocaine CPP. Student's t tests, *p < 0.05. G, PRMT1 activity was detected in nucleus and cytoplasm in NAc after cocaine CPP. The PRMT1 activity in nucleus was obviously increased. **p < 0.01. n.s., Not significant. n = 6 saline, n = 6 cocaine. Data are presented as the mean ± SEM. H, Levels of PRMT1 in cytoplasm and nuclear fractions in NAc from cocaine CPP mice were detected by Western blot. PRMT1 protein in nuclear extract was statistically increased. *p < 0.01. n.s., not significant, n = 4 saline, n = 4 cocaine.

Figure 3.

Intra-NAc injections of PRMT inhibitors regulate cocaine-induced behavioral plasticity. A, Intra-NAc MTA (500 μm, 1 μl/injection) attenuated cocaine CPP. *p < 0.05, **p < 0.01, ***p < 0.001. n = 9 Vehicle+Saline, n = 8 MTA+Saline, n = 8 Vehicle+Cocaine, n = 11 MTA+Cocaine. B, Intra-NAc AMI-1 (1 mm, 1 μl/injection) partially blocked cocaine CPP. *p < 0.05, ***p < 0.001. n = 13 Vehicle+Saline, n = 9 AMI-1+Saline, n = 12 Vehicle+Cocaine, n = 12 AMI-1+Cocaine. C, Intra-NAc AMI-1 (1 mm, 1 μl/injection) decreased cocaine CPP-induced H4R3me2a expression in NAc. *p < 0.05. n = 4 Vehicle+Saline, n = 4 AMI-1+Saline, n = 4 Vehicle+Cocaine, n = 4 AMI-1+Cocaine. D, Intra-NAc AMI-1 (1 mm, 1 μl/injection) did not influence basal PRMT1 expression in NAc. *p < 0.05. n.s., Not significant. n = 4 Vehicle+Saline, n = 4 AMI-1+Saline, n = 4 Vehicle+Cocaine, n = 4 AMI-1+Cocaine. Data are presented as the mean ± SEM.

Figure 4.

Discovery of a potent and selective inhibitor of PRMT1. A, Synthesis of the SKLB-639. B, The overall structure of homology hPRMT1 with SKLB-639 (light pink). Regions are colored as follows: SAM-binding site (yellow: residues His53, Arg62, Glu86, Glu 108, Glu 137); and active site (magenta: residues Glu152 and Glu161). C, Predicted binding modes of SKLB-639 in hPRMT1 homology model from docking and molecular dynamics simulation (Discovery Studio version 3.1). D, Substrate binding sites of SKLB-639 in PRMT1 were Glu161, Met154, Tyr47, His301, Arg361, and Tyr156. E, Concentration–response curve of SKLB-639 (blue) and AMI-1 (red) in the PRMT1 in vitro assay. F–J, Selectivity of SKLB-639 on five other protein arginine methyltransferases.

Figure 5.

SKLB-639 attenuates cocaine reward via specifically inhibiting modification of H4R3me2a in vivo. A, Timeline of the cocaine CPP experiment. B, Intra-NAc SKLB-639 (1 mm, 1 μl/injection) significantly attenuated cocaine CPP. *p < 0.05, ***p < 0.001. n.s., Not significant. n = 13 Vehicle+Saline, n = 10 SKLB-639+Saline, n = 11 Vehicle+Cocaine, n = 13 SKLB-639+Cocaine. C, SKLB-639 decreased expression of H4R3me2a in NAc. *p < 0.05, **p < 0.01. n.s., Not significant. n = 4 Vehicle+Saline, n = 4 Vehicle+Cocaine, n = 4 SKLB-639+Cocaine, n = 4 SKLB-639+Saline. D–F, SKLB-639 did not influence levels of H3R2me2a, H3R17me2a, and dimethyl (sym) in NAc in vivo, respectively. n = 4 Vehicle+Saline, n = 4 SKLB-639+Saline, n = 4 Vehicle+Cocaine, n = 4 SKLB-639+Cocaine. Data are presented as the mean ± SEM.

Figure 6.

NAc-specific downregulation of PRMT1 regulates cocaine-induced behavioral plasticity. A, Verification of anatomical placement and viral infection in NAc; diagram of the coronal brain slice was taken from the mouse brain atlas at 1.54 mm. B, One-way ANOVA revealed that PRMT1 downregulation significantly attenuated the cocaine reward of PRMT1. *p < 0.05, **p < 0.01, ***p < 0.001. n = 13 LV-GFP Saline, n = 13 LV-shPRMT1 Saline, n = 11 LV-GFP Cocaine, n = 12 LV-shPRMT1 Cocaine. C, LV-shPRMT1 significantly reduced the locomotor activity of mice treated with repeated cocaine injections. n = 11 LV-GFP Saline, n = 12 LV-shPRMT1 Saline, n = 12 LV-GFP Cocaine, n = 13 LV-shPRMT1 Cocaine. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with LV-shPRMT1 cocaine group. D, PRMT1 mRNA level in NAc of mice infected with LV-shPRMT1 after cocaine CPP. *p < 0.05, **p < 0.01. n = 6/condition. E, PRMT1 protein level in NAc of mice infected with LV-shPRMT1 after cocaine CPP. *p < 0.05, ***p < 0.001. n = 7 LV-GFP saline and LV-shPRMT1 saline group, n = 3 LV-GFP cocaine and LV-shPRMT1 cocaine group. F, Representative immunoblots demonstrating the downregulated expression of PRMT1 in NAc of mice with cocaine CPP test. G, Protein levels of H4R3me2a in NAc of mice injected with LV-GFP or LV-shPRMT1 after cocaine CPP. *p < 0.05, **p < 0.01. n.s., Not significant. n = 4/condition. H, Protein levels of acH3K9/K14 in NAc of mice infected with LV-GFP or LV-shPRMT1 after cocaine CPP. *p < 0.05. n.s., Not significant. n = 4/condition. All data are presented as the mean ± SEM.

Figure 7.

Histone modifications following repeated cocaine injections. A, CaMKII showed concerted changes in H4R3me2a and acH3K9/K14 enrichment following repeated cocaine injections. PR, promoter region; NPR, nonpromoter region. *p < 0.05. n = 3/condition, n = 4 animals pooled/condition. B, CaMKII was upregulated at the transcriptional level after repeated cocaine injections. *p < 0.05. n = 5/condition. C, Cdk5 promoter obviously increased the binding of H4R3me2 and acH3K9/K14. *p < 0.05, n = 3/condition, n = 4 animals pooled/condition. D, Cdk5 was increased in mRNA expression following repeated cocaine challenge. *p < 0.05. n = 5/condition. E, With suppression of PRMT1 in NAc, the expression of CaMKII and Cdk5 in the LV-shPRMT1 cocaine group was decreased compared with that of the LV-GFP cocaine group following CPP. *p < 0.05. n = 5/condition. F–H, Expressions of H4R3me2a, H3K9me2, and H3K36me3 in NAc after repeated noncontingent cocaine injections withdrawal at day 1 (D1), day 7 (D7), day 14 (D14), day 28 (D28), and day 42 (D42), respectively. Actin was used as the loading control. *p < 0.05 vs saline, n = 3/condition). Each bar represents the mean ± SEM.