Circadian transcription factor NPAS2 and the NAD+ -dependent deacetylase SIRT1 interact in the mouse nucleus accumbens and regulate reward

Abstract

Substance use disorders are associated with disruptions to both circadian rhythms and cellular metabolic state. At the molecular level, the circadian molecular clock and cellular metabolic state may be interconnected through interactions with the nicotinamide adenine dinucleotide (NAD⁺ )-dependent deacetylase, sirtuin 1 (SIRT1). In the nucleus accumbens (NAc), a region important for reward, both SIRT1 and the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) are highly enriched, and both are regulated by the metabolic cofactor NAD⁺ . Substances of abuse, like cocaine, greatly disrupt cellular metabolism and promote oxidative stress; however, their effects on NAD⁺ in the brain remain unclear. Interestingly, cocaine also induces NAc expression of both NPAS2 and SIRT1, and both have independently been shown to regulate cocaine reward in mice. However, whether NPAS2 and SIRT1 interact in the NAc and/or whether together they regulate reward is unknown. Here, we demonstrate diurnal expression of Npas2, Sirt1 and NAD⁺ in the NAc, which is altered by cocaine-induced upregulation. Additionally, co-immunoprecipitation reveals NPAS2 and SIRT1 interact in the NAc, and cross-analysis of NPAS2 and SIRT1 chromatin immunoprecipitation sequencing reveals several reward-relevant and metabolic-related pathways enriched among shared gene targets. Notably, NAc-specific Npas2 knock-down or a functional Npas2 mutation in mice attenuates SIRT1-mediated increases in cocaine preference. Together, our data reveal an interaction between NPAS2 and SIRT1 in the NAc, which may serve to integrate cocaine's effects on circadian and metabolic factors, leading to regulation of drug reward.

Keywords: NPAS2; SIRT1; circadian; cocaine; nucleus accumbens; reward.

Figure 1.. The molecular clock and regulators of cellular metabolic state show diurnal variation in the NAc.

(A) Core molecular clock gene expression of Npas2, Bmal1, Rev-erbα, and RORα show diurnal variation in the NAc, as well as the (B) metabolic genes Sirt1 and Nampt – measured using RT-qPCR. Relative expression normalized to the reference gene 18s. (C) HPLC analysis of NAD+ levels also shows diurnal variation in the NAc. (D) Rose plot of the observed peaks of expression illustrates peaks are primarily at ZT22 during the dark phase. Asterisks and bar indicate a significant main effect of time measured in a one-way ANOVA (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). NAc tissue was collected from saline-treated animals (14 days, i.p. at 10 ml/kg, between ZT4–8). Data represented as Mean ± SEM (n=5–6 / ZT). White stripe indicates lights on, black stripe indicates lights off. Grey shading on the rose plot indicates lights off, and the inset axis shows the number of genes. ZT 0 = 7 am.

Figure 2.. Cocaine exposure alters diurnal variation of the circadian molecular clock and cellular metabolic state in the NAc.

(A) Schematic outlining the 14-day injection paradigm. Mice were given either saline (S; 10 ml/kg, i.p.), chronic cocaine (C; 20 mg/kg, i.p.), or acute cocaine (A; 13 days x saline, 1-day x cocaine). NAc tissue was collected beginning 24 hours after the last injection for molecular assays. (B) Following cocaine exposure, diurnal variation of core molecular clock gene expression of Npas2, Bmal1, and ROR, as well as (C) the metabolic genes Sirt1 and Nampt, were significantly altered in the NAc – as indicated by significant interactions and main effects of time and/or treatment by 2-way ANOVA. (ROR blue #, p=0.0751; ROR red #, p=0.0732; Sirt1 @, p=0.0511). (D) HPLC analysis revealed cocaine exposure also significantly disrupts diurnal variation of NAD+ levels in the NAc, with significant main effects of time and treatment. (NAD+ @, p=0.0572). (E) Rose plot illustrates observed peaks in expression are primarily at ZT22 during the dark phase, but cocaine exposure advances more peaks of expression to the light phase. Saline data from Figure 1 are depicted as dotted lines for comparison purposes. Injections were administered between ZT4–8. White stripe indicates lights on, black stripe indicates lights off. Grey shading on rose plot indicates lights off, and the inset axis shows the number of genes. Asterisks, pounds, and at-signs above the plotted lines indicate a statistical difference relative to saline, revealed in a Bonferroni post-hoc analysis. Color of asterisk, pound, or at-sign corresponds to the treatment group compared. Data represented as Mean ± SEM (n=5–6 / ZT). (* p<0.05, ** p<0.01, *** p<0.001). ZT 0 = 7 am.

Figure 3.. NPAS2 and SIRT1 are important for cocaine preference.

(A) Schematic showing the cocaine conditioned place preference (CPP) paradigm. Mice were conditioned to saline (10 ml/kg, i.p.) or cocaine (5 mg/kg, i.p.) on conditioning days. (B) Npas2 mutant mice show decreased cocaine preference relative to wild-type (WT) littermates. (C) C57BL/6J mice were administered vehicle or resveratrol (20 mg/kg; i.p.) 30 minutes before conditioning. Mice administered resveratrol showed a significant increase in cocaine preference relative to vehicle controls. Data represented as Mean ± SEM (n= 8–11). CPP score is calculated by subtracting the initial pre-test time in the cocaine paired chamber from the same chamber’s test-day time (i.e., Test cocaine-paired time minus Pre-Test cocaine-paired time). Asterisk indicates significance relative to controls (* p<0.05). At-sign indicates a trend towards significance (@, p=0.0504).

Figure 4.. NPAS2 and SIRT1 interact in the NAc and share reward-relevant gene targets.

(A) Co-immunoprecipitation (Co-IP) of NPAS2 from whole NAc tissue and immunoblotting for SIRT1 revealed a band for SIRT1 at ~120kDa across time of day (left), while Co-IP of SIRT1 from whole NAc tissue and immunoblotting for NPAS2 revealed a band for NPAS2 at ~ 92kDa across time of day (right). Data represented as Mean ± SEM (n= 4–8). GAPDH was used as a negative control and was not detected (~37 kDa). (B) Cross analysis of chromatin immunoprecipitation sequencing (ChIP-seq) for NPAS2 and SIRT1 at ZT2, the time shared between the two datasets, revealed 2,347 and 3,189 genes bound (e.g., promoter or gene body) by NPAS2 and SIRT1, respectively, with 390 genes bound in common. (C) The top pathways enriched among these 390 shared genes include metabolic and reward-relevant biological functions. (D) Synaptic transmission, catecholamine transport, and other dopamine-relevant mechanisms are among the top biological processes enriched among NPAS2 and SIRT1’s shared gene targets. (E) Among the enriched shared biological processes, the catecholamine nodes and synaptic transmission nodes show a high degree of interconnectivity. Red line in bar graphs indicates significance threshold of p<0.05 or -log10(p-value) > 1.3.

Figure 5.. Both NPAS2 and SIRT1 regulate cocaine preference through their expression in the NAc.

(A) C57BL/6J mice were injected bilaterally into the NAc with either AAV2-Npas2-shRNA-GFP or scramble control, while Npas2 mutant or wild-type mice were injected with either AAV2-CMV-eGFP-2A-Sirt1 or eGFP control. The inset box depicts a representative image of virus placement and spread, as measured by GFP immunofluorescence. AC indicates anterior commissure, while arrow indicates virus injection path. (B) Following two weeks of recovery, C57BL/6J mice injected with either Npas2-shRNA or scramble were conditioned to saline or cocaine (5 mg/kg) and injected with vehicle or resveratrol (20 mg/kg, i.p.) 30 minutes before conditioning. Viral-mediated knock-down of Npas2 in the NAc attenuates resveratrol mediated increases in cocaine CPP – with both main effects of virus and treatment measured by a 2-way ANOVA. Expanding on the simple main effects, resveratrol significantly increased cocaine CPP in the scramble group (*, p<0.05), but not in the Npas2-shRNA group (n.s., p=0.5094). (C) Npas2 mutant mice overexpressing Sirt1 in the NAc conditioned to saline and cocaine (5 mg/kg) show an attenuated preference for cocaine in the CPP task relative to controls – with both a main effect of genotype and a trending effect of virus measured by a 2-way ANOVA (@, p=0.0512). Expanding on the simple main effects, Sirt1 overexpression increased cocaine CPP in the WT group (#, p=0.0943), but not in the Npas2 MUT group (n.s., p=0.2691). Horizontal or vertical bars indicate main effect comparisons. Symbols above plotted bars indicate within group statistical comparisons. Throughout, asterisks indicate significance (* p<0.05, ** p<0.01). Data represented as Mean ± SEM (n= 10–15).