Decreased voluntary alcohol intake and ventral striatal epigenetic and transcriptional remodeling in male Acss2 KO mice

Abstract

Metabolic-epigenetic interactions are emerging as key pathways in regulating alcohol-related transcriptional changes in the brain. Recently, we have shown that this is mediated by the metabolic enzyme Acetyl-CoA synthetase 2 (Acss2), which is nuclear and chromatin-bound in neurons. Mice lacking ACSS2 fail to deposit alcohol-derived acetate onto histones in the brain and show no conditioned place preference for ethanol reward. Here, we further explored the role of this pathway during voluntary alcohol intake. We found that Acss2 KO mice consume significantly less alcohol in a model of binge drinking, an effect primarily driven by males. Genome-wide transcriptional profiling of 7 key brain regions implicated in alcohol and drug use revealed that, following drinking, Acss2 KO mice exhibit blunted gene expression in the ventral striatum. Similarly to the behavioral differences, transcriptional dysregulation was more pronounced in male mice. Further, we found that the gene expression changes were associated with depletion of ventral striatal histone acetylation (H3K27ac) in Acss2 KO mice compared to WT. Taken together, our data suggest that ACSS2 plays an important role in orchestrating ventral striatal epigenetic and transcriptional changes during voluntary alcohol drinking, especially in males. Consequently, targeting this pathway could be a promising new therapeutic avenue.

Fig. 1.. Decreased voluntary alcohol intake in Acss2 KO mice.

(A) Schematic depicting the drinking-in-the-dark (DID) behavioral paradigm. Following habituation, mice had access to 15% (v/v) ethanol and water for 2–4 h during 4 days (2BC phase), then 15% (v/v) ethanol for 2–4 h during 4 days (1B phase). (B) Ethanol intake (g/kg) during the 2BC phase in female and male WT and Acss2 KO mice. (C) Cumulative ethanol intake during the 2BC phase of the DID paradigm. Two-way ANOVA revealed no significant effect of genotype (p = 0.0537). (D) Ethanol preference (%) in female and male WT and Acss2 KO mice. (E) Ethanol intake (g/kg) during the 1B phase in female and male WT and ACSS2 KO mice. During the 1B phase, three-way ANOVA revealed significant effects of both sex (F_1,34 = 4.159, p = 0.0493) and genotype (F_1,34 = 11.57, p = 0.0017). Post-hoc comparisons showed a significant decrease of alcohol intake on the final day of DID–1B in male mice (p-adj. = 0.0193), whereas no statistically significant differences were observed in female mice. (F) Cumulative ethanol intake during the 1B phase of the DID paradigm. Two-way ANOVA revealed significant effect of genotype (p = 0.0025), with post-hoc comparisons showing significantly decreased ethanol intake in male KOs (p-adj. = 0.0216). Symbols/columns and error bars represent the average ± SEM. Sample sizes are as follows: female WT (n = 8), female KO (n = 10), male WT (n = 10), male KO (n = 10). 2BC, 2 bottle choice; 1B, 1 bottle; KO, ACSS2 KO.

Fig. 2.. Transcriptional profiling across key brain regions following voluntary alcohol intake in WT and Acss2 KO mice.

(A) Heatmap showing differentially expressed genes (DEGs, n = 135) across seven brain regions investigated in Acss2 KO vs. WT male mice following voluntary alcohol consumption. Black bars (left) denote significant DEGs corresponding to each region (FDR<0.1). Magenta (2) indicates increased expression in Acss2 KO vs WT males, black (0) indicates no differential expression between Acss2 KO vs WT males, and green (−2) indicates decreased expression in Acss2 KO vs WT males. (B) Volcano plot of ventral striatal DEGs (n = 111) identified between Acss2 KO vs WT males. Each dot represents a gene. Red dots denote significantly upregulated genes, and blue dots denote significantly downregulated genes. (C) Gene Ontology (GO) analysis of ventral striatal DEGs between Acss2 KO vs WT males showing enrichment of genes linked to synaptic functions. (D) Gene expression tracks of the Chrm2 locus (chr6:36,353,001–36,563,497) across brain regions in male mice, showing specific significant decrease of Chrm2 expression in the ventral striatum. DEGs, differentially expressed genes; dHPC, dorsal hippocampus; dStriatum, dorsal striatum; PFC, prefrontal cortex; vHPC, ventral hippocampus; vStriatum, ventral striatum; GO, Gene Ontology. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3.. H3K27ac profiling of the ventral striatum following voluntary alcohol intake in male WT and Acss2 KO mice.

(A) Heatmaps showing 2 kb windows of significantly depleted (n = 8061) and significantly enriched (n = 4298) H3K27ac peaks in the ventral striatum of Acss2 KO vs WT male mice following voluntary alcohol consumption. (B) Violin plots showing positive correlation between H3K27ac enrichment changes (ChIP-seq, x-axis) and gene expression changes (RNA-seq, y-axis) in the ventral striatum of Acss2 KO vs WT male mice following voluntary alcohol consumption. (C) Tracks of the Sparc locus (chr11:55,365,250–55,455,549) showing decreased H3K27ac enrichment (top two rows) and decreased gene expression (bottom two rows) in the Acss2 KO male ventral striatum compared to that of WT. (D) De novo motif analysis showing the top 5 significantly enriched transcription factor binding motifs in H3K27ac depleted peaks. (E) Gene expression tracks of the Zic1 locus (chr9:91,357,1221–91,368,173).