Altered m6A methylation across eight reward- and motor-related brain regions in alcohol use disorder

N6-methyladenosine (m6A) is the most abundant internal mRNA modification, dynamically regulating post-transcriptional gene expression. Although m6A dysregulation has been implicated in addiction neurobiology, comprehensive characterization of m6A alterations in alcohol use disorder (AUD) remains unexplored. We profiled m6A methylomes across eight reward- and motor-related brain regions in postmortem tissue from 12 AUD subjects and 12 matched controls (192 samples total) using MazF-mediated enzymatic cleavage and microarray-based m6A site mapping. Region-specific differential analysis identified 1,403 differentially methylated m6A sites (P < 0.05, |fold change| ≥ 1.5): amygdala (n = 198), caudate nucleus (n = 91), cerebellum (n = 201), hippocampus (n = 89), nucleus accumbens (n = 276), prefrontal cortex (n = 362), putamen (n = 142), and ventral tegmental area (n = 241). Host transcripts were enriched for functional categories spanning intercellular communication, immune and inflammatory responses, RNA and protein synthesis, energy metabolism, and neurodegeneration. Gene set enrichment analysis revealed a coordinated inverse relationship between m6A methylation status and transcript abundance: hypermethylated transcripts were enriched among downregulated genes and hypomethylated transcripts among upregulated genes, consistent with m6A-mediated transcript destabilization. Cross-validation with Allen Human Brain Atlas region-specific expression profiles demonstrated that differential methylation patterns were primarily AUD-driven rather than secondary to regional transcriptional heterogeneity. These findings position m6A epitranscriptomics as a novel molecular signature of AUD and suggest that m6A regulatory machinery may serve as a therapeutic target. Future studies with larger validation cohorts and mechanistic interrogation are essential to establish causal relationships and facilitate translation to drug development.