Altered m ⁶ A methylation across eight reward- and motor-related brain regions in alcohol use disorder

N ⁶ -methyladenosine (m ⁶ A) is the most abundant internal mRNA modification, dynamically regulating post-transcriptional gene expression. Although m ⁶ A dysregulation has been implicated in addiction neurobiology, comprehensive characterization of m ⁶ A alterations in alcohol use disorder (AUD) remains unexplored. We profiled m ⁶ A 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 m ⁶ A site mapping. Region-specific differential analysis identified 1,403 differentially methylated m ⁶ A 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 m ⁶ A methylation status and transcript abundance: hypermethylated transcripts were enriched among downregulated genes and hypomethylated transcripts among upregulated genes, consistent with m ⁶ A-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 m ⁶ A epitranscriptomics as a novel molecular signature of AUD and suggest that m ⁶ A 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.