Elevated intron retention implicates neuroinflammation in brains of individuals with alcohol use disorder

Abstract Intron retention, a form of alternative RNA splicing, can occur as part of normal gene regulation or result from disruption of the splicing machinery. Retained introns can potentially form double-stranded RNA, activating innate immune sensors and inflammation. This mechanism has been implicated in cancer, but has not been studied in neuropsychiatric diseases like alcohol use disorder. We systematically analyzed transcriptome-wide intron retention events in post-mortem brain tissue from 142 individuals (66 with alcohol use disorder and 76 controls), encompassing 320 region-specific samples from the superior frontal cortex, nucleus accumbens, central nucleus, and basolateral amygdala. Analyses were adjusted for demographic, technical, and biological covariates. Validation was performed in alcohol-preferring (P) rats using long-read sequencing. In complementary experiments, immunofluorescent staining was used to detect double-stranded RNA in rat brain tissue, while single-cell RNA sequencing was performed to test activation of double-stranded RNA-sensing pathways in human brains. Brains from individuals with alcohol use disorder showed significantly higher total intron retention compared with controls, independent of age, with females showing greater increases than males. A total of 368 introns were positively associated with alcohol use disorder, and these introns were significantly longer and had weaker splice acceptor sites compared with non-associated introns. Genes harboring these intron retention events were enriched in Purkinje neurons, visual cortex neurons, and oligodendrocytes. Computational predictions indicated these long introns could form duplex RNA structures. Increased double-stranded RNA was confirmed experimentally in multiple brain regions of alcohol-consuming rats, where it co-localized primarily with neuronal nuclei and dendrites. In individuals with alcohol use disorder, we found that multiple pathways including double-stranded RNA responses, neuroinflammation, interferon and NF-κB signaling, adaptive immunity, and apoptosis were activated. In addition, NeuN-positive neuronal counts significantly decreased in both the prefrontal and visual cortices. Furthermore, single-cell analysis demonstrated upregulation of TICAM1, the target of double-stranded RNA sensor TLR3, in oligodendrocytes, as well as widespread activation of downstream inflammatory pathways across glial and neuronal cell types. These findings provide the first evidence that chronic alcohol consumption promotes an overall increase of intron retention in the brain and is associated with the presence of double-stranded RNA. Furthermore, the double-stranded RNA may contribute to neuronal loss and brain pathology by activating a neuroinflammatory response.