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We analyzed single-nucleus RNA-seq from the prefrontal cortex of 73 individuals (614,932 nuclei, 24 cell types), comparing AUD versus controls, smoking versus controls, and combined AUD+smoking versus controls. In excitatory neurons, neuroinflammatory, complement, GPCR, kinase, proteostatic, and extracellular-matrix pathways confined to one or two subtypes in either condition alone expanded across nearly all subtypes when AUD and smoking co-occurred. Inhibitory neurons showed a parallel but distinct expansion, additionally recruiting translational regulation, RNA splicing, and deubiquitination pathways. Smoking alone produced widespread repression of bioenergetic, proteostatic, and synaptic pathways in excitatory neurons, whereas AUD produced mixed pathway activation and repression in excitatory neurons and broad inflammatory activation in inhibitory neurons. In the combined condition, microglia exhibited paradoxical repression of phagocytosis, TYROBP signaling, and translational machinery, consistent with an exhaustion-like state, accompanied by an apparently compensatory shift in inflammatory signaling toward astrocytes, oligodendrocyte precursor cells, and oligodendrocytes, which showed coordinated inflammatory activation. Vascular cells showed an endothelial\u2013VLMC dichotomy, with endothelial repression of RNA processing and proteostasis contrasting with perivascular activation. Our analysis identifies bioenergetic and proteostatic stress as the predominant signature of smoking, whereas immune and metabolic activation characterize AUD. In AUD+Smoking, these alterations extend across more neuronal subtypes and include neuroinflammation, complement activation, altered signaling, proteostatic stress, and post-transcriptional remodeling associated with greater cortical dysfunction in co-occurring AUD and smoking.<\/jats:p>","DOI":"10.64898\/2026.09.24.753990","type":"posted-content","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T21:25:17Z","timestamp":1790717117000},"source":"Crossref","is-referenced-by-count":0,"title":["Alcohol Use Disorder and Smoking-Associated Molecular Alterations in Human Prefrontal Cortex in Single Nucleus (sn) RNA-Seq"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7334-9671","authenticated-orcid":false,"given":"Arpita","family":"Joshi","sequence":"first","affiliation":[{"name":"The Scripps Research Institute, San Diego, CA, 92109"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pietro Paolo","family":"Sanna","sequence":"additional","affiliation":[{"name":"The Scripps Research Institute, San Diego, CA, 92109"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026100120051555000_2026.09.24.753990v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-pharmtox-031323-115847"},{"key":"2026100120051555000_2026.09.24.753990v1.2","unstructured":"Alcohol Use Disorder (AUD) in the United States: Age Groups and Demographic Characteristics. https:\/\/www.niaaa.nih.gov\/alcohols-effects-health\/alcohol-topics\/alcohol-facts-and-statistics\/alcohol-use-disorder-aud-united-states-age-groups-and-demographic-characteristics. 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