PROJECT SUMMARY Astrocytes respond to CNS damage and disease with changes in gene expression and morphology and with immune activation to become reactive astrocytes. Alcohol Use Disorder is characterized in part by neuroimmune responses that increase with the progression of the disorder. Reactive astrocytes upregulate the expression of Tissue-type Plasminogen Activator (tPA) encoded by the gene Plat, which is involved in brain plasticity, the remodeling of the brain extracellular matrix, and neuroimmune responses including microglial activation and neuroinflammation. tPA is upregulated by ethanol in several brain areas and in astrocytes in several models of ethanol exposure. The main scientific questions driving the proposed studies are: 1) What are the changes in translating RNA occurring in astrocytes after Chronic Intermittent Ethanol-2 Bottle Choice (CIE-2BC)? 2) What are the changes in nuclear gene expression occurring in astrocytes after CIE-2BC? 3) Does Plat/tPA knock-down in astrocytes reduce escalation in drinking, neuroimmune responses, and synaptic changes induced by CIE-2BC? We hypothesize that: CIE-2BC induces changes in the translation of neuroimmune genes in astrocytes and that some, but not all, changes in translation are driven by changes in transcription. We also hypothesize that knocking down Plat selectively in astrocytes will attenuate escalation in drinking, induction of neuroimmune genes, and synaptic changes induced by CIE-2BC. We propose to use the Aldh1l1-EGFP-Rpl10a mouse model that allows the selective pull down of actively translating RNA from astrocytes by the translating ribosome affinity purification (TRAP) method. Moreover, this mouse line has GFP fluorescence in the nucleus that allows for the isolation of astrocyte-specific nuclei by Fluorescent-Activated Cell Sorting (FACS). In Specific Aim 1 we will study the translatome in amygdala and PFC of female and male Aldh1l1-EGFP-Rpl10a mice after CIE-2BC by TRAP-RNA-seq. Pathway analysis will be performed to determine enrichment in the biological processes affected by CIE-2BC. We will employ TRAP-qPCR, Western blot, and immunohistochemistry (IHC) to validate changes in neuroimmune gene translation and protein expression. In Specific Aim 2 we will study the nuclear transcriptome in the amygdala and PFC of female and male Aldh1l1-EGFP-Rpl10a mice after CIE-2BC by FACS sorting of astrocyte nuclei followed by RNA-seq. We will integrate transcriptome and translatome data to identify RNAs regulated by alcohol through transcription- dependent and transcription-independent mechanisms. We will employ FACS-qPCR and Fluorescence In Situ Hybridization (FISH)-RNAScope to validate changes in neuroimmune gene expression. In Specific Aim 3 we will investigate the hypothesis that tPA knock-down in astrocytes attenuates CIE-2BC-induced escalation of drinking, immune response, and synaptic changes. Inducible Aldh1l1-Cre/ERT2 mice will be crossed to floxed Plat mice for the selective kn...