Novel gene networks modulating progressive ethanol consumption in DO mice.
Over the past 3.5 years of support on this P50, our laboratory has used Diversity Outbred (DO) mice from The Jackson Laboratory to extend behavioral genetic and genomic studies on a model of progressive ethanol consumption using intermittent oral access. Behavioral studies identified narrow, highly significant loci associated with consumption phenotypes. We refined significant behavioral quantitative trait loci (bQTLs) associated with last week ethanol consumption (LWEC), first week ethanol preference (FWEP) and last week 30% ethanol choice (LW30C; 30% v/v vs. 15% v/v). Mechanistic features were characterized by RNAseq in prefrontal cortex and nucleus accumbens and co-expression network analysis, producing a defined list of positional candidate genes for bQTL, functional network associations with behavior and overlaps with human GWAS studies. Preliminary multivariate analysis of consumption across time identified fine structure of consumption behavior and associated genetic loci. The Car8 gene was identified as a high priority candidate gene influencing a Chr4 bQTL for LWEC. Car8 homologs exist in C. elegans and are currently being studied by Drs. Bettinger and Davies (Proj. 2) for ethanol behaviors. Based upon progress outlined above, we hypothesize that characterization of the progressive ethanol consumption microstructure will identify associated genomic signatures; that the Chr4 LWEC interval will modulate consumption in females and drive genome-specific expression patterns at a cellular level contributing to progressive consumption, and that Car8 may be a major candidate gene for such. We predict that the work will provide key findings relevant to mechanisms and treatment of AUD. Our specific aims include: 1) Genetic analysis of longitudinal drinking pattern microstructure in DO mice. This will use multivariate analysis to characterize microstructure to the progressive ethanol consumption and associated gene expression patterns; 2) Genetic and candidate gene analysis of the Chr4 behavioral QTL region for ethanol consumption. We will use Collaborative Cross (CC) mouse lines selected for specific genotype patterns on Chr4 to fully characterize the contribution of that region to ethanol behaviors, bulk RNAseq expression differences and networks and single-nuclei RNAseq to identify genotype x ethanol specific changes in cell type composition and cellular gene expression; and 3) Characterize the role of Car8 and other major candidate genes for LWEC, FWEP, 30% ethanol choice, and drinking endophenotypes (Aim 1) in intermittent ethanol access consumption. This aim will use transgenic animals and viral vector approaches to delete/over-express the Car8 gene or other candidates in medial prefrontal cortex to determine its role in progressive ethanol consumption and other ethanol behaviors. This novel gene discovery and network analysis effort will have major interactions with all other components of VCU ARC and will inform the field of alcohol research with understanding of mechanisms involved in the transition to compulsive drinking, and candidate genes/mechanisms for potential targeting by future therapeutic efforts.