Comparative gene pathway analysis during adolescent binge-EtOH exposure, withdrawal, and following abstinence

Abstract

Introduction: Alcohol use disorder (AUD) is a complex, chronic psychiatric disease. AUD manifests as having uncontrollable drinking patterns with detrimental effects. Excessive alcohol intake in the form of binge drinking, which is common among adolescents and young adults, is associated with increased risk of developing AUD. Here, we analyze RNA-seq data from hippocampi of Sprague Dawley rats to investigate temporal changes in gene expression. We used a rodent model of binge drinking, i.e., adolescent intermittent ethanol (AIE), to identify candidate genes that may play a role in the chronic changes in brain function and contribute to the development of AUD. Methods: At postnatal day (PND) 30 (adolescence), rats received chronic intermittent ethanol (5g/kg intragastrically (i.g.) 10 times across 16 days). We extracted total RNA from rat hippocampal tissue that was collected at three time points. RNA was sequenced on an Illumina HiSeq 2000 platform. We processed RNA-seq data (TrimGalore), compiled gene counts (HTSeq), and performed differential expression analysis (DESeq2). The full rank list of genes and the differentially expressed genes (DEGs) with nominal p<0.05 were used as inputs for pathway-based enrichment analyses GSEA and Cytoscape, respectively, at 3 timepoints in the presence or absence of ethanol (i.e., 3 comparisons). For each of these comparisons, GSEA was used to identify genes involved in enriched Gene Ontology (GO) terms while Cytoscape and its apps were used to identify networks of genes and, subsequently, subnetworks (i.e., clusters) of genes enriched by higher interaction. From clusters containing 15 or more nodes, we prioritized genes related with particular functions, cell types, or diseases, which were present in GO Processes, Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways, or Reactome Pathways. Results: Based on GSEA, the most impacted differential gene expression occurs within the potassium channels and receptor function but is heavily dependent on the time point at which the analysis occurs. Across both GSEA and Cytoscape pathway analyses, the most striking changes occur in genes that regulate neuroinflammatory processes and neuronal/synaptic remodeling and coincide with the enrichment of pathways involved in addiction processes. Conclusion: Identification of genes dysregulated by AIE may be useful in determining the underlying mechanism for acute and chronic effects of AIE exposure that contribute to neuronal remodeling and increased risk of developing AUD.