RiboTag-Seq Reveals the Activation of the Unfolded Protein Response in Striatal Microglia Induced by Ethanol Withdrawal

Repeated cycles of alcohol intoxication and withdrawal both induce profound changes in gene expression that can contribute to the physiological and behavioral consequences of ethanol. Since neuroinflammation is an important consequence of these changes, we used a novel strategy to investigate the impact of repeated cycles of chronic intermittent ethanol vapor and withdrawal on the RNAs actively undergoing translation in striatal microglia. RiboTag was selectively expressed in the microglia of transgenic mice and was used to immunopurify the RNA “translatome” from striatal microglia, yielding a snapshot of RNA translation during alcohol intoxication and after 8 hours of withdrawal. We obtained highly enriched microglial RNAs and analyzed these in individual animals by deep sequencing. We found a dramatic shift in gene expression during acute intoxication compared to air-exposed controls, with increases in genes and pathways associated with cytokine signaling, indicating increased neuroinflammation and microglial activation. After 8 hours of ethanol withdrawal, many inflammatory pathways remained upregulated but phagocytotic and proapoptotic pathways were increased. Using an unbiased bioinformatic method, weighted gene coexpression network analysis, multiple differentially expressed gene modules were identified. One in particular was differentially expressed in ethanol intoxicated vs. withdrawing animals, and there was a strong correlation between the centrality of the genes to this gene network and their individual statistical significance in differential expression. The unfolded protein response was over-represented in this network after withdrawal. The induction of this pathway in microglia is important since this cellular stress response can either lead towards restoration of normal function or apoptosis.