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

Repeated cycles of alcohol intoxication and withdrawal 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 microglia in striatum, a key region involved in the relapse to ethanol consumption. We performed deep sequencing of the "translatome" from striatal microglia of male and female RiboTag mice, yielding a snapshot of RNA translation during alcohol intoxication and after 8 hours of withdrawal. Chronic intermittent ethanol produced robust changes in the translatome, 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 and phagocytotic and proapoptotic pathways were increased. Using unbiased network analysis, we identified gene modules that were differentially expressed in ethanol intoxicated vs. withdrawing animals. Genes associated with the unfolded protein response (UPR) were over-represented in one such module after withdrawal, including the transcription factor Ddit3 (CHOP), an important mediator of the UPR. We tested the impact of conditional knockout of CHOP from microglia specifically; following withdrawal from chronic intermittent ethanol, these mice had reduced thermoregulatory disturbances, anxiety-like behavior, and voluntary ethanol consumption compared to wild-type littermates. We conclude that CHOP and the UPR in microglia may be important targets for reducing the impact of withdrawal from chronic ethanol exposure.