Prenatal ethanol exposure alters adult hippocampal VGLUT2 expression with concomitant changes in promoter DNA methylation, H3K4 trimethylation and miR-467b-5p levels

Background: Maternal consumption of alcohol during pregnancy is associated with a range of physical, cognitive and behavioural outcomes in the offspring which are collectively called foetal alcohol spectrum disorders. We and others have proposed that epigenetic modifications, such as DNA methylation and post-translational histone modifications, mediate the effects of prenatal alcohol exposure on gene expression and, ultimately, phenotype. Here we use an inbred C57BL/6J mouse model of early gestational ethanol exposure equivalent, developmentally, to the first 3-4 weeks of pregnancy in humans to examine the long-term effects on gene expression and epigenetic state in the hippocampus. Results: Gene expression analysis in the hippocampus revealed sex- and age-specific up-regulation of solute carrier family 17 member 6 (Slc17a6), which encodes vesicular glutamate transporter 2 (VGLUT2). Transcriptional up-regulation correlated with decreased DNA methylation and enrichment of histone H3 lysine 4 trimethylation, an active chromatin mark, at the Slc17a6 promoter. In contrast to Slc17a6 mRNA levels, hippocampal VGLUT2 protein levels were significantly decreased in adult ethanol-exposed offspring, suggesting an additional level of post-transcriptional control. MicroRNA expression profiling in the hippocampus identified four ethanol-sensitive microRNAs, of which miR-467b-5p was predicted to target Slc17a6. In vitro reporter assays showed that miR-467b-5p specifically interacted with the 3'UTR of Slc17a6, suggesting that it contributes to the reduction of hippocampal VGLUT2 in vivo. A significant correlation between microRNA expression in the hippocampus and serum of ethanol-exposed offspring was also observed. Conclusions: Prenatal ethanol exposure has complex transcriptional and post-transcriptional effects on Slc17a6 (VGLUT2) expression in the mouse hippocampus. These effects are observed following a relatively moderate exposure that is restricted to early pregnancy, modelling human consumption of alcohol before pregnancy is confirmed, and are only apparent in male offspring in adulthood. Our findings are consistent with the idea that altered epigenetic and/or microRNA-mediated regulation of glutamate neurotransmission in the hippocampus contributes to the cognitive and behavioural phenotypes observed in foetal alcohol spectrum disorders. Although further work is needed in both mice and humans, the results also suggest that circulating microRNAs could be used as biomarkers of early gestational ethanol exposure and hippocampal dysfunction.