Histone Methylation Mechanisms in the Adult Brain after Adolescent Alcohol Exposure

The developmental period of adolescence is characterized by profound changes in brain structure, function, and gene expression patterns that are at least partially epigenetically programmed. These biochemical alterations are responsible for the behavioral characteristics of adolescence, including increased impulsivity and the propensity to consume binge amounts of alcohol. Binge alcohol consumption during adolescence is associated with increased risk for alcohol use disorder (AUD) and comorbid psychiatric disorders such as depression and anxiety in adulthood. Rats exposed to adolescent intermittent ethanol (AIE) display increased alcohol consumption and increased anxiety-like behaviors in adulthood that are accompanied by changes to epigenetic and synaptic systems in the amygdala, a brain region that is crucial for the both expression of anxiety and the actions of ethanol. In this thesis, I focus on elucidating novel treatment targets for AUD and comorbid anxiety related to adolescent alcohol consumption in two separate but interrelated projects. In the first project, I examined the expression of microRNAs (miRNAs) in the amygdala of adult rats following AIE and found that microRNA-137 (miR-137) is increased in the AIE adult amygdala compared to saline-exposed (AIS) controls. The miR-137 target gene and epigenetic modifier lysine-specific demethylase 1 (Lsd1) and the neuron-specific splice variant Lsd1+8a show reduced mRNA levels in the AIE adult amygdala, and this is associated with an increase in repressive histone 3 lysine 9 dimethylation (H3K9me2) but unchanged H3K4me2. LSD1 levels locally at the exon IV promoter of brain-derived neurotrophic factor (Bdnf) are decreased in the AIE adult amygdala, correlating with decreased Bdnf IV mRNA expression. Infusion of an antisense antagomir of miR-137 directly into the central nucleus of the amygdala (CeA) rescues the increased alcohol drinking and anxiety-like behavior seen in AIE adult animals. miR-137 antagomir infusion in the CeA also attenuates Lsd1 and Lsd1+8a mRNA expression, levels of LSD1 at the Bdnf exon IV promoter, and Bdnf IV mRNA levels in the amygdala. These data suggest that upregulation of miR-137 is crucial to the long-lasting behavioral and molecular effects of adolescent alcohol exposure. In the second project, I found that synaptic density was reduced in the AIE adult amygdala compared to AIS rats, and that several synapse-associated genes including activity-regulated cytoskeleton-associated protein (Arc) show reduced mRNA levels in the AIE adult amygdala. AIE adult rats also show reduced occupancy of both lysine-specific demethylase 6B (KDM6B) and CREB binding protein (CBP), leading to increased H3K27me3 and decreased H3K27 acetylation (H3K27ac) at the Arc synaptic activity response element (SARE) site in the amygdala. The SARE site encodes an enhancer RNA (eRNA) upstream of Arc, and AIE leads to decreased Arc eRNA expression and increased binding of negative elongation factor (NELF) repressor complex to the Arc promoter in the amygdala which agrees with previous studies suggesting that Arc eRNA binds to NELF in order to de-repress Arc mRNA transcription. Acute ethanol challenge in adulthood normalizes the anxiety-like behavior seen in AIE adult rats, as well as the chromatin alterations at the Arc SARE site and promoter in the amygdala and altered Arc eRNA and mRNA expression. Knockdown of Kdm6b expression in the CeA provokes anxiety-like behavior in AIS rats and decreases Arc eRNA and mRNA expression, possibly via increased H3K27me3 and decreased H3K27ac observed at the SARE site and Arc promoter in the amygdala following Kdm6b small-interfering RNA (siRNA) infusion. Lastly, knockdown of the minus strand of Arc eRNA [Arc eRNA (-)] in the CeA leads to marked anxiety-like behavior in alcohol-naïve control rats as well as increased NELF repressor complex occupancy at the Arc promoter and decreased Arc mRNA expression in the amygdala. Therefore, Arc eRNA controls Arc mRNA expression in the amygdala and disruption of this system by adolescent alcohol exposure underlies the risk for anxiety-like behaviors seen in adulthood. In summary, adolescent alcohol exposure leads to an upregulation of miR-137 in the adult amygdala which alters LSD1-related chromatin remodeling pathways at Bdnf, as well as a decreased in Arc eRNA expression in the same brain region that is encoded by epigenetic alterations at the SARE site and downregulates Arc mRNA expression. These findings identify novel treatment targets and neurobiological pathways in the amygdala that contribute to the pathogenesis of AUD and comorbid anxiety. Finally, the work presented here furthers the knowledge of the epigenetic consequences of adolescent binge-like alcohol exposure in adulthood.