John Peyton Bohnsack

Adolescent binge drinking is a major risk factor for psychiatric disorders later in life including alcohol use disorder. Adolescent alcohol exposure induces epigenetic reprogramming at the enhancer region of the activity-regulated cytoskeleton-associated protein (Arc) immediate-early gene, known as synaptic activity response element (SARE), and decreases Arc expression in the amygdala of both rodents and humans. The causal role of amygdalar epigenomic regulation at Arc SARE in adult anxiety and drinking after adolescent alcohol exposure is unknown. Here, we show that dCas9-P300 increases histone acetylation at the Arc SARE and normalizes deficits in Arc expression, leading to attenuation of adult anxiety and excessive alcohol drinking in a rat model of adolescent alcohol exposure. Conversely, dCas9-KRAB increases repressive histone methylation at the Arc SARE, decreases Arc expression, and produces anxiety and alcohol drinking in control rats. These results demonstrate that epigenomic editing in the amygdala can ameliorate adult psychopathology after adolescent alcohol exposure.

Adolescent binge drinking is a serious public health concern and a risk factor for alcohol use disorder (AUD) and comorbid anxiety in adulthood. Chromatin remodeling mediated by epigenetic enzymes including lysine-specific demethylase 1 (LSD1) due to adolescent alcohol exposure may play a role in adult psychopathology. The mechanism by which adolescent alcohol exposure mechanistically regulates epigenetic reprogramming and behavioral changes in adulthood is unknown. We investigated the role of microRNA-137 (miR-137), which is crucial for normal neurodevelopment and targets LSD1, in adolescent intermittent ethanol (AIE) exposure-induced anxiety-like and alcohol-drinking behaviors and related epigenetic reprogramming in the amygdala in adulthood. Adolescent rats were exposed to 2 g/kg ethanol (2 d on/off; AIE) or adolescent intermittent saline (AIS) during postnatal days (PND)28–PND41 and allowed to grow to adulthood for analysis of behavior, miRNA expression, and epigenetic measures in the amygdala. Interestingly, miR-137 was increased and its target genesLsd1andLsd1 + 8awere decreased in the AIE adult amygdala. Infusion of miR-137 antagomir directly into the central nucleus of the amygdala (CeA) rescues AIE-induced alcohol-drinking and anxiety-like behaviors via normalization of decreasedLsd1expression, decreased LSD1 occupancy, and decreasedBdnf IVexpression due to increased H3K9 dimethylation in AIE adult rats. Further, concomitantLsd1small interfering RNA (siRNA) infusion into the CeA prevents the miR-137-mediated reversal of AIE-induced adult anxiety and chromatin remodeling at theBdnf IVpromoter. These novel results highlight miR-137 as a potential therapeutic target for anxiety and AUD susceptibility after adolescent alcohol exposure in adulthood.

Adolescent alcohol drinking is known to contribute to the development and severity of alcohol use disorders (AUDs) later in adulthood. Recent studies have shown that long non-coding RNAs (lncRNAs) are critical for brain development and synaptic plasticity. One such lncRNA is natural occurring brain-derived neurotrophic factor antisense (BDNF-AS) that has been shown to regulate BDNF expression. The role ofBDNF-ASlncRNA in the molecular mechanisms of AUD is unknown. Here, we evaluated the expression and functional role ofBDNF-ASin postmortem amygdala of either early onset or late onset alcoholics (individuals who began drinking before or after 21 years of age, respectively) and age-matched control subjects.BDNF-ASexpression is increased in early onset but not in late onset AUD amygdala and appears to be regulated epitranscriptomically via decreasedN6-methyladenosine onBDNF-AS. Upregulation ofBDNF-ASis associated with a significant decrease in BDNF expression and increased recruitment of EZH2, which deposits repressive H3K27 trimethylation (H3K27me3) at regulatory regions in theBDNFgene in the early onset AUD group. Drinking during adolescence also contributed to significant decreases in activity-regulated cytoskeleton-associated protein (ARC) expression which also appeared to be mediated by increased EZH2 deposition of repressive H3K27me3 at theARCsynaptic activity response element. These results suggest an important role forBDNF-ASin the regulation of synaptic plasticity via epigenetic reprogramming in the amygdala of AUD subjects who began drinking during adolescence.

Binge alcohol drinking in adolescence leads to increased risk for alcohol use and other psychiatric disorders in adulthood. The transcription factor cAMP-response element binding (CREB) protein is involved in the neuronal response to adult ethanol exposure, but its role in the enduring effects of adolescent alcohol exposure in adulthood is unknown. We exposed male rats to adolescent intermittent ethanol (AIE) or saline (AIS) during post-natal days 28–41 and evaluated the epigenetic regulation of CREB dynamics in the adult amygdala. A subset of these adult rats was exposed to an acute ethanol challenge. AIE decreased CREB, phosphorylated CREB, CREB-binding protein (CBP) and p300 protein levels in adult amygdaloid brain structures. AIE exposure also causes deficits in Creb1, Cbp, and p300 mRNA expression in the amygdala of AIE adult rats which are normalized after acute ethanol exposure. Interestingly, occupancy of acetylated histone H3K9/14 proteins at specific locations in the Creb1, Cbp, and p300 gene promoter regions was decreased in the amygdala of AIE adult rats and was normalized by acute ethanol exposure. These results suggest that AIE exposure epigenetically reduces CREB and other related transcriptional activators in the amygdala in adulthood that may be associated with the behavioral effects of adolescent alcohol exposure.

Alcohol use disorders are chronic debilitating diseases characterized by severe withdrawal symptoms that contribute to morbidity and relapse. GABA_A receptor (GABA_AR) adaptations have long been implicated in the chronic effects of alcohol and contribute to many withdrawal symptoms associated with alcohol dependence. In rodents, GABA_AR hypofunction results from decreases in Gabra1 expression, although the underlying mechanism controlling Gabra1 expression after chronic ethanol exposure is still unknown. We found that chronic ethanol exposure using either ethanol gavage or two-bottle choice voluntary access paradigms decreased Gabra1 expression and increased Hdac2 and Hdac3 expression. Administration of the HDAC inhibitor trichostatin A (TSA) after chronic ethanol exposure prevents the decrease in Gabra1 expression and function as well as the increase in Hdac2 and Hdac3 expression in both the cortex and the medial prefrontal cortex (mPFC). Chronic ethanol exposure and withdrawal, but not acute ethanol exposure or acute withdrawal, cause a selective upregulation of HDAC2 and HDAC3 associated with the Gabra1 promoter that accompanies a decrease in H3 acetylation of the Gabra1 promoter and the reduction in GABA_AR α1 subunit expression. TSA administration prevented each of these molecular events as well as behavioral manifestations of ethanol dependence, including tolerance to zolpidem-induced loss of righting reflex, reduced open-arm time in the elevated plus maze, reduced center-time and locomotor activity in the open-field assay, and TSA reduced voluntary ethanol consumption. The results show how chronic ethanol exposure regulates the highly prominent GABA_AR α1 subunit by an epigenetic mechanism that represents a potential treatment modality for alcohol dependence.

γ-Aminobutyric acid A receptors (GABA_A-Rs) mediate the majority of inhibitory neurotransmission in the adult brain. The α1-containing GABA_A-Rs are the most prominent subtype in the adult brain and are important in both homeostatic function and several disease pathologies including alcohol dependence, epilepsy, and stress. Ethanol exposure causes a decrease of α1 transcription and peptide expression both in vivo and in vitro, but the mechanism that controls the transcriptional regulation is unknown. Because ethanol is known to activate epigenetic regulation of gene expression, we tested the hypothesis that ethanol regulates α1 expression through histone modifications in cerebral cortical cultured neurons. We found that class I histone deacetylases (HDACs) regulate ethanol-induced changes in α1 gene and protein expression as pharmacologic inhibition or knockdown of HDAC1–3 prevents the effects of ethanol exposure. Targeted histone acetylation associated with the Gabra1 promoter using CRISPR (clustered regularly interspaced palindromic repeat) dCas9-P300 (a nuclease-null Cas9 fused with a histone acetyltransferase) increases histone acetylation and prevents the decrease of Gabra1 expression. In contrast, there was no effect of a mutant histone acetyltransferase or generic transcriptional activator or targeting P300 to a distant exon. Conversely, using a dCas9-KRAB construct that increases repressive methylation (H3K9me3) does not interfere with ethanol-induced histone deacetylation. Overall our results indicate that ethanol deacetylates histones associated with the Gabra1 promoter through class I HDACs and that pharmacologic, genetic, or epigenetic intervention prevents decreases in α1 expression in cultured cortical neurons.