Potential gene-editing treatment for anxiety and alcohol use disorder in adult rats who were exposed to binge drinking in their adolescence

Targeted epigenomic editing ameliorates adult anxiety and excessive drinking after adolescent alcohol exposure. John Peyton Bohnsack, Huaibo Zhang, Gabriela M. Wandling, Donghong He, Evan J. Kyzar, Amy W. Lasek and Subhash C. Pandey. Science Advances, May 4 2022. DOI: 10.1126/sciadv.abn2748

Abstract: 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.

Popular version: A study by researchers from the University of Illinois Chicago has found a potential gene-editing treatment for anxiety and alcohol use disorder in adults who were exposed to binge drinking in their adolescence. Binge drinking in adolescence alters brain chemistry in the enhancer region of the Arc gene and reduces Arc expression in the amygdala for both rodents and humans. This contributes to a predisposition to alcohol use disorder in adulthood. Rats that had been exposed to alcohol during adolescence showed fewer indicators of anxiety and lower alcohol consumption when Arc gene expression was increased using CRISPR technology. Normal rats showed more indicators of anxiety and increased their alcohol consumption when Arc gene expression was decreased.

DISCUSSION

Our results indicate that targeted epigenomic editing at the Arc SARE can bidirectionally modulate behavioral changes caused by adolescent alcohol exposure (Fig. 5). Furthermore, we demonstrate that these changes are largely due to an epigenetic circuit involving transcription of eRNAs from the Arc SARE, which causes epigenetic remodeling at the Arc promoter by looping of the chromatin to allow localized release of NELF. In addition, our results indicate that activating the Arc SARE site through histone acetylation using dCas9-P300 facilitates Arc eRNA transcription and long-range promoter-enhancer interactions and modulates transcription factor binding, which is consistent with other findings (25, 39, 40). Conversely, dCas9-KRAB–mediated epigenetic suppression at Arc SARE decreases eRNA transcription and increases NELF binding at the promoter without changing enhancer and promoter interactions and promotes anxiety and excessive drinking (Fig. 5). This contrasts with other studies in which KRAB is involved in heterochromatin spreading and transcriptional repression via alternative mechanisms (41). Here, we demonstrate that dCas9-KRAB increases repressive H3K27me3, which has previously been shown to be involved in repressing eRNA transcription (35). We observed no change in H3K9me3 occupancy at the Arc SARE site after dCas9-KRAB manipulations. Other studies have shown that targeting dCas9-KRAB to specific genomic loci increases H3K9me3, but these changes have been shown to be transitory and not sufficient for transcriptional repression (38, 42). While it is likely that increased H3K27me3 at the Arc SARE contributes to repressed Arc eRNA and mRNA expression and subsequent behavioral correlates, the current study did not investigate other repressive mechanisms induced by dCas9-KRAB.

Fig. 5. Model showing that adolescent alcohol exposure produces epigenetic reprogramming in the CeA as well as anxiety and excessive alcohol intake in adulthood.

dCas9-P300 infusion in the CeA was able to increase H3K27ac at Arc SARE, increase Arc eRNA and mRNA expression, and ameliorate anxiety and excessive alcohol intake induced by AIE exposure in adulthood. On the other hand, dCas9-KRAB increased repressive epigenetic marks, H3K27me3 at Arc SARE, leading to decreased Arc eRNA and mRNA expression and development of anxiety and alcohol drinking behaviors in control adult rats. These data causally link epigenetic modifications at an enhancer region of synaptic gene Arc to adult AUD and anxiety after adolescent alcohol exposure.

OPEN IN VIEWER

Preclinical and clinical data clearly suggest that adolescent alcohol consumption can increase the susceptibility of an individual to anxiety and AUD (4–7, 9, 10). Several studies in the field have shown that various biological mechanisms, including epigenetic changes, may be involved in the persistence of the effects of adolescent alcohol exposure into adulthood (9, 10, 43). It has been shown that epigenetic drugs such as histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors can attenuate adolescent alcohol exposure–induced anxiety-like and alcohol drinking behaviors in adult rats (28, 44). Similar to what was found in the current study using dCas9-P300, treatment with either a systemic HDAC inhibitor or DNMT inhibitor did not result in anxiolysis or reduce alcohol intake in AIS adult rats (28, 44). Here, there was a trend toward an increase in Arc expression and histone acetylation after dCas9-P300 treatment, but this was not statistically significant. Conversely, acute ethanol challenge in AIS adult rats increased Arc expression and produced anxiolysis that was associated with increased KDM6B/CBP, decreased H3K27me3, and increased H3K27ac occupancy at the Arc SARE site in the amygdala. These epigenetic modifications were associated with a significant increase in Arc expression in the amygdala of AIS rats (11). We therefore suggest that the dCas9-P300 manipulation did not reach the set point or biological threshold of Arc expression to induce anxiolysis, and this may be a possible explanation for the lack of observed effects of dCas9-P300 on anxiety and alcohol intake in AIS control rats.

The Arc gene is one of the most interactive genes in the altered synaptic gene network in the adult amygdala after adolescent alcohol exposure in rats (11). Arc interacts with a brain-derived neurotrophic factor (BDNF), and both are down-regulated via epigenetic mechanisms in the amygdala of rodents and humans in adulthood after adolescent alcohol consumption (11, 12, 30). Arc protein expression is lower in the CeA of alcohol-preferring rats as compared with alcohol nonpreferring rats, and this is most likely the result of increased HDAC2-mediated deficits in histone acetylation of Arc gene. Inhibition of HDAC2 expression in the CeA of preferring rats attenuated anxiety-like behaviors and excessive drinking and increased histone acetylation and Arc protein expression in the CeA (45). Furthermore, HDAC and DNMT inhibitors are also effective in preventing excessive drinking and alcohol self-administration in other animal models of AUD (46–48). These studies suggest that Arc expression is regulated via histone acetylation mechanisms and that pharmacological epigenetic agents are effective in attenuating anxiety and alcohol intake in several models of AUD. However, these studies do not establish the specific epigenetically regulated genomic regions implicated in these effects. Here, we used epigenomic editing (25–27) to modulate histone acetylation/methylation levels at a specific genomic locus regulating anxiety and excessive drinking.

AIE is associated with diminished synaptic events that are characterized by decreased mRNA and protein levels of Arc and decreased dendritic spines and synapses in the CeA in adulthood (11, 28). Our previous studies showed that up-regulation of EZH2 and down-regulation of KDM6B and CBP after AIE initiate repression of the Arc gene through increases in H3K27me3 and decreases in H3K27ac occupancy at the Arc SARE, providing an endogenous mechanism for epigenetic reprogramming in the amygdala of rodents (11) and AUD subjects with early age of onset (12). All these studies are correlative in nature, and we now causally demonstrate in detail how epigenetic activation and repression at the Arc SARE site can drive adult excessive drinking and anxiety induced by adolescent alcohol exposure. Our results further indicate that epigenetic editing using dCas9-P300 at the Arc SARE is sufficient for enhancer-mediated activation of Arc expression and can drive increased H3K27ac at the promoter site, most likely via chromatin looping (34, 39). There are few caveats to the present study. One caveat is that it was only performed in male animals, and since sex-specific effects of ethanol have been shown in the literature (49), a future study in female animals needs to be conducted. Another caveat is that we chose to mechanistically interrogate only the CeA, whereas previous studies have shown similar changes in Arc expression in the medial nucleus of amygdala (MeA) after AIE in adulthood (28). While the results of current study imply that epigenomic editing of Arc SARE in the CeA is sufficient to modulate anxiety-like and alcohol drinking behaviors, we cannot rule out the involvement of other brain regions that are implicated in these behaviors and addiction (2, 50).

Previous reports have indicated that the use of dCas9-induced epigenetic modulation at promoter regions can modulate behavior (51–53) as well as CRISPR-mediated chromosomal looping (54). Here, we demonstrate that this technology can also be used to investigate the role of noncoding RNAs, such as eRNAs, in regulating the interplay between three-dimensional chromatin structure, genetic regulatory elements, transcription factor binding, and RNA to influence adult anxiety and drinking after adolescent alcohol exposure. The current study focused on reversing epigenetic and behavioral changes in adulthood that were induced by adolescent alcohol exposure. However, we have previously observed that histone acetylation and behavioral changes also occur immediately after the AIE paradigm in adolescence and persist into adulthood (28). While the current study only focused on the long-term changes that persist until adulthood to better model what is seen in humans (4–7), the findings support the idea that activating the Arc SARE with dCas9-P300 could be applied as an intervention in adolescence. Our previous study showed that KDM6B siRNA infusion into the CeA of control adult rats produced anxiety-like behaviors, decreases in KDM6B, and increases in H3K27me3 occupancy at the Arc SARE site and suppressed Arc eRNA and mRNA expression in the amygdala (11). In addition, inhibition of Arc eRNA (−) levels in the CeA increased NELF binding, decreased Arc expression, and provoked anxiety-like behaviors in control adult rats (11). Here, we demonstrate that dCas9-KRAB infusion into CeA produces an increase in repressive H3K27me3 at the Arc SARE, decreases eRNA levels, increases NELF binding at the promoter, down-regulates Arc expression, and increases anxiety-like behavior and alcohol intake in control adult rats. Together, this suggests that epigenetic regulation at the Arc SARE site in the CeA is involved in modulating anxiety and alcohol intake. Thus, both developmental alcohol exposure–induced suppression of Arc expression and direct suppression of Arc expression in adulthood in the CeA appear to be crucial in anxiety and AUD.

Our findings regarding epigenomic editing allow for the analysis of gene- and amygdala nuclei–specific epigenetic changes that occur after adolescent alcohol exposure and persist until adulthood, driving complex behavior. The Cas9 system is easier to use and more modular than previous iterations [e.g., transcription activator–like effector nucleases (TALENs)] designed to alter precise epigenetic marks (55). In addition, the use of dCas9 to modulate gene expression, in lieu of a Cas9 enzyme with active endonuclease activity, could confer additional therapeutic and research benefit. Considerable effort has gone into reducing “off-target” double-stranded breaks that result in genome instability (56). The use of the dCas9 system avoids many of these off-target effects since DNA is not cut and not subject to error-prone nonhomologous end joining or spontaneous recombination events. The use of a dCas9 system is especially useful in the context of epigenetic regulation, and it has broad applications in the interrogation of long-lasting epigenetic changes that drive AUD and anxiety after adolescent alcohol exposure and the identification of tractable targets for the treatment of AUD and comorbid anxiety.