Distinguishing preexistent and induced epigenetic risk for alcohol use disorders

总结 每年有近88，000人死于与酒精有关的原因，使其成为第四大可预防的原因 死亡在美国。了解神经系统变化的分子机制 这对于开发新的、有效的酒精使用障碍（AUD）治疗至关重要。 为了实现这一目标，最近的研究已经确定了全基因组DNA甲基化（DNAm）信号， 酒精和非酒精的死后脑组织此外，差异DNAm（D-DNAm）的一个子集 信号与附近基因表达的改变有关，表明DNAm调节基因 与酒精依赖相关的表达模式。然而，目前尚不清楚是否有一些 在死后组织中检测到的DNA m信号是预先存在的，可能导致危险的风险。 饮酒，而不是由慢性酒精引起的。本研究旨在区分这两种可能性， 并确定酒精诱导的前额叶皮层（PFC）中DNA m的功能后果。的 这项研究利用了高度相关和充分表征的非人灵长类动物（NHP）酒精自身， 管理模式在这个模型中，恒河猴的自愿饮酒水平被测量 每天约12个月，使自然的“低”和“重”饮酒者的准确分类。在 此外，一小部分PFC在酒精摄入前进行活检， DNA m的基线测量。全基因组亚硫酸氢盐测序（GWBS）将用于确定酒精剂量 恒河猴PFC中依赖性差异甲基化胞嘧啶（DMC）和区域（DMR） 猕猴将使用相同的方法比较在给药前从猕猴获得的PFC中的DNAm。 酒精进入，使得能够检测预先存在的、酒精剂量相关的DNAm特征。此外，本发明还提供了一种方法， 将猕猴D-DNAm信号与从死后的人产生的D-DNAm数据进行比较 酒精PFC组织，识别在两种灵长类动物中复制的酒精相关DNA m。接下来， DMC/DMR相关基因的表达，包括完整基因和替代转录物 表达，将与DNAm数据相关。基于人类猕猴DNA的支持， 通过比较和相应的表达数据，将选择一个引人注目的新基因靶点子集 进行功能性研究。靶基因活性将使用药理学和基因表达来调节 在鼠模型中的修饰方法。目标调制的神经效应将使用 膜片钳电生理学分析，将使用间歇性酒精（2瓶）评价酒精使用情况 选择模型。总之，这项工作将确定预先存在的和酒精诱导的DNAm修饰， 灵长类PFC，并将澄清一个子集的DNAm连锁基因在促进酒精使用的作用。这些发现 本研究的结论将为设计有前景的新AUD治疗方法提供功能支持。

SUMMARY Nearly 88,000 people die from alcohol-related causes annually, making it the fourth leading preventable cause of death in the United States. Understanding the molecular mechanisms underlying the neurological changes that lead to alcohol dependence is crucial for developing new, effective alcohol use disorder (AUD) treatments. Towards this goal, recent studies have identified genome-wide DNA methylation (DNAm) signals distinguishing alcoholic and non-alcoholic post-mortem brain tissue. In addition, a subset of the differential DNAm (D-DNAm) signals was associated with the altered expression of nearby genes, suggesting that DNAm regulates the gene expression patterns associated with alcoholic dependence. However, it is unknown whether some of the DNAm signals detected in the post-mortem tissue were preexistent, potentially contributing risk for hazardous drinking, rather than being induced by chronic alcohol. This study aims to distinguish these two possibilities, and to pinpoint the functional consequences of alcohol-induced DNAm in the prefrontal cortex (PFC). The study makes use of the highly relevant and well-characterized nonhuman primate (NHP) alcohol self- administration model. In this model, voluntary alcohol consumption levels are measured in rhesus macaques daily for ~12 months, enabling the accurate classification of natural “low” and “heavy” alcohol drinkers. In addition, a small portion of the PFC is biopsied prior to alcohol access, providing opportunity to establish baseline measures of DNAm. Genome-wide bisulfite sequencing (GWBS) will be used to identify alcohol-dose dependent, differentially methylated cytosines (DMCs) and regions (DMRs) in the PFC of the rhesus macaques. The same approach will be used to compare DNAm in PFC obtained from the macaques prior to alcohol access, enabling the detection of preexistent, alcohol-dose associated DNAm signatures. In addition, the macaque D-DNAm signals will be compared to D-DNAm data generated from post-mortem human alcoholic PFC tissue, identifying alcohol-associated DNAm that is replicated across two primate species. Next, the expression of DMC/DMR-associated genes, including both whole gene and alternative transcript expression, will be correlated with the DNAm data. Based on support from the human-macaque DNAm comparison, and corresponding expression data, a subset of compelling, novel gene targets will be selected for functional study. Target gene activity will be modulated using pharmacological and gene expression modification approaches in a murine model. The neural effects of target modulation will be evaluated using patch-clamp electrophysiological analysis, and alcohol use will be evaluated using intermittent alcohol, 2 bottle choice models. In total, this work will identify preexistent and alcohol-induced DNAm modifications in the primate PFC and will clarify the role of a subset of DNAm-linked genes in promoting alcohol use. The findings of this study will provide functional support for the design of promising new AUD treatments.