Distinguishing preexistent and induced epigenetic risk for alcohol use disorders

摘要 每年有近8.8万人死于与酒精有关的原因，使其成为第四大可预防的原因 在美国的死亡。了解神经变化背后的分子机制 导致酒精依赖对于开发新的、有效的酒精使用障碍(AUD)治疗方法至关重要。 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 酒精和非酒精的尸检脑组织。另外，差分dNaM(D-dNaM)的一个子集 信号与附近基因的表达变化有关，这表明dNaM调节该基因 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 在身体组织中检测到的dNaM信号是预先存在的，这可能会增加危险的风险 饮酒，而不是被慢性酒精诱导。这项研究旨在区分这两种可能性， 并明确酒精诱导的前额叶皮质(PFC)dNaM的功能后果。这个 研究利用高度相关和特征良好的非人灵长类(NHP)酒精自身 管理模式。在这个模型中，我们测量了恒河猴自愿饮酒的水平。 持续约12个月的每日饮酒量，使自然“低度”和“重度”饮酒者得以准确分类。在……里面 此外，PFC的一小部分在酒精接触之前进行了活组织检查，从而提供了建立 DNaM的基线测量。全基因组亚硫酸氢盐测序(GWBS)将用于识别酒精剂量 恒河猴PFC中依赖的差异甲基化胞嘧啶(DMC)和区域(DMR) 猕猴。同样的方法将被用来比较从猕猴身上获得的PFC中的dNaM 酒精进入，能够检测到先前存在的与酒精剂量相关的dNaM签名。此外, 猕猴的D-dNaM信号将与尸检产生的D-dNaM数据进行比较 酒精PFC组织，识别与酒精相关的dNaM，该dNaM在两个灵长类物种中复制。下一首, DMC/DMR相关基因的表达，包括全基因和选择性转录本 表达式，将与dNaM数据相关联。基于人类猕猴dNaM的支持 比较，以及相应的表达数据，将选择引人注目的新基因靶点的子集 用于功能研究。靶基因活性将通过药物和基因表达来调节 小鼠模型中的修饰方法。目标调制的神经效应将使用 膜片钳电生理分析，酒精使用情况将使用间歇性酒精进行评估，2瓶 精选模特。总之，这项工作将确定先前存在的和酒精诱导的dNaM修饰 并将阐明dNaM连锁基因的子集在促进酒精使用中的作用。调查结果 这项研究将为设计有前景的AUD新疗法提供功能支持。

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.