Role of Cell Type-Specific Molecular Rhythm Disruption in Alcohol Use Disorder

项目总结/摘要 酒精使用是200多种健康状况的一个因果因素，估计占全球负担的5.1%。 疾病和伤害。尽管对公共卫生产生了巨大的影响，但我们仍然缺乏对 导致酒精使用障碍（AUD）。AUD的一个突出特征是昼夜节律 干扰，包括改变的生理节律和睡眠/觉醒周期。这种关系是双向的， 节律紊乱会加剧酒精消耗和典型昼夜节律基因的多态性 与酒精使用增加有关。然而，很少有人知道这些机制的基础， 关系，特别是在分子水平上与AUD个体的大脑。分子节律 由转录-翻译反馈环产生，以控制昼夜节律依赖性（近24小时）基因 表情值得注意的是，AUD与糖尿病患者典型昼夜节律基因的分子节律紊乱有关。 外周和啮齿类动物的研究表明，纹状体中分子节律的破坏，特别是 神经核（NAc）与改变的奖励调节和增加的物质使用风险有关。 然而，人类死后大脑中分子节律的测量在历史上一直具有挑战性， 每个大脑代表一个昼夜节律时间点。一项创新的分析使用“死亡时间”（TOD）来拟合 受试者在24小时“时钟”上;通过组合来自所有受试者的表达数据，可以重建分子生物学。 人类死后大脑的节律模式为了研究AUD相关的分子节律改变， 我们进行了一项初步研究，检查受试者NAc组织中大规模的转录变化， 与对照组相比。值得注意的是，核心昼夜节律基因（例如，PER 1，PER 2），其显示有节奏的 在对照组中的表达，在AUD中是无节律的，表明与行为节律的分子联系 在AUD患者中观察到的变化。然而，NAc由多种转录上不同的细胞组成， 在饮酒行为中具有不同作用的细胞类型，以及分子节律在这些细胞类型中的作用， 以前没有调查过。这个R21建议的中心假设是，分子节律是 在AUD受试者的NAc中以细胞类型特异性方式被破坏，并且这些破坏有助于 过度饮酒为了验证这一假设，我们将使用人类单核RNA测序（snRNA-seq）， 尸检NAc组织，以确定AUD中异质细胞群中分子节律如何改变 (Aim 1）。然后，我们将操纵特定NAc细胞类型的节律，以确定节律的因果作用 酒精饮料（目标2）。总之，这些研究将是第一次检查细胞类型特异性 人类纹状体的节律，它们如何在AUD中改变，以及它们对过度饮酒的贡献。 这些研究对于理解AUD昼夜节律紊乱的机制至关重要， 导致发现新的治疗靶点和/或未来治疗的时间依赖性策略。

PROJECT SUMMARY/ABSTRACT Alcohol use is a causal factor in over 200 health conditions and is estimated to account for 5.1% of global burden of disease and injury. Despite the enormous public health impact, we still lack a basic understanding of the mechanisms that contribute to alcohol use disorder (AUD). A prominent feature of AUD is circadian rhythm disturbances, including altered physiological rhythms and sleep/wake cycles. The relationship is bidirectional, as rhythm disruptions can exacerbate alcohol consumption and polymorphisms in canonical circadian genes associate with increased alcohol use. However, very little is known about the mechanisms underlying these relationships, especially at the molecular level in the brains of individuals with AUD. Molecular rhythms are generated by transcriptional-translational feedback loops to control circadian-dependent (near 24-hour) gene expression. Notably, AUD is associated with disrupted molecular rhythms of canonical circadian genes in the periphery and rodent studies have shown that disruptions to molecular rhythms in the striatum, particularly the nucleus accumbens (NAc), are associated with altered reward regulation and increased risk for substance use. Measures of molecular rhythms in human postmortem brain, however, have historically been challenging since each brain represents a single circadian timepoint. An innovative analysis uses “time of death” (TOD) to fit subjects on a 24-hr ‘clock’; by combining expression data from all subjects, it is possible to reconstruct molecular rhythm patterns in the human postmortem brain. To investigate AUD-associated molecular rhythm alterations, we performed a preliminary study examining large-scale transcriptional changes in bulk NAc tissue from subjects with AUD compared to controls. Notably, core circadian genes (e.g., CRY1, PER2), which display rhythmic expression in control subjects, were arhythmic in AUD, suggesting a molecular link to behavioral rhythm alterations observed in AUD patients. However, the NAc is composed of a variety of transcriptionally distinct cell types with differential roles in alcohol drinking behavior, and the role of molecular rhythms in these cell types has not been previously investigated. The central hypothesis of this R21 proposal is that molecular rhythms are disrupted in a cell-type specific manner in the NAc of AUD subjects, and that these disruptions contribute to excessive drinking. To test this hypothesis, we will use single nucleus RNA-sequencing (snRNA-seq) of human postmortem NAc tissue to determine how molecular rhythms are altered in heterogenous cell populations in AUD (Aim 1). We will then manipulate rhythms in specific NAc cell types to determine a causal role for rhythm disruption in alcohol drinking (Aim 2). Together, these studies will be the first to examine cell type-specific rhythms in human striatum, how they are altered in AUD, and their contribution to excessive alcohol consumption. These studies are central to understanding the mechanisms underlying circadian disruptions in AUD and may result in the discovery of novel therapeutic targets and/or time-dependent strategies for future treatments.