Cellular and genomic mechanisms of the impact of ethanol on human neural model

摘要和总结 酒精使用障碍（AUD）是一种复杂的行为，伴随着大量的发病率，死亡率和社会影响。 开销.遗传因素和环境因素都对AUD有影响。尽管人类遗传学的进步， AUD，特别是全基因组显著AUD遗传风险变异的鉴定，AUD的神经基础 在人类中是未知的。在过去的五年里，我们提供了令人信服的证据：1）人类 来源于诱导多能干（iPS）细胞的神经元细胞可以用作研究的易处理模型。 神经精神疾病，包括AUD; 2）乙醇暴露导致人类炎症反应 3）携带OPRM 1 A118 G次要基因变体的人神经元显示出对 阿片类药物和乙醇; 4）乙醇导致人类神经元和神经胶质细胞中的基因表达变化;以及5） 含有小胶质细胞的3D神经培养物可以成为研究神经炎性小胶质细胞-神经元 交互.这些前提为进一步的遗传和分子机制研究提供了基础 AUD在人类iPS细胞衍生的神经模型中的基础。我们还确定了知识的关键差距， 利用人类神经元作为模型系统来研究需要填充的AUD，特别是：1）乙醇如何 在人类神经环境中影响神经胶质相互作用尚不清楚; 2）乙醇如何影响神经发生 在3D环境中是未知的。此外，3）由于AUD的多基因性质，单个基因的贡献 基因对AUD风险可能很小，表型表现受个体遗传因素的强烈影响。 化妆.为了解决这些悬而未决的问题，我们假设乙醇通过以下途径损害神经功能： 影响神经胶质细胞的相互作用，这是由个人的多基因风险背景的影响。我们选择 36例多基因风险评分极高（PRS，前10%ile，n=18 AUD）或低 PRS（底部10% ile，n=18，无AUD）以检验该假设。我们将从这些细胞中分化出iPS细胞， 将受试者分成与人星形胶质细胞和人星形胶质细胞共培养的2D和3D（即脑类器官）神经元细胞 小胶质细胞在暴露于乙醇后，这些神经细胞受到形态学， 免疫细胞化学、电生理学、活细胞成像和基因组分析，以阐明机制 乙醇的潜在影响，重点是神经-星形-小胶质细胞的相互作用。在一个相对较大的收藏中 来自极端AUD PRS受试者的人iPS细胞（n=36株），我们希望解开 聚合表型和基因网络与极高或极低的AUD PRS有关。结果将 推进我们对AUD风险基因变异的致病作用以及 人类神经系统中的多基因风险背景。

Abstract and summary Alcohol use disorders (AUD) are complex behaviors accompanied by substantial morbidity, mortality and societal expense. Both genetic and environmental factors contribute to AUD. Despite progress in the human genetics of AUD, especially the identification of genome-wide significant AUD genetic risk variants, the neural basis of AUD in humans is largely unknown. Over the past five years, we have provided compelling evidence that: 1) Human neuronal cells derived from induced pluripotent stem (iPS) cells can be used as a tractable model to study neuropsychiatric disorders including AUD; 2) Ethanol exposure results in an inflammasome response in human neurons; 3) Human neurons carrying OPRM1 A118G minor gene variants showed enhanced sensitivity to opioids and ethanol; 4) Ethanol causes gene expression changes in both human neurons and glial cells; and 5) Microglia-containing 3D neural cultures can be a powerful system to study neuroinflammatory microglia-neuronal interactions. These premises provide a foundation for further mechanistic studies of the genetic and molecular underpinnings of AUD in human iPS cell-derived neural models. We also identified key gaps of knowledge in utilizing human neurons as a model system to study AUD that need to be filled, particularly: 1) how ethanol affects neuro-glial interactions in a human neural context is not known; and 2) how ethanol affects neurogenesis in a 3D context is not known. Moreover, 3) because of the polygenic nature of AUD, the contribution of single gene to AUD risk is likely small and the phenotypical manifestation is strongly influenced by individual’s genetic makeup. To address these outstanding questions, we hypothesize that ethanol impairs neuronal function via affecting neuroglial interactions, which is influenced by individual polygenic risk backgrounds. We have selected 36 subjects of both sexes with either extremely high polygenic risk score (PRS, top 10%ile, n=18 AUD) or low PRS (bottom 10%ile, n=18, no AUD) to test this hypothesis. We will differentiate iPS cells derived from these subjects into both 2D and 3D (i.e. brain organoids) neuronal cells co-cultured with human astrocytes and human microglia. Upon exposure to ethanol, these neural cells are subjected to a combination of morphological, immunocytochemical, electrophysiological, live cell imaging and genomic analyses to unravel the mechanism(s) underlying the impact of ethanol, focusing on neuro-astro-microglial interactions. In a relatively large collection of human iPS cells (n=36 lines) derived from subjects with extreme AUD PRSs, we hope to unravel the convergent phenotype and gene-networks that are linked to extreme high or low AUD PRSs. The results will advance our mechanistic understanding of the pathogenic role of AUD risk gene variants and the influence of polygenic risk background in a human neural system.