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

摘要与总结 酒精使用障碍(AUD)是一种复杂的行为，伴随着相当大的发病率、死亡率和社会性 费用。遗传因素和环境因素共同作用于AUD。尽管在人类遗传学方面取得了进展 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 在人类中的作用在很大程度上是未知的。在过去的五年里，我们提供了令人信服的证据：1)人类 来源于诱导多能干细胞(IPS)的神经细胞可以作为一种易于处理的模型来研究 包括AUD在内的神经精神障碍；2)酒精暴露导致人类的炎症体反应 携带OPRM1A118G微小基因变异的人神经元对 阿片类药物和乙醇；4)乙醇引起人类神经元和神经胶质细胞的基因表达变化；以及5) 含小胶质细胞的3D神经培养可作为研究神经炎性小胶质细胞-神经元的有力系统 互动。这些前提为进一步的遗传和分子机制研究奠定了基础。 AUD在人类诱导性多发性硬化细胞衍生神经模型中的基础。我们还确定了以下方面的主要知识差距 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-神经胶质相互作用尚不清楚；以及2)乙醇如何影响神经发生。 在3D环境中的位置是未知的。此外，由于AUD的多基因性质，单基因的贡献 基因对AUD的风险可能很小，表型表现受到个体遗传的强烈影响 化妆。为了解决这些悬而未决的问题，我们假设乙醇通过 影响神经胶质细胞的相互作用，这受个体多基因风险背景的影响。我们已经选择了 36名男性和女性受试者，具有极高的多基因风险得分(PRS，前10%的ILE，18 AUD)或低 PRS(最低的10%ILE，n=18，没有AUD)来检验这一假设。我们将从这些细胞中分化出iPS细胞 受试者进入2D和3D(即脑器官)神经细胞与人星形胶质细胞和人类共培养 小胶质细胞。一旦暴露在乙醇中，这些神经细胞就会受到形态、 免疫细胞化学、电生理、活细胞成像和基因组分析以揭示其机制(S) 乙醇的影响，重点放在神经-天文-小胶质细胞的相互作用上。在相对较大的集合中 来自极端AUD PRSS患者的人iPS细胞(n=36株)，我们希望解开 与极高或极低的AUD PRSS相关的收敛的表型和基因网络。结果将会是 促进我们对AUD危险基因变异致病作用及其影响的机械性理解 人类神经系统的多基因风险背景。

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.