Common schizophrenia variants functioning in developmental human cortical interneurons

摘要 精神分裂症（SCZ）是一种高度遗传和复杂的神经发育障碍。显著进步 最近在SCZ遗传学研究中，越来越多的风险位点达到全基因组范围， 然而，从这些基因座收集生物学见解一直具有挑战性。大部分SCZ风险 基因座位于非编码区。因此，假设它们通过调节远端基因而起作用。 通过3D染色质相互作用表达。然而，还有待确定哪些位点在 哪些细胞，在什么时间点，以及有什么影响。最近的基因组分析显示富集SCZ 在人类胎儿大脑而不是成人大脑中的遗传性，表明SCZ风险基因座在调节胎儿大脑中的作用。 增加SCZ风险。因此，阐明SCZ风险基因座在发育过程中的功能至关重要 了解基因对SCZ风险的影响。对基因表达的遗传影响（例如表达 数量性状基因座（eQTL））是细胞类型特异性的，并且有时根据细胞类型赋予相反的效果。 细胞类型，强调使用同质细胞群进行细胞类型特异性研究的重要性， 明确的机械理解。表达小清蛋白（PV）或生长抑素（SST）的内侧神经节 隆起（MGE）衍生的皮质中间神经元（CIN）在SCZ脑中始终受到影响。更重要的是， SCZ的遗传性被证明是丰富的MGE细胞在人类胎儿的大脑，有必要研究这些 细胞来了解SCZ风险位点的机制。虽然没有死后胎儿SCZ组织 机制研究，iPSC的体外分化-很好地再现了早期胚胎发育- 为发育中的SCZ脑细胞提供与患者大脑相同的基因组成。我们建立 从健康对照有效产生MGE衍生的cIN的同质群体的方法 (HC)和SCZ iPSC。我们还在体外和体内广泛验证了它们的功能和真实性， 包括强迁移和突触整合到宿主脑中，这导致有效的抑制性调节， 移植小鼠的宿主电路。使用前所未有的大量iPSC来提供同质的 对于HC与SCZ胎儿cIN的机制研究，我们将讨论我们的假设，即SCZ风险基因座 在发育中活跃的MGE型cIN通过3D染色质相互作用调节远端基因表达。 采用转录组分析、PrediXcan分析和Micro-C分析，我们将绘制SCZ风险基因座， 在发育过程中，它们在这些脆弱的细胞群体中调节的风险基因的功能是未知的。 发育cIN特异性遗传对基因表达的影响，基于多个品系的 确凿的证据，将使用CRISPRi/CRISPRa方法进行功能验证。这种不偏 来自发育MGE型cIN的全基因组综合数据集（具有功能验证）将提供 揭示发育SCZ风险的遗传基础的路线图，并帮助我们识别基于机制的 新的治疗靶点。

Abstract Schizophrenia (SCZ) is a highly heritable and complex neurodevelopmental disorder. Remarkable advances have been made recently in SCZ genetic studies with an increasing number of risk loci reaching genome-wide significance; however, gleaning biological insight from these loci has been challenging. The majority of SCZ risk loci are located in non-coding regions. As such, it is hypothesized that they function by regulating distal gene expression via 3D chromatin interactions. However, it has yet to be determined which loci are operational in which cells, at what time points, and with what impact. Recent genomic analyses showed enriched SCZ heritability in human fetal brains rather than adult brains, suggesting the role of SCZ risk loci in modulating fetal development for increased SCZ risks. Thus, unraveling SCZ risk loci function during development will be critical for understanding genetic influences on SCZ risks. Genetic influences on gene expression (e.g. expression quantitative trait loci (eQTLs)) are cell-type-specific, and sometimes confer opposing effects depending on the cell type, underscoring the importance of cell-type-specific studies using homogeneous cell populations for a clear mechanistic understanding. Parvalbumin (PV)- or somatostatin (SST)-expressing medial ganglionic eminence (MGE)-derived cortical interneurons (cINs) are consistently affected in SCZ brains. More importantly, SCZ heritability is shown to be enriched in MGE cells in human fetal brains, necessitating the study of these cells to understand the mechanisms of SCZ risk loci. Although there are no postmortem fetal SCZ tissues for mechanistic study, in vitro differentiation of iPSC—which well recapitulates early embryonic development— provides developmental SCZ brain cells with the same genetic makeup as patient brains. We established methods for the efficient generation of homogeneous populations of MGE-derived cINs from healthy control (HC) and SCZ iPSCs. We also extensively validated their functionality and authenticity both in vitro and in vivo, including robust migration and synaptic integration into host brains that results in efficient inhibitory regulation of host circuitry in transplanted mice. Using an unprecedentedly large number of iPSCs to provide homogeneous populations of HC vs SCZ fetal cINs for mechanistic studies, we will address our hypothesis that SCZ risk loci active in developmental MGE-type cINs regulate distal gene expression via 3D chromatin interactions. Employing transcriptome analysis, PrediXcan analysis, and Micro-C analysis, we will map SCZ risk loci with unknown functions to the risk genes they regulate in these vulnerable cell populations during development. Developmental cIN-specific genetic influences on gene expression, identified based on multiple lines of corroborating evidence, will be functionally validated using CRISPRi/CRISPRa approaches. This unbiased genome-wide comprehensive data set from developmental MGE-type cINs with functional validation will provide a road map for unravelling the genetic basis of developmental SCZ risks and help us identify mechanism-based novel therapeutic targets.