Uncovering mechanisms of CHD2-associated epilepsy using human cortical organoids

项目总结/摘要 CHD 2基因突变与发育性癫痫性脑病（DEE）有关， 儿童癫痫CHD 2属于ATP的染色体结构域解旋酶DNA结合（CHD）家族 已知依赖性染色质重塑通过染色质组织在神经发育中起关键作用 和基因调控;并且是其他9种CHD（CHD 1-CHD 9）家族蛋白中唯一一种导致大脑 限制性表型表明其在神经发育障碍中的非冗余作用。对小鼠的研究表明， 显示在胚胎皮质中敲低CHD 2降低放射状胶质细胞（RGC）的扩增， 促进中间祖细胞（IP）的产生。此外，Chd 2 +/-小鼠，唯一已知的 迄今为止模拟人类CHD 2单倍不足的杂合小鼠系， GABA能中间神经元，破坏发育中前脑的细胞增殖，并显示神经元 兴奋性此外，使用内侧神经节隆起（MGE）样祖细胞的2D培养的研究， 来自人胚胎干细胞（hESC）的皮质中间神经元已经显示，CHD 2缺陷 损害中间神经元的发育并改变其功能。尽管这些研究对我们的 基本了解CHD 2在神经发育中的作用，无论是小鼠还是2D神经元培养物 代表了人类大脑发展的全部广度，并在有关人类的知识方面留下了关键的空白。 与CHD 2突变相关的特定细胞和分子机制。为了填补这一空白，我们建议使用 患者来源的诱导多能干细胞（iPSC）分化成人皮质类器官（hCO）。HCO 密切模仿人类前脑的发育，是研究患者特异性的创新模型系统。 导致疾病突变的细胞和分子机制。根据我们的初步数据， 在hCO中的单细胞RNA测序（scRNA-seq）中，我们假设CHD 2突变导致了 神经发育过程中皮质祖细胞的增殖和分化有助于改变 皮层回路和癫痫为了验证我们的假设，我们将1）确定CHD 2对细胞的影响 使用免疫组织化学（Aim 1）和2）描述皮质发育和癫痫中的突变 CHD 2基因突变在神经发育和癫痫中的分子机制 （scRNA-seq + scATAC-seq）（目的2）。建议的研究不仅会增加我们对 儿童癫痫和相关的神经发育障碍，但也提供了更深入的了解新的体外 皮质类器官模型来研究一些致病性变体。据我们所知，这是首次 已经提出了分化成皮质类器官的患者来源的iPSC，以研究人的CHD 2突变。 在我们的模型系统中，我们将能够识别以前未知的细胞和分子 CHD 2相关癫痫的潜在机制为开发更好的治疗方法铺平了道路 干预策略。

PROJECT SUMMARY/ABSTRACT Mutations in CHD2 gene are associated with developmental and epileptic encephalopathy (DEE), a severe form of childhood epilepsy. CHD2 belongs to the chromodomain helicase DNA binding (CHD) family of ATP dependent chromatin remodelers known to play a critical role in neurodevelopment via chromatin organization and gene regulation; and is the only one among 9 other CHD (CHD1-CHD9) family proteins that causes a brain restricted phenotype suggesting its non-redundant role in neurodevelopmental disorders. Studies in mice have shown that knockdown of CHD2 in embryonic cortex decreases the amplification of radial glial cells (RGCs) and promotes the generation of intermediate progenitor cells (IPs). Moreover, Chd2+/- mice, the only known heterozygous mouse line to date to mimic CHD2 haploinsufficiency in human, exhibits reduced number of GABAergic interneurons, disrupts cell proliferation in the developing forebrain and displays a shift in neuronal excitability. Additionally, a study using 2D culture of medial ganglionic eminence (MGE) like progenitors and cortical interneurons derived from human embryonic stem cells (hESCs) has shown that CHD2 deficiency impairs interneuron development and alters its functions. Though these studies have been critical towards our fundamental understanding of the role of CHD2 in neurodevelopment, neither mice nor 2D neuronal cultures represent the full breadth of human brain development and leave a critical gap in knowledge about the human specific cellular and molecular mechanisms associated with CHD2 mutation. To fill this gap, we propose to use patient derived induced pluripotent stem cells (iPSCs) differentiated into human cortical organoids (hCO). hCO closely mimic the development of human forebrain and is an innovative model system to study patient specific cellular and molecular mechanisms underlying a disease-causing mutation. Based on our preliminary data using single cell RNA sequencing (scRNA-seq) in hCO, we hypothesize that CHD2 mutation leads to defects in cortical progenitor proliferation and differentiation during neurodevelopment contributing to altered cortical circuitry and epilepsy. To test our hypothesis, we will 1) determine the cellular impact of CHD2 mutation in cortical development and epilepsy using immunohistochemistry (Aim 1) and 2) delineate the molecular mechanisms of CHD2 mutation in neurodevelopment and epilepsy by performing multiome analysis (scRNA-seq + scATAC-seq) (Aim 2). The proposed study will not only increase our understanding of the childhood epilepsy and related neurodevelopmental disorders but also provide greater insight into novel in vitro cortical organoid models to study a number of pathogenic variants. To our knowledge, this is the first time that patient derived iPSCs differentiated into cortical organoids have been proposed to study CHD2 mutation in humans, and in our model system, we will be able to identify previously unknown cellular and molecular mechanisms underlying CHD2 associated epilepsy paving the way for development of better therapeutic intervention strategies.