Uncovering mechanisms of CHD2-associated epilepsy using human cortical organoids

利用人类皮质类器官揭示 CHD2 相关癫痫的机制

基本信息

批准号：
10791545
负责人：
Vanesa Nieto Estevez
金额：
$ 41.25万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-15 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10791545
关键词：
Address Affect Animal Model Biological Models Brain CHD1 gene CHD4 gene CHD7 gene Catalogs Cell Proliferation Cells Chromatin DNA Binding Data Defect Development Developmental Delay Disorders Disease Embryo Enzymes Epilepsy Exhibits Family Forebrain Development Functional disorder Gene Expression Regulation Generations Genes Goals Heterozygote Histones Human Immunohistochemistry Impairment In Vitro Intellectual functioning disability Interneurons Intervention Knowledge Medial Modeling Molecular Mus Mutation NOR Mouse Neocortex Neurodevelopmental Disorder Neuroglia Neurons Nucleosomes Organoids Pathogenicity Patient Recruitments Patients Phenotype Play Proliferating Proliferation Marker Prosencephalon Protein Family Radial Role Testing Therapeutic Therapeutic Intervention Time Variant autism spectrum disorder cell type childhood epilepsy chromatin remodeling disease phenotype disease-causing mutation epileptic encephalopathies helicase human embryonic stem cell induced pluripotent stem cell innovation insight knock-down mouse model multiple omics mutant neocortical nerve stem cell nervous system disorder neural neurodevelopment neuronal excitability new technology novel progenitor single cell analysis single-cell RNA sequencing stem cell differentiation stem cells transcription factor

项目摘要

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.

项目概要/摘要 CHD2 基因突变与发育性癫痫性脑病 (DEE) 相关，这是一种严重的形式儿童癫痫症。 CHD2 属于 ATP 染色质结构域解旋酶 DNA 结合 (CHD) 家族已知依赖染色质重塑剂通过染色质组织在神经发育中发挥关键作用和基因调控；并且是其他 9 种 CHD (CHD1-CHD9) 家族蛋白中唯一一种会导致大脑限制表型表明其在神经发育障碍中的非冗余作用。对小鼠的研究有研究表明，胚胎皮质中 CHD2 的敲低会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，Chd2+/- 小鼠是唯一已知的迄今为止，模拟人类CHD2单倍体不足的杂合小鼠系表现出减少的数量 GABA能中间神经元，破坏发育中前脑的细胞增殖，并显示神经元的变化兴奋性。此外，一项使用内侧神经节隆起 (MGE) 的 2D 培养（如祖细胞和来自人类胚胎干细胞 (hESC) 的皮质中间神经元表明，CHD2 缺陷损害中间神经元发育并改变其功能。尽管这些研究对我们来说至关重要对 CHD2 在神经发育中的作用的基本了解，无论是小鼠还是 2D 神经元培养物代表了人类大脑发育的全部范围，并在关于人类的知识上留下了一个关键的空白与 CHD2 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) 分化为人类皮质类器官 (hCO)。二氧化碳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致病突变的细胞和分子机制。根据我们的初步数据使用 hCO 中的单细胞 RNA 测序 (scRNA-seq)，我们假设 CHD2 突变导致缺陷神经发育过程中皮质祖细胞的增殖和分化导致改变皮质电路和癫痫。为了检验我们的假设，我们将 1) 确定 CHD2 的细胞影响使用免疫组织化学（目标 1）和 2）描述皮质发育和癫痫的突变通过多组分析研究CHD2突变在神经发育和癫痫中的分子机制（scRNA-seq + scATAC-seq）（目标 2）。拟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，而且还提供了对新型体外实验的更深入的了解皮质类器官模型来研究许多致病变异。据我们所知，这是第一次已提出将患者来源的 iPSC 分化为皮质类器官来研究 CHD2 突变人类，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 CHD2相关癫痫的潜在机制为开发更好的治疗方法铺平道路干预策略。