Function of MeCP2 in hESC-derived neurons

描述（由申请人提供）：Rett综合征（RTT）是一种严重的神经发育障碍，由MeCP2突变引起，MeCP2是一种与甲基化DNA结合的转录抑制因子。目前尚不清楚MeCP2突变如何导致RTT中的神经系统功能障碍，并且没有有效的RTT治疗方法。本提案的总体目标是表征由人胚胎干细胞（hESC）体外分化衍生的人神经元的性质，并阐明由MeCP2在人神经元中的损失引起的细胞和生理损伤。我们建议利用不同的NIH注册的人胚胎干细胞系来产生人类神经元，并采用小发夹RNA（shRNA）敲低策略来产生缺乏MeCP2的神经元，并可用于研究MeCP2缺乏对神经元基因表达和基本神经元特征（如形态发生、突触发生、电特性和突触功能）的影响。该提案的总体目标是确定人类神经元中MeCP 2缺乏引起的分子、细胞和生理损伤。提出了三个具体目标：具体目标1将采用HSF 1和HSF 6 hESC系来产生人神经祖细胞（hNPC）和神经元，并使用shRNA敲低和慢病毒感染来产生MeCP 2缺陷或表达RTT中发现的MeCP 2突变形式的hNPC和神经元。具体目标2将确定MeCP 2缺乏对基本神经元特征的影响，例如形态发生、突触发生、电特性和突触功能。具体目标3将分析基因表达和表观遗传状态（例如，DNA甲基化、组蛋白修饰和miRNA表达），并将使用ChIP芯片分析鉴定MeCP2靶基因。通过这种方法，我们将开始连接MeCP 2缺陷直接MeCP 2靶基因及其下游基因的表达的改变，这两者都可能与神经元表型。总的来说，这些实验将提供对MeCP2在人类神经元中与啮齿动物神经元相比的功能的初步了解。如果在小鼠和人类神经元之间观察到MeCP2减少的影响的根本差异，我们的方法将允许分析这些差异的基础。相反，如果没有观察到这种差异，我们的数据将为更广泛地使用小鼠突变体研究RTT提供理论基础。总的来说，拟议实验的结果不仅将为RTT开发新的疾病模型，而且还有助于揭示使用人类神经元的RTT机制，具有识别治疗靶点的潜力。公共卫生相关性：Rett综合征是一种由MeCP2基因突变引起的使人衰弱的发育性大脑疾病。在大脑中，已知MeCP2调节基因表达，但MeCP2突变如何损害Rett综合征的大脑尚不清楚，也没有有效的治疗方法。我们在这里建议使用人类胚胎干细胞产生缺乏MeCP 2的人脑细胞，并研究这种缺乏对这些脑细胞的发育和功能的影响，希望结果有助于更好地了解Rett综合征，并促进Rett综合征治疗的发展。

DESCRIPTION (provided by applicant): Rett syndrome (RTT) is a severe neurodevelopmental disorder that is caused by mutations in MeCP2, a transcriptional repressor that binds to methylated DNA. It is unclear how MeCP2 mutations lead to dysfunction of the nervous system in RTT, and no effective treatments for RTT are available. The overall goal of the present proposal is to characterize the properties of human neurons derived by in vitro differentiation of human embryonic stem cells (hESCs), and to elucidate the cellular and physiological impairments that are caused by the loss of MeCP2 in human neurons. We propose to utilize different NIH-registered hESC lines to produce human neurons, and to employ a small-hairpin RNA (shRNA) knockdown strategy to generate neurons that are deficient in MeCP2 and that can be used to study the effect of MeCP2 deficiency on neuronal gene expression and basic neuronal characteristics, such as morphogenesis, synaptogenesis, electrical properties, and synaptic function. The overall goal of the proposal is to determine the molecular, cellular and physiological impairments that are caused by MeCP2 deficiency in human neurons. Three Specific Aims are proposed: Specific Aim 1 will employ HSF1 and HSF6 hESC lines to produce human neural progenitor cells (hNPCs) and neurons, and to use shRNA knockdown and lentivirus infection to generate hNPCs and neurons that are deficient in MeCP2 or that express mutant forms of MeCP2 found in RTT. Specific Aim 2 will establish the effects of MeCP2 deficiency on fundamental neuronal characteristics, such as morphogenesis, synaptogenesis, electrical properties and synaptic function. Specific Aim 3 will analyze gene expression and epigenetic status (e.g., DNA methylation, histone modifications and miRNA expression) of MeCP2 deficient human neurons, and will identify MeCP2 target genes using ChIP-on-chip analysis. Through this approach, we will begin to link MeCP2 deficiency to alterations in the expression of direct MeCP2 target genes and their downstream genes, both of which may be associated with the neuronal phenotype. Overall, these experiments will provide initial insights into the function of MeCP2 in human neurons compared to rodent neurons. If fundamental differences in the effect of the MeCP2 decrease are observed between mouse and human neurons, our approach will allow analysis of the basis for these differences. If no such differences are observed, conversely, our data will provide a rationale for a wider use of mouse mutants for studying RTT. Collectively, the results of the proposed experiments will not only develop new disease models for RTT, but also help revealing the mechanisms of RTT using human neurons with the potential of identifying therapeutic targets. PUBLIC HEALTH RELEVANCE: Rett syndrome is a debilitating developmental brain disorder caused by mutations in a gene called MeCP2. In the brain, MeCP2 is known to regulate expression of genes, but how MeCP2 mutations impair the brain in Rett syndrome is unclear, and no effective treatments are available. We propose here to use human embryonic stem cells to generate human brain cells with a deficiency in MeCP2, and to study the effect of this deficiency on the development and functions of these brain cells, with the hope that the results will aid a better understanding of Rett syndrome, and facilitate the development of therapies for Rett syndrome.