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是一种结合甲基化DNA的转录抑制因子。目前尚不清楚Mecp2突变如何导致RTT神经系统功能障碍，也没有有效的RTT治疗方法。本提案的总体目标是表征人类胚胎干细胞（hESCs）体外分化产生的人类神经元的特性，并阐明由于人类神经元中Mecp2缺失而引起的细胞和生理损伤。我们建议利用不同的nih注册hESC细胞系产生人类神经元，并采用小发夹RNA （shRNA）敲低策略产生Mecp2缺失的神经元，并可用于研究Mecp2缺失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、电学性质和突触功能）的影响。总的来说，这些研究将导致hESCs体外分化衍生的人类神经元特性的定义，以及Mecp2缺陷对人类神经元的影响，从而允许基于人类神经元的RTT疾病模型的发展。目的1将确定4种不同的nih批准的hESC系（HSF1 （x/y）、HSF6 （x/x）、H1 （x/y）和H9 (x/x)）是否具有相似或不同的神经元亚型特异性分化潜能，以确定不同的hESC系是否针对不同的细胞命运进行了预编程。这一目标将提供通过体外分化获得的人类神经元的全面分析，这不仅将阐明人类神经元的基本特性，如突触传递，而且还将为使用这些神经元来研究人类疾病奠定基础。目的2将通过慢病毒介导的shRNA敲低方法，结合微阵列分析，建立和表征Mecp2水平降低的人类神经元，以揭示这些神经元中的基因表达。目的3将验证Mecp2的缺失是否改变了神经元前体细胞向不同亚型神经元的分化和/或这些神经元的功能特性。总的来说，我们相信这些实验将提供Mecp2在人类神经元中与啮齿动物神经元相比的功能的初步见解。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，我们的研究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来研究RTT提供理论依据。此外，我们的实验将建立人类神经元RTT的模型系统，这可能有助于开发新的RTT药物和治疗方法。

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. Overall, these studies will lead to a definition of the properties of human neurons derived by in vitro differentiation of hESCs and of the effects of the Mecp2 deficiency on human neurons, permitting the development of RTT disease models that are based on human neurons. Three specific aims are proposed: Aim 1 will determine whether 4 different NIH-approved hESC lines (HSF1 (x/y), HSF6 (x/x), H1 (x/y), and H9 (x/x)) have similar or different neuronal subtype-specific differentiation potentials to address whether different hESC lines are pre-programmed for distinct cell fates. This aim will provide a comprehensive analysis of human neurons derived by in vitro differentiation, which will not only shed light onto fundamental neuronal properties such as synaptic transmission in specifically human neurons, but also form the basis for the use of such neurons to investigate human diseases. Aim 2 will establish and characterize human neurons with decreased levels of Mecp2 via a lentiviral-mediated shRNA knockdown approach in combination with micro- array analyses to reveal gene expression in these neurons. Aim 3 will test the hypothesis that deletion of Mecp2 alters the differentiation of neuronal precursor cells into different subtypes of neurons and/or the functional properties of these neurons. Overall, we believe that 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 studies 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. Moreover, our experiments will establish a model system for RTT in human neurons that might be helpful in the development of new drugs and therapeutic approaches for RTT. 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.