Function of MeCP2 in hESC-derived neurons

MeCP2 在 hESC 衍生神经元中的功能

• 批准号: 8259221
• 负责人: YI EVE SUN
• 金额: $ 37.19万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259221
• 关键词: Astrocytes; Binding; Brain; Brain Diseases; Candidate Disease Gene; Cell Separation; ChIP-on-chip; Characteristics; Coculture Techniques; DNA; DNA Methylation; Data; Defect; Development; Disease model; Epigenetic Process; Functional disorder; Gene Expression; Gene Targeting; Genes; Glutamates; Goals; HSF1; Human; Impairment; In Vitro; Infection; Lead; Lentivirus Infections; Life; Link; Messenger RNA; Methods; Methyl-CpG-Binding Protein 2; MicroRNAs; Microarray Analysis; Molecular; Morphogenesis; Mus; Mutant Strains Mice; Mutation; Nervous system structure; Neurodevelopmental Disorder; Neurons; Pathway interactions; Phenotype; Physiological; Property; Resistance; Rett Syndrome; Rodent; Synapses; Transcription Repressor/Corepressor; UC06; United States National Institutes of Health; abstracting; base; brain cell; chromatin immunoprecipitation; effective therapy; electrical property; histone modification; human embryonic stem cell; human embryonic stem cell line; insight; mutant; nerve stem cell; protein expression; research study; small hairpin RNA; synaptic function; synaptogenesis; therapeutic target; therapy development

Project Summary/Abstract 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.

项目摘要/摘要 RETT综合征（RTT）是一种严重的神经发育障碍，是由MECP2突变引起的 与甲基化DNA结合的转录阻遏物。目前尚不清楚MECP2突变如何导致功能障碍 RTT中的神经系统的，并且没有有效的RTT治疗方法。总体目标 目前的建议是表征通过人类体外分化得出的人类神经元的特性 胚胎干细胞（HESC），并阐明由细胞和生理障碍 人类神经元中MECP2的丧失。我们建议利用不同的NIH注册的hESC线生成 人类神经元，并采用小发pin RNA（shRNA）敲低策略来产生神经元 缺乏MECP2，可用于研究MECP2缺乏对神经元基因表达的影响 和基本的神经元特征，例如形态发生，突触发生，电性能和 突触功能。该提案的总体目标是确定分子，细胞和生理 由人类神经元中MECP2缺乏引起的损伤。提出了三个具体目标： 特定的目标1将采用HSF1和HSF6 HESC系来产生人类神经祖细胞（HNPC） 和神经元，并使用shRNA敲低和慢病毒感染产生HNPC和神经元 RTT中发现的MECP2的MECP2缺乏或表达突变体形式。具体目标2将确定 MECP2缺乏对基本神经元特征的影响，例如形态发生， 突触发生，电性能和突触功能。特定目标3将分析基因表达和 MECP2缺乏的表观遗传状态（例如DNA甲基化，组蛋白修饰和miRNA表达） 人类神经元，并将使用芯片片分析鉴定MECP2靶基因。通过这种方法，我们 将开始将MECP2缺乏症与直接MECP2靶基因表达的变化联系起来 下游基因，两者都可能与神经元表型有关。总体而言，这些实验 与啮齿动物神经元相比，将提供对MECP2功能的初步见解。如果 在小鼠和人之间观察到MECP2降低效应的基本差异 神经元，我们的方法将允许分析这些差异的基础。如果没有这样的差异 相反，我们的数据将为更广泛使用小鼠突变体用于研究RTT提供理由。 总的来说，提出的实验的结果不仅将开发RTT的新疾病模型，而且还将 还可以帮助使用人类神经元来揭示RTT的机制，以鉴定治疗性 目标。