Studying the glial contribution to RTT pathogenesis using patient-specific iPSCs

描述(由申请人提供)：使用患者特有的IPSCs研究神经胶质在RTT发病中的作用抽象Rett综合征(RTT)是一种主要影响女性的自闭症谱系障碍(ASD)。X-连锁MECP2(甲基-CpG结合蛋白2)基因突变被鉴定为RTT的原因，导致了研究疾病机制的小鼠模型的建立。然而，小鼠模型在模拟人类RTT突变和药物筛选方面存在局限性。诱导多能干细胞(IPSCs)已经从RTT患者中产生，作为一个体外人类模型来验证和扩展从小鼠研究中获得的知识。目前，所有涉及RTT IPSCs的研究都集中在研究星形胶质细胞缺失时的神经元病理学。作为大脑中的另一种主要细胞类型，星形胶质细胞也表达MeCP2。有强有力的证据表明，在RTT小鼠模型中，星形胶质细胞在疾病进展中起着关键作用。因此，我们假设，从突变的RTT-IPSC系分化的星形胶质细胞与神经元一起培养时，将显著损害神经元的生长和成熟。在目前的提案中，我们将重点揭示神经元/星形胶质细胞共培养系统中胶质细胞对RTT病理的贡献。为了最大限度地减少不同个体产生的不同遗传背景造成的表型差异，我们从相同的女性RTT患者(携带常见或罕见的RTT突变)皮肤细胞中建立了几对等基因的IPSC系，这些细胞克隆表达MECP2基因的野生型拷贝或突变拷贝(但不是两者)。利用这些独特的工具，我们计划测试1)突变型iPSC来源的星形胶质细胞是否会在神经元/星形胶质细胞共培养中损害野生型IPSC来源的神经元的生长和成熟；2)野生型IPSC来源的星形胶质细胞是否可以挽救神经元/星形胶质细胞共培养中突变型IPSC来源的神经元的生长和成熟缺陷；以及3)潜在的星形胶质细胞的影响是通过细胞间的接触还是由分泌的分子介导的。更好地了解胶质细胞在RTT病理中的作用不仅有助于深入了解RTT的发病机制，还将为未来利用RTT iPSC来源的神经元和/或星形胶质细胞进行药物筛选奠定基础。此外，鉴于最近有证据表明，在许多自闭症患者中，MECP2可以在基因组水平和表达水平上发生改变，因此在研究RTT时吸取的经验教训和使用的实验方法也可能有助于对自闭症的一般理解。

DESCRIPTION (provided by applicant): Studying the Glial Contribution to RTT Pathogenesis Using Patient-Specific iPSCs Abstract Rett syndrome (RTT) is an autism spectrum disorder (ASD) that predominantly affects females. The identification of mutations in the X-linked MECP2 (methyl-CpG binding protein 2) gene as the cause of RTT has led to the creation of mouse models for studying disease mechanism. However, mouse models have limitations in mimicking human RTT mutations and in drug screening. Induced pluripotent stem cells (iPSCs) have been generated from RTT patients as an in vitro human model to validate and extend knowledge obtained from mouse studies. Currently, all studies involving RTT iPSCs have focused on studying neuronal pathologies in the absence of astrocytes. As the other major cell type in the brain, astrocytes also express MeCP2. There is strong evidence, from work in RTT mouse models, that astrocytes play a critical role in disease progression. Thus, we hypothesize that astrocytes differentiated from mutant RTT-iPSC lines will significantly impair neuronal growth and maturation when cultured together with neurons. In the current proposal, we will focus our attention on revealing the glial contribution to RTT pathology in a neuron/astrocyte co-culture system. To minimize phenotypic variation caused by different genetic backgrounds across iPSC lines generated from different individuals, we have established several pairs of isogenic iPSC lines from the same female RTT patients (carrying either common or rare RTT mutations) skin cells that clonally express either the wild type copy or the mutant copy (but not both) of the MECP2 gene. Using these unique tools, we plan to test 1) whether mutant iPSC-derived astrocytes may impair the growth and maturation of wild type iPSC-derived neurons in the neuron/astrocyte co-culture; 2) whether wild type iPSC-derived astrocytes may rescue the growth and maturation defects of mutant type iPSC-derived neurons in the neuron/astrocyte co-culture; and 3) whether the potential astrocyte influnce is mediated by cell-cell contact or secreted molecules. Better understanding of the glial contribution to RTT pathology will not only provide insight into disease mechanisms, but also lay the groundwork for future drug screens using RTT iPSC-derived neurons and/or astrocytes. Furthermore, in light of the recent evidence that MECP2 may be altered at both the genomic level and the expression level in many autism patients, the lessons learned and the experimental approaches used in studying RTT might also benefit the general understanding of autism.