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

摘要Rett综合征（RTT）是一种主要影响女性的自闭症谱系障碍（ASD）。鉴定出x连锁MECP2（甲基- cpg结合蛋白2）基因突变是RTT的病因，从而建立了用于研究疾病机制的小鼠模型。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患者身上产生，作为体外人类模型，以验证和扩展从小鼠研究中获得的知识。目前，所有涉及RTT iPSCs的研究都集中在缺乏星形胶质细胞的情况下研究神经元病理。作为大脑中另一种主要的细胞类型，星形胶质细胞也表达MeCP2。在RTT小鼠模型中，有强有力的证据表明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RTT-iPSC细胞系分化的星形胶质细胞与神经元一起培养时，会显著损害神经元的生长和成熟。在目前的建议中，我们将重点关注揭示神经元/星形胶质细胞共培养系统中神经胶质对RTT病理的贡献。为了尽量减少由不同个体产生的不同遗传背景引起的表型变异，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（但不是两者都表达）。利用这些独特的工具，我们计划测试1)突变型ipsc衍生的星形胶质细胞是否会损害野生型ipsc衍生的神经元在神经元/星形胶质细胞共培养中的生长和成熟；2)野生型ipsc来源的星形胶质细胞是否可以在神经元/星形胶质细胞共培养中修复突变型ipsc来源的神经元生长和成熟缺陷；3)星形胶质细胞的潜在影响是通过细胞间接触还是分泌分子介导的。更好地了解神经胶质对RTT病理的贡献不仅有助于深入了解疾病机制，而且为未来使用RTT ipsc衍生的神经元和/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可能在基因组水平和表达水平上都发生了改变，研究RTT的经验教训和实验方法也可能有助于对自闭症的普遍理解。

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.