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

使用患者特异性iPSCs研究神经胶质对RTT发病机制的贡献摘要Rett综合征（RTT）是一种主要影响女性的自闭症谱系障碍（ASD）。X连锁MECP 2（甲基-CpG结合蛋白2）基因突变的鉴定是RTT的原因，这导致了用于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（iPSC）已从RTT患者中产生，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 iPSC的研究都集中在研究不存在星形胶质细胞的神经元病理学。作为大脑中的另一种主要细胞类型，星形胶质细胞也表达MeCP 2。从RTT小鼠模型的工作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，我们假设当与神经元一起培养时，从突变RTT-iPSC系分化的星形胶质细胞将显著损害神经元的生长和成熟。在目前的建议中，我们将集中我们的注意力揭示神经胶质细胞的RTT病理在神经元/星形胶质细胞共培养系统的贡献。为了最小化由不同个体产生的iPSC系之间的不同遗传背景引起的表型变异，我们已经建立了来自相同女性RTT患者（携带常见或罕见RTT突变）皮肤细胞的几对等基因iPSC系，其克隆表达MECP 2基因的野生型拷贝或突变体拷贝（但不是两者）。使用这些独特的工具，我们计划测试1）突变iPSC衍生的星形胶质细胞是否可以损害神经元/星形胶质细胞共培养物中野生型iPSC衍生的神经元的生长和成熟; 2）野生型iPSC衍生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物中突变型iPSC衍生的神经元的生长和成熟缺陷;以及3）潜在的星形胶质细胞影响是否由细胞-细胞接触或分泌分子介导。更好地理解胶质细胞对RTT病理学的贡献不仅可以深入了解疾病机制，还可以为未来使用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，鉴于最近的证据表明，MECP 2可能在许多自闭症患者的基因组水平和表达水平上都发生了改变，在研究RTT中所吸取的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。 公共卫生相关性：使用患者特异性iPSCs项目研究神经胶质对RTT病理发生的贡献X连锁人类MECP 2基因（甲基-CpG结合蛋白2）中的叙述性突变导致Rett综合征（RTT），这是一种主要影响女性的自闭症谱系发育障碍。为了充分理解RTT的分子机制，重要的是研究星形胶质细胞在人类细胞疾病进展中的作用。该项目的成功完成将大大推进对RTT疾病机制的理解，并促进未来治疗RTT的疗法的开发。首先，该提案旨在研究人类RTT神经元和星形胶质细胞中的RTT发病机制，这是RTT患者中受影响的两种主要细胞类型。这是必要的，因为老鼠不是人类。在小鼠研究中获得的任何知识或在小鼠模型中显示有效性的任何治疗都应在人类系统中得到验证。其次，该提案旨在揭示神经胶质对RTT病理学的贡献，这将开辟一个尚未在人类细胞中探索的研究领域。这对于治疗RTT是非常重要的，因为如果神经胶质缺陷不治疗，旨在纠正神经元缺陷的策略可能无法有效地工作。第三，该提案将建立RT诱导多能干细胞（iPSC）衍生的神经元和星形胶质细胞作为未来药物筛选的体外平台，这是将实验室发现转化为床旁治疗的下一步。最后，由于RTT和自闭症谱系障碍之间的临床特征有相当大的重叠，以及最近关于自闭症患者MECP 2改变的报道，研究RTT的经验教训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. PUBLIC HEALTH RELEVANCE: Studying the Glial Contribution to RTT Pathologenesis Using Patient-Specific iPSCs Project Narrative Mutations in the X-linked human MECP2 gene (methyl-CpG binding protein 2) cause Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To fully understand the molecular mechanism of RTT, it is important to study the role of astrocytes in disease progression in human cells. The successful completion of this project will significantly advance the understanding of RTT disease mechanism and facilitate future development of therapies to treat RTT. First, the proposal aims at studying RTT pathogenesis in human RTT neurons and astrocytes, the two major cell types affected in RTT patients. This is necessary because mice are not humans. Any knowledge obtained in mouse studies or any treatment showing efficacy in mouse models should be validated in human systems. Second, the proposal aims at revealing the glial contribution to RTT pathology, which will open up a research area that has not been explored in human cells. This is very important for treating RTT, because strategies aimed at correcting neuronal defects may not work efficiently if glial defects are left untreated. Third, the proposal will establish RT induced pluripotent stem cell (iPSC)-derived neurons and astrocytes as in vitro platforms for future drug screens, which is the next step to translate discoveries at the bench side to treatments at the bed side. Finally, because of the considerable overlap in clinical features between RTT and autism spectrum disorders and the recent reports of MECP2 alterations in autism patients, the lessons learned studying RTT might also benefit the general understanding of autism.