Modeling Neural Development Using Human iPSCs from TSC Patients

描述（申请人提供）：神经系统的发育需要神经元的适当分化、迁移和形态发生。单个神经元的形态分化和组成人类神经系统的数万亿神经元连接的组装是通过轴突和树突的引导延伸发生的。神经元发育环境中的分子引导线索引导位于延伸轴突和树突尖端的神经元生长锥。mtor介导的生长锥内新蛋白的局部合成已成为控制轴突引导的重要机制。参与局部蛋白质合成的基因突变是几种人类自闭症谱系障碍的原因，包括脆性X综合征和结节性硬化症（TSC）。虽然已知在一些动物模型系统中，mtor依赖性蛋白合成的调节在吸引和排斥轴突引导的下游都是必需的，但尚不清楚类似的机制是否在发育中的人类神经元中起作用。该提案将首先测试来自人诱导多能干细胞（hiPSCs）的人视网膜神经节细胞（RGCs）是否使用mtor介导的蛋白质合成来响应正、负轴突引导信号。在Aim 2中，我们将测试TSC1/TSC2复合物的作用，TSC1/TSC2复合物是mTOR的关键上游负调节因子。为此，我们将通过重编程来自TSC患者的成纤维细胞来产生新的hiPSCs系。虽然这项提议将重点放在RGCs上，但使用这些新的hiPSC系可以研究各种其他细胞和神经元类型。因此，这些重要的新细胞系将成为许多研究人员的宝贵资源，并将通过WiCell进行分发。在Aim 3中，我们将测试TSC hiPSCs衍生的RGCs是否对Aim 1中测试的轴突引导线索表现出异常反应。

DESCRIPTION (provided by applicant): The development of the nervous system requires the proper differentiation, migration and morphogenesis of neurons. The morphological differentiation of individual neurons and assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. Molecular guidance cues in the environment of developing neurons guide neuronal growth cones at the tips of extending axons and dendrites. mTOR-mediated local synthesis of new proteins within growth cones as emerged as an important mechanism that controls axon guidance. Mutations in genes involved local protein synthesis are responsible for several human autism spectrum disorders, including Fragile X syndrome and Tuberous Sclerosis Complex (TSC). While modulation of mTOR-dependent protein synthesis is known to be required downstream of both attractive and repulsive axon guidance in several animal model systems, it is unknown if similar mechanisms function in developing human neurons. This proposal will first test whether human Retinal Ganglion Cells (RGCs) derived from human induced pluripotent stem cells (hiPSCs) use mTOR-mediated protein synthesis to respond to positive and negative axon guidance cues. In Aim 2 we will test the role of the TSC1/TSC2 complex, which is a key upstream negative regulator of mTOR. For this we will generate new lines of hiPSCs by reprograming fibroblast cells from patients with TSC. While this proposal will focus on RGCs, a wide variety of other cell and neuronal types can be studied using these new hiPSC lines. Therefore, these important new cell lines will be a valuable resource for many investigators and will be made available for distribution through WiCell. In Aim 3 we will test whether RGCs derived from TSC hiPSCs exhibit abnormal response to axon guidance cues tested in Aim 1.