Mechanisms of mTOR-independent axon growth and guidance defects in TSC2 mutant human neurons

越来越多的证据表明，与其他神经发育障碍一样，结节性硬化症(TSC)患者在发育过程中形成的神经元连接也存在错误连接。这些 神经元连接缺陷可能导致TSC的症状，如认知障碍、自闭症和 癫痫。然而，只有从大脑成像才能发现人类神经元的轴突引导有缺陷。 作为研究误导发育中的人类神经元的分子基础的模型，研究还没有 发展起来的。为了直接解决这些基本问题，我们将研究人类神经元的发育 我们从TSC患者来源的细胞中分化出人类诱导多能干细胞(HiPSC)，并 他们的基因工程对应物。利用一系列细胞行为和分子信号分析，我们 将在体外和体内比较TSC2突变神经元和它们的基因校正、同基因对照神经元 三维前脑球体。我们将研究TSC2中mTORC1和mTORC2信号通路的变化 以确定每条信号通路对神经元发育的相对贡献。 而mTOR依赖的蛋白质合成的调节被认为需要在下游 在几个动物模型系统中，吸引轴突引导和排斥轴突引导是否相似尚不清楚。 这些机制在人类神经元发育过程中发挥作用。我们令人惊讶的初步数据表明，TSC2 在生长锥体中独立于mTOR的功能，直接调节细胞骨架，控制轴突引导。 在这个方案中，我们将确定TSC2功能的丧失如何改变人类前脑的发育 神经元，目前的重点是轴突延伸和对关键轴突指导线索的敏感性，这是两个重要的 TSC2功能异常的细胞后果。我们将确定下游的分子机制 并从功能上测试这些信号通路如何导致异常的轴突延伸和 引导提示反应。从长远来看，我们相信我们的研究可能有助于确定关键的可用药靶点 在TSC患者中。

Growing evidence suggests that patients with Tuberous Sclerosis Complex (TSC), like other neuro-developmental disorders, have mis-wiring of neuronal connections that form during development. These defects in neuronal connectivity likely contribute to symptoms of TSC, such as cognitive deficits, autism and epilepsy. However, defective axon guidance by human neurons has only been suggested from brain imaging studies, as models to study the molecular basis for mis-guidance of developing human neurons have not been developed. To directly address these fundamental questions, we will study the development of human neurons that we differentiate from human induced pluripotent stem cells (hiPSCs) from TSC patient-derived cells and their genetically engineered counterparts. Using a series of cell behavior and molecular signaling assays, we will compare TSC2 mutant neurons with their gene-corrected, isogenic control neurons both in vitro and within 3D forebrain spheroids. We will examine changes in mTORC1 and mTORC2 signaling pathways in TSC2 mutant neurons to determine the relative contributions of each signaling pathway to neuronal development. While modulation of mTOR-dependent protein synthesis has been suggested 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. Our surprising preliminary data suggest that TSC2 functions independent of mTOR in growth cones to directly regulate the cytoskeleton to control axon guidance. In this proposal, we will determine how loss of TSC2 function alters the development of human forebrain neurons, with a current focus on axon extension and sensitivity to key axon guidance cues, two important cellular consequences of abnormal TSC2 function. We will determine the molecular mechanisms downstream of TSC2 and test functionally how these signaling pathway contribute to abnormal axon extension and guidance cue responses. Over the long term, we believe our research may help identify key druggable targets in patients with TSC.