Regulation of Neurogenesis in TSC by mTORC1 and mTORC2

描述(申请人提供)：结节性硬化症(TSC)是一种由TSC1或TSC2基因突变引起的遗传性疾病。患者通常有严重的中枢神经系统症状，包括癫痫、智能障碍和自闭症。TSC患者大脑中的皮质畸形(“结节”)通常被认为是导致这些严重的神经表现的原因。之前使用人类组织、转基因小鼠和斑马鱼模型进行的研究引导我们将重点放在神经前体/干细胞上。神经前体细胞的异常可以很好地解释TSC患者脑内细胞数量增加、非典型分化和神经元异位的原因。一些信号通路在TSC中被认为是失调的，尽管对mTOR激酶的控制似乎是最关键的。最近的发现支持在两个不同的蛋白质复合体mTORC1和mTORC2中mTOR的异常。这些改变是不同的，因为我们发现TSC1缺陷的小鼠神经前体细胞似乎增加了mTORC1信号，但减少了mTORC2信号。然而，mTORC1和mTORC2信号在TSC发病机制中的相对作用尚不清楚。这项建议的首要目标是确定mTORC1和mTORC2信号在TSC发病机制中的作用。这一目标直接针对NINDS战略计划，寻求增加我们对正常大脑和神经系统如何发育的知识，这些过程在疾病中是如何被颠覆的，以及这些信息是否可以导致更好的神经疾病治疗。为了实现这一目标，我们将使用以小鼠TSC1基因失活为特征的转基因小鼠模型，以及我们从由于TSC1或TSC2基因功能突变而导致的TSC患者中产生的诱导多能干细胞(IPSC)。对小鼠和人类神经前体细胞的直接比较是这一提议的一个关键方面，因为在大脑中控制mTOR信号的根本差异可能出现在进化过程中。本项目的具体目标是1)确定TSC1/2突变的人和TSC1缺失的神经前体细胞是否能产生过多的神经元；2)确定TSC1缺失和TSC2缺失的人和小鼠神经前体细胞是否能够获得下层或上层的身份，以及抑制mTORC1信号是否可以恢复这些身份；3)。确定mTORC2信号的降低是否足以导致小鼠和人类神经前体细胞的异常分化。这些研究将大大增加我们对小鼠TSC1和人类TSC基因功能的理解。我们的建议有望定义小鼠和人类细胞中异常的mTORC1和mTORC2信号通路，并导致为TSC患者开发更有效的治疗方法。

DESCRIPTION (provided by applicant): Tuberous Sclerosis Complex (TSC) is a genetic disease caused by mutation of the TSC1 or TSC2 genes. Patients frequently have severe CNS manifestations including epilepsy, intellectual disabilities and autism. Cortical malformations ("tubers") in the brains of patients with TSC are generally accepted as responsible for these severe neurological manifestations. Previous studies using human tissues and transgenic mouse and zebrafish models have led our focus on neural progenitor/stem cells. Abnormalities of neural progenitor cells may readily explain the increased cell number, atypical differentiation and ectopic position of neurons seen within the brain of patients with TSC. Several signaling pathways are known to be dysregulated in TSC though control of the mTOR kinase appears most critical. Recent findings support abnormalities of mTOR within two distinct protein complexes, mTORC1 and mTORC2. These alterations differ as we have found that Tsc1- deficient mouse neural progenitor cells appear to have increased mTORC1 but decreased mTORC2 signaling. However, the relative contribution of mTORC1 and mTORC2 signaling during the pathogenesis of TSC remains poorly understood. The overarching goal of this proposal is to determine the role of mTORC1 and mTORC2 signaling in the pathogenesis of TSC. This goal directly addresses the NINDS Strategic Plan by seeking to increase our knowledge of how the normal brain and nervous system develop, how these processes are subverted in disease and whether this information can lead to better treatments of neurological disorders. To achieve this goal we will use both transgenic mouse models featuring the inactivation of the mouse Tsc1 gene as well as induced pluripotent stem cells (iPSC) we have generated from patients with TSC due to loss of function mutations of the TSC1 or TSC2 genes. The direct comparison of mouse and human neural progenitor cells is a key aspect of this proposal as fundamental differences in the control of mTOR signaling in the brain likely arose during evolution. The Specific Aims of this project are to 1) Determine if TSC1/2-mutant human and Tsc1-deficient mouse neural progenitor cells generate excessive numbers of neurons; 2) Determine if neurons generated from TSC1-deficient and TSC2-deficient human and Tsc1-deficient mouse neural progenitors are able to acquire lower or upper layer identity and whether inhibition of mTORC1 signaling can restore these identities; 3). Determine if decreased mTORC2 signaling alone is sufficient to cause abnormal differentiation of mouse and human neural progenitor cells. These studies should greatly increase our understanding of the function of the mouse Tsc1 and human TSC genes. Our proposal is expected to define abnormal mTORC1 and mTORC2 signaling pathways in mouse and human cells and lead to the development of much more effective therapies for patients with TSC.