Elucidating the origins of cortical tuber cells using human brain organoid models of TSC

项目摘要 多发性硬化症（TSC）是一种多系统发育障碍， TSC 1或TSC 2基因。这些基因的蛋白质产物形成一个复合体， mTORC 1信号转导的调节因子。在缺乏功能性TSC 1/2复合物的情况下，mTORC 1信号传导被抑制。 放松管制和组成活跃。虽然TSC的表现可以影响几个不同的器官， 系统，疾病的神经和精神方面是最沉重的照顾者， 最不容易理解。这些包括早发性癫痫，不同程度的智力残疾，以及高 自闭症谱系障碍和其他行为状况的患病率。TSC的一个标志性病理是 皮质结节的存在，皮质结节是皮质中扩大的发育不良的神经元和神经胶质的病灶区域， 在胚胎发育期间形成。皮质结节可以成为癫痫灶，在某些情况下， 在患有顽固性癫痫的患者中手术切除。皮层块茎的大小和数量是可变的 皮质结节负荷增加与更差的结局相关，包括更严重的 癫痫和认知障碍。 该项目的目标是确定TSC 1或TSC 2突变的分子机制。 TSC 2导致皮质块茎细胞的形成。为了做到这一点，我们将使用我们最近建立的人类大脑 TSC的类器官模型，其中我们在TSC 1或TSC 2中设计了功能丧失突变。这些 人脑类器官模型鲁棒地再现了皮质块茎的关键细胞特征， 神经元、反应性星形胶质细胞和巨/气球细胞。此外，我们还观察到， 以TSC脑类器官中的神经元为代价产生神经胶质细胞系细胞， 患者块茎样本的观察结果。在这里，我们将定义改变皮质细胞的分子基础， 发展由于TSC 1/2突变，并研究如何产生块茎细胞影响的功能， 周围的皮层网络。在目标1中，我们将探讨两种可能的假说， 脑类器官中的TSC 1/2突变细胞：1）星形胶质细胞转录程序的过早激活， 干扰正常神经发生和/或2）新生神经元的存活和发育受损。测试 这些假设，我们将使用药理学，shRNA和CRISPRi操作来测试 候选路径。在目标2中，我们将使用不同的策略来操纵mTORC 1信号传导和特异性 下游武器的途径，以测试这些是否可以防止或挽救改变细胞的发展。在 目的3，我们将进行功能分析，以确定皮质结节细胞的存在如何影响 周围皮层网络的活动。这些目标的结果将产生新的见解， 导致皮质块茎形成的分子和细胞机制以及这些细胞最终如何 影响皮层功能

PROJECT SUMMARY Tuberous Sclerosis Complex (TSC) is a multi-system developmental disorder caused by mutations in the TSC1 or TSC2 genes. The protein products of these genes form a complex that is an essential negative regulator of mTORC1 signaling. In the absence of a functional TSC1/2 complex, mTORC1 signaling is deregulated and constitutively active. While the manifestations of TSC can affect several different organ systems, the neurological and psychiatric aspects of the disease are the most burdensome for caregivers and least well understood. These include early-onset epilepsy, varying degrees of intellectual disability, and a high prevalence of autism spectrum disorder and other behavioral conditions. A hallmark pathology of TSC is the presence of cortical tubers, which are focal regions of enlarged, dysplastic neurons and glia in the cortex that form during embryonic development. Cortical tubers can become epileptic foci and in some cases are surgically removed in individuals with intractable seizures. The size and number of cortical tubers is variable between patients and increased cortical tuber load is associated with worse outcomes including more severe epilepsy and cognitive impairment. The goal of this project is to determine the molecular mechanism(s) by which mutations in TSC1 or TSC2 lead to the formation of cortical tuber cells. To do this we will use our recently established human brain organoid models of TSC in which we have engineered loss of function mutations in TSC1 or TSC2. These human brain organoid models robustly reproduce key cellular features of cortical tubers including dysmorphic neurons, reactive astrocytes, and giant/balloon cells. In addition, we have observed a strong bias towards the production of glial-lineage cells at the expense of neurons in TSC brain organoids, which recapitulates observations from patient tuber samples. Here we will define the molecular basis for altered cortical cell development due to TSC1/2 mutations and investigate how the resulting tuber cells impact the function of the surrounding cortical network. In Aim 1 we will explore two potential hypotheses for altered differentiation of TSC1/2 mutant cells in brain organoids: 1) premature activation of astrogenic transcription programs that interfere with normal neurogenesis and/or 2) impaired survival and development of newborn neurons. To test these hypotheses we will use pharmacological, shRNA, and CRISPRi manipulations to test the contribution of candidate pathways. In Aim 2 we will use different strategies to manipulate mTORC1 signaling and specific downstream arms of the pathway to test whether these can prevent or rescue altered cellular development. In Aim 3, we will perform functional analyses to determine how the presence of cortical tuber cells impacts the activity of the surrounding cortical network. Together the results of these aims will generate new insights into the molecular and cellular mechanisms leading to cortical tuber formation and how these cells ultimately impact cortical function.