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

项目总结 结节性硬化症(TSC)是一种由基因突变引起的多系统发育障碍 TSC1或TSC2基因。这些基因的蛋白质产物形成了一种复合体，这是一种本质上的负面影响 MTORC1信号调节因子。在没有功能的TSC1/2复合体的情况下，mTORC1信号是 放松管制，在宪法上很活跃。而TSC的表现可以影响到几个不同的器官 系统，这种疾病的神经和精神方面是照顾者和 最不为人所知。这些包括早发性癫痫，不同程度的智力残疾，以及 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，这是皮质中扩大的、发育不良的神经元和胶质细胞的焦点区域， 在胚胎发育过程中形成。皮质结节可成为癫痫灶，在某些情况下 对于难治性癫痫发作的患者，手术切除。皮质结节的大小和数量是可变的 患者和皮质结节负荷的增加与更糟糕的结果相关，包括更严重 癫痫和认知障碍。 这个项目的目标是确定TSC1或TSC1基因突变的分子机制(S) TSC2诱导皮层块茎细胞的形成。为了做到这一点，我们将使用我们最近建立的人脑 我们设计了TSC1或TSC2功能突变丧失的TSC器官模型。这些 人脑器官模型强健地再现了包括畸形在内的皮质结节的关键细胞特征 神经元、反应性星形胶质细胞和巨细胞/气球细胞。此外，我们还观察到一种强烈的偏向 以TSC脑器官中神经元为代价产生胶质细胞系细胞，这是重述 对患者块茎样本的观察。在这里，我们将定义改变的皮质细胞的分子基础 TSC1/2突变所致的发育，并研究由此产生的结节细胞如何影响 围绕着大脑皮层网络。在目标1中，我们将探索改变分化的两个潜在假说 脑有机体中的TSC1/2突变细胞：1)导致星形转录程序过早激活， 干扰正常的神经发生和/或2)损害新生神经元的存活和发育。为了测试 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处理mTORC1信号和特定的 以测试它们是否可以阻止或挽救改变的细胞发育。在……里面 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 周围皮质网络的活动。这些目标的结果加在一起将产生新的见解 导致皮质块茎形成的分子和细胞机制以及这些细胞最终是如何 影响皮质功能。

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.