Identifying mTOR Dependent Periods During Brain Development

结节性硬化症(TSC)是一种由TSC1或TSC2突变引起的多器官疾病 基因。TSC是一种具有挑战性的疾病，因为有许多涉及的器官系统， 有明显的症状发作、疾病进展的特征，在某些情况下甚至稳定或 称为错构瘤的良性肿瘤的消退。这些不同时间的多个例子 每个器官系统的进程强烈表明，TSC1/TSC2基因控制细胞信号传递 具有组织特异性和发育调节的通路，导致损伤 出现在患者一生中的不同时期。这些途径当然包括mTOR 激酶信号，但在发育过程中关键的上游和下游调节因子 在特定组织中的调控过程仍然知之甚少。多发性硬化症的神经学表现 TSC通常在很小的时候就很严重，包括癫痫，智力残疾，自闭症， 和行为/精神障碍。最近在人类细胞衍生模型系统中的发现表明 TSC的神经发育受到干扰，mTOR信号的适当调节是 与其他哺乳动物相比，在人类大脑中尤为重要。然而，蜂窝 TSC1/2突变和该突变的表型结果之间的联系机制不是很好 明白了。该项目将使用患者来源的细胞和单细胞蛋白质测量和 RNA用于测量人脑不同细胞类型中改变的信号通路，也可用于 这些异常如何影响特定的发育阶段。检查阶段将跨越早期 在出生后的大脑中发现神经前体细胞向更成熟的神经元转化。人类诱因 携带TSC2突变患者的多能干细胞(IPSCs)将被用来产生 定向祖细胞和分化的神经元和神经胶质细胞。我们还将使用新鲜的 切除的人类块茎以及先前切除的固定和储存的人类块茎， 并将使用定制设计的计算管道来比较发展轨迹 从TSC2突变细胞到匹配的对照和更大的已发表数据集。使用尖端电池 成像和分析方案，我们将检验最重要的假设，即患者的结节 在TSC和干细胞衍生的情况下，神经细胞和组织具有mTOR依赖和 与mTOR无关的信号异常是受血统和时间限制的。最后，我们 将定量比较特定发育阶段和谱系中的信号动力学 TSC2突变细胞和来自第二个mTOR病的细胞之间存在重叠，但 临床表现不尽相同，解剖本病不同成分的功能 神经发育和发病机制中的通路及其揭示 携带TSC2或DEPDC5突变的细胞治疗后的代偿信号。

Tuberous Sclerosis Complex (TSC) is a multi-organ disorder caused by mutations in the TSC1 or TSC2 genes. TSC is a challenging disease to approach as there are many involved organ systems, which have distinct profiles of symptom onset, disease progression, and in some cases even stability or regression of the benign tumors known as hamartomas. These multiple examples of distinct time courses in each organ system strongly suggest that the TSC1/TSC2 genes control cell signaling pathways that are tissue specific and developmentally regulated, resulting in lesions that present at different times in the lifetime of the patient. These pathways certainly include mTOR kinase signaling, but critical upstream and downstream regulators of this developmentally regulated process in specific tissues remain poorly understood. The neurological manifestations of TSC are typically severe at very early ages and include epilepsy, intellectual disability, autism, and behavioral/psychiatric disorders. Recent findings in human cell-derived model systems suggest that neural development is disrupted in TSC, and that proper regulation of mTOR signaling is especially important in human brain in comparison to other mammals. However, the cellular mechanisms connecting TSC1/2 mutation and the phenotypic outcomes of this mutation are not well understood. This project will use patient-derived cells and single-cell measurements of protein and RNA to measure altered signaling pathways in various cell types of the human brain and also address how these abnormalities impact specific developmental stages. Examined stages will span early neural progenitor cells to more mature neurons found in the postnatal brain. Human induced pluripotent stem cells (iPSCs) from patients carrying TSC2 mutations will be used to generate lineage-committed progenitors and differentiated neurons and glia. We will also use freshly resected human tubers as well as previously resected human tubers that have been fixed and stored, and will employ custom-designed computational pipelines to compare the developmental trajectories of TSC2-mutant cells to matched controls and larger published datasets. Using cutting edge cell imaging and analysis protocols, we will test the overarching hypothesis that tubers from patients with TSC and stem cell derivative neural cells and tissues have mTOR-dependent and mTOR- independent signaling abnormalities that are lineage- and temporally-restricted. Finally, we will quantitatively compare signaling dynamics in specific developmental stages and lineages between TSC2 mutant cells and cells derived from a second “mTORopathy” with overlapping but non-identical clinical features, to dissect the function of different components of this pathway in neural development and pathogenesis and reveal compensatory signaling after treatment of cells carrying TSC2 or DEPDC5 mutations.