Identifying mTOR Dependent Periods During Brain Development

多发性硬化症（TSC）是由TSC 1或TSC 2突变引起的多器官疾病 基因. TSC是一种具有挑战性的疾病，因为涉及许多器官系统， 有不同的症状发作，疾病进展，在某些情况下，甚至稳定或 称为错构瘤的良性肿瘤的消退。这些不同时间的例子 每个器官系统的病程强烈表明，TSC 1/TSC 2基因控制细胞信号传导， 组织特异性和发育调节的途径，导致病变， 在患者一生中的不同时间出现。这些途径当然包括mTOR 激酶信号，但关键的上游和下游调控这一发展 在特定组织中的调控过程仍然知之甚少。的神经学表现 TSC通常在很小的年龄就很严重，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， 神经发育在TSC中被破坏，并且mTOR信号的适当调节是 与其他哺乳动物相比，在人类大脑中尤其重要。然而，Cellular TSC 1/2突变与该突变的表型结果之间的联系机制尚不清楚 明白该项目将使用患者来源的细胞和蛋白质的单细胞测量， RNA来测量人脑各种细胞类型中改变的信号通路， 这些异常是如何影响特定的发育阶段的检查阶段将跨越早期 神经前体细胞到出生后大脑中发现的更成熟的神经元。人诱导 来自携带TSC 2突变的患者的多能干细胞（iPSC）将用于产生 谱系定向祖细胞和分化的神经元和神经胶质。我们还将使用新鲜的 切除的人块茎以及先前切除的已经固定和储存的人块茎， 并将采用定制设计的计算管道来比较发展轨迹 TSC 2突变细胞与匹配的对照组和更大的已发表数据集进行比较。使用尖端电池 成像和分析协议，我们将测试的总体假设，从病人的块茎 对于TSC和干细胞衍生物，神经细胞和组织具有mTOR依赖性， mTOR-非依赖性信号传导异常，其是谱系和时间限制性的。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 在TSC 2突变细胞和来源于第二种“mTOR病”的细胞之间， 不相同的临床特征，解剖不同组成部分的功能， 神经发育和发病机制的途径，并揭示 在处理携带TSC 2或DEPDC 5突变的细胞后的补偿信号传导。

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.