Mapping the Time Course of mTORC1-Driven Tumorigenesis in the Developing Brain

摘要 哺乳动物靶标雷帕霉素复合体1(MTORC1)信号通路调节细胞大小和生长 在疾病中经常发生突变，包括在一类被称为 “mTORopathies”一种这样的疾病，结节性硬化症(TSC)，几乎每6000人中就有1人受到影响 以全身良性肿瘤的生长为特征。TSC由一种 编码mTORC1负调控因子的基因发生失活突变，导致蛋白质丢失 功能，mTORC1的过度激活，并通过核糖体的磷酸化促进细胞增殖 蛋白S6(p-S6)和真核细胞翻译起始因子4E结合蛋白1(p-4EBP1)。20%的 TSC患者发生大肿瘤，优先出现在脑室腹侧区域附近- 脑室下区(V-SVZ)，成人大脑中最大的神经干细胞生态位。神经的最新研究进展 V-SVZ中的干细胞表现出不同的转录和功能能力，与细胞的 V-SVZ背腹轴的位置，包括mTORC1的差异激活和易感性 对TSC肿瘤形成的影响。此外，壁龛中不同群体的神经干细胞在有丝分裂中活跃。 在出生前和出生后神经发育的不同时期。在出生后，mTORC1已被证明是 在调节神经干细胞静止中的重要作用，但mTORC1信号在产前V-SVZ中及其作用 胚胎神经干细胞静止期的研究尚未见报道。这个项目的目标是 确定脑脊束背腹轴出现差异mTORC1活动的发育阶段 V-SVZ和失调的mTORC1信号改变细胞命运的程度。的中心假说 这个项目是mTORC1依赖的p-4EBP1的水平，而不是p-S6的水平，决定了产前神经干细胞 细胞的有丝分裂活性和对TSC肿瘤发展的敏感性。为了验证这一假设，一只可诱导的小鼠 模型和药理学试剂将被用于在胚胎发育过程中操纵mTORC1信号。至 绘制出健康和疾病状态下mTORC1活性的差异，每个细胞水平 MTORC1依赖的磷酸化事件将通过成像和流式细胞仪分析进行量化 胚胎神经干细胞。为了跨物种和平台比较结果，mTORC1依赖的信号转导 将在由TSC来源的诱导多能干细胞生长的脑器官模型中进行量化 病人。通过这个项目，我将在我之前的培训的基础上评估细胞信号和分化 人类和小鼠的疾病模型。这项工作的结果将决定mTORC1在调控中的作用 产前神经干细胞在健康和疾病中的命运。在临床上，这项工作将提供更多的了解 导致V-SVZ肿瘤围生期发展的信号失控机制及鉴定 对TSC和其他疾病患者进行治疗干预的潜在合适时间点。

SUMMARY The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway regulates cell size and growth and is frequently mutated in disease, including in a class of neurodevelopmental disorders known as “mTORopathies.” One such disorder, Tuberous Sclerosis Complex (TSC), affects nearly 1 in every 6,000 newborns and is characterized by the growth of benign tumors throughout the body. TSC is caused by an inactivating mutation in the genes that encode the negative regulators of mTORC1, leading to protein loss of function, hyperactivation of mTORC1, and increased cell proliferation through phosphorylation of ribosomal protein S6 (p-S6) and eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1). Twenty percent of TSC patients develop a large tumor that preferentially presents near the ventral region of the ventricular- subventricular zone (V-SVZ), the largest neural stem cell niche in the adult brain. Recent studies of neural stem cells in the V-SVZ revealed variable transcriptional and functional capabilities corresponding to a cell’s position along the dorsoventral axis of the V-SVZ, including differential activation of mTORC1 and susceptibility to TSC tumor formation. Further, different populations of neural stem cells in the niche are mitotically active at different times throughout pre- and postnatal neural development. Postnatally, mTORC1 has been shown to be important in regulating neural stem cell quiescence, but mTORC1 signaling in the prenatal V-SVZ and its effect on quiescence in embryonic neural stem cells has not been investigated. The goal of this project is to determine the developmental stage when differential mTORC1 activity emerges along the dorsoventral axis of the V-SVZ and the extent to which dysregulated mTORC1 signaling alters cell fate. The central hypothesis of this project is that levels of mTORC1-dependent p-4EBP1, but not p-S6, determine both a prenatal neural stem cell's mitotic activity and susceptibility to TSC tumor development. To test this hypothesis, an inducible mouse model and pharmacologic agents will be used to manipulate mTORC1 signaling during embryogenesis. To map the emergence of differences in mTORC1 activity in healthy and disease states, per-cell levels of mTORC1-dependent phosphorylation events will be quantified via imaging and flow cytometry analyses of embryonic neural stem cells. To compare results across species and platforms, mTORC1-dependent signaling will be quantified in cerebral organoid models grown from induced pluripotent stem cells derived from TSC patients. Through this project, I will build upon my prior training to evaluate cell signaling and differentiation in human and mouse models of disease. Results of this work will determine the role of mTORC1 in regulating prenatal neural stem cell fate in health and disease. Clinically, this work will provide a greater understanding of the dysregulated signaling mechanisms that lead to perinatal development of tumors in the V-SVZ and identify potential suitable time points for therapeutic interventions for patients with TSC and other mTORopathies.