Mechanisms of autophagy in TSC1-deficient neural stem cells

项目总结 结节性硬化症是一种遗传性疾病，其特征是 脑部和身体许多其他部位的非癌(良性)肿瘤。这种疾病影响的人数多达 美国有5万人。TSC常累及脑部和室管膜下巨大星形细胞瘤(SEGA)。 会导致严重或危及生命的并发症。TSC1或TSC2基因突变导致 其肿瘤抑制功能的丧失控制mTOR的活性是其发病机制的基础 台糖公司。MTOR刺激S6K和4EBP1的活性，是自噬的负调节因子。然而， 最近的发现，包括我们的发现，揭示了TSC1缺陷神经元中更高的自噬水平和 能量应激下的TSC1缺陷乳腺肿瘤细胞mTOR活性高，提示 TSC1缺陷细胞中自噬的未知功能。为了进一步了解复杂的相互作用 在自噬和mTOR信号之间，我们建立了一种新的双条件基因敲除(CKO) 小鼠模型缺失TSC1和FIP200，自噬诱导复合体中的一个重要成分，在 神经干细胞(NSC)。该模型也为研究自噬机制提供了一种独特的工具。 TSC缺陷神经干细胞中SEN/SEGA的异常发育和肿瘤发生我们的预赛 结果表明，FIP200介导的自噬对于维持高活性是必不可少的。 在TSC1基因缺失的神经干细胞中，mTOR和SEN/SEGA在肿瘤的发生中起关键作用。此外，我们 发现自噬被用来在TSC1缺陷细胞中维持高水平的mTOR活性 糖酵解抑制，这是一个众所周知但尚未完全澄清的现象。这些发现构成了 基于我们的假设，即自噬满足TSC1缺陷的NSCs的高能量需求 有限的能源供应，以维持其高mTOR活性和肿瘤发生。在这件事上 提议，我们将检测诱导自噬的分子事件，以及 自噬调节信号通路和代谢活性以维持高mTOR活性 体外培养TSC1、CKO和TSC1/FIP200双重CKO小鼠的神经干细胞。在AIM2中，我们将采用 抑制TSC1基因缺陷神经干细胞自噬和糖酵解的药理学方法研究 SEN/SEGA对TSC1 CKO小鼠肿瘤预防作用的研究在这些研究结束后，我们将扩大我们的 TSC发病机制的知识，确定候选的关键信号通路和代谢变化， 并开发新的治疗概念，为继续研究治疗一个重要的 无序。

PROJECT SUMMARY Tuberous sclerosis complex (TSC) is a genetic disorder characterized by the growth of numerous noncancerous (benign) tumors in brain and many other parts of the body. This disease affects as many as 50,000 people in US. TSC often affects brain and the tumors of subependymal giant astrocytoma (SEGA) can cause serious or life-threatening complications. The mutations of Tsc1 or Tsc2 gene leading to the loss of their tumor suppressor functions to control the activity of mTOR underlie the pathogenesis of TSC. mTOR stimulates the activity of S6K and 4EBP1 and is a negative regulator of autophagy. However, recent findings, including ours, revealed a higher autophagy level in TSC1-deficient neurons and TSC1-deficient mammary gland tumor cells under energy stress with high mTOR activity, suggesting unrecognized functions of autophagy in TSC1-deficient cells. To further understand the complex interplay between autophagy and mTOR signaling, we established a novel double conditional knockout (cKO) mouse model to delete TSC1 and FIP200, an essential component in autophagy induction complex, in neural stem cells (NSCs). This model also provides a unique tool to study the mechanism of autophagy in abnormal development and tumorigenesis of SEN/SEGA from TSC-deficient NSCs. Our preliminary findings indicated that FIP200 mediated autophagy was indispensable to maintain the high activity of mTOR and was crucial for the tumorigenesis of SEN/SEGA in TSC1-deficient NSCs. In addition, we found that autophagy was used to sustain high mTOR activity in TSC1-deficient cells under the context of glycolysis inhibition, which is a well-known but not totally clarified phenomenon. These findings form the basis of our hypothesis that autophagy satisfies the high energy demanding in TSC1-deficient NSCs with limited energy source supply to maintain their high mTOR activity and tumorigenesis. In Aim1 of this proposal, we will exam the molecular events to induce autophagy, as well as the mechanism of autophagy to regulate signaling pathways and metabolic activity to maintain high mTOR activity using the in vitro cultured NSCs from TSC1 cKO and TSC1/FIP200 double cKO mice. In Aim2, we will adopt pharmacological methods to inhibit autophagy and glycolysis in TSC1-deficient NSCs to test the feasibility of tumor prevention of SEN/SEGA in TSC1 cKO mice. At the end of these studies, we will expand our knowledge of TSC pathogenesis, identify candidate key signaling pathways and metabolic alterations, and develop new therapeutic concepts for continued investigation into the treatment of an important disorder.