Mechanisms of autophagy in TSC1-deficient neural stem cells

项目摘要 多发性硬化症（TSC）是一种遗传性疾病，其特征是大量的 大脑和身体其他部位的非癌性（良性）肿瘤。这种疾病影响多达 美国有5万人。TSC常累及脑及室管膜下巨大星形细胞瘤（SEGA） 可能导致严重或危及生命的并发症。Tsc 1或Tsc 2基因突变导致的 它们控制mTOR活性的肿瘤抑制功能的丧失是肿瘤的发病机制的基础。 TSC。mTOR刺激S6K和4EBP 1的活性，并且是自噬的负调节剂。然而，在这方面， 最近的研究结果，包括我们的研究结果，揭示了TSC 1缺陷神经元的自噬水平更高， TSC1缺陷型乳腺肿瘤细胞在能量应激下具有高mTOR活性，表明 TSC1缺陷细胞中未被认识的自噬功能。为了进一步了解 在自噬和mTOR信号传导之间，我们建立了一种新的双重条件性敲除（cKO） 小鼠模型，以删除TSC 1和FIP 200，自噬诱导复合物中的一种重要成分， 神经干细胞（NSC）。该模型也为研究自噬机制提供了一个独特的工具， 从TSC缺陷的NSC中的SEN/SEGA的异常发育和肿瘤发生。我们的初步 研究结果表明，FIP200介导的自噬对于维持细胞的高活性是必不可少的。 在TSC 1缺陷的NSC中，mTOR对SEN/SEGA的肿瘤发生至关重要。另外我们 发现自噬被用于在TSC1缺陷细胞中维持高mTOR活性， 糖酵解抑制，这是一种众所周知但尚未完全阐明的现象。这些发现构成了 我们假设自噬满足了TSC 1缺陷型NSC的高能量需求， 有限的能量源供应以维持其高mTOR活性和肿瘤发生。在这方面， 建议，我们将检查分子事件诱导自噬，以及机制， 自噬以调节信号传导途径和代谢活性，从而使用免疫调节剂维持高mTOR活性。 体外培养的NSCs来自TSC1 cKO和TSC1/FIP200双cKO小鼠。在目标2中，我们将采用 药理学方法来抑制TSC 1缺陷的NSC中的自噬和糖酵解，以测试可行性 SEN/SEGA在TSC 1 cKO小鼠中的肿瘤预防。在这些研究结束时，我们将扩大我们的 了解TSC发病机制，鉴定候选关键信号通路和代谢改变， 并开发新的治疗概念，以继续研究治疗一个重要的 disorder.

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.