Roles of autophagy-mediated pathways in the pathogenesis and treatment of TSC

描述（由申请人提供）：TSC患者发生多系统疾病，包括脑、心脏、皮肤和肾脏的良性肿瘤。到10岁时，80%的TSC儿童患有肾血管平滑肌脂肪瘤和/或肾囊肿。哺乳动物雷帕霉素靶蛋白复合体1（mTOR 1）在结节性硬化症复合体（TSC）中被激活，是细胞生长、细胞代谢和自噬的主要调节因子。使用TORC 1抑制剂治疗可部分减少TSC相关脑和肾脏病变的大小，但当治疗停止时，它们会重新生长。 我们的中心假设是，自噬和细胞代谢的失调在TSC的发病机制中起着关键作用，并在TSC相关的肾脏病变的TORC 1靶向治疗的反应。 在目标1和2中，我们将检验以下假设：TSC 2缺陷细胞中低水平的自噬导致“代谢饥饿”表型，使TSC 2缺陷细胞对进一步的自噬抑制超敏感。根据这一假设，我们 已经发现抑制自噬诱导代谢失调并降低TSC 2缺陷细胞的体内生长。在目标3中，我们将讨论p62/螯合体1依赖的信号网络促进TSC 2缺陷细胞的生长和存活的假设。与这一假设一致，我们发现p62在人血管平滑肌脂肪瘤和其他TSC 2缺陷细胞中积累是低自噬的结果，并且p62的shRNA下调抑制TSC 2缺陷细胞的体内生长。我们的体内策略（目的2和3）包括TSC 2缺陷型血管平滑肌脂肪瘤衍生细胞和Tsc 2 +/-小鼠中的肾囊肿/囊腺瘤定量。 在整个提案中，我们将利用创新的最先进的技术，包括使用“海马”系统实时监测生物能量参数，与Broad研究所合作进行代谢组学分析，高通量合成致死性药物筛选，以及血管平滑肌脂肪瘤细胞的体内生物发光检测。将生成新试剂，包括2种新小鼠模型（Tsc 2 +/-Atg 5 +/-和Tsc 2 +/-p62-/-）。和血管平滑肌脂肪瘤来源的细胞与关键分子的shRNA下调。 该项目的意义在于，它将首次揭示自噬依赖的细胞网络如何有助于TSC中肾脏疾病的发病机制。我们希望这个项目能产生很大的影响，因为TSC相关的病变在儿童和成人中都有毁灭性的后果，并且因为TORC 1信号网络在其他人类疾病中失调，包括多囊肾病。

DESCRIPTION (provided by applicant): TSC patients develop multi-system disease including benign tumors of the brain, heart, skin and kidney. By age 10, 80% of children with TSC have renal angiomyolipomas and/or renal cysts. Mammalian target of rapamycin (mTOR) complex 1 (TORC1), which is activated in tuberous sclerosis complex (TSC), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment with TORC1 inhibitors partially decreases the size of TSC-associated brain and kidney lesions, but they regrow when treatment is stopped. Our central hypothesis is that dysregulation of autophagy and cellular metabolism plays a critical role in the pathogenesis of TSC and in the response of TSC-associated renal lesions to TORC1-targeted therapy. In Aims 1 and 2, we will test the hypothesis that low levels of autophagy in TSC2-deficient cells lead to a "metabolic starvation" phenotype, making TSC2-deficient cells hypersensitive to further autophagy inhibition. Consistent with this hypothesis, we have found that inhibiting autophagy induces metabolic dysregulation and decreases the in vivo growth of TSC2-deficient cells. In Aim 3, we will address the hypothesis that p62/sequestosome1-dependent signaling networks promote the growth and survival of TSC2-deficient cells. Consistent with this hypothesis, we have found that p62 accumulates in human angiomyolipomas and other TSC2-deficient cells as a consequence of low autophagy, and that shRNA down regulation of p62 inhibits the in vivo growth of TSC2-deficient cells. Our in vivo strategy (Aims 2 and 3) includes TSC2-deficient angiomyolipoma-derived cells and renal cyst/cystadenoma quantitation in Tsc2+/- mice. Throughout the proposal, we will utilize innovative, state-of-the-art technology including real-time monitoring of bioenergetic parameters using the "Seahorse" system, metabolomic profiling in collaboration with the Broad Institute, high throughput synthetic-lethality drug screening, and in vivo bioluminescent detection of angiomyolipoma cells. Novel reagents will be generated, including 2 new mouse models (Tsc2+/-Atg5+/- and Tsc2+/-p62-/-.) and angiomyolipoma-derived cells with shRNA down regulation of key molecules. The significance of this project is that it will reveal for the firsttime how autophagy-dependent cellular networks contribute to the pathogenesis of renal disease in TSC. We expect this project to have high impact because TSC-associated lesions have devastating consequences in both children and adults and because the TORC1 signaling network is dysregulated in other human diseases including polycystic kidney disease.