Regulation of Lipid Catabolism by Autophagy in Neural Stem Cell of Tuberous Sclerosis Complex

多发性硬化症（TSC）是一种遗传性疾病，在美国影响多达5万人。TSC 通常通过癫痫发作、自闭症、智力不稳定和非癌性（良性）肿瘤的生长影响大脑。 这些缺陷在TSC患者的新生儿和成人大脑中持续存在。Tsc 1或Tsc 2基因突变 导致其控制mTORC 1活性的肿瘤抑制功能丧失， TSC的发病机制。mTORC 1是一种已确立的细胞稳态的主调节因子，并刺激 但负调节自噬，一种保守分解代谢过程， 溶酶体中的细胞质成分和细胞器。有趣的是，我们最近的研究结果显示， 在能量应激条件下，Tsc 1缺陷细胞中的自噬活性更高，导致建立 一种新型双条件敲除（cKO）小鼠模型，用于删除TSC 1和FIP 200（Fak相互作用蛋白） 200 KD，自噬诱导复合物中的重要组分）在出生后神经干细胞（NSC）中的作用。 利用这种独特的模型，我们揭示了自噬维持高mTORC 1活性的基本功能 和Tsc 1缺陷型NSC的出生后发育异常。此外，我们发现自噬是 所需的线粒体氧化磷酸化，以抵御能量应激条件，最有可能提供 游离脂肪酸从细胞内脂质储存中产生ATP。这些试点结果构成了我们的基础， 自噬介导Tsc 1缺陷NSC中脂质降解β-氧化和ATP的假说 生产以维持其高mTORC 1活性，这为TSC提供了有价值的代谢靶点 患者在本提案的目标1中，我们将检查自噬的分子和代谢机制， 脂质降解和噬脂作用调节高mTORC 1活性的信号传导途径的功能， 使用原始和差分NSC。在Aim 2中，我们将使用我们新开发的FIP 200条件 敲入小鼠，其与Atg 13结合缺陷，以产生Tsc 1/Fip 200 2cKI小鼠。这个基因工具 将进一步阐明Tsc 1缺陷的NSC的噬脂机制。我们还将采用药理学 靶向自噬介导的脂质催化剂以治疗出生后Tsc 1缺陷型NSC缺陷的方法。在 目的3，我们将产生新的诱导型小鼠模型，在出生后特异性地消耗Tsc 1和Fip 200。 国家安全委员会我们还将使用这些小鼠模型来治疗现有的SEN/SEGA，这与临床更相关， 在Aim 2中补充研究。在这些研究的最后，我们将扩大我们的发病机制的知识， TSC缺陷的NSC，通过过度活化的NSC，识别候选信号通路和代谢改变， mTORC 1，并开发新的治疗概念，以继续研究脑 TSC患者的发育障碍。

Tuberous sclerosis complex (TSC) is a genetic disorder affects as many as 50,000 people in US. TSC often affects brain by seizures, autism, intelligence instability and growth of noncancerous (benign) tumors. These defects persist in neonatal and adult brains of TSC patients. The mutations of Tsc1 or Tsc2 gene leading to the loss of their tumor suppressor functions to control the activity of mTORC1 underlie the pathogenesis of TSC. mTORC1 is an established master regulator of cellular homeostasis and stimulates the activity of translation but negatively regulate autophagy, a conserved catabolic process that degrades cytoplasmic constituents and organelles in the lysosome. Interestingly, ours recent findings revealed a higher autophagy activity in Tsc1-deficient cells under energy stress conditions, leading to establishment of a novel double conditional knockout (cKO) mouse model to delete TSC1 and FIP200 (Fak interaction protein of 200 KD, an essential component in autophagy induction complex) in postnatal neural stem cells (NSC). Using this unique model, we revealed the essential functions of autophagy to sustain high mTORC1 activity and in abnormal postnatal development of Tsc1-deficient NSC. In addition, we found that autophagy was required for mitochondria oxidative phosphorylation to resist energy-stress conditions, most likely providing free fatty acids to generate ATP from intracellular lipid storage. These pilot findings form the basis of our hypothesis that autophagy mediates the lipids degradation in Tsc1-deficient NSC for β-oxidation and ATP production to maintain their high mTORC1 activity, which provide a valuable metabolic target for TSC patients. In Aim1 of this proposal, we will exam the molecular and metabolic mechanisms of autophagy in lipid degradation and the functions of lipophagy to regulate signaling pathways for high mTORC1 activity, using primitive and differentiating NSC. In Aim2, we will use our newly developed FIP200 conditional knockin mouse which is defective in binding with Atg13 to generate Tsc1/Fip200 2cKI mice. This genetic tool will further clarify the mechanisms of lipophagy in Tsc1-deficient NSC. We will also adopt pharmacological methods to target autophagy mediated lipid catabolism to treat defects in postnatal Tsc1-deficient NSC. In Aim3, we will generate novel inducible mouse models to specifically deplete Tsc1 and Fip200 in postnatal NSC. We will also use these mouse models to treat existing SEN/SEGA, which is more clinically relevant, to complement studies in Aim2. At the end of these studies, we will expand our knowledge of pathogenesis in TSC-deficient NSC, identify candidate signaling pathways and metabolic alterations by hyperactivated mTORC1, and develop new therapeutic concepts for continued investigation in the treatment of brain development disorders in TSC patients.