Regulation of mTORC1 signaling by mROS and ATM

描述(申请人提供)：共济失调-毛细血管扩张症(A-T)是由编码共济失调-毛细血管扩张症突变激酶(ATM)的ATM基因突变引起的。A-T的许多症状，包括神经退行性变和癌症，可能会因ROS增加而导致的慢性氧化应激而加剧。在氧化磷酸化过程中，当电子过早地转移到氧气中时，线粒体显著增加了细胞的ROS。赞助商的实验室已经将线粒体功能障碍与A-T患者细胞中ROS产生增加联系起来，从而导致了这一提议的总体前提，即线粒体ROS(MRO)增加有助于A-T病理。具体地说，这一提议将检验以下假设：1)MRO被ATM感知以调节哺乳动物雷帕霉素复合体1(MTORC1)介导的促生长和应激反应信号；2)促进Akt的活性，这两者都可能导致A-T病理。与其在DNA损伤信号中的作用无关，ATM被ROS激活，并通过激活AMP激活的蛋白激酶(AMPK)来抑制mTORC1。由于mTORC1信号刺激生长并抑制抗逆性和自噬通路，因此，目标1的假设是ATM通常感知MRO，以抑制mTORC1抑制细胞生长，提高抗逆性能力。实验将使用从ATMFLOX/FLOX小鼠制备的小鼠胚胎成纤维细胞(MEF)进行，其中ATM基因将使用慢病毒-CRE系统敲除，AMPK和mTORC1信号的急性变化将通过激活的激酶及其下游靶点的蛋白质印迹来评估。MRO的贡献将通过监测线粒体抗氧化酶(MCAT和MnSOD)过表达或不过表达的ATMflx/FLOX MEF中mTORC1的激活来确定。最后，为了确定ATM是否感觉到MRO和/或线粒体功能障碍，ATM的激活将根据增加MRO、抑制呼吸或两者兼而有之的药理药物进行测量。AKT是一种参与细胞生长、增殖、存活和代谢途径的激酶。在ATM-/-小鼠的胸腺细胞和ATM-/-小鼠的多个组织中观察到高活性的Akt。AKT由PI3K信号激活，而PI3K信号受肿瘤抑制因子PTEN的负调控。Akt过度激活的另一个潜在机制是它不能被多泛素化和降解。在目标2中，将通过监测PTEN活性和/或Akt的合成和稳定性的变化来确定ATM-/-MEF中Akt的激活机制。最后，由于过度活跃的Akt可能通过抑制TSC2上调mTORC1，因此将按照目标1中的描述进行Akt的药理学和基因敲除抑制以及mTORC1信号参数的分析，以确定ATM-/-MEF中是否存在这种情况。该项目的长期目标是更好地了解MRO在ATM和mTORC1信号中的作用，这可能有助于开发新的A-T治疗策略。

DESCRIPTION (provided by applicant): Ataxia-Telangiectasia (A-T) is caused by mutations in the ATM gene, which encodes the Ataxia-Telangiectasia Mutated kinase (ATM). Many symptoms of A-T, including neurodegeneration and cancer, are likely exacerbated by chronic oxidative stress due to increased ROS. Mitochondria add significantly to cellular ROS when electrons are prematurely transferred to oxygen during oxidative phosphorylation. The Sponsor's lab has linked mitochondrial dysfunction to increased ROS production in A-T patient cells, leading to the overall premise of this proposal that increased mitochondrial ROS (mROS) contribute to A-T pathology. Specifically, this proposal will test the hypotheses that mROS 1) are sensed by ATM to regulate mammalian target of rapamycin complex 1 (mTORC1)-mediated pro-growth and stress-response signaling and 2) contribute to increased activity of Akt, both of which likely contribute to A-T pathology. Independent of its role in DNA damage signaling, ATM is activated by ROS and inhibits mTORC1 via activation of AMP-activated protein kinase (AMPK). Since mTORC1 signaling stimulates growth and inhibits stress resistance and autophagy pathways, the hypothesis of Aim 1 is that ATM normally senses mROS in order to inhibit mTORC1 to repress cell growth and increase stress resistance capacity. Experiments will be carried out using mouse embryonic fibroblasts (MEFs) prepared from ATMflox/flox mice in which the ATM gene will be knocked out using a lentiviral- Cre system and acute changes in AMPK and mTORC1 signaling will be assessed by western blot of activated kinases and their downstream targets. The contribution of mROS will be determined by monitoring mTORC1 activation in ATMflox/flox MEFs that do or do not overexpress mitochondrial antioxidant enzymes (mCAT and MnSOD). Finally, to determine if ATM senses mROS and/or mitochondrial dysfunction, ATM activation will be measured in response to pharmacological agents that increase mROS, inhibit respiration, or both. Akt is a kinase involved in cell growth, proliferation survival, and metabolism pathways. Hyperactive Akt is observed in thymocytes in ATM-/- mice and in multiple tissues in ATM-/- mice. Akt is activated by PI3K signaling, which is negatively regulated by the tumor suppressor PTEN. Another potential mechanism of Akt hyperactivation is its inability to be polyubiquitinated and degraded. In Aim 2, the mechanism of Akt activation in ATM-/- MEFs will be determined by monitoring for changes in PTEN activity and/or in the synthesis and stability of Akt. Finally, since hyperactive Akt may upregulate mTORC1 through inhibition of TSC2, pharmacological and gene knockdown inhibition of Akt and analysis of mTORC1 signaling parameters will be carried out as described in Aim 1 to determine if this is the case in ATM-/- MEFs. The long-term goal of this project is to better understand the role of mROS in ATM and mTORC1 signaling, which may allow development of new therapeutic strategies for A-T.