Regulation of mTORC1 signaling by mROS and ATM

描述（由申请人提供）：共济失调-毛细血管扩张症（A-T）是由ATM基因突变引起的，该基因编码共济失调-毛细血管扩张突变激酶（ATM）。许多A-T症状，包括神经退行性变和癌症，可能会因ROS增加而加剧慢性氧化应激。在氧化磷酸化过程中，当电子过早转移到氧中时，线粒体显著增加细胞ROS。发起人的实验室已经将线粒体功能障碍与A-T患者细胞中ROS生成增加联系起来，从而导致该提案的总体前提是线粒体ROS （mROS）增加有助于A-T病理。具体来说，本研究将验证以下假设：1)mROS被ATM感知以调节哺乳动物雷帕霉素靶蛋白1 （mTORC1）介导的促生长和应激反应信号；2)促进Akt活性增加，这两个假设都可能导致A-T病理。除了在DNA损伤信号传导中的作用外，ATM可被ROS激活，并通过活化amp活化的蛋白激酶（AMPK）抑制mTORC1。由于mTORC1信号通路刺激生长，抑制抗逆性和自噬途径，Aim 1的假设是ATM通常感知mROS，从而抑制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活化激酶及其下游靶点的western blot进行评估。mROS的作用将通过监测是否过表达线粒体抗氧化酶（mCAT和MnSOD）的ATMflox/flox mef中mTORC1的激活来确定。最后，为了确定ATM是否感知到mROS和/或线粒体功能障碍，将测量ATM的激活情况，以响应增加mROS、抑制呼吸或两者兼有的药物。Akt是一种参与细胞生长、增殖、存活和代谢途径的激酶。在ATM-/-小鼠胸腺细胞和ATM-/-小鼠的多个组织中观察到Akt过度活跃。Akt受PI3K信号激活，而PI3K信号受肿瘤抑制因子PTEN负调控。Akt过度激活的另一个潜在机制是其不能被多泛素化和降解。在Aim 2中，Akt在ATM-/- MEFs中的激活机制将通过监测PTEN活性的变化和/或Akt的合成和稳定性来确定。最后，由于过度活跃的Akt可能通过抑制TSC2而上调mTORC1，因此我们将按照Aim 1的描述，对Akt进行药理学和基因敲低抑制，并分析mTORC1信号参数，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mROS在ATM和mTORC1信号传导中的作用，这可能有助于开发新的治疗A-T的策略。

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.