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（A-T）由编码共济失调-毛细血管扩张突变激酶（ATM）的ATM基因突变引起。A-T的许多症状，包括神经变性和癌症，可能会因ROS增加而导致的慢性氧化应激而加剧。当电子在氧化磷酸化过程中过早地转移到氧时，线粒体显著地增加细胞ROS。申办方实验室已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，从而得出本提案的总体前提，即线粒体ROS（mROS）增加有助于A-T病理学。具体而言，该提议将测试以下假设：mROS 1）被ATM感知以调节雷帕霉素复合物1（mTORC 1）介导的促生长和应激反应信号转导的哺乳动物靶标，以及2）有助于Akt活性的增加，这两者都可能有助于A-T病理学。ATM不依赖于其在DNA损伤信号传导中的作用，被ROS激活并通过激活AMP活化蛋白激酶（AMPK）来抑制mTORC 1。由于mTORC 1信号刺激生长并抑制抗应激和自噬途径，因此Aim 1的假设是ATM通常感知mROS以抑制mTORC 1来抑制细胞生长并增加抗应激能力。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，并且AMPK和mTORC 1信号传导的急性变化将通过活化激酶及其下游靶标的蛋白质印迹来评估。将通过监测过表达或不过表达线粒体抗氧化酶（mCAT和MnSOD）的ATMflox/flox MEF中的mTORC 1活化来确定mROS的贡献。最后，为了确定ATM是否感测mROS和/或线粒体功能障碍，将响应于增加mROS、抑制呼吸或两者的药理学试剂来测量ATM活化。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过度活跃的Akt。Akt由PI 3 K信号传导激活，PI 3 K信号传导由肿瘤抑制因子PTEN负调控。Akt超活化的另一个潜在机制是其不能被多聚泛素化和降解。在目的2中，ATM-/- MEFs中Akt活化的机制将通过监测PTEN活性和/或Akt合成和稳定性的变化来确定。最后，由于过度活跃的Akt可通过抑制TSC 2上调mTORC 1，因此将如目的1中所述进行Akt的药理学和基因敲低抑制以及mTORC 1信号传导参数的分析，以确定ATM-/-MEF中是否是这种情况。该项目的长期目标是更好地了解mROS在ATM和mTORC 1信号传导中的作用，这可能有助于开发A-T的新治疗策略。 公共卫生相关性：该提案中描述的研究与公共卫生直接相关，因为它侧重于共济失调-毛细血管扩张症（一种毁灭性的人类疾病）和氧化应激（与阿尔茨海默氏症、帕金森氏症和其他与年龄相关的神经退行性疾病有关）。该项目将测试线粒体活性氧（mROS）参与mTORC 1和Akt介导的生长，抗应激和自噬途径的调节的假设，这两者都可能有助于A-T病理学。了解mROS如何促进共济失调-毛细血管扩张症的氧化应激，可能有助于开发这种毁灭性疾病的新治疗策略。

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. PUBLIC HEALTH RELEVANCE: Research described in this proposal is directly related to public health, because it focuses on both Ataxia- Telangiectasia, a devastating human disease, and oxidative stress, which has been implicated in Alzheimer's, Parkinson's, and other age-related neurodegenerative diseases. This project will test the hypothesis that mitochondrial reactive oxygen species (mROS) are involved in the regulation of growth, stress resistance, and autophagy pathways mediated by mTORC1 and Akt, both of which likely contribute to A-T pathology. Understanding how mROS contribute to oxidative stress in Ataxia-Telangiectasia may allow development of new therapeutic strategies for this devastating disease.