Nutrient signaling at ER-Mitochondrial contacts and age-related mitochondrial dysfunction

项目摘要 线粒体功能改变是衰老的标志，导致衰老相关疾病的部分原因是精力充沛 失败了。线粒体能量学的维持需要适当的裂变和融合，而这反过来又受到 内质网-线粒体接触处的蛋白质(又名线粒体相关膜或MAM)。 禁食刺激线粒体脂肪酸的利用，人们认为禁食的好处是 线粒体动力学和呼吸作用的适应性变化。禁食如何影响线粒体动力学和 以及这些基本机制是否会随着年龄和年龄的变化而改变 饮食压力仍不清楚。上游信号级联调控线粒体的机制 动力学正在成为一个非常感兴趣的领域，最近的研究表明AMPK是裂变的调节器。我们的 新的数据显示，典型的营养反应mTORC2被禁食反常地重新激活，而且 MTORC2的重新激活支持线粒体的分裂。事实上，mTORC2监管机构Rictor Show的肝脏保持沉默 分裂受损，呼吸受抑；随着年龄的增长，mTORC2活性受到抑制。这些数据引发了一个 要点：由于临床上可用的mTOR抑制剂(如雷帕霉素)同时阻断mTORC1和mTORC2，我们 认为雷帕霉素长期抑制mTOR的益处被线粒体抵消 伴随的mTORC2抑制导致的功能障碍。这构成了探索是否阻止mTORC1-in的基础 与保持mTORC2活动相结合-将导致健康和寿命延长。磷酸化 是线粒体动力学调控中一个公认的信号。我们的磷蛋白质组学数据表明 禁食期间mTORC2激酶的反常重新激活通过磷酸化刺激线粒体分裂 针对绵羊的新蛋白靶点，包括NDRG1，N-Myc下游调控1.有趣的是，NDRG1S336A 磷酸化缺陷突变体重述mTORC2缺失对线粒体的影响 分裂/呼吸作用，而拟磷NDRG1S336D刺激线粒体分裂。耐人寻味的NDRG1，但 而不是NDRG1S336A磷酸化缺陷突变体，它与CDC42相互作用，CDC42是一种细胞动力学蛋白，具有内在的 线粒体分裂所需的GTP水解酶活性。根据我们的初步数据，我们建议 MTORC2磷酸化NDRG1Ser336-CDC42轴在巨噬细胞上的激活调节线粒体分裂 在禁食期间，mTORC2-NDRG1Ser336-CDC42轴的失调导致与年龄相关的 线粒体分裂和呼吸作用。提出了描述线粒体的三个目标(目标1) 衰老和应激肝脏的动力学和MAM；(目标2)确定mTORC2是否以及如何失调- NDRG1Ser336-CDC42轴导致与年龄相关的线粒体分裂丧失；和(目标3)探索是否协同使用 雷帕霉素和模拟磷脂酶NDRG1S336D的共表达将导致更大的寿命延长 与单独使用雷帕霉素相比。这里提出的实验将严格检验一组新的假设 MTORC2信号的改变是与年龄相关的线粒体功能丧失的主要因素。

Project Summary Altered mitochondrial function, a hallmark of aging, contributes to age-related diseases, in part, due to energetic failure. Maintenance of mitochondrial energetics requires proper fission and fusion that are in turn regulated by proteins at endoplasmic reticulum-mitochondria contacts (a.k.a. mitochondria-associated membranes or MAMs). Fasting stimulates mitochondrial fatty acid utilization, and it is thought that the benefits of fasting are due to adaptive changes in mitochondrial dynamics and respiration. How fasting impacts mitochondrial dynamics and MAMs in intact mammalian systems, and whether these fundamental mechanisms are altered with age and dietary stress remain unknown. The mechanisms by which upstream signaling cascades regulate mitochondrial dynamics is becoming an area of great interest, with recent work showing AMPK as a regulator of fission. Our new data show that the typically nutrient-responsive mTORC2 is paradoxically reactivated by fasting, and that mTORC2 reactivation supports mitochondrial fission. Indeed, livers silenced for mTORC2 regulator Rictor show impaired fission and dampened respiration; and mTORC2 activity is suppressed with age. These data raise a key point: since clinically available mTOR inhibitors (e.g., rapamycin) block both mTORC1 and mTORC2, we argue that benefits of long-term mTOR inhibition with rapamycin are counterbalanced by mitochondrial dysfunction due to concomitant mTORC2 inhibition. This forms that basis to explore if blocking mTORC1—in conjunction with maintaining mTORC2 activity—will lead to healthspan and lifespan extension. Phosphorylation is a well-established signal in regulation of mitochondrial dynamics. Our phosphoproteomics data suggest that paradoxical reactivation of mTORC2 kinase during fasting stimulates mitochondrial fission by phosphorylating novel protein targets at MAMs, including NDRG1, N-Myc downstream regulated 1. Interestingly, the NDRG1S336A phosphorylation-deficient mutant recapitulates the effects of loss of mTORC2 on mitochondrial fission/respiration, while phosphomimetic NDRG1S336D stimulates mitochondrial fission. Intriguingly NDRG1, but not the NDRG1S336A phosphorylation-deficient mutant, interacts with Cdc42, a cytokinetic protein with the intrinsic GTP hydrolysis activity required for mitochondrial fission. Based on our preliminary data, we propose that activation of the mTORC2-phosphorylated NDRG1Ser336-Cdc42 axis at MAMs regulates mitochondrial fission during fasting, and that dysregulation of the mTORC2-NDRG1Ser336-Cdc42 axis leads to age-related loss of mitochondrial fission and respiration. Three aims are proposed that will characterize (Aim 1) mitochondrial dynamics and MAMs in aged and stressed livers; (Aim 2) determine if and how dysregulation of the mTORC2- NDRG1Ser336-Cdc42 axis causes age-related loss of mitochondrial fission; and (Aim 3) explore if synergistic use of rapamycin and co-expression of phosphomimetic NDRG1S336D will lead to greater lifespan extension when compared to use of rapamycin alone. Experiments proposed here will rigorously test a new set of hypotheses that altered mTORC2 signaling is a major contributing factor to age-related loss of mitochondrial function.