Role of skeletal muscle mitophagy in healthy aging

项目摘要 随着年龄的增长，骨骼肌中的线粒体质量逐渐下降，导致组织功能障碍。 和疾病。多条证据表明，老年动物骨骼肌中线粒体质量较差 人类在很大程度上是由于降解受损/功能失调的能力受损或不足所致 线粒体通过有丝分裂。运动促进线粒体质量，导致健康衰老，但潜在的 机制以及它们如何随年龄的变化没有很好的定义，特别是在吞丝分裂、抑制 制定有效的干预措施。在我之前工作的基础上证明了锻炼确实 促进骨骼肌中的有丝分裂，但只有一小部分线粒体网状结构，我在这里展示 AMPK依赖机制的证据，可以区分受损和健康的脑区 随年龄增长而消失的线粒体网状结构。此外，我还证明了下游与有丝分裂相关的关键因素 在运动中被招募到骨骼肌线粒体中的蛋白质是线粒体质量所必需的 黑腹蛇的健康衰老。这项拟议的研究验证了这样一种假设，即识别损坏 老年小鼠骨骼肌对运动作出反应的线粒体网状结构区域受损，变钝 局部招募关键的有丝分裂蛋白，导致线粒体质量较差。这些研究将提供 洞察运动对骨骼肌有丝分裂的新调控，并为 开发有针对性的干预措施来提高骨骼肌中线粒体的质量，以改善组织 功能与健康衰老。在指导阶段，我将使用最先进的双光子显微镜来 对骨骼肌线粒体AMPK活性进行活体和体外荧光寿命显微镜观察 以确定AMPK对持续收缩的依赖年龄的局部反应 和线粒体损伤。此外，我将继续我的专业和科学发展，为 在我的指导委员会的持续指导下，进入了独立阶段。在独立阶段期间， 我将在幼年和老年野生型小鼠的骨骼肌以及骨骼肌中采用共体基因转移。 肌肉特异的、有条件的Ulk1基因敲除小鼠确定年龄依赖的招募调节 运动对骨骼肌线粒体下游吞丝相关因子的影响。还有，我会的 开发新的模拟磷酸化的构建体，以结构性地激活或抑制吞丝相关因子Atg9和 ATG2在幼年和老年小鼠骨骼肌中的表达及其对分解的必要性和充分性的研究 通过最先进的高分辨率蛋白质组学研究线粒体蛋白质和维持线粒体质量 和共聚焦成像方法。总的来说，这些学习和职业发展活动将促进我的 持续的科学和专业培训，导致成功的独立、学术研究计划。

Project Summary Mitochondrial quality in skeletal muscle progressively declines with advancing age, leading to tissue dysfunction and disease. Several lines of evidence suggest poor mitochondrial quality in skeletal muscle of old animals and humans is due in large part to an impaired or insufficient capacity to degrade damaged/dysfunctional mitochondria via mitophagy. Exercise promotes mitochondrial quality leading to healthy aging but the underlying mechanisms and how they differ with age is not well defined, particularly in regards to mitophagy, restraining development of effective interventions. Building upon my previous work demonstrating that exercise does indeed promote mitophagy in skeletal muscle but only of a small fraction of the total mitochondrial reticulum, I show here evidence of an Ampk-dependent mechanism that may distinguish damaged vs. healthy regions of the mitochondrial reticulum that is lost with age. Additionally, I show that key downstream mitophagy-related factors that are recruited to mitochondria in skeletal muscle with exercise are required for mitochondrial quality and healthy aging in d. melanogaster. The proposed research tests the hypothesis that recognition of damaged regions of the mitochondrial reticulum in response to exercise is impaired in skeletal muscle of old mice, blunting local recruitment of key mitophagy proteins, leading to poor mitochondrial quality. These studies will provide insight into novel regulation of skeletal muscle mitophagy in response to exercise and lay a foundation for the development of targeted interventions to promote mitochondrial quality in skeletal muscle for improved tissue function and healthy aging. During the mentored phase, I will employ state-of-the-art two-photon microscopy to perform intravital and ex vivo fluorescent lifetime microscopy of Ampk activity on mitochondria in skeletal muscle of young and old mice to determine the age-dependent, localized response of Ampk to sustained contraction and mitochondrial damage. Also, I will continue my professional and scientific development in preparation for the independent phase with continuous guidance from my mentoring committee. During the independent phase, I will employ co-somatic gene transfer in skeletal muscle of young and old wild-type mice as well as skeletal muscle-specific, conditional Ulk1 knock-out mice to determine the age-dependent regulation for the recruitment of downstream mitophagy-related factors to mitochondria in skeletal muscle in response to exercise. Also, I will develop novel phospho-mimetic constructs to constitutively activate or inhibit mitophagy-related factors Atg9 and Atg2 in young and old mouse skeletal muscle and investigate their necessity and sufficiency for the breakdown of mitochondrial proteins and maintenance of mitochondrial quality, via state-of-the-art high resolution proteomic and confocal imaging approaches. Collectively, these studies and career development activities will foster my continued scientific and professional training, leading to a successful independent, academic research program.