Removal of damaged mitochondria by alternative autophagy

通过替代自噬去除受损的线粒体

• 批准号: 9978602
• 负责人: Junichi Sadoshima
• 金额: $ 60.1万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2021-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978602
• 关键词: Address; Affect; Autophagocytosis; Autophagosome; Biogenesis; Cardiac; Cardiac Myocytes; Cell Death; Detection; Development; Electron Microscopy; Excision; Fasting; Goals; Golgi Apparatus; Heart; Heart failure; Homeostasis; Injury; Intervention; Ischemia; Knock-in Mouse; Knockout Mice; Knowledge; Lead; Maintenance; Mediating; Membrane; Metabolism; Mitochondria; Molecular; Myocardial Ischemia; Myocardial dysfunction; Organelles; Pathologic; Patients; Phosphorylation; Play; Production; Quality Control; RIPK1 gene; Reactive Oxygen Species; Reporter; Reporting; Role; Starvation; Stimulus; Stress; Testing; Vesicle; cardioprotection; cell type; heart cell; heart function; ischemic injury; loss of function; mitochondrial autophagy; mitochondrial dysfunction; mouse model; mutant; novel; novel strategies; parkin gene/protein; recruit; release factor; response; scaffold

Autophagy is a major mechanism of degradation of damaged mitochondria. Without elimination of damaged mitochondria, depolarized mitochondria and reactive oxygen species (ROS) rapidly affect healthy mitochondria, leading to wide-spread mitochondrial dysfunction and cell death. Understanding how damaged mitochondria are removed will provide a key to achieving healthier mitochondria in cardiomyocytes (CMs) and developing novel treatments for heart failure. Although it is believed that damaged mitochondria are degraded primarily by Pink1-Parkin-mediated mitophagy, we have discovered that CMs degrade mitochondria through an Atg7-independent and Ulk1-dependent form of autophagy that is homologous to the “alternative” autophagy previously reported by Nishida, and that this form of autophagy plays a significant role in the elimination of damaged mitochondria in response to starvation. However, neither the molecular mechanism nor the functional significance of mitophagy mediated through alternative autophagy has been clearly established in CMs yet. Thus, the goal of this project is to demonstrate the functional significance of alternative autophagy in eliminating damaged mitochondria in the heart in response to relevant stresses and to elucidate the underlying molecular mechanisms. Hypothesis 1: In response to myocardial ischemia, the heart activates Atg7- independent/Ulk1-dependent alternative autophagy, which plays an essential role in mediating the clearance of damaged mitochondria and protects the heart from myocardial ischemia. Hypothesis 2: Ulk1 phosphorylated at Ser555 acts as a scaffold to induce Rab9 interaction for autophagosome formation and phosphorylation of Drp1 at Ser616 for mitochondrial fission, both of which are important in mediating mitochondrial autophagy in response to myocardial ischemia. We will: Aim 1: Demonstrate that the atg7-independent and ulk1-dependent alternative autophagy is activated by myocardial ischemia. Evaluate whether alternative autophagy protects the heart during myocardial ischemia. To this end, we will use cardiac-specific atg7- and ulk1-knockout mice, electron microscopy, specific reporters of alternative autophagy and mitochondrial autophagy, and functional analyses of mitochondria. We will show that damaged mitochondria are degraded primarily through alternative autophagy during myocardial ischemia. Aim 2: Evaluate whether phosphorylation of Ulk1 at Ser555 plays an essential role in mediating alternative autophagy and cardioprotection during myocardial ischemia by stimulating interaction with Rab9 and Ser616-phosphorylated Drp1. To this end, we will use loss-of-function and knock-in mouse models and unique and reliable reporters for alternative autophagy and lysosomal degradation of mitochondria. The knowledge obtained from this aim should lead to development of specific interventions to modulate mitophagy during myocardial ischemia. In summary, our study will demonstrate that alternative autophagy is a novel and predominant mechanism of mitochondrial degradation, which is essential for the maintenance of mitochondrial quality in the heart during ischemia.

自噬是受损线粒体降解的主要机制。未消除损坏 线粒体、去极化线粒体和活性氧 (ROS) 会迅速影响健康 线粒体，导致广泛的线粒体功能障碍和细胞死亡。了解损坏程度 去除线粒体将为心肌细胞（CM）中实现更健康的线粒体提供关键 开发治疗心力衰竭的新疗法。尽管人们相信受损的线粒体会被降解 主要通过 Pink1-Parkin 介导的线粒体自噬，我们发现 CM 通过 不依赖 Atg7 且依赖 Ulk1 的自噬形式，与“替代”自噬同源 Nishida 之前报道过，这种形式的自噬在消除 饥饿导致线粒体受损。然而，无论是分子机制还是功能 通过替代自噬介导的线粒体自噬的重要性在 CM 中已得到明确证实。 因此，该项目的目标是证明替代自噬在 消除心脏中受损的线粒体以应对相关压力并阐明潜在的原因 分子机制。假设 1：响应心肌缺血，心脏激活 Atg7- 独立/依赖 Ulk1 的替代自噬，在介导清除 受损的线粒体并保护心脏免受心肌缺血。假设2：Ulk1磷酸化 Ser555 作为支架诱导 Rab9 相互作用，促进自噬体形成和磷酸化 Drp1 Ser616 负责线粒体裂变，两者对于介导线粒体自噬都很重要 对心肌缺血的反应。我们将： 目标 1：证明 atg7 独立且 ulk1 依赖 心肌缺血会激活替代自噬。评估替代自噬是否具有保护作用 心肌缺血时的心脏。为此，我们将使用心脏特异性 atg7 和 ulk1 基因敲除小鼠， 电子显微镜、选择性自噬和线粒体自噬的特异性报告基因以及功能性自噬 线粒体分析。我们将证明受损的线粒体主要通过替代途径降解 心肌缺血时的自噬。目标 2：评估 Ulk1 Ser555 处的磷酸化是否发挥作用 心肌缺血期间介导替代自噬和心脏保护的重要作用 刺激与 Rab9 和 Ser616 磷酸化 Drp1 的相互作用。为此，我们将使用功能丧失 和敲入小鼠模型以及用于替代自噬和溶酶体的独特且可靠的报告基因 线粒体的降解。从这个目标中获得的知识应该导致具体的开发 心肌缺血期间调节线粒体自噬的干预措施。总之，我们的研究将证明 替代自噬是线粒体降解的一种新颖且主要的机制，这是至关重要的 用于在缺血期间维持心脏中的线粒体质量。