Removal of damaged mitochondria by alternative autophagy

自噬是受损线粒体降解的主要机制。不消除损害 线粒体、去极化线粒体和活性氧（ROS）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。了解如何损坏 去除线粒体将为在心肌细胞（CM）中获得更健康的线粒体提供关键， 开发治疗心力衰竭的新方法尽管人们认为受损的线粒体会被降解 主要通过Pink 1-Parkin介导的线粒体自噬，我们已经发现CM通过一种 Atg 7-独立和Ulk 1-依赖的自噬形式，与“替代性”自噬同源 Nishida先前报道，这种形式的自噬在消除 受损的线粒体。然而，无论是分子机制还是功能机制， 通过选择性自噬介导的线粒体自噬在CM中的重要性已经被清楚地确立。 因此，该项目的目标是证明替代性自噬在细胞内的功能意义。 消除心脏中受损的线粒体以响应相关的压力，并阐明潜在的 分子机制假设1：在对心肌缺血的反应中，心脏激活Atg 7- 独立/Ulk 1依赖的替代性自噬，其在介导细胞的清除中起重要作用。 受损的线粒体和保护心脏免受心肌缺血。假设2：Ulk 1磷酸化 在Ser 555处作为支架诱导Rab 9相互作用，以形成自噬体和磷酸化 Drp 1在Ser 616处进行线粒体分裂，这两者在介导线粒体自噬中都是重要的。 对心肌缺血的反应。目标1：证明atg 7独立和ulk 1依赖 心肌缺血可激活交替性自噬。评估替代性自噬是否能保护 心肌缺血时的心脏。为此，我们将使用心脏特异性atg 7和ulk 1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 线粒体的分析。我们将证明受损的线粒体主要通过替代性的 心肌缺血时的自噬。目的2：评估Ulk 1在Ser 555处的磷酸化是否起作用。 在心肌缺血期间介导替代性自噬和心脏保护中的重要作用 刺激与Rab 9和Ser 616-磷酸化Drp 1的相互作用。为此，我们将使用功能丧失 和基因敲入小鼠模型以及用于替代性自噬和溶酶体 线粒体降解。从这一目标中获得的知识应导致制定具体的 干预以调节心肌缺血期间的线粒体自噬。总之，我们的研究将证明， 选择性自噬是线粒体降解的一种新的和主要的机制， 用于在局部缺血期间维持心脏中的线粒体质量。

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.