Role of the Endosomal-Lysosomal Pathway in Mitochondrial Quality Control

项目摘要 心肌细胞线粒体功能障碍发生在心力衰竭发病的早期。在心中， 线粒体的主要功能是通过提供能量来满足跳动的肌细胞的高能量需求 ATP通过氧化磷酸化。然而，线粒体可以迅速转变为促进死亡的 细胞器为了应对细胞内环境的变化，它们可以成为过量的 活性氧并释放促死亡蛋白。毫不奇怪，细胞已经发展出防御能力， 细胞修复自身的能力， 防止不必要的死亡在有丝分裂后细胞如不能被 很容易被取代。研究发现，功能失调的线粒体可以被自噬体隔离 并随后递送至溶酶体进行降解。然而， 线粒体的去除没有得到很好的表征，以及线粒体的其他机制是否 目前尚不清楚是否存在清除。我们以前已经发现E3泛素连接酶Parkin起着重要的作用。 在清除心脏中功能失调的线粒体以应对压力和缺乏帕金导联中的重要作用 与心肌梗死后功能障碍的线粒体堆积有关。帕金以诱导 但我们的初步研究发现，帕金也可以促进线粒体的自噬， 通过自噬非依赖性机制清除线粒体。在本建议书中，我们将探讨 假设小GTCRab 5和内体降解途径在 清除肌细胞中功能失调的线粒体。我们将以两个目标来检验这些假设。目标1将 定义Rab 5和内体介导的线粒体清除在心肌细胞中的功能重要性， 体外和体内。我们还将研究内体途径与 心脏中的传统/替代自噬途径。在目标2中，我们将描述Beclin 1在以下方面的作用： 启动内体途径以响应线粒体损伤。我们将研究Beclin 1 调节内体降解途径的激活，以响应细胞应激，通过形成特异性的 与Rab 5和Vps 34的前内体复合物。使用独特的药物进行体外和体内功能丧失研究 心脏特异性诱导型Beclin 1缺陷小鼠将用于证实Beclin 1的功能重要性 响应线粒体损伤和应激而启动早期内体的形成。这些研究将 提供了重要的新见解，了解功能失调和潜在危险的线粒体是如何被清除的， 心脏这些研究还将为该途径中新的潜在治疗靶点提供见解。

Project Summary Mitochondrial dysfunction in cardiac myocytes occurs early in the pathogenesis of heart failure. In the heart, the primary function of mitochondria is to meet the high energy demand of the beating myocytes by providing ATP through oxidative phosphorylation. However, mitochondria can quickly change into death–promoting organelles. In response to changes in the intracellular environment, they can become excessive producers of reactive oxygen species and release pro-death proteins. Not surprisingly, cells have developed defense mechanisms against aberrant mitochondria that can cause harm to it. The ability of a cell to repair itself and prevent unnecessary death is particularly important in a post-mitotic cell such as a myocyte that cannot be easily replaced. Studies have found that dysfunctional mitochondria can be sequestered by autophagosomes and subsequently delivered to lysosomes for degradation. However, the mechanism and regulation of mitochondrial removal are not well characterized and whether additional mechanisms of mitochondrial clearance exist is currently unclear. We have previously found that the E3 ubiquitin ligase Parkin plays an important role in clearing dysfunctional mitochondria in the heart in response to stress and lack of Parkin leads to accumulation of dysfunctional mitochondria after a myocardial infarction. Parkin is known to induce autophagy of mitochondria but our preliminary studies have uncovered evidence that Parkin can also promote clearance of mitochondria via an autophagy-independent mechanism. In this proposal, we will explore the hypothesis that the small GTPase Rab5 and the endosomal degradation pathway play an important role in clearing dysfunctional mitochondria in myocytes. This hypotheses will be tested with two aims. Aim 1 will define the functional importance of Rab5 and endosomal-mediated mitochondrial clearance in myocytes in vitro and in vivo. We will also examine the relationships between the endosomal pathway and traditional/alternative autophagy pathways in the heart. In Aim 2, we will delineate the role of Beclin1 in initiating the endosomal pathway in response to mitochondrial damage. We will examine whether Beclin1 regulates activation of the endosomal degradation pathway in response to cellular stress by forming a specific pro-endosomal complex with Rab5 and Vps34. Loss-of-function studies in vitro and in vivo using unique cardiac specific inducible Beclin1 deficient mice will be utilized to confirm the functional importance of Beclin1 initiating formation of early endosomes in response to mitochondrial damage and stress. These studies will provide important novel insight into how dysfunctional and potentially dangerous mitochondria are cleared in the heart. These studies will also provide insights into new potential therapeutic targets in this pathway.