Neddylation and mitophagy in cardiac aging

项目摘要 线粒体质量和活性的下降与正常衰老有关， 一系列与年龄有关的疾病的发展。因此，恢复线粒体功能或 改善线粒体质量控制可能是对抗衰老的有效策略。线粒体自噬是一种重要的 线粒体质量控制机制，介导受损线粒体的溶酶体清除。 越来越多的证据表明，增强线粒体自噬在各种疾病中具有延长寿命的作用。 模式生物有趣的是，最近的研究表明，增加线粒体自噬可能会抵消衰老- 相关的心功能不全。因此，鉴定可以调节细胞凋亡的更有效和特异性的试剂， 通过线粒体自噬清除有缺陷的线粒体可能具有显著的治疗益处。我们 进行了高含量的基于图像的测定线粒体自噬调节剂使用pH依赖性荧光 线粒体自噬记者凯玛山我们确定了选择性neddylation抑制剂MLN 4924，是最 有效的线粒体自噬激活剂。Neddylation是一种翻译后修饰， NEDD 8通过NEDD 8特异性E1-E2-E3酶与蛋白靶点结合。值得注意的是，我们的机械研究表明 MLN 4924可有效阻断Cullin 2的neddylation，Cullin 2是延伸蛋白B/C-Cullin 2-Rbx 1（Ring-Box）的组分 1)-VHL（Von Hippel-Lindau蛋白）E3泛素连接酶复合物（CRL 2 VHL）。这种抑制导致了 缺氧诱导因子1 α亚基（HIF 1 α）（一种CRL 2 VHL底物）的蓄积，以及随后的 BCL 2相互作用蛋白3（BNIP 3）的激活，BNIP 3是一种线粒体自噬诱导受体。这些 结果提供了neddylation和线粒体自噬之间的新联系。这个项目的目的是描绘小说 线粒体自噬和neddylation之间的机制联系，并确定线粒体自噬是否代表一种新的 治疗年龄相关性心功能不全的机制和治疗靶点。将促进这些研究 通过我们最近描述的mt-Keima小鼠模型来监测体内心脏线粒体自噬。此外，我们将 利用一套创新的试剂来遗传和非遗传调节neddylation。直接评估 为了研究neddylation在心脏中的作用，我们培育了心肌细胞特异性缺失NAE 1的小鼠， 编码E1奈迪化激活酶的亚基。在拟议研究的目标1中，我们的目标是 确定抑制neddylation调节心肌细胞和心脏线粒体自噬的机制。 在拟议研究的目标2中，我们将在成人中遗传和非遗传操纵neddylation。 使用缺乏NAE 1或用MLN 4924处理的小鼠模型的心脏。我们将决定是否恢复 线粒体自噬通过抑制neddylation改善年龄相关的心功能障碍。完成所提出的 研究将对线粒体自噬在与年龄相关的病理条件中的作用产生重要的见解， 从根本上推进我们对心脏线粒体质量控制机制的理解。

PROJECT SUMMARY A decline in mitochondrial quality and activity has been associated with normal aging and correlated with the development of a wide range of age-related diseases. Therefore, rejuvenating mitochondrial function or improving mitochondrial quality control might be an effective strategy to combat aging. Mitophagy is an essential mitochondrial quality control mechanism that mediates the lysosomal clearance of damaged mitochondria. Increasing lines of evidence have established the longevity-extending effects of enhanced mitophagy in various model organisms. Interestingly, recent studies suggest that augmented mitophagy may counteract aging- associated cardiac dysfunction. Therefore, identifying more efficient and specific agents that can modulate the clearance of defective mitochondria via mitophagy are likely to have significant therapeutic benefits. We conducted high-content image-based assays for mitophagy modulators using the pH-dependent fluorescent mitophagy reporter, mt-Keima. We identified the selective neddylation inhibitor, MLN4924, to be the most effective mitophagy activator. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Of note, our mechanistic studies suggest MLN4924 effectively blocks neddylation of Cullin 2, a component of the elongins B/C-Cullin 2-Rbx1 (Ring-Box 1)-VHL (Von Hippel-Lindau protein) E3 ubiquitin ligase complex (CRL2VHL). The inhibition leads to an accumulation of the Hypoxia Inducible Factor 1 Subunit Alpha (HIF1α), a CRL2VHL substrate, and subsequent activation of the BCL2-interacting protein 3 (BNIP3), a mitochondrial receptor for mitophagy induction. These results provide a novel connection between neddylation and mitophagy. This project aims to delineate the novel mechanistic link between mitophagy and neddylation, and to determine whether mitophagy represents a novel mechanism and therapeutic target for treating age-related cardiac dysfunction. These studies will be facilitated by our recently described mt-Keima mouse model to monitor in vivo cardiac mitophagy. Additionally, we will utilize a set of innovative reagents to genetically and pharmacologically modulate neddylation. To directly assess the role of neddylation in the heart, we have generated mice with the cardiomyocyte-specific deletion of NAE1, encoding a subunit of the E1 neddylation activating enzyme. In Aim 1 of the proposed studies, our goal is to determine the mechanisms by which inhibiting neddylation regulates mitophagy in cardiomyocytes and the heart. In Aim 2 of the proposed studies, we will genetically and pharmacologically manipulate neddylation in the adult heart using mouse models that lack NAE1 or are treated with MLN4924. We will determine whether restoring mitophagy via inhibiting neddylation ameliorates age-related cardiac dysfunction. Completing the proposed studies will produce critical insights into the role of mitophagy in age-related pathological conditions, and will fundamentally advance our understanding of the mechanisms of mitochondrial quality control in the heart.