Neddylation and mitophagy in cardiac aging

项目总结 线粒体质量和活性的下降与正常衰老有关，并与 各种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。有丝分裂是必不可少的 线粒体质量控制机制，调节受损线粒体的溶酶体清除。 越来越多的证据已经证实，在不同的疾病中，增强的有丝分裂吞噬作用可以延长寿命 模型生物。有趣的是，最近的研究表明，增强的有丝分裂可能会抵消衰老-- 相关的心脏功能不全。因此，找出更有效和更具体的药物来调节 通过吞噬有丝分裂来清除有缺陷的线粒体可能会有显著的治疗效果。我们 使用pH依赖的荧光对有丝分裂调节剂进行了高含量的基于图像的分析 有丝分裂记者，凯马山。我们发现选择性代谢抑制剂MLN4924是最多的 有效的有丝分裂激活剂。去甲基化是一种结合泛素样蛋白的翻译后修饰 NEDD8通过NEDD8特异性的E1-E2-E3酶与蛋白质靶标结合。值得注意的是，我们的机械学研究表明 MLN4924有效地阻断了细长蛋白B/C-cullin 2-Rbx1(Ring-Box)的组成成分cullin 2的脱氢酶 1)-VHL(Von Hippel-Lindau蛋白)E3泛素连接酶复合体(CRL2VHL)。这种抑制导致了一个 低氧诱导因子1亚单位α(HIF1α)的积累，CRL2VHL底物，以及随后的 激活BCL2相互作用蛋白3(BNIP3)，这是一种线粒体受体，用于诱导有丝分裂。这些 结果提供了一种新的连接核变性和有丝分裂之间的联系。这个项目的目的是描绘这部小说 有丝分裂与新陈代谢之间的机械联系，以及确定有丝分裂是否代表一种小说 治疗老年性心功能不全的机制和治疗靶点。这些研究将得到便利 通过我们最近描述的Mt-Keima小鼠模型来监测体内心脏的吞噬作用。此外，我们还将 利用一系列创新试剂从基因和药物上调节糖尿病。直接评估 在心脏代谢中的作用，我们产生了心肌细胞特异性的NAE1缺失的小鼠， 编码E1脱氢酶激活酶的一个亚基。在拟议研究的目标1中，我们的目标是 确定抑制核代谢调节心肌细胞和心脏有丝分裂的机制。 在拟议研究的目标2中，我们将从遗传学和药理学方面对成人糖尿病进行操作。 使用缺乏NAE1或使用MLN4924治疗的小鼠模型的心脏。我们将确定是否恢复 丝裂原吞噬通过抑制代谢改善与年龄相关的心功能障碍。完成建议的 研究将对吞噬有丝分裂在年龄相关病理条件中的作用产生批判性的见解，并将 从根本上促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.