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 ...