Project Summary/Abstract Mitochondria are best known as the “powerhouses” of the cell, due to their predominant role in generating cellular energy through the tricarboxylic acid cycle, fatty acid metabolism, and oxidative phosphorylation (OXPHOS). Beyond energy generation, these essential organelles also play central roles in apoptosis, calcium handing, innate immunity, cell signaling, and iron-sulfur cluster assembly. Human health therefore depends critically on mitochondrial function. The research program proposed here seeks to understand three homeostatic mechanisms that regulate mitochondrial health. The first mechanism is mitochondrial fusion and fission dynamics. Mitochondria are dynamic organelles whose physiology is regulated by the balance between the opposing processes of membrane fusion and fission. Second, the quality of the mitochondrial population is maintained by selective degradation of excessive or defective mitochondria through mitophagy, the autophagic pathway that shuttles mitochondria to the lysosome for destruction. Finally, mitochondrial quantity and function are regulated by biogenesis programs that control expression of mitochondrial genes, ensuring that appropriate levels of mitochondria are maintained in response to a specific cellular state. This research project targets key gaps in knowledge in each of these fundamental homeostatic mechanisms. For mitochondrial fusion and fission, we are using mouse genetics and cell biology to understand how mitochondrial dynamics regulates mitochondrial function. The least understood of the core mitochondrial dynamics genes is Fis1. We have found that Fis1 plays essential functions in neurons, astrocytes, and oligodendrocytes in the central nervous system. We are pinpointing the cellular functions of Fis1 and determining which function is responsible for the in vivo phenotypes. Our preliminary data suggest that Fis1 serves as a link between mitochondria and the actin cytoskeleton. Moreover, we are determining the elusive mechanism through which the balance between fusion and fission is regulated. There is evidence that each fusion event is coupled to a future fission event, and we will determine the molecular mechanism of this coupling. To study mitochondrial degradation, we are performing whole- genome CRISPR interference screens and have discovered the integrated stress response as a key regulator of mitophagy. We will elucidate the molecular mechanism through which this cell stress pathway tunes the level of mitophagy. Finally, we are using CRISPR interference screens to identify how mitochondrial density and function are regulated. These studies have identified two chromatin remodeling complexes as critical for mitochondrial function. By determining how these chromatin remodeling complexes regulate mitochondrial biogenesis through control of gene expression, we will gain insight into how cells dynamically adjust mitochondrial density to fit cellular demands. All together, these ...