PROJECT SUMMARY/ABSTRACT Mitochondrial dysfunction is a pathological feature of many neurodegenerative diseases. We recently discovered that mutations in the mitochondrial protein ATAD3A (ATPase family, AAA domain containing 3A) cause HAREL- YOON syndrome, a disease characterized by peripheral neuropathy, optic atrophy, cardiomyopathy, and brain malformation. ATAD3A is implicated in other neurological genetic diseases, including hereditary spastic paraplegia, and congenital pontocerebellar hypoplasia, with abnormal cholesterol metabolism suggested as an underlying cause. Although ATAD3A is known to influence mitochondrial biology and lipid metabolism, how mutations in ATAD3A cause disease is unknown. There is an urgent need to fill this knowledge gap in order to prevent or treat ATAD3A-associated and other mitochondrial diseases, most of which have no known cure. Our long-term goal is to discover new therapeutic targets and strategies for mitochondrial diseases. The objective of our proposal is to uncover the mechanisms by which ATAD3A controls mitochondrial functions, using Drosophila and ATAD3A patient-derived induced pluripotent stem cells. Our CENTRAL HYPOTHESIS is that ATAD3A regulates mitochondrial membrane lipid homeostasis, and thus mitochondrial membrane dynamics, mitochondrial DNA (mtDNA) replication, and lipid metabolism, based on the following compelling evidence. First, ATAD3A plays a role in the formation of ER-mitochondria contact sites (EMCS) which are essential for import and synthesis of phospholipids. Second, ATAD3A is essential for importing cholesterol into mitochondria as well as maintaining cholesterol-rich mitochondria membrane structures. Lastly, patients carrying ATAD3A mutations exhibit increased 3-methylglutaconic acid excretion, which is often caused by a deficiency in mitochondrial respiratory complexes secondary to defects in cardiolipin remodeling. We expect that mechanistic insight into the consequences of ATADA3 mutations derived from our studies will reveal unanticipated therapeutic targets for prevention or treatment of a variety of mitochondrial diseases. We will test our central hypothesis by performing the following Specific Aims: (1) Elucidate how ATAD3A regulates mitochondrial membrane dynamics; (2) Determine how ATAD3A promotes mtDNA replication in neurons; and (3) Determine how ATAD3A regulates proper heart function. Upon conclusion of our studies, we expect to uncover how ATAD3A modulates mitochondria membrane dynamics, biogenesis, and heart function. Understanding how ATAD3A links diverse aspects of mitochondrial biology is expected to have a positive impact by revealing the molecular basis, as well as novel therapeutic targets and strategies for ATAD3A-associated diseases and other disorders caused by mitochondrial dysfunction.