Summary Mitochondrial Lon is an ATP-powered protease that plays a pivotal role in regulating mitochondrial proteostasis, metabolism and cell stress responses. Biallelic mutations in the LONP1 gene cause a broad spectrum of rare developmental diseases presenting during early development, which include CODAS syndrome- characterized by cerebral, ocular, dental, auricular and skeletal anomalies, profound neurological dysfunction and depletion of mitochondrial DNA (mtDNA). This project aims at filling a fundamental knowledge gap pertaining to the mechanistic impact of Lon binding to mtDNA and its role in regulating mtDNA integrity and expression. We will employ isogenic patient-derived induced pluripotent cell (iPSCs) with homozygous mutations in the LONP1 gene causing CODAS syndrome c.2161C>G, (p.Arg721Gly), or severe neurologic dysfunction c.2282 C>T, (p.Pro761Leu). Using iPSC-derived cell types expressing Lon-WT, Lon-R721G and Lon-P761L we will: (1) characterize differences in mitochondrial protein turnover, energetics and cell stress responses; (2) determine mtDNA-binding by wild-type and mutant Lon proteins using chromatin immunoprecipitation with sequencing (ChIP-seq); and (3) analyze the biogenesis of mtRNA transcripts using single-molecule fluorescence in situ hybridization (smFISH). The innovative approach of smFISH will allow us to determine differences in mtRNA synthesis, half-lives (i.e., degradation rates) and spatial localization within mitochondria. This project will provide new mechanistic insights into the importance of Lon in regulating mtDNA maintenance, transcription and translation, which has broader implications for its critical roles during normal physiology and common diseases such as cancer, neurodegeneration and cardiac dysfunction.