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Introduction: Cardiolipin (CL) is a glycerophospholipid found exclusively in the inner mitochondrial membrane consisting of a double glycerophosphate backbone and 4 acyl side chains; the profile of these side chains is largely tissue-specific.CL is involved in critical functions in the mitochondria, including organization of the respiratory chain, initiation of apoptosis, activation of mitophagy, and maintenance of bioenergetic homeostasis.Two major disorders of CL metabolism include Barth Syndrome (BTHS MIM 302060), an X-linked disorder caused by pathogenic variants in the gene TAFAZZIN resulting in abnormal CL remodeling and Combined Oxidative Phosphorylation Deficiency 57 (COXPD57 MIM 620167), an autosomal recessive disorder of defective nascent CL synthesis caused by pathogenic variants in CL Synthase, (CRLS1).Both disorders are ultra-rare; BTHS has an estimated prevalence of 1/400,000-1/1,000,000 and COXPD57 has been described in 4 patients to date.BTHS and COXPD57 are associated with multiorgan involvement; however, there is a strikingly disparate neurologic involvement between both disorders.COXPD57 is a severe neurodevelopmental disorder characterized by progressive infantile encephalopathy, hearing loss, and maculopathy, whereas individuals with BTHS typically have a relatively mild neurodevelopmental profile, characterized by learning disabilities, spatial memory challenges, and cognitive fatigue.Recent studies in CRLS1-KO and TAZ-KD mice have shown defective hippocampal neurogenesis and impaired spatial memory, respectively, however few studies have investigated the impact of defective CL synthesis (CRLS1) and remodeling (TAZ) on the central nervous system in human cells.Methods: The goals of this study were to generate CRLS1-deficient human iPSC models using CRISPR base and prime editing and to identify convergent vs disease-specific molecular mechanisms between the CRLS1 mutant cells and an established TAZ-knockout (TAZ-KO) iPSC model already under investigation in our lab.We generated two CRLS1 mutant cell lines with homozygosity for disease-causing variants that were reported in affected patients: p.Ile109Asn and p.Leu217Phe.Results: Both CRLS1 mutant lines demonstrated increased CRLS1 transcript expression and protein abundance in the setting of pathognomonic lipid abnormalities including decreased cardiolipin content and increased cardiolipin precursors (eg, phosphatidylglycerol).The CRLS1 mutant iPSCs had a strikingly abnormal respiratory chain phenotype including abnormal expression of mtDNA encoded OXPHOS subunits including ND-1, ND-3, CYB, CO-1, and ATP6, as well as abnormal respiratory chain supercomplex (RSC) distribution, assembly, and abundance.Finally, we observed a disturbance in proteins involved in canonical mitophagy protein levels including LC3, indicating impaired basal mitophagy in CL deficient cells.Conclusion: Based on our findings, we conclude that CRLS1-deficient (COXPD57) and TAZ-KO (Barth syndrome) iPSCs share a core mitochondrial phenotype characterized by respiratory chain supercomplex instability (especially CIII/CIV), impaired CIV activity, disrupted mitochondria-ER contacts, fragmented/dysmorphic mitochondrial morphology, and mitophagy dysregulation in the setting of CL defects and phosphatidylglycerol (PG) accumulation.Ultimately, understanding the role of CL metabolism in the brain will help to uncover therapeutic targets for both BTHS and COXPD57 and, from a broader perspective, may help shed light on treatment for other neurodegenerative disorders where aberrant CL metabolism has been implicated, including Alzheimer's Disease, Parkinson's Disease, Traumatic Brain Injury, and Amyotrophic Lateral Sclerosis.This work ultimately may uncover fundamental insights into mitochondrial pathology relevant to both rare and common neurodegenerative diseases.