Abstract 1321 Defective mitochondrial and peroxisomal fission disrupts neurogenesis in the rare disease EMPF1

Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase DRP1 (dynamin-related protein 1). Patients with de novo heterozygous missense mutations in DRP1 present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) – a devastating rare neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations lead to developmental defects, we are using induced pluripotent stem cell (iPSC)-derived models from patients who present with clinically disparate conditions and have mutations in different domains of DRP1. Our studies with patient fibroblasts revealed that in addition to the hyperfused mitochondrial morphology and lack of fission, patient-derived cells display lower coupling efficiency, increased proton leak, and upregulation of glycolysis. Mitochondrial hyperfusion is also associated with aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisome morphology is severely elongated in patient cells and is associated with accumulation of very long chain fatty acids. Accordingly, metabolomic analyses revealed impaired fatty acid oxidation pathways. Given that EMPF1 patients present with a spectrum of neurodevelopmental abnormalities, we then explore the effects of DRP1 mutations on neural fate by introducing mutations found in EMPF patients into iPSCs, using CRISPR/Cas9. These iPSCs were differentiated into neural progenitor cells and neural organoids. We find that DRP1 mutant neural progenitor cells express lower levels of critical identity transcription factors, such as PAX6 and TBR2. Intriguingly, neural organoids with mutations in the stalk domain of DRP1 take on a choroid plexus identity instead of following a cortical development trajectory. Our results show that EMPF1 associated DRP1 mutations lead to metabolic dysregulation, which may cause changes in neural cell fate during early corticogenesis. We are currently investigating how dysregulated mitochondrial/peroxisomal fission and aberrant metabolic pathways may be central to the modulation of neural identity. Understanding these mechanisms will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment, as well as the mechanisms underlying the rare EMPF1 disease. This work was supported by 1R35GM128915-01 (VG), 1RF1MH123971-01 (VG); F99NS125829 (GLR); Northwestern University Pulmonary and Critical Care Department Cugell fellowship (RPC). Metabolomics Core at Robert H. Lurie Comprehensive Cancer Center: NIH1R35CA197532 and 5R01AI148190 ( NSC). Vanderbilt Cell Imaging Shared Resource: NIH1S10OD012324-01 and1S10OD021630-01.