Understanding the effects of mitochondrial fission disruption during early cortical development

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Summary With the advent of exome sequencing, a growing number of children are being identified with de novo loss of function mutations in the large GTPase essential for mitochondrial fission - Dynamin Related Protein 1 (DRP1); these mutations result in severe neurodevelopmental phenotypes such as developmental delay, optic atrophy, and epileptic encephalopathies. Though it is established that mitochondrial fission is an essential precursor to the rapidly changing metabolic needs of the developing cortex, it is not understood how identified mutations in different domains of DRP1 uniquely disrupt this process. F-actin and the endoplasmic reticulum (ER) form a complex to prime the mitochondria for fission by pre-constricting the mitochondrial membrane prior to formation of DRP1 oligomers. The effect of DRP1 mutations on protein interactions with F-actin and the ER has never been studied in cell types of the developing cortex. This proposal focuses on testing the mechanism of DRP1 dysfunction both on protein interactions at sites of fission as well as downstream effects on cortical neuron differentiation and maturation. We aim to approach these gaps by leveraging the power of induced pluripotent stem cells (iPSCs) harboring DRP1 mutations in either the GTPase or stalk domains to model cell-fate changes associated with early cortical development. We will functionally assess the capacity for these iPSCs with mutant DRP1 to adopt a neural progenitor fate and progress to active cortical neurons using quantitative analysis of neurite outgrowth and branching, calcium transient recording, and synchronous synaptic firing. To understand how mutant forms of DRP1 interact with F-actin and the ER during fission, we will use live super-resolution Airyscan microscopy paired with in-cell immunoprecipitation to capture changes in the assembly and disassembly of this fission machinery. Successful completion of these aims will improve our understanding of the role of mitochondrial fission during cortical development and at which stages of this process perturbations become highly pathogenic. Furthermore, these results could help shed light on variable patient symptoms and outcomes based on specific DRP1 mutations, possibly leading to individualized therapeutics for mitochondrial disease.