AbstractBackground Mutations in parkin are the most common cause of early onset Parkinson’s disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in parkin mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from parkin mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Methods Fibroblasts from 4 controls and 4 parkin mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Results Direct conversion of control and parkin mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Conclusions Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches.
Oxidative switch drives mitophagy defects in dopaminergic parkin mutant patient neurons