Oxidative switch drives mitophagy defects in dopaminergicparkinmutant patient neurons

Abstract

22Background Mutations in parkin are the most common cause of early onset Parkinson's disease. Parkin 23 is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in parkin 24 mutant models. Patient derived neurons are a promising model in which to study pathogenic 25 mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from parkin 26 mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial 27 phenotypes as neurons undergo a metabolic switch during differentiation. Methods Fibroblasts from 28 4 controls and 4 parkin mutant patients were transformed into induced neuronal progenitor cells and 29 subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and 30 mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species 31 production quantification. Results Direct conversion of control and parkin mutant patient fibroblasts 32 results in induced neuronal progenitor and their differentiation yields high percentage of 33 dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons 34 undergo a metabolic switch during differentiation. Our results show that when pre-neurons are 35 glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons 36 become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant 37 patient neurons. These changes correlate with changes in mitochondrial function and morphology; 38 resulting in lower neuron yield and altered neuronal morphology. Conclusions Induced neuronal 39 progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial 40 phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only 41 when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are 42 identified. These data provide insight into cell specific effects of PRKN mutations, in particular in 43 relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic 44 approaches. 45 46 47 48 Parkinson's disease (PD) is the second most common neurodegenerative disease, with 49 approximately 10 million people affected worldwide. Only symptomatic treatment options are 50 available. Mutations in PRKN are the most common cause of early onset PD (EOPD). Parkin is an E3 51 ubiquitin ligase and functions in the mitophagy pathway 1 . Mitochondrial dysfunction is well 52 established in both familial and sporadic forms of PD (recently reviewed 2 ). Mitochondrial 53 abnormalities are present in both PRKN null Drosophila 3 and mice 4 . We and others have shown 54 mitochondrial abnormalities in peripheral cells from patients with PRKN mutations 5-8 ; these include 55 cellular ATP defects, mitochondrial membrane potential deficiencies, complex I defect and altered 56 mitochondrial morphology. Recent work suggests mitophagy is...