Lysosomal perturbations in human dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

Justyna Okarmus,Brent James Ryan,Helle Bogetofte,Sissel Ida Schmidt,Silvia García-López,Alberto Martínez-Serrano,Poul Hyttel,Morten Meyer,Matias Ryding

doi:10.1038/s41598-020-67091-6

Abstract

Mutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson’s disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. We used such cells to investigate the impact of PARK2 KO on the lysosomal compartment and found a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology with enlarged electron-lucent lysosomes and an increased lysosomal content, which was exacerbated by mitochondrial stress and could be ameliorated by antioxidant treatment. We also found decreased lysosomal enzyme activity and autophagic perturbations, suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, β-glucocerebrosidase, was increased, suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons and reveal an important new connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

Highlights

Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting 1–2% of the population
To study the disease mechanism underlining PARK2-mediated PD, we analysed two isogenic induced pluripotent stem cell (iPSC) lines that were created from a healthy control iPSC line, where KO of the PARK2 gene was created by zinc finger nuclease gene editing technology[30]
Both lines showed comparable expression levels with no significant differences. These data indicate that PARK2 KO does not affect the neuronal differentiation potential of the iPSC-derived neuronal stem cells (NSCs), as both the PARK2 KO and isogenic control lines were efficient in generating midbrain dopaminergic neurons

Summary

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting 1–2% of the population. Recent studies have documented mitochondria-lysosome membrane contact sites, which enable bidirectional regulation of mitochondrial and lysosomal dynamics, and have demonstrated how mitochondrial impairment supresses autophagic flux, suggesting a complex mutual relationship between these two cellular compartments[24,25,26,27,28]. By studying the lysosomal compartment and function in the context of parkin deficiency, we sought to address whether chronic mitochondrial dysfunction causes lysosomal impairment, contributing to PD pathogenesis. For this purpose, we studied isogenic iPSC-derived neuronal cultures with and without PARK2 mutation, which as we have recently shown, leads to several mitochondrial defects[29]. Parkin deficiency resulted in a number of perturbations including altered lysosomal content, morphology, and function as well as autophagic changes. This indicates a link between parkin deficiency and lysosomal disturbances

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