Mitochondrial dysfunction in Parkinson disease: evidence in mutant PARK2 fibroblasts.

Maria C Zanellati,Enza M Valente,Alberto Albanese,Chiara Barzaghi,Barbara Garavaglia,Valentina Monti,Daniele Ghezzi,Chiara Reale,Nardo Nardocci

doi:10.3389/fgene.2015.00078

Maria C Zanellati, Enza M Valente + Show 7 more

Open Access

https://doi.org/10.3389/fgene.2015.00078

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Abstract

Mutations in PARK2, encoding Parkin, cause an autosomal recessive form of juvenile Parkinson Disease (JPD). The aim of the present study was to investigate the impact of PARK2 mutations on mitochondrial function and morphology in human skin fibroblasts. We analyzed cells obtained from four patients clinically characterized by JPD, harboring recessive mutations in PARK2. By quantitative PCR we found a reduction (<50%) of PARK2 transcript in all patients but one; however Western Blot analysis demonstrated the virtual absence of Parkin protein in all mutant fibroblasts. Respiration assays showed an increment of oxygen consumption, which was uncoupled to ATP cellular levels. This finding was probably due to presence of altered mitochondrial membrane potential (ΔΨm), confirmed by JC-1 analysis. The mitochondrial network was comparable between mutant and control cells but, interestingly, a “chain-like” network was found only in mutant fibroblasts. Dissipation of ΔΨm usually leads to mitochondrial fragmentation in healthy cells and eventually to mitophagy; however, this behavior was not observed in patients' fibroblasts. The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy. This condition should increase the oxidative stress and lead to cellular dysfunction and death. Our results suggest that PARK2 mutations cause mitochondrial impairment, in particular reduction in ATP cellular levels and alteration of ΔΨm, even in non-neuronal cells and confirm the hypothesis that Parkin holds a pivotal role in pro-fission events.

Highlights

Parkinson’s disease (PD) is the second most common neurodegenerative disorder
Till the studies performed on PARK2 mutant fibroblasts in order to explore the impact of Parkin on mitochondrial functionality, have remain elusive (Mortiboys et al, 2008; Grünewald et al, 2010; Pacelli et al, 2011; van der Merwe et al, 2014); these reports highlighted how fibroblasts derived from PD patients may be a reliable model system to study mitochondrial dysfunction
Fibroblasts Derived from Patients with Mutations in PARK2 have Reduced Level of PARK2 mRNA and Parkin Protein Compared to Controls To examine the possible effect of PARK2 mutations on splicing, a PCR amplification was performed on mRNA extracted from patients’ fibroblasts

Summary

Introduction

Its etiology is mainly elusive, it has been shown that some substances with toxic effect on mitochondrial functioning (e.g., MPTP and rotenone) can induce parkinsonism in human and animal model (Langston et al, 1983; Nicklas et al, 1985), suggesting an important role of mitochondria in the pathophysiology of PD. Even if environment factors play an important role in PD, mutations in a number of genes have been found to cause inherited forms of PD, with both. Mitochondrial dysfunction in PARK2 fibroblasts autosomal dominant (e.g., LRRK2, SNCA) and recessive transmission (e.g., PARK2, PINK1, DJ1). Many of these PD geneproducts have been proven to influence mitochondrial bioenergetics and dynamics, including membrane potential, respiratory activity, cristae structure, calcium homeostasis, mitochondrial DNA (mtDNA) integrity and clearance of dysfunctional mitochondria (reviewed in Sai et al, 2012). PARK2 mutations range from single base pair substitutions, splice site mutations and small nucleotide deletions, to large deletions or duplications of one or more PARK2 exons; albeit through different mechanisms, all these variants probably have a “loss of function” effect

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