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The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease
Published on: October 3, 2012
Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts
Anne Grünewald1, Lisa Voges, Aleksandar Rakovic
1Section of Clinical and Molecular Neurogenetics, Department of Neurology, University of Lübeck, Lübeck, Germany.
Background:
Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy.
Methodology/Principal Findings:
In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7), as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T) and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and controls under basal conditions and decreased to a similar extent under paraquat-induced stress.
Conclusions:
Our results indicate that Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.
Insights
Parkin mutations, common in Parkinson disease (PD), impair mitochondrial function and morphology. This study in human cells shows decreased ATP and altered mitochondrial potential in PD patients with Parkin mutations.
Area of Science:
- Neuroscience
- Cell Biology
- Genetics
Background:
- Mutations in the Parkin gene are a primary cause of autosomal recessive Parkinson disease (PD).
- Parkin, an E3 ubiquitin-protein ligase, is crucial for mitochondrial health, influencing respiratory chain function, mitochondrial dynamics, and mitophagy.
- Understanding Parkin's role is vital for elucidating PD pathogenesis.
Purpose of the Study:
- To investigate the impact of specific Parkin mutations on mitochondrial function and morphology in a human cellular model.
- To analyze cellular and mitochondrial responses under basal and oxidative stress conditions in fibroblasts from Parkinson disease patients.
Main Methods:
- Utilized fibroblasts from Parkinson disease patients with defined Parkin mutations and healthy controls.
- Assessed ATP synthesis, mitochondrial membrane potential, complex activities, oxidative stress markers, and mitochondrial network morphology.
- Employed luminometry, spectrophotometry, membrane potential assays, OxyBlot, and immunocytochemistry.
Main Results:
- Parkin-mutant fibroblasts exhibited reduced ATP production and concentration, alongside increased mitochondrial mass.
- Oxidative stress induced a significant decrease in mitochondrial membrane potential in patient cells compared to controls.
- Elevated levels of oxidized proteins were observed in Parkin-mutant cells under both basal and stress conditions.
Conclusions:
- Parkin mutations lead to significant abnormalities in mitochondrial function and morphology.
- These findings highlight the critical role of Parkin in maintaining cellular and mitochondrial homeostasis, even in non-neuronal cells.
- The study provides insights into the cellular mechanisms underlying Parkinson disease.
