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Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

ABSTRACT: 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.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.Our results indicate that Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.

SUBMITTER: Grunewald A

PROVIDER: S-EPMC2946349 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

Grünewald Anne A Voges Lisa L Rakovic Aleksandar A Kasten Meike M Vandebona Himesha H Hemmelmann Claudia C Lohmann Katja K Orolicki Slobodanka S Ramirez Alfredo A Schapira Anthony H V AH Pramstaller Peter P PP Sue Carolyn M CM Klein Christine C

PloS one 20100927 9

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cel ...[more]

PMID: 20885945

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Project description:PurposeOxidative stress is a risk factor for the onset and progression of primary open-angle glaucoma (POAG), but the exact molecular basis remains unknown. Here, we investigated the mechanisms for Pro370Leu mutant myocilin to induce mitochondrial dysfunction and subsequent reactive oxygen species (ROS) generation in trabecular meshwork (TM) cells obtained from POAG individuals.MethodsPrimary non-diseased human TM cultures were transfected with pIRES-EGFP (Mock), pIRES-wild-type (WT), or pIRES-Pro370Leu mutant myocilin. Transfection efficiency and myocilin subcellular localization were determined by polymerase chain reaction (PCR), western blot analysis, and confocal microscopy. ROS levels as well as free Ca(2+) concentrations in cytoplasm ([Ca(2+)](c)) and mitochondria ([Ca(2+)]m) were examined by 2'7'-dichlorofluorescein diacetate (H(2)-DCF-DA), Fluo-3 acetoxymethyl ester (Fluo-3/AM), and Rhod-2 acetoxymethyl ester (rhod-2/AM), respectively, using flow cytometry. Mitochondrial functions were revealed by changes in mitochondrial membrane potential (DeltaPsim) and ATP production, which were found by fluorescent probe 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively.ResultsBoth WT and Pro370Leu mutant myocilin are localized in the mitochondria of TM cells as indicated using confocal microscopy and western blot analysis. Overexpression of WT myocilin decreases DeltaPsim, which is further reduced by Pro370Leu mutant myocilin. TM cells that overexpressed Pro370Leu mutant myocilin have greater cell death, higher endogenous ROS, [Ca(2+)](c), and [Ca(2+)](m) levels, and lower ATP production, and yet, these effects are not seen in the overexpression of WT myocilin.ConclusionsOur findings suggested that Pro370Leu mutant myocilin causes mitochondrial defects, which may lead to TM cell dysfunction and even cell death. Therefore, preventive measures targeting mitochondrial protection may delay the onset of glaucoma in individuals carrying the Pro370Leu myocilin mutation.

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Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

Publications

Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

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