title: Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.
abstract: 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.
  
  
  RESULTS
  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.
authors.affiliation: Section of Clinical and Molecular Neurogenetics, Department of Neurology, University of Lübeck, Lübeck, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Grünewald
authors.forename: Lisa
authors.initials: L
authors.lastname: Voges
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Rakovic
authors.forename: Meike
authors.initials: M
authors.lastname: Kasten
authors.forename: Himesha
authors.initials: H
authors.lastname: Vandebona
authors.forename: Claudia
authors.initials: C
authors.lastname: Hemmelmann
authors.forename: Katja
authors.initials: K
authors.lastname: Lohmann
authors.forename: Slobodanka
authors.initials: S
authors.lastname: Orolicki
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ramirez
authors.forename: Anthony H V
authors.initials: AH
authors.lastname: Schapira
authors.forename: Peter P
authors.initials: PP
authors.lastname: Pramstaller
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Sue
authors.forename: Christine
authors.initials: C
authors.lastname: Klein
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008875:Middle Aged; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D018384:Oxidative Stress; D010300:Parkinson Disease / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein