title: Low levels of ALS-linked Cu/Zn superoxide dismutase increase the production of reactive oxygen species and cause mitochondrial damage and death in motor neuron-like cells.
abstract: Mutations of Cu/Zn superoxide dismutase (SOD1) are found in patients with familial amyotrophic lateral sclerosis (FALS). A cellular model of FALS was developed by stably transfecting the motor neuron-like cell line NSC-34 with human wild type (wt) or mutant (G93A) SOD1. Expression levels of G93ASOD1 were close to those seen in the human disease. The presence of G93ASOD1 did not alter cell proliferation but toxicity was evident when the cells were in the growth plateau phase. Flow cytometry analysis indicated that, in this phase, G93ASOD1 significantly lowered viability and that the level of reactive oxygen species was significantly higher in living G93ASOD1 cells compared to wt SOD1 cells. Biparametric analysis of mitochondrial membrane potential and viability of transfected cells highlighted a peculiar population of damaged cells with strong mitochondrial depolarization in the G93ASOD1 cells. Mitochondrial function seemed related to the level of the mutant protein since MTT conversion decreased when expression of G93ASOD1 doubled after treating cells with sodium butyrate. The mutant protein rendered G93ASOD1 cells more sensitive to mitochondrial dysfunction induced by stimuli that alter cellular free radical homeostasis, like serum withdrawal, depletion of glutathione by ethacrynic acid or rotenone-mediated inhibition of complex I of the mitochondrial electron transport chain. In conclusion, even a small amount of mutant SOD1 put motor neurons in a condition of oxidative stress and mitochondrial damage that causes cell vulnerability and death.
authors.affiliation: Laboratory of Molecular Pathology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
authors.forename: Milena
authors.initials: M
authors.lastname: Rizzardini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mangolini
authors.forename: Monica
authors.initials: M
authors.lastname: Lupi
authors.forename: Paolo
authors.initials: P
authors.lastname: Ubezio
authors.forename: Caterina
authors.initials: C
authors.lastname: Bendotti
authors.forename: Lavinia
authors.initials: L
authors.lastname: Cantoni
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology*; D015153:Blotting, Western; D016923:Cell Death; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000201:enzymology* / Q000473:pathology*; D009046:Motor Neurons / Q000201:enzymology* / Q000473:pathology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
chemical_list: D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D013482:Superoxide Dismutase; C022616:thiazolyl blue