title: Effects of Cellular Pathway Disturbances on Misfolded Superoxide Dismutase-1 in Fibroblasts Derived from ALS Patients.
abstract: Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.
authors.affiliation: Department of Pharmacology and Clinical Neurosciences, Umeå University, Umeå, Sweden.
authors.forename: Isil
authors.initials: I
authors.lastname: Keskin
authors.affiliation: Department of Pharmacology and Clinical Neurosciences, Umeå University, Umeå, Sweden.
authors.forename: Elin
authors.initials: E
authors.lastname: Forsgren
authors.affiliation: Department of Neurology, Hospital for Special Surgery and Weill Cornell Medical Center, New York, NY, United States of America.
authors.forename: Dale J
authors.initials: DJ
authors.lastname: Lange
authors.affiliation: Neuromusucular Diseases Unit/ALS Clinic, Kantonsspital St.Gallen, St. Gallen, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Weber
authors.affiliation: Department of Pharmacology and Clinical Neurosciences, Umeå University, Umeå, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Birve
authors.affiliation: Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, Tübingen, Germany.|German Research Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Tübingen, Germany.
authors.forename: Matthis
authors.initials: M
authors.lastname: Synofzik
authors.affiliation: Department of Pharmacology and Clinical Neurosciences, Umeå University, Umeå, Sweden.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Gilthorpe
authors.affiliation: Department of Pharmacology and Clinical Neurosciences, Umeå University, Umeå, Sweden.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Andersen
authors.affiliation: Department of Medical Biosciences, Clinical Chemistry, Umeå University, Umeå, Sweden.
authors.forename: Stefan L
authors.initials: SL
authors.lastname: Marklund
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D017668:Age of Onset; D000375:Aging / Q000378:metabolism; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D000069286:Bortezomib / Q000494:pharmacology; D000073885:C9orf72 Protein; D016022:Case-Control Studies; D002478:Cells, Cultured; D042622:DNA Repeat Expansion; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors; D059865:Endoplasmic Reticulum Stress; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000201:enzymology* / Q000473:pathology; D005838:Genotype; D006801:Humans; D009154:Mutation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D066263:Protein Aggregation, Pathological; D017510:Protein Folding; D011506:Proteins / Q000235:genetics; D059748:Proteolysis; D012402:Rotenone / Q000494:pharmacology; D012995:Solubility; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1
chemical_list: D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D011480:Protease Inhibitors; D011506:Proteins; C000606290:SOD1 protein, human; D012402:Rotenone; C025946:3-methyladenine; D000069286:Bortezomib; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D046988:Proteasome Endopeptidase Complex; D042967:Electron Transport Complex I; D000225:Adenine