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.
Highlights
Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset degeneration of upper and lower motor neurons
In this study we have performed the first comprehensive analysis of misfolded, aggregated and total superoxide dismutase-1 (SOD1) in a variety of fibroblast cell lines obtained from ALS patients carrying mutations in SOD1, as well as from patients carrying the GGGGCC-repeatexpansion in C9orf72 and from non-disease controls
We found elevated levels of misfolded SOD1 in fibroblast lines that expressed mutant SOD1 compared to the non-disease controls
Summary
Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset degeneration of upper and lower motor neurons. There are no apparent clinical (e.g. age of onset, survival time) or post-mortem histological differences between patients carrying missense mutations and disruptive mutations [5,6,7]. This suggests that a common cytotoxic mechanism originates from misfolded SOD1 species. The most disrupted truncated mutants are present at 100-fold lower levels [7, 10]. These findings suggest that minute subfractions of misfolded, not total, mutant SOD1 are the relevant pathogenic species for ALS
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