Abstract
Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O2 is required for formation of the bond, we reasoned that low O2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low O2 tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1. However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression.
Highlights
Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset progressive degeneration of upper and lower motor neurons (MN)
To determine whether levels of copper chaperone for superoxide dismutase (CCS) or glutaredoxin-1 were affected by low O2 tension and responsible for increased disordered superoxide dismutase 1 (SOD1), we analyzed their levels in motor neuron and astrocyte cultures (MNACs) grown at 19% versus 2% O2
By comparing the responses of a large panel of patientderived cell lines carrying genetically and biochemically distinct SOD1 variants, we have found that low O2 tension leads to remarkably large increases in SOD1 C57–C146 disulfide bond reduction and disorder
Summary
Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset progressive degeneration of upper and lower motor neurons (MN). Most of the truncation mutations disrupt both the β-barrel core and the stabilizing intrasubunit C57–C146 disulfide bond of the protein. These mutants are intrinsically disordered, since they cannot fold correctly, and are rapidly degraded [13, 45, 50, 51, 68]. Based on these observations, it is likely that any common ALS-causing SOD1 species is disordered, lacks the C57–C146 disulfide bond, and is present at very low levels
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