A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis.

One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.