Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1‐linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin‐proteasome pathway in the mutant SOD1‐related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild‐type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild‐type, while only mutants were co‐immunoprecipitated with poly‐ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non‐ionic detergent‐insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS‐resistant dimer formation. Furthermore, the treatment of hSOD1‐expressing cells with hydrogen peroxide promoted the oligomerization, and detergent‐insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly‐ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin‐like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non‐motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1‐linked ALS.

Recent studies suggest that superoxide dismutase (SOD1) may represent a major target of oxidative damage in neurodegenerative diseases. To test the possibility that oxidized species of wild‐type (WT) SOD1 might be involved in pathogenic processes, we analyzed the properties of the WT human SOD1 protein after its oxidation in vivo or in vitro by hydrogen peroxide (H₂O₂) treatment. Using transfected Neuro2a cells expressing WT or amyotrophic lateral sclerosis‐linked SOD1 species, we show that exposure to H₂O₂ modifies the properties of WT SOD1. Western blot analysis of immunoprecipitates from cell lysates revealed that, like mutant SOD1, oxidized WT SOD1 can be conjugated with poly‐ubiquitin and can interact with Hsp70. Chromogranin B, a neurosecretory protein that interacts with mutant SOD1 but not with WT SOD1, was co‐immunoprecipitated with oxidized WT SOD1 from lysates of Neuro2a cells treated with H₂O₂. Treatment of microglial cells (line BV2) with either oxidized WT SOD1 or mutant SOD1 recombinant proteins induced tumor necrosis factor‐α and inducible nitric oxide synthase. Furthermore, exposure of cultured motor neurons to oxidized WT SOD1 caused dose‐dependent cell death like mutant SOD1 proteins. These results suggest that WT SOD1 may acquire binding and toxic properties of mutant forms of SOD1 through oxidative damage.

There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants linked to amyotrophic lateral sclerosis (ALS) and for neurotoxicity of extracellular mutant SOD1. This evidence led us to test immunization protocols aiming to reduce the burden of extracellular SOD1 mutants in nervous tissue of mice models of ALS, by using bacterially purified recombinant SOD1 mutant protein as an immunogen. First, a vaccination was tested on a G37R SOD1 mouse strain with late-onset disease exhibiting levels of mutant SOD1 protein at 4-fold higher than normal SOD1 levels. Repeated injections of adjuvant/SOD1 mutant with a final booster injection before symptoms at 6 months of age were effective in delaying disease onset and extending the life span of G37R SOD1 mice by >4 weeks. Western blot analysis with a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In contrast, this vaccination approach failed to confer significant protection in G93A SOD1 mice with extreme overexpression of mutant SOD1. Nonetheless, a passive immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic minipump succeeded in alleviating disease symptoms and prolonging the life span of G93A SOD1 mice. From these results, we propose that immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations.