Destabilized Soluble SOD1 Species as Potential Determinants of Disease Severity in Familial Amyotrophic Lateral Sclerosis.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are linked to familial amyotrophic lateral sclerosis (ALS), yet the identity of the toxic molecular species remains unclear. We investigated the relationship between protein misfolding and pathogenicity by expressing GFP-tagged wild-type and mutant SOD1 (A4V, H46R, G93A) in mouse hippocampal HT22 cells. Western blotting under nonreducing conditions suggested that A4V, associated with rapid disease progression, was largely depleted of properly folded soluble SOD1 and instead produced highly destabilized soluble species. In contrast, H46R, associated with a milder phenotype, showed a moderate reduction in properly folded soluble SOD1 and generated partially folded/native-like conformers. G93A exhibited biochemical characteristics intermediate between those of A4V and H46R. A4V also showed a pronounced loss of GFP fluorescence, indicating severe structural destabilization; the extent of fluorescence loss in A4V, G93A, and H46R broadly correlated with clinical severity. Neither CuATSM nor ebselen─targeting metal binding and disulfide formation, respectively─rescued fluorescence, suggesting broader defects in SOD1 maturation. Nevertheless, both compounds inhibited ferroptosis, a nonapoptotic form of cell death characterized by iron-dependent lipid peroxidation, in HT22 cells, indicating alternative neuroprotective mechanisms. These findings identify destabilized soluble SOD1 species as a key toxic entity in ALS and highlight the utility of GFP-tagged constructs for evaluating folding status and screening therapeutic candidates.