Non-native Soluble Oligomers of Cu/Zn Superoxide Dismutase (SOD1) Contain a Conformational Epitope Linked to Cytotoxicity in Amyotrophic Lateral Sclerosis (ALS)

Abstract

Soluble misfolded Cu/Zn superoxide dismutase (SOD1) is implicated in motor neuron death in amyotrophic lateral sclerosis (ALS); however, the relative toxicities of the various non-native species formed by SOD1 as it misfolds and aggregates are unknown. Here, we demonstrate that early stages of SOD1 aggregation involve the formation of soluble oligomers that contain an epitope specific to disease-relevant misfolded SOD1; this epitope, recognized by the C4F6 antibody, has been proposed as a marker of toxic species. Formation of potentially toxic oligomers is likely to be exacerbated by an oxidizing cellular environment, as evidenced by increased oligomerization propensity and C4F6 reactivity when oxidative modification by glutathione is present at Cys-111. These findings suggest that soluble non-native SOD1 oligomers, rather than native-like dimers or monomers, share structural similarity to pathogenic misfolded species found in ALS patients and therefore represent potential cytotoxic agents and therapeutic targets in ALS.

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

“…Additionally, the recently discovered epitope of the C4F6 Ab (18), which preferentially binds the SOD1 trimer (Fig. S4A) as well as several other disease-linked SOD1 species (15,16) over the native dimer or monomer, is completely exposed on the surface of the trimer structure ( Fig. S4B), further supporting our model and suggesting the potential toxicity of the SOD1 trimer.…”

“…These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15)(16)(17)(18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).…”

“…We transfected cells with expression constructs for each of our designed SOD1 mutants, WT SOD1, and A4V-SOD1, an aggressive ALS-linked mutation responsible for the majority of familial ALS cases in North America (30,31), which we use as a positive control for ALS-relevant cell death. The A4V mutation has previously been observed to promote the formation nonnative SOD1 trimer (15). We observed significantly increased cell death in NSC-34 hybrid motor neuron cells expressing A4V-SOD1 (positive control) or our designed trimerstabilizing SOD1 mutants (Fig.…”

“…Our rationale is that to demonstrate agreement with limited proteolysis results, each proteolytic cut site residue should make as few contacts with other residues as possible, because its ability to be cleaved by proteolytic enzymes denotes its solvent accessibility and lack of participation in secondary structure interactions. However, although the proteolytic cleavage sites should demonstrate a minimum of structural interaction, SEC results (15) indicate that the structure should be a compact, associated trimer. We therefore use a combination of proteolytic cut site contacts and trimer radius of gyration (R g ) to select a pool of candidate structures from all REX simulation trajectories.…”

“…For determination of surface-accessible sites in the SOD1 trimer in limited proteolysis studies, we isolated and purified SOD1 from human erythrocytes, as previously described (26). To generate SOD1 mutants for aggregation time courses using SEC, and WT SOD1 for comparison of comparable species, we performed cloning, expression, and purification of human recombinant SOD1 in Saccharomyces cerevisiae as described elsewhere (15,26,38). We determined the concentration of purified SOD1 species by measuring the A at 280 nm with an extinction coefficient of 10,800 M −1 ·cm −1 .…”