Experimental Mutations in Superoxide Dismutase 1 Provide Insight into Potential Mechanisms Involved in Aberrant Aggregation in Familial Amyotrophic Lateral Sclerosis.

Mutations in more than 80 different positions in superoxide dismutase 1 (SOD1) have been associated with amyotrophic lateral sclerosis (fALS). There is substantial evidence that a common consequence of these mutations is to induce the protein to misfold and aggregate. How these mutations perturb native structure to heighten the propensity to misfold and aggregate is unclear. In the present study, we have mutagenized Glu residues at positions 40 and 133 that are involved in stabilizing the ß-barrel structure of the native protein and a critical Zn binding domain, respectively, to examine how specific mutations may cause SOD1 misfolding and aggregation. Mutations associated with ALS as well as experimental mutations were introduced into these positions. We used an assay in which mutant SOD1 was fused to yellow fluorescent protein (SOD1:YFP) to visualize the formation of cytosolic inclusions by mutant SOD1. We then used existing structural data on SOD1, to predict how different mutations might alter local 3D conformation. Our findings reveal an association between mutant SOD1 aggregation and amino acid substitutions that are predicted to introduce steric strain, sometimes subtly, in the 3D conformation of the peptide backbone.

Loss of charge mutations in solvent exposed Lys residues of superoxide dismutase 1 do not induce inclusion formation in cultured cell models.

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis (fALS) induce the protein to misfold and aggregate. Missense mutations at more than 80 different amino acid positions have been associated with disease. How these mutations heighten the propensity of SOD1 to misfold and aggregate is unclear. With so many mutations, it is possible that more than one mechanism of aggregation may be involved. Of many possible mechanisms to explain heightened aggregation, one that has been suggested is that mutations that eliminate charged amino acids could diminish repulsive forces that would inhibit aberrant protein:protein interactions. Mutations at twenty-one charged residues in SOD1 have been associated with fALS, but of the 11 Lys residues in the protein, only 1 has been identified as mutated in ALS patients. Here, we examined whether loss of positively charged surface Lys residues in SOD1 would induce misfolding and formation of intracellular inclusions. We mutated four different Lys residues (K30, K36, K75, K91) in SOD1 that are not particularly well conserved, and expressed these variants as fusion proteins with yellow fluorescent protein (YFP) to assess inclusion formation. We also assessed whether these mutations induced binding to a conformation-restricted SOD1 antibody, designated C4F6, which recognizes non-natively folded protein. Although we observed some mutations to cause enhanced C4F6 binding, we did not observe that mutations that reduce charge at these positions caused the protein to form intracellular inclusions. Our findings may have implications for the low frequency of mutations at Lys residues SOD1 in ALS patients.

Direct and indirect mechanisms for wild-type SOD1 to enhance the toxicity of mutant SOD1 in bigenic transgenic mice.

Co-expression of wild-type human superoxide dismutase 1 (WT-hSOD1) with ALS mutant hSOD1 accelerates disease onset relative to mice expressing only mutant protein. Here, we analyzed the effect of co-expressed WT-hSOD1 in two established mutant mouse models (L126Z and G37R), and a new model that expresses the first 102 amino acids of SOD1 with mutations at histidines 46, 48 and 63 to eliminate Cu binding (Cu-V103Z). A subset of Cu-V103Z mice developed paralysis between 500 and 730 days. Similar to mice expressing L126Z-SOD1, the spinal cords of this new model showed SOD1 immunoreactive fibrillar inclusions. Co-expression of WT-hSOD1 with Cu-V103Z SOD1 moderately accelerated the age to paralysis, similar in magnitude to WT/L126Z mice. In either combination of these bigenic mice, the severity of fibrillar inclusion pathology was diminished and unreactive to antibodies specific for the C terminus of WT protein. Co-expression of WT-hSOD1 fused to yellow fluorescent protein (WT-hSOD1:YFP) with G37R-hSOD1 produced earlier disease, and spinal cords of paralyzed bigenic mice showed YFP fluorescent inclusion-like structures. In bigenic L126Z/WT-hSOD1:YFP mice, disease was not accelerated and WT-hSOD1:YFP remained diffusely distributed. A combination of split luciferase complementation assays and affinity capture-binding assays demonstrated that soluble G37R-hSOD1 efficiently and tightly bound soluble WT-hSOD1, whereas soluble forms of the Cu-V103Z and L126Z variants demonstrated low affinity. These data indicate that WT-hSOD1 may indirectly augment the toxicity of mutant protein by competing for protective factors, but disease onset seems to be most accelerated when WT-hSOD1 interacts with mutant SOD1 and becomes misfolded.

Features of wild-type human SOD1 limit interactions with misfolded aggregates of mouse G86R Sod1.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is well established that mutations in SOD1, associated with fALS, heighten the propensity of the protein to misfold and aggregate. Although aggregation appears to be a factor in the toxicity of mutant SOD1s, the precise nature of this toxicity has not been elucidated. A number of other studies have now firmly established that raising the levels of wild-type (WT) human SOD1 (hSOD1) proteins can in some manner augment the toxicity of mutant hSOD1 proteins. However, a recent study demonstrated that raising the levels of WT-hSOD1 did not affect disease in mice that harbor a mouse Sod1 gene (mSod1) encoding a well characterized fALS mutation (G86R). In the present study, we sought a potential explanation for the differing effects with WT-hSOD1 on the toxicity of mutant hSOD1 versus mutant mSod1. In the cell culture models used here, we observe poor interactions between WT-hSOD1 and misfolded G86R-mSod1, possibly explaining why over-expression of WT-hSOD1 does not synergize with mutant mSod1 to accelerate the course of the disease in mice.

Role of disulfide cross-linking of mutant SOD1 in the formation of inclusion-body-like structures.

BACKGROUND: Pathologic aggregates of superoxide dismutase 1 (SOD1) harboring mutations linked to familial amyotrophic lateral sclerosis (fALS) have been shown to contain aberrant intermolecular disulfide cross-links. In prior studies, we observed that intermolecular bonding was not necessary in the formation of detergent- insoluble SOD1 complexes by mutant SOD1, but we were unable to assess whether this type of bonding may be important for pathologic inclusion formation. In the present study, we visually assess the formation of large inclusions by fusing mutant SOD1 to yellow fluorescent protein (YFP). METHODOLOGY/PRINCIPAL FINDINGS: Experimental constructs possessing mutations at all cysteine residues in SOD1 (sites 6, 57, 111, and 146 to F,S,Y,R or G,S,Y,R, respectively) were shown to maintain a high propensity of inclusion formation despite the inability to form disulfide cross-links. Interestingly, although aggregates form when all cysteines were mutated, double mutants of the ALS mutation C6G with an experimental mutation C111S exhibited low aggregation propensity. CONCLUSIONS/SIGNIFICANCE: Overall, this study is an extension of previous work demonstrating that cysteine residues in mutant SOD1 play a role in modulating aggregation and that intermolecular disulfide bonds are not required to produce large intracellular inclusion-like structures.