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. 2009 May 12;106(19):7774-9.
doi: 10.1073/pnas.0902505106. Epub 2009 Apr 30.

Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS

Celeste M Karch  1 , Mercedes Prudencio, Duane D Winkler, P John Hart, David R Borchelt
Affiliations

Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS

Celeste M Karch et al. Proc Natl Acad Sci U S A. .

Abstract

Transgenic mice that model familial (f)ALS, caused by mutations in superoxide dismutase (SOD)1, develop paralysis with pathology that includes the accumulation of aggregated forms of the mutant protein. Using a highly sensitive detergent extraction assay, we traced the appearance and abundance of detergent-insoluble and disulfide cross-linked aggregates of SOD1 throughout the disease course of SOD1-fALS mice (G93A, G37R, and H46R/H48Q). We demonstrate that the accumulation of disulfide cross-linked, detergent-insoluble, aggregates of mutant SOD1 occurs primarily in the later stages of the disease, concurrent with the appearance of rapidly progressing symptoms. We find no evidence for a model in which aberrant intermolecular disulfide bonding has an important role in promoting the aggregation of mutant SOD1, instead, such cross-linking appears to be a secondary event. Also, using both cell culture and mouse models, we find that mutant protein lacking the normal intramolecular disulfide bond is a major component of the insoluble SOD1 aggregates. Overall, our findings suggest a model in which soluble forms of mutant SOD1 initiate disease with larger aggregates implicated only in rapidly progressing events in the final stages of disease. Within the final stages of disease, abnormalities in the oxidation of a normal intramolecular disulfide bond in mutant SOD1 facilitate the aggregation of mutant protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Detergent-insoluble SOD1 accumulates to high levels near disease endstage. The amount of SOD1 present in the detergent-insoluble protein fraction was quantified by measuring the band intensity of SOD1 in each lane using a Fuji imaging system (FUJIFILM, LifeScience). Aggregation was quantified by calculating the ratio of band intensity in the insoluble fraction at each time point to the insoluble fraction at disease endstage (set at 1). The SEM was calculated for aggregation of each sample (Table S2) and graphed (A, G93A; B, G37R; C, H46RH48Q). Timelines of pathologic changes were graphed with aggregation. GA, Golgi apparatus.
Fig. 2.
Fig. 2.
The appearance of disulfide cross-linked SOD1 is coincident with the accumulation of detergent-insoluble mutant protein. G93A spinal cords were extracted in 1% SDS (A and B) or in 0.5% Nonidet P-40 (C and D) in the presence of 100 mM IA. Fractions were electrophoresed in the absence of βME. Immunoblots were probed with m/hSOD1 antiserum (representative image from 3 repetitions). NTg, nontransgenic. (A) Insoluble in 1% SDS (20 μg). (B) Soluble in 1% SDS (5 μg). (C) Insoluble in 0.5% Nonidet P-40. (D) Soluble in 0.5% Nonidet P-40. R, reduced disulfide bond (C57-C146). O, oxidized disulfide bond (C57-C146). *, disease endstage (paralysis); ·, fresh spinal cord tissue.
Fig. 3.
Fig. 3.
SOD1 aggregates are largely composed of disulfide-reduced forms of SOD1 in fresh spinal cord tissue. Spinal cords from freshly harvested mice were extracted in nonionic detergent and IA. Gels were incubated in reducing buffer before transfer. (Lanes 1–4) Detergent-soluble (40-μg protein). (Lanes 5–8) Detergent-insoluble (40-μg protein). Immunoblots were probed with m/hSOD1 antiserum. R, reduced. O, oxidized. #, possible nonnatively oxidized bond. For additional repetitions, see Fig. S7.
Fig. 4.
Fig. 4.
Reduced hSOD1 protein is preferentially incorporated into detergent-insoluble aggregates. HEK293FT cells were transfected with vectors for WT, A4V, G37R, G93A, and H46R/H48Q hSOD1 proteins, harvested after 48 h, and extracted in buffers with 0.5% Nonidet P-40 and 100 mM IA (representative experiment from 4 repetitions). Detergent-insoluble (A) and soluble fractions (B) from cells transfected with each construct were electrophoresed in the absence of βME (lanes 2–8). Lane 1 of each panel contains purified WT SOD1 that was reduced before electrophoresis (*). This lane was separated from the remaining samples by a lane containing marker proteins and an empty space to prevent reducing agent from diffusing into other samples (these intervening lanes have been cropped out of the gel image). Lane 8 of each panel contains purified WT SOD1 that was verified to have in intact intramolecular disulfide bond (·). Purified WT SOD1 proteins were treated with 100 mM IA prior boiling in Sample buffer and electrophoresis. To enhance detection of oxidized forms of hSOD1, gels were incubated in transfer buffer containing 2% βME for 10 min before electrotransfer to nitrocellulose membranes. UT, untransfected cells; R, reduced disulfide bond (C57-C146). O, oxidized disulfide bond (C57-C146).
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