Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochondria

Abstract

Twenty percent of the familial form of amyotrophic lateral sclerosis (ALS) is caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1) through the gain of a toxic function. The nature of this toxic function of mutant SOD1 has remained largely unknown. Here we show that WT SOD1 not only hastens onset of the ALS phenotype but can also convert an unaffected phenotype to an ALS phenotype in mutant SOD1 transgenic mouse models. Further analyses of the single- and double-transgenic mice revealed that conversion of mutant SOD1 from a soluble form to an aggregated and detergent-insoluble form was associated with development of the ALS phenotype in transgenic mice. Conversion of WT SOD1 from a soluble form to an aggregated and insoluble form also correlates with exacerbation of the disease or conversion to a disease phenotype in double-transgenic mice. This conversion, observed in the mitochondrial fraction of the spinal cord, involved formation of insoluble SOD1 dimers and multimers that are crosslinked through intermolecular disulfide bonds via oxidation of cysteine residues in SOD1. Our data thus show a molecular mechanism by which SOD1, an important protein in cellular defense against free radicals, is converted to aggregated and apparently ALS-associated toxic dimers and multimers by redox processes. These findings provide evidence of direct links among oxidation, protein aggregation, mitochondrial damage, and SOD1-mediated ALS, with possible applications to the aging process and other late-onset neurodegenerative disorders. Importantly, rational therapy based on these observations can now be developed and tested.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Kaplan–Meier plots showing the age of onset and cumulative survival of the single- and double-transgenic mice. (Top) hwtSOD1 exacerbates the disease in SOD1G93A (shown as G93A) transgenic mice. The G93A/hwtSOD1 double-transgenic mice (red line; n = 21) have an earlier onset of disease (76.8 ± 8.7 days vs. 103.2 ± 9.9 days; log-rank test χ2 = 97.63, P < 0.0001) (Left) and a shorter lifespan (117.7 ± 7.7 days vs. 127.4 ± 7.6 days; log-rank test χ2 = 20.81, P < 0.0001) (Right) than the G93A single-transgenic mice (blue line; n = 58, sibling control). (Middle) hwtSOD1 converts an unaffected phenotype of a SOD1A4V (shown as A4V) transgenic mouse line to an ALS-like phenotype. The A4V transgenic mice do not develop disease in their lifetime (>600 days; n = 23). However, the A4V/hwtSOD1 double-transgenic mice (n = 6) de novo develop ALS-like disease (227.7 ± 50.7 days; χ2 = 41.91, P < 0.0001) (Left) with a shorter lifespan (318.5 ± 41.7 days; χ2 = 41.01, P < 0.0001) (Right). (Bottom) hwtSOD1 exacerbates the disease in SOD1L126Z (shown as L126Z) transgenic mice. The L126Z/hwtSOD1 double-transgenic mice have a much earlier onset of disease [178.3 ± 19.1 days (n = 7) vs. 336.3 ± 42.7 days (n = 18); χ2 = 34.38, P < 0.0001] (Left) and a much shorter lifespan than the single-transgenic mice (201.4 ± 14.5 days vs. 359 ± 41.1 days; χ2 = 34.38, P < 0.0001) (Right).

Fig. 2.

Immunohistochemical staining of the spinal cord sections of various SOD1 transgenic mice. (a–c) SOD1L126Z mice, affected. (a) Distribution of SOD1-immunoreactive aggregates (arrow) in an entire spinal cord section, stained with the f-SOD1 antibody (not counterstained with hematoxylin for clarity of the distribution of the SOD1 aggregates). (Scale bar: 100 μm.) (b) c-SOD1 antibody: no aggregates and a light diffuse staining (arrow). (Scale bar: 35 μm.) (c) hs-SOD1 antibody, SOD1 aggregates of SOD1L126Z, a component of the aggregates. A loss of the large neurons in the anterior horns is apparent in panels a–c. (d) hwtSOD1 transgenic mice, unaffected. c-SOD1 antibody: diffusely distributed SOD1 immunostaining (arrow), predominantly in large cells, and no SOD1 aggregates in large neurons of the anterior horn of the spinal cord. (e) SOD1L126Z/hwtSOD1 double-transgenic mice, affected. c-SOD1 antibody: pronounced SOD1 aggregates (arrow), indicating the presence of wtSOD1 in the aggregates.

Fig. 3.

Detergent-insoluble form of both mutant and WT SOD1 in spinal cord of affected transgenic mice. (a) SOD1 in the supernatant fraction detected with hs-SOD1 (Top) and f-SOD1 (Middle) antibodies in different mouse lines. NT, nontransgenic mice. β-Actin was used as a control (Bottom). Mouse endogenous SOD1 is indicated by an arrow. Human SOD1 is used as a standard (stSOD1). (b) SOD1 in the pellet fraction detected with f-SOD1 antibody. A certain amount of hwtSOD1 could be detected in hwtSOD1 transgenic mice (Upper, arrowhead), but this hwtSOD1 is not detergent-insoluble (Lower). The detergent-insoluble form of SOD1 was detected in the affected SOD1A4V/hwtSOD1 double-transgenic mice but not in the unaffected SOD1A4V single-transgenic mice. The truncated SOD1L126Z protein was detected only in the insoluble fraction and was present in a detergent-insoluble form (arrow). Different from the hwtSOD1 in the hwtSOD1 transgenic mice, the hwtSOD1 in the SOD1L126Z/hwtSOD1 double-transgenic mice is detergent-insoluble (arrowhead). Similar results were obtained by using the hs-SOD1 antibody.

Fig. 4.

ImmunoGold electron microscopy of the spinal cord sections with a SOD1 antibody (f-SOD1) showing altered mitochondrial morphology. The morphology of the cristae in gold-particle-dense areas was severely altered in the affected SOD1G93A (a) and SOD1L126Z (b) mice. Some gold particles were deposited in close association with the swollen cristae (arrowheads). Some cristae in the gold-particle-dense areas were severely damaged (large arrows). Such damage was not observed in the relatively normal mitochondria without (a) or with fewer (b) gold particles in the same mice as indicated by small arrows.

Fig. 5.

Disulfide bond-mediated SOD1 dimers and multimers in the affected SOD1G93A, SOD1L126Z, and SOD1L126Z/hwtSOD1 mice. Shown are the detergent-insoluble mitochondrial fractions from transgenic mice overexpressing hwtSOD1, SOD1G93A, SOD1L126Z/hwtSOD1, and SOD1L126Z. A soluble fraction from hwtSOD1 transgenic mice [hwtSOD1 (soluble)] was also loaded to show the position of the human (Hu) and mouse (Mo) SOD1 monomers in Right. Left was detected with the antibody f-SOD1, and Right was detected with c-SOD1. Protein molecular weight markers are indicated on the right. The unaffected human hwtSOD1 transgenic mice did not show SOD1 dimers and multimers, but affected SOD1G93A mice showed ladder-like SOD1 molecules, with the molecular sizes equivalent to dimer, trimer, tetramer, and so on, as labeled between Left and Right. The patterns of the dimers and multimers were similar, although two different antibodies (f-SOD1 and c-SOD1) were used. The affected SOD1L126Z mice showed a predominant dimer band when antibody f-SOD1 was applied. As expected, this dimer band was not detected by using the antibody c-SOD1 in Right. The SOD1L126Z/hwtSOD1 mice showed a unique pattern of dimers and multimers. The dimer group consisted of three dimer bands. The dimer band with the lower molecular weight (large arrow) in the SOD1L126Z/hwtSOD1 mice was similar to the dimer band in SOD1L126Z mice, in both molecular size and antigenecity with antibody c-SOD1, indicating that it is composed of the truncated SOD1L126Z protein. The dimer band with the higher molecular weight (small arrow) had a molecular size similar to that of the dimer in SOD1G93A mice, suggesting that two molecules of hwtSOD1, but not the truncated SOD1L126Z, are involved in this dimer formation. The dimer band in the middle (arrowhead) was smaller than SOD1G93A dimer band but larger than SOD1L126Z dimer band in molecular size. It was positive when both the f-SOD1 and c-SOD1 were applied, but its intensity was greatly reduced with antibody c-SOD1. This finding suggests that it is a heterodimer, including one molecule unit of SOD1L126Z and one molecule unit of hwtSOD1. Apparently, each multimer group also consists of various combinations of hwtSOD1 and SOD1L126 multimers. Very small amounts of SOD1 monomers could also be seen in longer time exposure as shown in Right.