The Elliott lab uses excess CCS as a sort of genetic magnifying glass to amplify the pathology of mSOD1 in mitochondria. Though SOD1 is mostly cytosolic, a small fraction is normally found in the mitochondrial intermembrane space, where it appears to aid mitochondrial manganese SOD2 in cleaning up reactive oxygen species (Sturtz et al., 2001). But some mSOD1 mice have mitochondrial pathology, including vacuolization of the mitochondria in many cell types (reviewed in Dupuis et al., 2004).

In yeast, CCS influences the cellular partitioning of SOD1 between the cytosol and mitochondria. When Son and Elliott overexpressed CCS in mSOD1 mice, “It had the most amazing effect on disease course,” Elliott said. CCS/SOD1-G93A mice develop tremors, ataxia, and spasticity at an average age of 11 days, compared to the six months it takes SOD1-G93A single mutants to exhibit symptoms (Son et al., 2007).

There are more than 100 distinct SOD1 mutations that have been linked to ALS, but only some, such as G93A and G37R, cause severe mitochondrial pathology. Other SOD1 mutations, such as G86R, have minimal vacuolization of mitochondria. Son used the CCS magnifying glass to probe the different mechanisms of four different SOD1 mutations: G93A, G37R, G86R, and L126Z. Both the G93A and G37R mutations responded strongly to additional CCS, making disease significantly worse in mice. Yet G86R and L126Z mice were not affected by overexpressed CCS.

Son looked to the redox state of mSOD1 as a potential explanation for the different phenotypes. In wild-type mice, approximately 15 percent of SOD1 has its disulfide bond in the reduced state. Elliott’s lab recently found that overexpressed CCS favors oxidation of the two cysteine amino acids that form the bond in wild-type SOD1, oxidizing 100 percent of the protein. However, extra CCS favors reduction of the same amino acids in SOD1-G93A (Proescher et al., 2008). Like SOD1-G93A, SOD1-G37R is also more likely to be reduced in mice overexpressing CCS (approximately 30 percent of SOD1 was reduced) than mutant SOD1 alone (10 percent reduced in young animals). In contrast, SOD1-G86R and SOD1-L126Z are fully reduced in single mutants and excess CCS does not affect their redox state, explaining why overexpressed CCS does not exacerbate disease in SOD1-G86R and SOD1-L126Z animals. Son and Elliott suggest that mSOD1’s redox state may be important in determining disease progression and pathology.

The explanation for the toxicity of disulfide-reduced mitochondrial SOD1 is still a mystery. Elliott hypothesizes that reduced SOD1 gums up the works by forming disulfide bonds with other essential mitochondrial proteins. Son and Elliott recently showed that CCS overexpression in SOD1-G93A mice results in lowered levels of cytochrome c oxidase (COX), which catalyzes the last electron transfer in the respiratory electron transport chain (Son et al., 2008). COX assembly is a delicate process requiring several proteins, Elliott said, so it’s possible that the reduced SOD1 interacts with and prevents those proteins from doing their job, cutting respiration and disabling the mitochondria.

Also at the SfN meeting, Elliott lab member Krishna Puttaparthi presented a poster outlining a screen to identify molecules that enhance COX levels. Puttaparthi dissected spinal cord slices from CCS/SOD1-G93A mice and cultured them with different treatments. Puttaparthi identified three compounds that increased COX levels. One such compound, resveratrol, increased survival by an average of 22 days when given to SOD1-G93A mice.

By amplifying CCS to exacerbate the mitochondrial phenotype of mSOD1 animals, Elliott and Culotta hope to zero in on the molecular mechanism of mitochondrial pathology in familial ALS caused by SOD1 mutations. For example, Culotta plans to use crystallography to collect “snapshots” of the incompletely developed SOD1 in cells overexpressing CCS.

The interactions between CCS and SOD1 are an example of how sensitive cells can be to protein levels. “You really need to have the right amount of CCS and the right amount of SOD1 to make the magic work,” Culotta said.—Amber Dance.