Chromogranins A and B are secretory vesicle components of unknown function in the nervous system. Julien’s group became interested in the proteins when a yeast two-hybrid screen using mSOD1 as bait pulled out chromogranin A. Both chromogranins immunoprecipitated with mutant SOD1, but not wild-type SOD1 (see ARF related news story on Urushitani et al., 2006). The chromogranins appear to require a domain similar to heat-shock protein sequences to bind mSOD1, suggesting they may recognize the mutant protein’s improper conformation. In human studies by another group, chromogranins colocalized with SOD1 in intracellular aggregates in tissue from people who had ALS (Schrott-Fischer et al., 2009). Chromogranins have also been found in Aβ plaques in Alzheimer disease (Marksteiner et al., 2000), and in prion deposits in Creutzfeld-Jakob disease (Rangon et al., 2003).

To further investigate the chromogranin-mSOD1 interaction in disease, Julien and colleagues engineered mice with the chromogranin A gene driven by the human Thy1 promoter, which is active in the nervous system. The mice express twice the normal amount of chromogranin A in neurons. They crossed these mice with animals expressing human SOD1-G37R. The double mutant mice exhibited symptoms of motor neuron disease one month earlier than their mSOD1 single-mutant parents. The double mutants had increased degeneration of motor neurons, compared to SOD1-G37R mice, as well.

However, the excess chromogranin A did not speed up the final stages of the disease; the double mutant mice survived for the same length of time as single mutants. Julien hypothesized that early on, the animal’s full complement of motor neurons produced plenty of chromogranin A and mSOD1, accelerating disease onset. But as motor neurons died, there were fewer of them to produce the damaging proteins. In later stages, then, the rate of disease slowed to normal.

How does chromogranin A accelerate motor neuron degeneration? Using an antibody specific for misfolded SOD1, the researchers found that the double mutants had more of the malformed species than did single mutant mice. Julien hypothesized that chromogranin A binds and stabilizes the misfolded protein, allowing it to hang around longer and do more damage. Alternatively, he suggested, the excess chromogranin A may promote secretion of misfolded SOD1 into the extracellular space where it is cytotoxic.

Previous results indicated that chromogranins promote secretion of mSOD1 (Urushitani et al., 2006), and aggregated mSOD1 is found in the endoplasmic reticulum and Golgi, along the secretory roadway (Urushitani et al., 2008). Abou Ezzi suggested that the increased chromogranin A might capture mSOD1, causing it to aggregate in the ER and Golgi, leading to ER stress. Other researchers have shown that motor neurons are especially vulnerable to endoplasmic reticulum stress (see ARF related news story on Saxena et al., 2009), so stressing the ER or secretory pathways could be a common theme in the disease, Julien suggested.

The scientists also crossed the SOD1-G37R mouse with a chromogranin A knockout strain. These double mutants showed little difference in disease phenotype from single SOD1 mutants, perhaps because chromogranin B compensates for the loss of chromogranin A, Julien suggested. However, the double mSOD1/chromogranin A knockout mutants did have more motor axons in the ventral root than their mSOD1 counterparts at a late stage of disease, suggesting reduced motor neuron degeneration.

Do these findings in mice have any bearing on human disease? Julien and colleagues also looked for evidence of chromogranin involvement in people with ALS. They discovered a chromogranin B variant, not yet published, that was present at higher levels in a French ALS population than in controls. This variant appears to be a risk factor for the disease, and to cause onset seven years earlier. “It resembles very much ApoE in Alzheimer’s,” Julien said. The researchers are currently working with cell cultures and mice to decipher the mechanism behind the increased risk.

The research supports current hypotheses about mSOD1’s damaging effects, said Christine Vande Velde of the University of Montréal, who was not involved in the study. “It lends more strength to the idea that misfolded SOD1, if you let it stick around or if you stabilize it, then it is more toxic,” she said.—Amber Dance.