Mar. 19, 2002 After almost 10 years of research with cells and animals to learn what makes a certain enzyme act as a "bad guy" in the progressive and fatal disease amyotrophic lateral sclerosis (ALS), Johns Hopkins scientists report that a leading candidate -- copper -- is off the hook.
In mice that develop ALS-like paralysis because of a malfunctioning enzyme called superoxide dismutase (SOD1), the researchers found that taking away copper had no effect on the pace of the disease, even though it is normally required for the enzyme's activity. The results appear in the current Advanced Online Publication section of Nature Neuroscience.
"Since the 1993 discovery of the enzyme's involvement in ALS, it's been proposed that copper might be to blame," explains Philip Wong, Ph.D., associate professor of pathology and neuroscience. "However, the long and short of it is that preventing copper from getting to SOD1 didn't make any difference in the onset of disease or survival of these ALS mice."
Because free copper is deadly to cells, the metal is carried to and fro by proteins called chaperones. A few years ago, researchers from Hopkins and elsewhere discovered the chaperone that delivers copper to SOD1 and named it CCS. In the new study, postdoctoral fellow Jamuna Subramaniam crossed mutant-SOD1 mice, which develop ALS-like symptoms, and mice that lack CCS to see how things changed if copper couldn't get to the enzyme.
The research team also tested other potential influences on symptoms, such as changes in the amounts of SOD1 present, and did experiments to see if copper was sneaking through to SOD1 by a back door. The sum of their results shows that copper and CCS play no role in the disease in these mice, says Wong.
"This was a direct test, in animals, of the copper hypothesis in SOD1-associated ALS, so we're sort of back to square one to examine other possible pathways," he says.
Not only did the disease pathway not change, but just as much mutant SOD1 enzyme was made in mice without CCS, an important result because levels of SOD1 affect timing of symptom progression. Furthermore, Jonathan Gitlin at Washington University in St. Louis used radioactive copper (copper-64) to "trace" distribution of the metal in the mice and proved that copper isn't reaching SOD1.
"Taking away CCS dramatically decreased the copper available to mutant SOD1, and yet the disease course didn't change," says Wong. "The results strongly argue against a role for copper in these ALS mice and imply that the same may be true in people with ALS who have mutations in SOD1."
With copper out of the way, the researchers will continue investigating SOD1 and will examine the SOD1-CCS mice for other explanations for the mutant enzyme's effects on motor neurons. The normal enzyme, which is found in all eukaryotes (animals and plants), is a key antioxidant inside cells, changing potentially damaging "superoxide" molecules into less damaging hydrogen peroxide. The hydrogen peroxide is then turned into harmless water by another enzyme.
"Initially we thought finding out what was happening with SOD1-related disease would be a piece of cake because the enzyme was so well known, but it's not turned out that way," says Wong.
Mutations in SOD1 are responsible for about one-fifth of familial, or inherited, ALS, and some non-inherited cases as well. The 60 or so mutations in SOD1 that lead to familial ALS are "autosomal dominant," which means that inheriting a single mutated copy will lead to disease, even if the other copy is normal. (A copy of each gene is inherited from each parent.) Familial ALS accounts for roughly 10 percent of all cases.
### The research was funded by the National Institutes of Health, the Amyotrophic Lateral Sclerosis Foundation, and The Spinal Cord Research Foundation.
Authors on the report are Subramaniam, Wong, Ernest Lyons, Jeffrey Rothstein and Donald Price, all of Hopkins; Jian Liu and Don Cleveland of the Ludwig Institute of Cancer Research at the University of California at San Diego; and Thomas Bartnikas and Jonathan Gitlin of Washington University in St. Louis.
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