Study links protein "clumps" with severity of disease in ALS model
Scientists studying amyotrophic lateral sclerosis (ALS) have long wondered why the fatal disease attacks only the nervous system, and more specifically, why it focuses on motor neurons-cells associated with movement-within the spinal cord.

Now investigators with the Center for ALS Research at Johns Hopkins have taken a small but definite step on the path to an explanation. Using a surprisingly simple technique, the researchers have shown, in mice engineered to carry the human gene for a specific, less common, form of ALS that large complexes of mutant protein accumulate in the spinal cords of the animals. Though the mutant protein-versions of a normal enzyme called SOD1- exists in many other cells of the body, it appears to form complexes only in the spinal cord and, to a lesser extent, in the brain.

The researchers have also shown that the spinal cord build-up apparently increases as symptoms of the disease worsen. Mice who had more severe paralysis, they say, had greater amounts of the protein complex. Before this study, scientists have observed protein clumping under the microscope, in sections of spinal cord from people with a heritable, or familial, form of ALS. They've also seen the aggregated protein in mice engineered with various forms of the mutant human SOD1 gene. But in this work, the researchers were able, for the first time, to measure the clumped protein in animals at different stages of the disease. They also discovered that the protein appears in large complexes, rather than just as a build-up of single mutated SOD1 molecules.

"It's clear to us," says David R. Borchelt, Ph.D., one of the researchers, "that the clumping isn't related so much to the amount of protein. It appears, instead, that something in the nervous system makes it less capable of preventing mutant SOD1 from aggregating. Cells in the spinal cord are particularly inept."

In their study, the researchers analyzed various mouse tissues, including brains and spinal cords, all containing human mutant SOD1 genes. Three different populations of mice were in the study, each with one of three of the commonest SOD1 mutations.

SOD1 mice have long been used as models of the specific familial form of ALS because they readily develop the same progressive paralysis and other symptoms that humans with ALS suffer. Researchers believe that the mice also provide important insights for the more common forms of ALS-that they have a similar downward spiral though the triggers may be somewhat different.

Tissues were taken from mice at all stages of the disease and liquidized, then poured through cellulose acetate-a type of synthetic membrane-as a filter. The acetate has tiny pores that lets smaller molecules through but that traps the large complexes of mutant SOD1. Tagged antibodies to human SOD1 identified the trapped protein. Further tests on the protein showed just how large the complexes were. The simple filtering technique has been used by others studying abnormal proteins in Huntington's disease, but, usually, proteins aren't trapped by cellulose acetate. Only very large complexes-which hadn't been suspected-appear to get caught. "We were really pleased to see this technique worked for SOD1," says Borchelt. "We've tried for a long time to find some straightforward way to isolate the protein so we could measure it."

As for what's going on within the cells, the researchers suggest something may go wrong with a normal "housekeeping" process in nerve cells to remove the clumped protein. It involves structures called proteasomes. "But that's just speculation," says researcher Jiou Wang, Ph.D. "We also don't know if the clumps are a result of the disease process or are a cause. And we also don't know why the large aggregates appear confined to the nervous system. But now we have a better way of studying it.”

Protein aggregation is also a hallmark of Alzheimer's disease and some other neurological disorders. There's a growing idea that the clumping may reflect some common response nerve cells have to disease. Guilian Xu, Ph.D. was also a member of the research team. Funding for the study came from the National Institutes of Health, from the ALS Association and the Center for ALS Research at Johns Hopkins.

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