ALS STUDIES FOCUS ON POSSIBLE CAUSES

Inadequate "cleanup" of glutamate from around nerve cells may contribute to amyotrophic lateral sclerosis (ALS), say researchers at Johns Hopkins University in Baltimore and Emory University in Atlanta. The team, which included MDA grantee Jeffrey Rothstein at Johns Hopkins, found much less of the protein GLT-1 in the brains and spinal cords of the ALS patients they studied compared to their control group. GLT-1 is supposed to clear away glutamate. The researchers compared autopsy samples from 19 people who died of sporadic ALS and three people who died of familial ALS with samples from 17 people who died of causes other than neurologic disease.

MDA researchers have considered toxic buildup of glutamate as a possible cause, or at least a contributing factor, in ALS for several years. The drugs riluzole (Rilutek) and gabapentin (Neurontin), which are being studied for use in ALS, have anti- glutamate effects. The GLT-1 deficiency findings support the use of this type of medication.

Riluzole received approval from a U.S. Food and Drug Administration advisory committee in September. Gaba-pentin is approved for treating epilepsy.

Doctors have long noted an above-average incidence of motor neuron diseases that resemble ALS on the Pacific island of Guam. One explanation has been that abundant heavy metals in the soil and water on Guam might be poisoning the central nervous system. But researchers at the Mayo Clinic and Foundation in Rochester, Minn., found no differences in blood or urine levels of aluminum, arsenic, cadmium, copper, iron, manganese, mercury or zinc in 12 Guamanians with neurodegenerative disease compared with 12 people from the island without these diseases. They found slightly higher levels of lead in the patient group, but no level in either group was outside the normal range. Similar results were found in tests of urine, nails and hair. The scientists say the results fail to support heavy metal toxicity as a cause of motor neuron disease on Guam.

MYOTONIC DYSTROPHY DEFECT MAY AFFECT MORE THAN ONE GENE

In 1992, MDA researchers identified an expanded stretch of DNA on chromosome 19 that causes myotonic dystrophy (MMD), a complex disease in which symptoms can include not only difficulty relaxing muscles (myotonia) and weakness, but cataracts, infertility, diabetes, sleep disorders and mental abnormalities. The size of the expanded DNA section roughly matches with the number of symptoms and their severity, with larger expansions usually causing more severe disease.

Since 1992, researchers have found that the gene for an enzyme recently dubbed DMPK (myotonic dystrophy protein kinase) is almost certainly affected by the expanded DNA. MDA grantee Keith Johnson of Charing Cross and Westminster Medical School in London now reports that a gene near the DMPK gene may be affected in at least some people and may be responsible for the eye problems in this disease. A Dutch and German group that included MDA grantee Bé Wieringa at the University of Nijmegen in the Netherlands has located a gene they're calling DMR-N9 that could also be affected by the expansion. This gene, if altered, could cause the mental and testicular symptoms sometimes seen in DM, the researchers say.

MICE MAY SPEED FSHD RESEARCH

Mice with a genetic muscle disease that resembles human facioscapulohumeral muscular dystrophy (FSHD) were first identified in 1976. At that time, the mouse disease was found to be caused by a defect on mouse chromosome 8 and was not thought to match any human disease. The mice have muscle abnormalities in their back legs, dystrophic changes in their muscle biopsies and hearing loss.

A research group at the University of Iowa that included MDA grantee Katherine Mathews now says these "myd" mice may have a disease that's directly related to FSHD, because the region of mouse chromosome 8 where their defect lies matches the region of human chromosome 4 where the FSHD gene flaw is located. Isolation of the mouse gene may speed isolation of the human gene and, if the genes match, the mice could be used to test potential therapies for FSHD. The "mdx" mouse, with a defect in the dystrophin gene, serves this purpose for Duchenne dystrophy.

INFLAMMATION EYED IN THREE DISEASES

Inflammation, part of the body's response to injury or infection, may play an important role in Duchenne muscular dystrophy (DMD) and facioscapulohumeral muscular dystrophy (FSHD), even though the primary defect in both these conditions is a gene flaw. Inflammation may play a less starring role than previously thought in causing inclusion body myositis (IBM), one of the inflammatory myopathies.

Inflammation is a sequence of events triggered when the body's immunologic defenses go on alert, usually in response to an invading bacterium, virus or other organism, or to a foreign body (such as a knife cutting the skin). Usually, inflammation is a helpful process that sets the stage for tissue repair. However, it can also injure tissues.

A team led by MDA grantee Michael Hudecki at the State University of New York at Buffalo says mast cells, which take part in inflammation, may play a role in muscle degeneration in DMD. Hudecki's group made genetically engineered mice with excess mast cell activity in addition to a dystrophin defect (mdx/Tsk mice). The mice developed a disease that closely resembles DMD, showing more muscle degeneration and progressive weakness than mdx mice have. Hudecki will be collaborating with MDA grantees at the University of Rochester (N.Y.) to study the role of steroid drugs like prednisone and deflazacort. These drugs may slow the course of muscle degeneration in DMD by combating mast-cell- related inflammation, Hudecki says.

Inflammation may play a significant role in FSHD, say Japanese researchers, including MDA-supported Kiichi Arahata at the National Institute of Neuroscience in Tokyo. The group examined eight muscle biopsy samples from people with FSHD and found the number of dead fibers paralleled the number of inflammatory cells. Combining these findings with other information, the investigators say inflammation probably contributes to fiber degeneration in FSHD and probably isn't just the body's effort to clean up debris. The investigators note that steroids have been given to patients with muscle inflammation and weakness of the face, shoulder and upper arm muscles (FSHD pattern), but they only helped temporarily.

Starchlike deposits in muscle fibers and inflammation around the fibers characterize IBM, one of the inflammatory myopathies. But unlike the other inflammatory myopathies, polymyositis and dermatomyositis, IBM generally doesn't respond well to anti- inflammatory therapy with steroids like prednisone. A new study by researchers at the University of Texas Southwestern Medical Center in Dallas, the U.S. Air Force Medical Center in San Antonio and Ohio State University in Columbus confirms these findings. The team, which included MDA grantee Jerry Mendell at Ohio State, studied eight people with IBM and found muscle weakness actually worsened and fibers with abnormal deposits increased after prednisone treatment. The drug did suppress inflammation, however. The researchers concluded that inflammation may play a secondary role in IBM, which may be a different kind of disorder from the other types of myositis.

MYOBLAST TRANSFER BACK TO THE MOUSE

Since 1990, transplanting immature muscle cells (myoblasts) from healthy relatives into boys with Duchenne muscular dystrophy (DMD) has been tried as a way to give the boys new dystrophin genes. Myoblasts play an important role in the early development of muscle tissue when they fuse and form muscle fibers. Once a muscle is mature, myoblasts sit outside the fiber in a dormant state, waiting to be "called in" should muscle repairs (fiber regeneration) be needed. Because of their ability to carry out repairs, researchers had high hopes for them as therapy for muscle disease. Unfortunately, clinical trials of myoblast transfer have been disappointing (see "Clinical Trials: Where Are We Now?," file 5QST02.TXT), but researchers have gone back to the drawing board the mouse to see how they might improve the procedure.

A team at Laval University in Quebec treated myoblasts with fibroblast growth factor before injecting them into dystrophin- deficient (mdx) mice. When they studied six mice 28 days after they received growth-factor-treated myoblasts in one leg muscle and untreated myoblasts in the other, they found an average of five times as many muscle fibers with dystrophin in the leg with the treated myoblasts. Injecting fibroblast growth factor separately from the myoblasts wasn't helpful. The researchers suggest further study of this and other growth-promoting factors.

STEM CELL TRANSFER

Researchers at Case Western Reserve University in Cleveland are trying a different approach to cell transplantation in DMD and possibly other forms of muscular dystrophy. A group that included MDA-supported Arnold Caplan isolated cells from bone marrow that can become muscle, bone, cartilage or tendon. These "stem" cells aren't yet committed to becoming a certain kind of tissue but can give rise to myoblasts. According to Caplan, the finding is enticing for at least three reasons. First, it's much easier to get cells for transplant from bone marrow than from muscle. Second, it's easier to grow enormous numbers of the stem cells in the laboratory than it is to grow myoblasts. (Caplan's group has produced billions of cells.) Third, because the cells are in an undeveloped state, Caplan believes they're probably able to respond to signals in their environment and become precisely the type of muscle into which they're transplanted (for example, limb or diaphragm). Subtle differences in these muscles are important for function. Caplan's group is trying the transplants in rodents, surgically inserting sheets of stem-cell-implanted meshwork over the animals' muscles. Caplan says the new approach can be thought of as tissue engineering.

MINIGENES LOOK GOOD FOR DMD GENE THERAPY

Viruses, mainly the adenovirus, are among the leading candidates to deliver dystrophin genes to the muscles of boys with Duchenne dystrophy (DMD). But, dystrophin genes are too large to fit into adenoviruses. Researchers around the world are racing to perfect adenoviruses that are safe, have room for at least a small version of a dystrophin gene, and can still do their job as carriers. At the same time, they're trying to miniaturize the dystrophin gene.

MDA researchers in three laboratories have experimented with dystrophin minigenes designed to fit into adenoviruses. MDA grantees in two independent research groups recently tested the same type of minigene. Jeffrey Chamberlain and Michael Hauser at the University of Michigan were part of a group at that institution, and Kevin Campbell at the University of Iowa joined with British researchers to study the effects of the so-called Becker minigene. The gene is based on one isolated in 1990 from a British patient with Becker muscular dystrophy, a less severe disease than Duchenne. It codes for a shortened version of dystrophin that both teams found nearly eliminated signs of muscular dystrophy in dystrophin-deficient (mdx) mice. The researchers say the Becker gene fits inside an adenovirus, but they didn't deliver it to the mice that way. Instead, they inserted it into one-celled mouse embryos, a procedure not applicable to humans.

MDA grantee Paula Clemens at Baylor College of Medicine in Houston was part of a third team, which tested four types of minigenes (not including the Becker gene). The group found some minigenes were better than others at producing dystrophin. Two of them produced more dystrophin (although as a shortened protein) in laboratory-grown cells than the full-length dystrophin gene. The group tested one minigene by viral delivery in mdx mice and found the dystrophin it produced was in the right place but amounts varied between muscle fibers. The researchers say each minigene must be tested for its distinct effects.