Research Updates

• MOUSE MODEL FOR LGMD 2B, MIYOSHI IS SURPRISE
• STEM CELLS JOIN PIPELINE IN FIGHT AGAINST DMD
• GENES IN FSHD FOUND TO BE 'MISREGULATED'
• MG NASAL SPRAY WORKS IN RATS
• POTENTIAL ALS TREATMENT INEFFECTIVE
• FAMILIES WITH DMD, BMD NEEDED FOR STUDY
  

MOUSE MODEL FOR LGMD 2B, MIYOSHI IS SURPRISE

Animal models of human diseases can be very important tools for understanding disease processes. Often, after a genetic defect is identified, the next step in understanding the disease is to try to duplicate the disease in mice by "knocking out," or rendering nonfunctional, the equivalent mouse gene. This process can be expensive and time-consuming.

Sometimes, however, Mother Nature steps in and speeds things along. Just last year, defects in the gene for a protein named dysferlin were found responsible for both a recessive form of limb-girdle muscular dystrophy (LGMD) called type 2B, and for a distal myopathy called Miyoshi myopathy (MM). More recently, researchers discovered that the dysferlin protein is in the cell membrane, but it isn't known how the lack of it leads to muscle weakness in the disorders.

Now, in a surprising turn of events, creating a mouse model of LGMD-2B/MM has proved unnecessary with the discovery that a strain of mice with a faulty dysferlin gene already exists. Reginald Bittner of the University of Vienna in Austria has determined that an existing strain of mice called SJL mice, long studied in the laboratory for their susceptibility to inflammatory muscle disease, has a mutation in the mouse version of the dysferlin gene.

As a result of the mutation, the mice appear to make only 15 percent of the normal amount of dysferlin in their muscles. Bittner says that this reduction is similar to that seen in some humans with LGMD2B. Further, the mice do have detectable muscle weakness as early as three weeks after birth, and microscopic changes in their muscle tissue are consistent with those seen in a progressive muscular dystrophy.

Bittner predicts that the SJL mouse will become a useful tool for understanding the mechanism by which reduced dysferlin leads to LGMD2B or MM, and for developing therapies to slow or reverse muscle weakness in these disorders. The findings were in the October issue of Nature Genetics.

STEM CELLS JOIN PIPELINE IN FIGHT AGAINST DMD

In the Sept. 23 issue of Nature, MDA researchers Louis Kunkel and Emanuella Gussoni of Children's Hospital in Boston announced that primitive cells isolated from the bone marrow or muscles of healthy donors can help rebuild the muscles of mice with muscular dystrophy.

Louis Kunkel

Although researchers have long known that stem cells in the bone marrow can give rise to all different types of blood cells, it wasn't until last year that anyone realized that these cells could form different tissues as well. Since that time, scientists have been able to coax these cells into becoming bone, cartilage, muscle and even nerve cells.

Now, Kunkel, who first discovered that defects in the dystrophin gene cause Duchenne muscular dystrophy, has adapted a method from the bone marrow field to isolate stem cells from muscle. He tested the potential of these muscle-derived stem cells to help rebuild the muscles of mice that lack dystrophin.

In the same way that bone marrow transplants are performed in humans, the researchers first prepared dystrophin-lacking mice by irradiating them to destroy their bone marrow. Then these mice were infused with a solution containing stem cells from either the bone marrow or muscles of healthy donor mice.

	MAKING NEW MUSCLE Stem cells, removed from either the muscle or bone marrow of a healthy donor, are injected into the bloodstream of a mouse that lacks dystrophin. In response to injury signals (green arrow) from the muscle, the dystrophin-containing stem cells gradually migrate out of the bloodstream and into all of the muscles (they may have to spend time in the bone marrow first). To make the process safer and more efficient, researchers hope to learn what signals from the muscle are required to attract the stem cells.

Weeks later, the researchers found that both types of transplanted cells were able to completely regenerate the bone marrow of the dystrophic mice and, even better, that every muscle examined now contained some muscle cells de-rived from the healthy donor. And some of these "donor-derived" muscle cells were indeed making the dystrophin protein.

Kunkel says it's "amazing" that the donor cells "contributed enormously" to rebuilding the dystrophin-deficient muscles of the recipient mice, and that they did so by entering the muscles from the bloodstream. "We've got a cell that has the capability of remodeling [rebuilding] muscle from the circulation of the organism and delivering gene product [dystrophin] to that remodeled muscle."

Although the approach is promising, there are still some issues that need to be ironed out to make this a practical treatment. First, 12 weeks after the transplant, only 4 percent of the mouse muscle cells were positive for dystrophin — not enough to make a difference in the clinical course of the disease in the mice or in humans. A method must be found for increasing the number of donor cells that become incorporated into muscle.

Second, Kunkel speculates that, in order to attract stem cells out of the circulation and into the muscle, the muscle itself must be damaged. In the current experiment, the muscles of the mice were damaged both by a lack of the dystrophin protein and by the irradiation procedure used to destroy the bone marrow. Kunkel is quick to caution that his group doesn't plan to irradiate children with Duchenne to damage their muscles and bone marrow, but would instead like to identify the damage signals.

"What we're trying to do now is create the signals from a diseased muscle that make cells go from the circulation without having to destroy tissues with radiation. So you could just inject the cells into the circulation and there would be sufficient signal from the muscle saying 'I am injured, come help me' that the cells home to those muscles," he says.

If the technique can be made safe and efficient, it may have implications for treatment in all forms of degenerative muscle disease.

GENES IN FSHD FOUND TO BE 'MISREGULATED'

People with facioscapulohumeral muscular dystrophy (FSHD) are making the wrong amounts of many different muscle proteins, reports a group led by Michael Green of the University of Massachusetts Medical School and Howard Hughes Medical Institute in Worcester. The surprise finding may shed light on the mechanism by which FSHD causes muscle weakness.

A common form of muscular dystrophy, FSHD affects the muscles of the face and shoulders, although generalized weakness can occur with time. Unlike most other neuromuscular disorders for which the genetic defects have been found, the problem in the DNA that leads to FSHD doesn't seem to cause mutations in a specific gene.

Instead, researchers have found that people with FSHD lack regions of their DNA on chromosome 4 that normally contain multiple copies of a particular sequence of nucleotides (the building blocks of DNA). Oddly, these repeated sequences don't seem to code for proteins, and it isn't known how lacking them causes problems in the muscles of those affected by FSHD.

One theory held by many researchers is that the lack of these repetitive sequences (known as 3.3kb repeats) somehow changes the shape of chromosome 4 in such a way that genes in neighboring regions of DNA are affected negatively. Researchers are studying several nearby genes to see whether their activity is affected by the loss of the 3.3kb repeats.

Now Green reports that many muscle-specific genes are over- or under-active in people with FSHD. This conclusion was reached after comparing the activity of the genes in muscle samples from people with FSHD, Becker muscular dystrophy, amyotrophic lateral sclerosis and healthy people. Only the samples from people with FSHD demonstrated what the authors describe as a "profound" misregulation of gene expression.

Green speculates that global misregulation of muscle genes in FSHD may be caused when the loss of 3.3kb repeats interferes with the normal function of a muscle-specific "transcription factor." Transcription factors are specialized proteins that bind to DNA and activate the expression of other proteins. If the activity of a transcription factor isn't normal, then the activity of all of the other genes it regulates may also be abnormal.

MG NASAL SPRAY WORKS IN RATS

Myasthenia gravis (MG) is an acquired autoimmune disease in which the body turns against and destroys receptors that are important for conveying signals from the nervous system to the muscles. The loss of these receptors, called acetylcholine (Ach) receptors, results in overwhelming fatigue as a day progresses.

Current treatments depend on general suppression of the immune system and are plagued by unwelcome side effects. Researchers have been searching for ways to "reprogram" the immune systems of people with MG to no longer recognize Ach receptors as the enemy. Now, MDA researcher Sara Fuchs of the Weizmann Institute in Rehovot, Israel, has met with some success using a nasal spray to trick the immune system into ignoring Ach receptors. The results are reported in the July 6 issue of the Proceedings of the National Academy of Sciences.

The strategy takes advantage of the fact that the T cells of the immune system, which normally carry out the attack against the Ach receptors, must be stimulated in two ways to become activated against a molecule in the body. If the T cells are exposed to a molecule without the appropriate co-stimulation, they self-destruct instead of becoming activated, effectively preventing an immune response.

It turns out that the cells lining the gastric system and nasal passage lack these co-stimulatory signals. Research-ers have theorized that if these cells could be made to display pieces of the Ach receptor on their surfaces, the T cells that responded to this "bait" wouldn't receive co-stimulatory signals and would self-destruct. Eventually, many of the T cells that would normally attack Ach receptors might be weeded out in the nasal passage.

Fuchs took advantage of this knowledge to develop a nasal spray containing millions of tiny pieces of the human Ach receptor — bits that were carefully chosen to attract T cells. When the pieces of Ach receptor entered the nasal passage, they were taken up by the lining mucosal cells and displayed on their surfaces.

The group found that rats treated with the Ach receptor nasal spray were less likely than untreated rats to develop MG under experimental laboratory conditions that normally trigger the disease. Even better, rats that already had symptoms of MG experienced some improvement.

One of the dangers of such a treatment strategy is that a greater immune response against the Ach receptors may be triggered accidentally. Fuchs reports, however, that her nasal preparation is remarkably free of immune-stimulating activity.

POTENTIAL ALS TREATMENT INEFFECTIVE

The drug gabapentin (brand name Neurontin, manufactured by the Parke-Davis division of Warner-Lambert) failed to slow the progress of amyotrophic lateral sclerosis, a paralyzing disease in which muscle-controlling nerve cells (motor neurons) die. In a nine-month trial completed by 128 patients, gabapentin failed to show any advantage over the placebo on measures of arm strength, rate of decline of breathing muscle function, decline in quality of life measures or any other evaluated aspect of ALS.

The trial was a phase 3 study and follows an earlier study (phase 2) that led researchers to believe that gabapentin might have some benefit in ALS. The drug is already on the market for seizures. The ALS study was supported by MDA, the Food and Drug Administration and Warner-Lambert.

FAMILIES WITH DMD, BMD NEEDED FOR STUDY

MDA grantee Veronica Hinton, a neuropsychologist at Columbia University's Sergievsky Center in New York, continues to recruit participants for her study of learning styles in boys with Duchenne or Becker muscular dystrophy. Her research team is studying these muscular dystrophies to see whether there are any particular ways of learning and thinking associated with DMD or BMD. The team is seeking boys with DMD or BMD between 5 and 12 years old and, where possible, with an unaffected brother or sister.

The children must speak English and be interested in participating. Parents may speak English or Spanish.

For the children, the study involves a three-hour, mostly paper-and-pencil test. A blood sample will be needed from each child.

Parents will be asked to fill out a questionnaire on the child's developmental history and behavior.

For more information, contact:

Robert Fee
(212) 305-2394
e-mail: feerobe@sergievsky.cpmc.columbia.edu

Veronica Hinton
(212) 305-2512
e-mail: hintonv@sergievsky.cpmc.columbia.edu