Two Research Teams Treat CMD Mice

Recently published findings from two independent groups have suggested possible treatment pathways for the merosin-deficient and integrin-deficient forms of congenital muscular dystrophy (CMD).

Doxycycline fights cell death and lessens disease severity in merosin-deficient mice

The first finding, published online Dec. 11, 2008, in Annals of Neurology, shows doxycycline, normally used as an infection-fighting antibiotic but known to have other properties, increased survival time, improved growth and delayed the onset of paralysis of the back legs in merosin-deficient mice with a CMD-like disease.

MDA grantee Jeffrey Boone Miller of Harvard Medical School in Boston and Boston Biomedical Research Institute in Watertown, Mass., coordinated the merosin-deficient CMD research team, which also included Mahasweta Girgenrath, lead author on the report and an MDA grantee at Boston University.

This type of CMD is caused by mutations in the gene for laminin alpha 2, a protein strand in a larger protein called merosin (also known as laminin 2), which connects muscle fibers to their surrounding tissue. Without the laminin alpha 2 strand, the normally three-stranded merosin protein can't perform this connective function or carry out other roles, and severe neuromuscular dysfunction results.

MDA grantee Jeffrey Boone Miller
MDA grantee Jeffrey Boone Miller

Earlier research has shown that at least one mechanism by which merosin-deficient CMD leads to weakness, paralysis and premature death is the inappropriate induction of a "cell death program" (apoptosis) in skeletal muscles and the nerve cells controlling them.

Because doxycycline and related antibiotics have been reported to interfere with apoptosis, Girgenrath and colleagues decided to see how it might affect disease progression in merosin-deficient CMD mice.

They randomly assigned some of the CMD mice to receive doxycycline in their drinking water starting one to three days after birth and others to receive water without doxycycline.

In the doxycycline-treated group, half the mice were still alive at about 70 days after birth, while in the untreated group, half had died at about 32 days.

And at 3.5 weeks after birth, doxycycline-treated mice weighed 30 percent to 40 percent more than untreated mice.

In marked contrast to the back leg paralysis seen in all the untreated mice four to six weeks after birth, the majority of the treated mice didn't have this paralysis at age 10 to 12 weeks. At age 6 weeks, doxycycline-treated merosin-deficient mice stood on their hind legs about as often as mice with normal merosin levels, while untreated merosin-deficient mice did this less than one-third as often.

In addition, the muscles of the treated mice showed less inflammation and fewer indicators of apoptotic cell death than those in the untreated mice.

"Our study demonstrated that disease due to loss of laminin alpha 2 was indeed significantly ameliorated by oral administration of doxycycline," the researchers write.

However, they note two warnings: it’s not known if doxycycline or similar therapies would still be effective if started later in life than they were in these experiments; or if other drugs in the same family as doxycycline might be more effective than this medication. They also caution that extensive repair of injured skeletal muscles requires formation of new blood vessels and that doxycycline appears to interfere with this repair mechanism.

The researchers say a pharmacological therapy to slow disease progression in human merosin-deficient CMD would be of considerable benefit and that additional inhibitors of apoptosis should be tested.

Laminin 111 restores muscle repair process in integrin-deficient mice

A research team coordinated by Dean Burkin at the University of Nevada School of Medicine in Reno has found a way to improve muscle repair in a mouse model of integrin-deficient CMD. The team’s findings were published in the January 2009 issue of the American Journal of Pathology.

Like merosin, integrins are part of the apparatus that helps anchor muscle fibers to their surroundings. Evidence also shows that some integrin proteins, such as alpha 7 integrin, are needed to maintain normal levels of laminin alpha 2 and merosin and apparently activate the muscle repair and regeneration process.

Children with mutations in the gene for alpha 7 integrin have a deficiency of this protein, with muscle abnormalities, delayed developmental milestones and impaired mobility. Mice lacking alpha 7 integrin develop a CMD-like disease with muscle and blood-vessel defects.

To see whether alpha 7 integrin is important for skeletal muscle regeneration and repair, Burkin, with colleagues at the University of Nevada and the University of Washington-Seattle, purposely damaged muscle tissue in alpha-7-integrin-deficient mice and found that regeneration in response to injury was defective.

They say the regenerative capacity of skeletal muscle depends on an "intricate interplay" between cells that carry out muscle repair and their normally laminin-rich environment, leading them to test the hypothesis that injecting a laminin protein might improve muscle repair.

Prior to injury, they injected integrin-deficient mouse muscle with laminin 111, normally produced in muscle during embryonic development. After the mice were injured, their muscle repair was restored to normal.

"One possible explanation for the improved muscle regneration ... is that injection of laminin 111 may [reactivate] an embryonic myogenic program in adult skeletal muscle," the researchers write, which may in turn result in improved muscle repair.

They also note that, because loss of regenerative capacity has been implicated in a variety of muscular dystrophies, including merosin-deficient CMD and Duchenne muscular dystrophy, they're now investigating whether laminin 111 protein therapy might also be beneficial in other forms of MD.

The Muscle-Fiber Membrane and Its Proteins

Normally, laminin 2 (merosin) connects proteins nestled in the muscle-fiber membrane with the surrounding tissue. Loss of either integrin or merosin interferes with this connection and results in congenital muscular dystrophy (CMD).

In one recent study, doxycycline appeared to save merosin-deficient muscle fibers by stopping a cell-death program. In another, an embryonic form of laminin restored the ability of muscle fibers to repair themselves in the absence of integrin. Both approaches could become avenues for CMD treatment development.

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