Research Updates November-December 2005

Article Highlights:

News on the latest research as of October 2005

by Margaret Wahl on November 1, 2005 - 10:22am

QUEST Vol. 12, No. 6

This article reports research news about congenital MD * Duchenne MD * limb-girdle MD * spinal muscular atrophy * myasthenia gravis * Charcot-Marie-Tooth disease type 2 * Friedreich's ataxia

Genes for agrin treat mice lacking laminin

Chunping Qiao Xiao Xiao

MDA-supported research at the University of Pittsburgh shows that a miniaturized version of the gene for agrin can markedly improve the health and longevity of mice with congenital muscular dystrophy (CMD) resulting from a lack of the crucial laminin alpha-2 chain of the laminin protein.

This type of CMD, usually called merosin-deficient CMD by physicians, generally begins at or near birth. The absence of merosin (laminin), a protein linking the muscle cell membrane to structures outside the cell, probably weakens a sheath around each muscle cell (the basal lamina), and destabilizes the membrane.

MDA grantee Chunping Qiao and colleagues, including senior investigator Xiao Xiao, who has received MDA funding for the development of viral gene transporters, published their results in the Aug. 23 issue of Proceedings of the National Academy of Sciences. Their hypothesis was that high levels of the muscle protein agrin would help compensate for the missing laminin.

In merosin-deficient congenital MD, laminin (merosin) is partially or completely missing, which breaks the connection between alpha-dystroglycan and the basal lamina. Adding agrin to merosin-deficient muscle cells seems to partially compensate for the lack of merosin.

The Pittsburgh researchers performed a set of experiments, delivering a miniaturized agrin (miniagrin) gene by two methods, each of which used a slightly different viral transporter.

In one experiment, they delivered miniagrin genes, each inserted into the shell of an AAV2 (adeno-associated virus 2) directly into the leg muscles of mice with laminin-deficient CMD. Two months after the injections, the treated leg muscles showed more agrin, less scarring and general signs of improvement in muscle health compared to the same muscles in untreated mice.

In another experiment, they delivered the same genes, this time inserted into AAV1 (adeno-associated virus 1) shells, injected into the abdominal cavity of the mice as a form of systemwide therapy.

The systemic delivery resulted in far-reaching therapeutic effects. About four months after treatment, the mice showed increased agrin levels and improvement in the appearance of several skeletal muscle groups, as well as the respiratory diaphragm, other breathing muscles and the heart.

The treated muscle fibers were larger and didn’t show the scarring that reflects dystrophy-related muscle damage. The hearts of the treated mice were nearly indistinguishable from those of normal mice, the investigators say.

Only 50 percent of untreated mice with laminin-deficient CMD were alive at the age of 4 weeks, while 50 percent of those systemically treated with the miniagrin genes were alive at more than 17 weeks.

Xiao said the results are encouraging and could bode well for future development of agrin-based therapy for patients with merosin-deficient CMD.

The University of Pittsburgh is one of three muscular dystrophy centers of excellence co-funded by MDA and the National Institutes of Health.

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Compounds up utrophin, help DMD-affected mice

Sabine de la Porte, an MDA research grantee at a branch of the Centre National de la Recherche Scientifique in Gif-Sur-Yvette, France, and colleagues, have demonstrated that mice with a disease resembling Duchenne muscular dystrophy (DMD) benefit from treatments that increase utrophin, a protein similar to dystrophin, which is missing in DMD.

The researchers, who published their findings in the October issue of Neurobiology of Disease, say that treating the mice with L-arginine resulted in a twofold to threefold increase in utrophin levels in the muscles of the mice. Molsidomine, another compound, had similar effects.

Sabine de la Porte Elisabeth Barton
Sabine de la Porte Elisabeth /Barton

The study’s authors say they believe both compounds raise utrophin levels by increasing production of nitric oxide (NO). Bernard Jasmin, an MDA grantee at the University of Ottawa (Canada), says preliminary observations in his lab support these findings.

In another study, published online Aug. 22 in Muscle & Nerve, MDA-supported Elisabeth Barton at the University of Pennsylvania in Philadelphia also noted the benefits of L-arginine in DMD-affected mice.

Her group found that when L-arginine was injected into the abdomens of the mice or delivered via an implanted pump for at least four weeks, it increased levels of utrophin and improved resistance to contraction-related muscle fiber injury.

The Philadelphia investigators say they think L-arginine may have activated muscle fiber repair mechanisms, as well as stabilizing their membranes and perhaps improving their handling of calcium.

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Modifier genes change severity of limb-girdle MD

In some types of limb-girdle MD, one of the four sarcoglycan proteins is missing from the cell membrane, which destabilizes the rest of this group of proteins.

MDA grantee Elizabeth McNally, a cardiologist and molecular geneticist at the University of Chicago, recently led a research group that found that genes other than those directly responsible for two forms of limb-girdle muscular dystrophy (LGMD) can influence disease severity.

The investigators, who published their findings in the October issue of Neuromuscular Disorders, studied mice with a type of LGMD resulting from an absence of gamma-sarcoglycan, a protein in the muscle-fiber membrane, and other mice missing delta-sarcoglycan, another such protein. The mouse muscular dystrophies resulting from these missing proteins correspond to the human disorders known as LGMD2C and 2F, respectively.

They studied these two forms of sarcoglycan-related LGMD in mice from several different genetic families (strains) to see whether the various strains would respond differently to a sarcoglycan deficiency.

Elizabeth McNally
Elizabeth McNally

In LGMD2C-affected mice, the resulting dystrophy was least severe in mice from a strain called 129 and most severe in mice from the DBA strain. They measured the severity of the disease by whether the cells were permeable to a blue dye (an indication of muscle cell membrane fragility), and by how much scarring there was in the muscle tissue.

In the 129 strain, mice missing delta-sarcoglycan fared worse with respect to membrane fragility and scarring than did mice missing gamma-sarcoglycan.

But in another strain, called C57, there was no difference between the gamma- and delta-sarcoglycan-deficient animals.

“Uncovering these other gene modifiers will help us predict who will be more or less severely affected by their muscular dystrophy,” McNally said.

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Two genes better than one in mice with Duchenne MD

Simultaneously delivering genes for the muscle protein dystrophin and for the muscle-specific form of insulin-like growth factor 1 (IGF1) rescues muscle fibers of mice with a disease closely resembling Duchenne muscular dystrophy (DMD) more effectively than does either compound alone, say researchers at the University of Washington in Seattle.

In Duchenne MD, muscle cells lack dystrophin, which affects the stability of the rest of the proteins in the membrane cluster. Utrophin can stand in for dystrophin, but there isn't much of it in mature cells.

The investigators, who published their results in the September issue of Molecular Therapy, found that miniaturized genes for dystrophin (microdystrophin) helped muscle fibers resist mechanical injury from muscle contraction, while genes for the muscle form of IGF1 made the muscles larger. Together, the dystrophin and IGF1 made from the two genes increased muscle mass and strength and increased resistance to contraction-related injury.

The researchers inserted the genes into an AAV6 (adeno-associated virus type 6) and injected the viral particles into the leg muscles of the mice.

Team members Paul Gregorevic and Jeffrey Chamberlain have MDA funding for closely related work. Chamberlain directs the University’s Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, which is co-funded by MDA and the National Institutes of Health.

“Microdystrophin delivery is showing great promise as a potential treatment for muscular dystrophy,” Chamberlain said. “However, the smaller dystrophins are not quite as effective as the normal-sized gene. This new study shows that delivering muscle-building agents such as IGF1 with them leads to a further improvement in muscle strength.”

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SMA cells get boost from hydroxyurea

MDA grantee Ching Wang, a pediatric neurologist at Stanford (Calif.) University, was the senior investigator on a research team that recently demonstrated that adding the drug hydroxyurea to white blood cells taken from people with spinal muscular atrophy (SMA) increased the ability of these cells to produce SMN1, a protein needed but deficient in SMA.

Ching Wang
Ching Wang

Wang’s team added the drug to blood cells from five people with type 1 SMA (the most severe), five with type 2 (medium severity), five with type 3 (least severe) and five without SMA. It was effective at increasing SMN1 in all groups, although the increase was most significant in the SMA-affected cells.

The investigators, who published their results in the August issue of Annals of Neurology, say they believe the increase in SMN1 protein resulted from a change in the output from SMN2 genes, which carry almost identical instructions for making the SMN protein but can only produce a small amount of it. SMN2 genes mostly produce a closely related, but shorter, SMN protein.

The hydroxyurea apparently caused the SMN2 genes, which people with SMA have, to produce more of the longer, SMN1 protein, which those with SMA normally lack because they don’t have functional SMN1 genes.

“We are very encouraged by our findings that hydroxyurea is able to increase SMN protein production in white blood cells isolated from SMA patients,” Wang said. “We hope that we can see the same effects when we use hydroxyurea to treat SMA patients.”

Wang and colleagues are testing hydroxyurea in people younger than 10 years with all types of SMA. For more information, contact Tony Trela at (650) 498-7658, or; or visit

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Heart overgrowth blocker found

Investigators at the University of Wurzburg (Germany) and the Frankfurt branch of Sanofi-Aventis, a multinational pharmaceutical firm, have found that the protein NAB1 prevents the kind of damaging cardiac overgrowth, or hypertrophy, characterized by a dangerous thickening of the heart muscle wall (myocardium), at least in mice.

Cardiac hypertrophy is a significant problem in some neuromuscular diseases, particularly Friedreich’s ataxia.

When Monica Buitrago and colleagues, who published their findings in the August issue of Nature Medicine, bred mice that produced extra NAB1 (NGFIA binding protein 1), they found the mice showed significantly less hypertrophy in response to abnormal situations, such as pressure overload, while at the same time exhibiting normal heart growth during development and in response to exercise.

The paper’s authors say that NAB1 “represents an especially promising target to prevent maladaptive cardiac hypertrophy, as it leaves physiological growth unaffected.”

Targets like these are what the biotech industry looks for as a first step in drug development.

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New compound protects hearts from DMD damage

Investigators at the University of Michigan in Ann Arbor have found that a synthetic compound known as poloxamer 188 (p188) can protect heart muscle cells in mice lacking the protein dystrophin, which have a muscle disease resembling Duchenne muscular dystrophy (DMD).

Soichiro Yasuda and colleagues, who published their results online July 17 in Nature, found that when they added p188 to heart muscle cells from dystrophin-deficient mice, the cells’ resistance to stress matched that of cells from healthy mice. They believe the drug may shore up the fragile cell membranes seen in DMD.

Next, they gave some of the mice an intravenous infusion of dobutamine, a drug that increases heart rate and blood pressure, and another group an infusion of dobutamine preceded by intravenous p188.

John Quinlan
John Quinlan

Several of the 10 DMD-affected mice in the first group experienced acute heart failure, which didn’t occur in the mice that received p188.

“If issues of dosing and long-term safety can be addressed, our results indicate that membrane-sealing poloxamers could represent a new class of therapeutic agents” for heart muscle damage associated with DMD and possibly other types of MD involving defects in the muscle-cell membrane, the authors say.

John Quinlan, an MDA grantee at the University of Cincinnati who is studying cardiac problems in DMD-affected mice and is also interested in p188, said, “This work is exciting and cause for hope. The Michigan team has provided us with a better understanding of how DMD attacks cardiac function on a cellular level. Most importantly, they showed how p188 has both beneficial action on reversing cellular damage and improving heart function under stressed conditions.”

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Blocking immune system's IL1 helps mice with MG

Erdem Tuzun and Premkumar Christadoss, both MDA-funded investigators at the University of Texas Medical Branch in Galveston, were part of a group that recently demonstrated the potential value of blocking the action of interleukin 1 (IL1) in treating myasthenia gravis (MG).

In most cases of MG, the patient’s immune system mistakenly attacks the part of each muscle cell that receives signals from the nervous system — the acetylcholine receptor. IL1 and several other substances associated with the immune system combine to destroy or block acetylcholine receptors, causing the fluctuating weakness of MG.

Christadoss and colleagues, who published their report in the Aug. 1 issue of the Journal of Immunology, gave multiple daily injections of an IL1-blocking compound for two weeks to mice destined to develop a laboratory-induced form of MG. (The mice had been immunized against their own acetylcholine receptors.)

The injections reduced the number of cases of MG that developed in the mice, and in those cases that did develop, the symptoms were less severe than in the mice that didn’t receive the IL1 blocker.

The study authors say that compounds that block IL1 activity, perhaps in combination with etanercept, which blocks the activity of another immune-system compound (see “Clinical Trials and Studies,” March-April 2005), might be beneficial in the treatment of human MG. Because current treatments for MG often involve the use of highly toxic medications, the search is on for better strategies.

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MFN2 confirmed as a major cause of CMT2

Flaws (mutations) in a chromosome 1 gene that instructs for the mitofusin 2 protein account for approximately 20 percent of type 2 Charcot-Marie-Tooth disease (CMT) cases, says a study in the July 26 issue of Neurology.

The mitofusin 2, or MFN2, gene was first linked to CMT2 last year (see “Research Updates,” July-August 2004). It affects the behavior of mitochondria, the energy-producing units in cells.

Now, researchers in the laboratory of Kevin Flanigan at the University of Utah School of Medicine in Salt Lake City have identified mutations in the MFN2 gene in three large families with CMT2.

Type 2 CMT results from abnormalities in the nerve fibers (axons), while type 1 results from defects in the insulating sheath surrounding the axon and containing proteins and carbohydrates (myelin sheath). These are distinguished by nerve conduction testing.

The investigators suggest that genetic testing of CMT2 patients begin with a screen of the MFN2 gene. (Such screening is commercially available.)

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