New type of MMD mouse adds to understanding of human disease
MDA grantee Thomas Cooper at Baylor College of Medicine, with colleagues there and in France, has added a new piece to the puzzle of type 1 myotonic dystrophy (MMD1) that may help explain some of the differences between it and type 2 myotonic dystrophy (MMD2) and could ultimately lead to treatment advances.
Cooper, a professor of pathology and of molecular and cellular biology, coordinated the research team, which published its findings online Feb. 11 in Proceedings of the National Academy of Sciences.
The investigators first bred mice with an expanded stretch of DNA in the so-called DMPK gene, the same defect in the same location as the one that causes human MMD1. They say these mice mimic the human disease better than any other “mouse model” of MMD1 created so far because, in addition to myotonia (inability to relax muscles) and characteristic molecular abnormalities, these mice exhibit severe muscle wasting (atrophy), as observed in the human disease.
Previously developed mouse models of MMD1 have added the expanded DNA (which consists of chains of repeated DNA sequences) to a gene other than DMPK; or have inserted high numbers of normal-length repeated DNA pieces instead of a long, repeated DNA expansion; or have mimicked a secondary effect of the DNA expansion, the depletion of a protein known as MBNL1.
Cooper and colleagues say these other models exhibit some of the features and molecular events seen in human MMD1, but not all. They say their new model is the only one to mimic the muscle wasting that patients have and to show elevated levels of a protein called CUGBP1 in muscle cells, another characteristic of human MMD1.
They note that people with MMD2, which involves an expanded stretch of repeated DNA sequences in a gene other than DMPK, don’t have high levels of CUGBP1 and typically have milder muscle wasting than people with MMD1.
The increase in CUGBP1 levels, which has deleterious effects on muscle tissue and correlates with the severe muscle atrophy, seem to occur only when the expanded DNA sections are in the DMPK gene and not when they’re in other genes.
The study challenges a view widely held until now that the location of the expanded DNA stretch isn’t important in either type of MMD and that its existence in any location would cause roughly the same problems.
“Muscle atrophy is the primary cause of disability and death in individuals with MMD1,” Cooper said. “Having an animal model that reproduces this aspect of the disease provides an important tool to understand the process and to test therapies. This model has already given us a reason to think the CUGBP1 protein is involved, and this is an important lead to follow. We’re now testing how important CUGBP1 is to muscle wasting. If it’s a key factor, it gives us another target for therapies.”
Flaws in FHL1 gene implicated in rare myopathies
Mutations (flaws) in an X-chromosome gene for a muscle protein called FHL1 have been implicated in a range of rare myopathies (muscle disorders) affecting skeletal and cardiac muscles. Until now, these myopathies have lacked an identifiable genetic cause.
The FHL1 protein is found in skeletal and cardiac muscles and is thought to play a role in the sarcomere, the contracting part of a muscle cell, and in the sarcolemma, the membrane surrounding the cell.
In January and February, three separate research groups, two of which had MDA funding, announced they had identified mutations in the FHL1 gene as underlying a muscle disorder.
In the January issue of the American Journal of Human Genetics, a team coordinated by Michio Hirano at Columbia University Medical Center in New York that included MDA-supported Catarina Quinzii at that institution described how a mutation in the FHL1 gene is the cause of weakness in the shoulder and lower leg muscles in a large Italian-American family.
In the same issue of the journal, a group led by Christian Windpassinger at the University of Toronto and the Medical University of Graz in Austria identified two mutations in the FHL1 gene, different from the one identified by Hirano and colleagues and from each other, as causing weakness of the shoulder and lower leg muscles with cardiac involvement in an Austrian and a U.K. family.
And in February, MDA-supported Carsten Bonnemann at Children’s Hospital of Philadelphia and colleagues described four additional mutations in the FHL1 gene as being responsible for “reducing body” myopathy, a rare muscle disease characterized by progressive weakness and the presence of abnormal protein deposits called reducing bodies in the muscle cells.
Bonnemann’s group analyzed muscle samples from four families in the United States and the United Kingdom and published its findings online Feb. 14 in the Journal of Clinical Investigation.
The Hirano and Windpassinger groups used a method called linkage analysis, in which a region of DNA difference in affected versus unaffected family members is used to identify a disease-causing gene.
The Bonnemann group, however, used a new method to reach its conclusions. Instead of starting with DNA analysis, which is the usual approach, they analyzed the content of the abnormal protein deposits in muscle samples from people with reducing body myopathy and found that the FHL1 protein was the largest component. They then analyzed the FHL1 gene in the four families and found it was abnormal in the affected patients.
When they put the abnormal FHL1 genes into cells in a lab dish, they saw the formation of reducing bodies just as they had in the patients’ muscles.
They say this new “laser microdissection proteomics” approach may become important in identifying the cause of other rare diseases that have prominent cellular changes.
Progress in SMA Research
Spinal muscular atrophy (SMA), a disease in which muscle-controlling nerve cells (motor neurons) in the spinal cord are lost, is caused by a lack of full-length SMN, a protein normally produced from DNA instructions in the SMN1 gene. People with SMA lack SMN1 genes but have SMN2 genes, from which the majority of SMN protein molecules produced are relatively short compared to full-length SMN and are nonfunctional. Efforts to coax nerve cells to read SMN2 instructions as if they were SMN1 instructions are the main focus of current research in SMA.
Gene-repair strategy
Researchers in the laboratory of Eric Kmiec at the University of Delaware in Newark say they’ve developed a gene repair method that has the potential to improve the prognosis in SMA.
Using a molecular “bandage” called a sequence-specific oligonucleotide, the investigators changed the way cells interpreted the DNA in the SMN2 gene and made them interpret it as if it were an SMN1 gene. They published their results Feb. 15 in Experimental Cell Research.
The investigators conducted their experiments on skin cells taken from a child with type 1 SMA, the most severe form of the disease, in which a severe deficiency of full-length SMN leads to respiratory muscle weakness and early death.
They added the oligonucleotide bandage to the cells in a laboratory dish and found that they began making more full-length, functional SMN. (Elsewhere, medications are being tested that may also boost full-length protein production from SMN2 genes.)
The researchers say further studies are now under way to test this gene-repair method in cells from patients with types 2 and 3 SMA, in which SMN levels are higher than in type 1, leading to a less severe disease. They’re also exploring methods to deliver the oligonucleotides to patients’ nerve cells.
If such a treatment, which they’ve called “targeted gene alteration,” could be delivered to these cells, the authors say, it would likely improve SMN protein levels and delay muscle atrophy.
“Our initial studies in animals are consistent with the positive results we obtained in the patient’s cells,” Kmiec said. “We’re hoping to conduct more detailed animal studies in the near future.” He also noted that combining this gene repair strategy with compounds called HDAC inhibitors is a possibility.
Kmiec and colleagues are working with the biotechnnology company OrphageniX (www.orphagenix.com) in Wilmington, Del.
Raising acidity level also may help in SMA
Treating cells taken from patients with SMA with a compound known as a sodium-hydrogen exchanger inhibitor significantly increased production of full-length SMN protein molecules from SMN2 genes.
Jan-Gowth Chang at Kaohsiung Medical University Hospital in Kaohsiung, Taiwan, and colleagues, who published their results in the January issue of Annals of Neurology, used a compound called EIPA, which increases the hydrogen ion concentration, thereby raising the acidity level, of the environment inside cells. They’re not certain whether the mechanism for the increase in full-length SMN protein production is the increase in acidity or another effect of EIPA, but they may have uncovered an important new direction in SMA research. |
Three New Studies Shed Light on Myostatin Blocking to Treat MD
Three sets of laboratory experiments investigating the effects of interfering with myostatin, a protein that limits muscle growth, have shown that this approach may have to be individualized with respect to different types and stages of muscular dystrophy, and that some myostatin suppression strategies may be better than others.
The findings come on the heels of the announcement by Wyeth Pharmaceuticals (Madison, N.J.) earlier this month that it will not continue development of MYO-029, an antibody (immune-system protein) that blocks myostatin, for muscular dystrophy. (See “Wyeth won’t continue”.)
Responses vary with disease
In the March issue of Muscle & Nerve, Tejvir Khurana at the University of Pennsylvania, with MDA-supported Sasha Bogdanovich at that institution and Elizabeth McNally at the University of Chicago, announced that blocking myostatin with a Wyeth-supplied myostatin antibody in mice with type 2C limb-girdle muscular dystrophy (LGMD2C) improved some aspects of muscle health but failed to improve others. (Khurana and McNally have MDA research grants but were not specifically funded for this work.)
The investigators say they observed an “uncoupling” of effects on muscle physiology and effects on muscle-fiber appearance and structure in these LGMD2C-affected mice, which, like humans with this disease, lack the muscle protein gamma-sarcoglycan.
The treated mice received intraperitoneal (abdominal) injections of mouse myostatin antibodies weekly for three months starting at the age of 4 weeks. Untreated mice were injected with saline (salt solution).
The mice treated with the myostatin antibodies showed increases in muscle bulk, body weight and muscle-fiber size, as well as improvement in their ability to stay on a rotating rod. However, their muscle tissue looked the same as it did in the untreated mice, and there was no reduction in the level of serum creatine kinase, an enzyme that leaks out of damaged fibers.
The average number of fibers in a leg muscle remained relatively constant, leading the researchers to conclude that any increase in muscle bulk was due to enlargement of individual fibers rather than generation of new fibers.
They note that previous studies have shown more benefit from myostatin blocking in dystrophin-deficient mice with Duchenne muscular dystrophy (DMD) and in mice with early-stage type 2F limb-girdle MD (LGMD2F) resulting from a deficiency of delta-sarcoglycan, than it has in mice with late-stage LGMD2F or merosin-deficient congenital MD.
They say that, because of differences in study designs, it isn’t possible to make direct comparisons of these results, but that it’s likely the benefits of myostatin blocking are limited by the age at which treatment is started and the natural history and severity of the disease being treated.
Boosting follistatin may be better than blocking myostatin with antibodies
In a different set of experiments, reported online March 11 in Proceedings of the National Academy of Sciences, Brian Kaspar at Nationwide Children’s Hospital Research Institute in Columbus, Ohio, and colleagues describe the benefits in DMD-affected mice of a gene-therapy approach to inhibition of myostatin. (MDA is supporting Kaspar and Jerry Mendell, also on this study team and also at Nationwide, for other types of muscle-directed gene therapy.)
First, these investigators injected genes for the protein follistatin inside an adeno-associated viral shell into upper and lower leg muscles in 3-week old mice with DMD. Follistatin is known to inhibit myostatin activity. The mice, divided into high-dose and low-dose treatment groups and an untreated (control) group, were then observed for five months.
The mice treated with follistatin genes developed larger bodies and larger, heavier muscles, with the high-dose group showing the greatest effects. Follistatin was detected in the bloodstream of low- and high-dose-treated mice, and it affected muscles far from the injection sites.
The investigators observed an increase in the size of muscle fibers in mice receiving the gene therapy but not in fiber numbers.
Both groups of treated mice showed reduced levels of creatine kinase, indicating less leakiness of muscle-fiber membranes compared to control mice. The researchers speculate that the treated fibers became less susceptible to damage.
The investigators then injected 7-month-old DMD-affected mice with follistatin genes in viral shells. These older mice showed increases in strength about two months after the injections, which persisted for the more than 18 months during which the mice were evaluated.
At the end of the study, the treated mice had substantially fewer groups of dead muscle fibers, fewer inflammatory cells in their muscles, and less scar tissue than did untreated mice, and their muscle fibers were larger in diameter than those of the control group.
The improvements were sustained and well tolerated over more than two years.
The investigators note that the follistatin gene transfer was beneficial in aged dystrophin-deficient mice even after they had undergone multiple rounds of muscle degeneration and regeneration, implying that this type of therapy could have potential for treating older DMD patients.
They note that follistatin not only suppresses myostatin but also affects various cell signaling pathways and reduces inflammation. They conclude that “the striking ability of follistatin to provide gross and functional long-term improvement to dystrophic muscles in aged animals warrants its consideration for clinical develoment to treat musculoskeletal diseases, including older DMD patients.”
Tendons may need myostatin to stay supple
New results from the University of Michigan reveal a previously unrecognized downside of myostatin blocking.
John Faulkner and colleagues, who published their results Jan. 8 in Proceedings of the National Academy of Sciences, have found that mice bred to lack myostatin from birth have tendons that are 14 times stiffer than tendons in mice that produce myostatin.
Tendons attach muscles to bone, and their flexibility plays a role in protecting muscle fibers from contraction-associated injuries. Muscle fibers in boys with DMD are particularly susceptible to this type of injury.
It isn’t yet known whether myostatin blocking has the same effect on human tendons as it does on mouse tendons, or whether blocking myostatin months to years after birth would be different from stopping its production before birth. However, the findings are a caveat about strategies to block myostatin as a treatment for muscular dystrophy. |
