By David N. Leff
In this fateful Y2K year of the 20th century, some 4 million infants will be born alive in the U.S. About half of them will be males. Of their number, about 1,140 boy babies, in the third year of the 21st century, will start to stumble when they walk, or rather waddle.
These 3-year-old children will tire easily, and have a hard time climbing stairs. The bulging calves of their legs will look extremely muscular, but their pediatric neurologists will have to inform their parents that fat and connective tissue build up, not musculature, accounts for the impressive limb appearance.
These youngsters will be diagnosed with Duchenne muscular dystrophy (DMD), an X-linked inherited disease that strikes one live male birth in 3,500. Its cause is the total absence of dystrophin from their muscle cells. At 2.7 million base pairs in length, the dystrophin gene is the largest disease-related gene in the human genome.
The young patients' difficulties will creep slowly upward from wasting limb muscles to the musculature of diaphragm, jaw, arms and heart. By age 12, most of them will be wheelchair bound; by 20, dead of respiratory and heart failure. (See BioWorld Today, Jan. 6, 1999, p. 1.)
"People have known for a number of years," recalled pediatric cardiologist R. Mark Grady, at Washington University in St. Louis, that if you don't have dystrophin, you get DMD. But it hasn't been clear why, without dystrophin, your muscles fall apart. What's the connection? Why does the muscle need that protein? What's it actually doing in the muscle cell?
"Traditionally," Grady went on, "when the dystrophin protein was first discovered about 15 year ago, it was known to be associated with seven other muscle-cell proteins, now called the dystrophin glycoprotein complex (DGC). And people have thought the primary role of this dystrophin, together with its other proteins in the DGC, is a structural one. It's acting much like the girders in a skyscraper; holding the muscle together - creating a bridge from the interior of the muscle cell to its exterior. Severing this bridge, or disrupting this linkage in any way, either by removing dystrophin or some of these other proteins in the complex, basically breaks up the backbone of the muscle cell. The muscle then, because it's having to move, to contract, gets torn up, and the cell dies.
"Although that's not been convincingly proven," Grady observed, "it is the leading hypothesis - that dystrophin and the other proteins in this complex, keep the muscle together."
However, a knockout (KO) mouse deprived of another key muscle protein in that complex, a-dystrobrevin, saw that hypothesis and raised it one. "A finding from this KO mouse," Grady said, "suggests that the DGC may be keeping the muscle together through other ways not previously thought about." Grady is lead author of a report in the Aug. 1999 issue of Nature Cell Biology, which describes those ways. Its title: "Role for a-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies."
Muscular Civil Engineering - Plus Communication
"We suggest in our paper," Grady pointed out, "that the complex may be holding the muscle together, not just through the structural way - standing there like an inert I-beam - but also through a more subtle signaling pathway. It's actually signaling to other areas of the cell, to a number of different proteins, turning genes on and off. Somehow, we propose, this signaling process is also important for keeping the muscle together."
As for that falling-apart process, Grady explained, "It appears that in a Duchenne patient the muscle cell eventually develops holes or tears in its membrane. Then calcium, and other things from the outside that are not supposed to be there, flood into the cell, which leads to cell death. And it's always been thought that there's a way that that process occurs, because once you've taken away the structural support to this muscle cell, its membrane becomes very vulnerable to being torn."
Grady continued, "We initially got interested in this dystrobrevin protein because we thought that it may have some role in forming synapses at the neuromuscular junction. That led us to making mice that had it knocked out. And it turned out, much to my surprise, when I looked at the muscle of those dystrobrevin-minus mice, that they had muscular dystrophy.
"Several years back," he recounted, "we had made a KO of utrophin, which is a muscle protein very similar to dystrophin. As it turned out, utrophin has been a popular model for people researching MD [muscular dystrophy]. They have shown, at least in mice, that if you increase levels of utrophin in a Duchenne mouse, you can actually reverse the disease."
Triple-Threat Rodent Exposed Signal Protein
"So we made a triple-KO mouse deficient in all three proteins - dystrophin, utrophin and dystrobrevin. We found that the animals' MD was severe, but no more so than in a mouse that was already lacking the first two proteins. Thus, we showed, that dystrobrevin works through the DGC to help hold the muscle together.
"What we then showed," Grady went on, "was that when we took away dystrobrevin, we also took away, or reduced, the level of nitric oxide synthase (NOS) that's attached to the DGC complex as well. And we made the point that, as NOS, the precursor of nitric oxide (NO), is pretty much everybody's favorite signaling molecule, when we take away dystrobrevin, we think that this strengthens the case that this muscle protein must be working through some signaling pathway."
"There are patients out there," he observed, "who have DMD that lack not only dystrophin but the whole BGC, which disappears along with dystrobrevin. So we think that the MD that you see in a patient with Duchenne is a combination of losing not only the structural arm but also the signaling arm.
"So that's another mechanism in which the muscle can fall apart." Grady made the point, "Anytime you can identify a new pathway or mechanism, that gives you another way to try and prevent it from happening." As for circumventing the loss of the signaling pathway, he concluded, "perhaps through either some drug or gene transfer, we can make the disease not quite as severe. But you never know."