Newly Discovered Protein May Be Key To Muscular Dystrophy

Scientists at UCSF's Ernest Gallo Clinic and Research Center discovered in an animal model of the disease that during periods of intense muscle activity, muscles remain excited too long and degenerate if the protein fails to transport the neurotransmitter acetylcholine away from the nerve-muscle synapse. Muscle degeneration is the hallmark of muscular dystrophy, one of the most common genetic diseases.

The study was carried out in the roundworm, C. elegans, an animal which has provided early clues to the role of a number of important molecules in the human nervous system. The researchers expect that the protein, which they showed is an acetylcholine transporter, plays the same role in humans as it does in C. elegans, identifying a potential new route for treatment of muscular dystrophy.

The research is being reported in the August 19 issue of the journal Nature.

Normally, a nerve cell induces a muscle cell to contract by releasing the neurotransmitter acetylcholine at the synapse -- the junction where the two cells meet. Researchers have identified transporters for most neurotransmitters, such as serotonin, dopamine and glutamate. The transporters remove neurotransmitters from the synapse, in effect providing an "off" switch to the neurotransmitter's potent effects. It was thought that normal enzymatic breakdown of acetylcholine was so effective that a transporter wasn't needed to clear excess acetylcholine from the synapse. But the scientists found that during periods of intense muscle activity, a transporter must clear acetylcholine from the synapse.

"Discovering a transporter for acetylcholine was quite a surprise," said Steven McIntire, MD, PhD, UCSF assistant professor of neurology. "Identification of an acetylcholine transporter in mammals could lead to useful therapeutics to treat neuromuscular diseases as well as disorders of the central nervous system." McIntire is a principal investigator at the UCSF-affiliated Gallo Clinic and Research Center, and is senior author on the Nature paper.

Transporters have proven to be important drug targets, McIntire said. Prozac, for example, modulates a transporter that regulates serotonin concentration. Besides carrying messages from nerve cells to muscle cells, acetylcholine triggers communication between neurons in the brain, and is involved directly or indirectly in many diseases, including Alzheimer's and diseases of peripheral nerves. Discovery of the acetylcholine transporter could lead to therapies for some of these diseases based on altering acetylcholine levels, he said.

Duchenne muscular dystrophy, the most common form of the disease, primarily affects boys -- about one in 3,500 male births. It typically begins in early childhood. Children often experience leg weakness, falling and progressive loss of movement, eventually becoming wheel chair-dependent. Many die in their late teens or early twenties. Currently, there is no cure.

Scientists already knew that muscular dystrophy results from genetic defects in components of a network of proteins, known as the dystrophin-glycoprotein complex (DGC),that extends through the entire muscle cell membrane, linking the framework inside the cell to the region outside of the cell. The complex is found in C. elegans, in mice and in humans. In each species, defects in the complex can cause muscle degeneration, but the exact role of the complex in disease has not been clear.

In a genetic screen, the scientists identified 12 mutations which produced defects in coordinated movement just like those found in C. elegans DGC mutants -- a model for human muscular dystrophy. While seven of the mutants involved defective DGC function, five others resulted from variations in a previously unidentified gene. The team cloned this gene, snf-6, and found that its sequence was similar to genes in a family of mammalian neurotransmitter transporters. Because analysis of mutants indicated defects in acetylcholine activity, they tested whether the expressed protein, SNF-6, transports acetylcholine in mammalian cells grown in culture. They found that it is indeed an acetylcholine transporter.

The team used fluorescence techniques to determine that the distribution of SNF-6 was altered in DGC mutants. They found that the DGC, imbedded in the muscle cell membrane, maintains the neurotransmitter transporter at the neuromuscular synapse. In this location, the transporter is available to clear excess acetylcholine.

Further studies confirmed that when acetylcholine transporter function is disrupted, whether by defects in the DGC protein complex that maintains the transporter, or by genetic defects in the transporter itself, the result is muscle degeneration typical of muscular dystrophy.

"We hope that these findings will ultimately lead to an effective treatment for common forms of muscular dystrophy," McIntire said.

Lead author on the Nature paper is Hongkyun Kim, PhD, a postdoctoral scientist in McIntire's lab. Co-authors are Matthew J. Rogers, BS, research assistant in the lab; and Janet E. Richmond, PhD, assistant professor of biology at the University of Illinois in Chicago.

The research was supported in part by the Muscular Dystrophy Association.