In an article posted June 8, 2003, in the online Nature Neuroscience, pharmacologists Alexander Finn and Ann Marie Pendergast, and neurobiologist Guoping Feng reported that a protein enzyme switch called Abl kinase is a key to activating the pathway that prepares the “receivers” on the muscle side of the neuromuscular junction to react to a nerve impulse. Finn is a graduate student in the M.D./Ph.D. program; Pendergast is an associate professor of pharmacology and cancer biology, and Feng is an assistant professor of neurobiology. Their research is sponsored by the National Institutes of Health, March of Dimes Birth Defects Foundation and the Medical Scientist Training Program.
Nerve cells trigger both muscles and other nerve cells by launching explosive bursts of chemicals called neurotransmitters across connections known as synapses. Although much has been learned about the function of the neuromuscular junction -- including how malfunctions can cause disease -- important details remained unknown, said Pendergast.
For example, it was known that a large protein “organizing” signal known as agrin in the synapse triggers a receptor called MuSK on the muscle cell surface to induce receptors for the neurotransmitter acetylcholine to cluster on the receiving surface, or postsynaptic membrane of the muscle cells. This receptor clustering prepares the muscle cell for the nerve impulse necessary to trigger a contraction. Also, previous researchers had found, a “linker” molecule called rapsyn was necessary for the process. “Even though it was known that agrin activated MuSK, it was a big question mark how this activation led to the aggregation of acetylcholine receptors, which is where we came in,” said Pendergast.
Understanding the detailed machinery of this aggregation is important, said Pendergast, because mutations in rapsyn or nicotinic acetylcholine receptor -- or antibodies that attack muscle acetylcholine receptors or MuSK in autoimmune disease -- are known to lead to neuromuscular diseases such as myasthenia gravis. Thus, drugs that affect this pathway might prove useful in treating these diseases. More broadly, she said, it is also unknown how postsynaptic receptor assembly occurs after agrin activates MuSK.
Work by Pendergast and her colleagues and other researchers on Abl kinase had revealed it to be a critical “on-switch” for a number of processes that take place at the cell membrane and involve cell surface receptors. Many of these processes resemble the reorganization of the cellular “cytoskeleton” that takes place when acetylcholine receptors are aggregated in the neuromuscular junction. Thus, reasoned the authors, Abl kinase -- and the closely related enzyme Arg -- might also play a role in the neuromuscular junction.
Using antibodies that could detect the Abl and Arg kinases, Finn showed in slices of mouse muscle tissue that the kinases were indeed localized to the postsynaptic neuromuscular junction. He also showed that that inhibiting Abl and Arg in cell cultures of mouse muscle cells inhibited the ability of acetylcholine receptor clusters to form. In further experiments with the cell cultures, the researchers also demonstrated that agrin triggers Abl and MuSK to form a specific interacting complex with one another that triggers the signaling required for receptor clusters to form.
In their paper, the authors concluded in the paper that their findings "have broad implications regarding the role of Abl kinases in synapse formation.” While previous studies have shown that Abl kinases are important for guiding the nerve cells to reach their targets, theirs is the first evidence that the enzymes also have a postsynaptic function. According to Pendergast, the findings could not only be important for drug development, but might also raise caution on the use of Abl kinase inhibitors.
“If it should prove that neuromuscular diseases can be linked to defects in this pathway, it might be feasible to use drugs to modulate that pathway by affecting the Abl kinases or their targets, to treat the disease,” said Pendergast. “And, of course, the more you know about the pathway, the better you are equipped for such pharmacological intervention.
“These studies also raise a caution about giving compounds that inhibit Abl function, particularly during development, because the formation of the nervous system requires functional Abl kinases,” she said.
In this regard, genetic inactivation of both Abl and Arg kinases in mice by knockout technology, results in embryonic death, and the embryos display abnormal closure of the neural tube, among many other defects. Feng and Pendergast plan to broaden their studies to identify the direct targets of the Abl kinases in the postsynaptic regions of the neuromuscular junction, and the physiological consequences of conditionally inactivating Abl kinases in muscle using mouse models.
Journal
Nature Neuroscience