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New Insight Into Machinery of Neuromuscular Junction Assembly

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Duke Health News 919-660-1306

DURHAM, N.C. -- Duke University Medical Center researchers
have discovered a new component of the mechanism by which
nerves trigger muscles to contract. Their finding yields a
better understanding of the machinery of this "neuromuscular
junction" and could lead to drugs to treat such neuromuscular
disorders as myasthenia gravis, said the researchers.

In an article posted June 8, 2003, in the online Nature Neuroscience,
pharmacologists Alexander Finn and Ann Marie Pendergast, Ph.D.,
and neurobiologist Guoping Feng, Ph.D., 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.

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