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And a Little Mollusk Shall Lead Us
By Ying Ju Sung, PhD
For the estimated 50 million Americans who suffer from chronic
pain, time can appear to pass slowly as they wait anxiously
for a cure. It is ironic, then, that one of nature's lowest
animals, the sea slug Aplysia californica, may hold the key
that unlocks the answer to chronic pain. Through the efforts
of Nobel laureate Eric Kandel, Aplysia is a well-known model
organism for studying learning and memory formation. However,
the pioneering work of Dr. Richard Ambron of Columbia University
and Dr. Edgar Walters of the University of Texas has amply
demonstrated that Aplysia can also be used to investigate
a particular facet of chronic pain called long-term hyperexcitability
(LTH).
LTH develops days after a nerve is injured as an increased
sensitivity to electrical or mechanical stimuli to the cell
bodies and axons of the injured nerve cells. Thus, stimuli
that normally would not produce a response in uninjured nerves
result in a burst of electrical discharges known as action
potentials in the injured nerve cells (neurons). Because neurons
communicate with each other, the action potentials from the
injured neurons are transmitted to other neurons. They are
then activated and the signal eventually reaches the brain
where it is perceived as pain. What makes LTH even more important
is that it requires the expression of new genes. The genes
direct the synthesis of proteins that actually alter the structure
of the neuron, which explains why the patient experiences
persistent pain.
The Injury Site Communicates to the Cell Body
When I first began working on the cause of LTH there was only
a rudimentary knowledge of its basis. It was a mystery because
most injuries occur far from the cell body, where proteins
are synthesized. The problem was figuring out how the injury
site communicated to the cell body. An interesting idea was
that one or more proteins from the site of injury were transmitted
down the axons by an unknown vehicle to the nerve cell body.
Once there, they would activate another signaling molecule,
which would then enter the cell's nucleus and either turn
on, or turn off the expression of specific nerve cell proteins.
The resulting actions ultimately would cause LTH. However,
while some of the molecules that were involved in LTH had
been identified, the details of the pathway were largely unknown.
My initial interest in this problem led me to a protein called
cyclic GMP-dependent protein kinase or PKG. PKG is an enzyme
that, when activated, marks or tags other enzymes, thereby
either activating or inhibiting them. PKG was activated after
crushing Aplysia nerves. Significantly, active PKG was found
only in crushed nerves; it originated at the site of the injury
and then was transported down axons to neuron cell bodies.
In fact, nerve crush initiates a cascade at the site of injury
that begins with the activation of an enzyme called nitric
oxide synthase (NOS). NOS produces nitric oxide (NO). NO,
in turn, activates a soluble guanylyl cyclase (sGC), resulting
in the formation of cGMP, which activates PKG. Blocking the
activation of NOS, the sGC, or PKG prevented the appearance
of LTH. These and other experiments helped establish the PKG-pain
pathway in Aplysia.
The Mammalian Model
Although Aplysia have a number of properties that make them
a favorable system to investigate the molecular basis of pain
and we believe that LTH is an evolutionarily conserved mechanism,
the real proof of PKG's involvement in pain needed to come
from a mammalian model. In a paper that was recently published
in Neuroscience, we demonstrated that the major outlines of
the PKG-pain pathway in Aplysia are also found in the rat.
The mammalian nervous system of course is much more complex
than Aplysia, which in this case was helpful because it enabled
us to show that PKG is present predominantly in a class of
small nociceptive (pain sensing) neurons, exactly what one
would expect if PKG was associated with pain. In contrast,
PKG was largely absent from motor nerves, which control movement.
Again, we found that crushing nerves, specifically the rat's
sciatic nerve, resulted in the activation of PKG. Again, we
demonstrated that the active PKG was transported back to the
cell bodies and that the commercially available PKG inhibitor
blocked the activation and the transport of PKG. Using rats
allowed us to take our work a step farther. Specifically,
we knew from previously published studies by other investigators
that nerve inflammation causes LTH in rats and is associated
with a number of chronic pain conditions such as ileitis,
cystitis and osteoarthritis. We therefore injected an inflammatory
agent called CFA (an agent used to induce osteoarthritis in
rats) into the hindpaws of rats to produce an inflammation.
We found that after the inflammation developed, PKG was activated
and then carried back to the neuron exactly as it was following
nerve injury.
Our results suggest that PKG may be activated by a variety
of pain-producing stimuli. This role for PKG is further supported
by studies showing that inhibiting PKG after nerve compression,
which can occur in spinal cord injuries, reduces both LTH
and the sensitivity to temperature that accompanies this type
of injury. In addition, the response to inflammatory pain
is greatly reduced in mice that have been genetically altered
not to produce PKG. Thus, PKG is essential for communicating
information that is ultimately perceived as chronic pain.
Because of this, it is currently a very attractive candidate
for developing therapeutic drugs. In addition, the vehicle
that transports active PKG from the injured or inflamed site
and the signaling molecule(s) that are activated by PKG (they
enter the nucleus and turn on or off the genes that give rise
to LTH) are also targets for chronic pain drug development.
We are now carrying out experiments to identify these components.
Can PKG-specific inhibitors potentially treat CRPS?
The studies from Aplysia and the rat
suggest that the answer is yes, but we are still at the threshold
of beginning to answer whether inhibiting PKG or the downstream
targets of the PKG-pain pathway may treat any chronic pain
states in humans. However, I found it extremely interesting
that one of the underlying mechanisms that have been suggested
to explain CRPS is neurogenic inflammation. Since sensory
neurons release the substances that trigger neurogenic inflammation,
regulating their excitability should inhibit the release of
these agents. Ultimately, this should decrease neurogenic
inflammation. Thus, amid pain there is hope.
Ying Ju Sung is currently an Assistant Professor in the
Department of Anatomy and Cell Biology of Columbia University.
Updated December 14, 2006 |
