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Switching on Two Genes Activates Significant Regeneration of Spinal Cord Axons

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Duke Health News 919-660-1306

DURHAM, N.C. - In experiments using cell cultures and
gene-altered mice, researchers have found that switching on
just two genes can induce considerable regeneration of damaged
nerve fibers in the spinal cord. Their finding suggests that
genetic therapy or drugs that activate perhaps only a handful
of genes might be enough to induce regeneration of spinal cords
in humans with spinal cord injury or other central nervous
system damage.

In addition, the scientists said their in vitro method of
testing the effects of such treatments on cultured nerve cells
should speed research on such therapies.

In an article in the January 2001 issue of Nature
Neuroscience, Duke University Medical Center neurobiologist
Pate Skene and his colleagues reported that inserting into
transgenic mice the genes for the regulatory proteins GAP-43
and CAP-23 induced neurons to grow the elongated nerve fibers
called axons that are characteristic of nerves that are
successfully regenerating. In contrast, they found, inserting
either gene alone produced a more restricted, highly branching
growth that could enhance the local development of connections
between neurons, but which is not sufficient for regrowth over
long distances.

In an accompanying News & Views article in the journal,
Clifford Woolf of Massachusetts General Hospital and Harvard
Medical School called the finding "a major advance in the
understanding of which molecules are required to induce injured
axons to grow over long distances."

"This was a very happy surprise," Skene said. "We had not
made such an experiment a priority because it seemed hard to
believe that expressing only one or two genes could have such a
significant impact on neuronal regeneration.

The number could have been closer to 50 or a hundred.
Fortunately, however, we decided it was time to find out the
effect of expressing the important genes we already
understood."

Other co-authors on the paper are Duke researchers Howard
Bomze, Ketan Bulsara and Bermans Iskandar and Pico Caroni of
the Friedrich Miescher Institute in Basel, Switzerland.

Their research was supported by the National Institutes of
Health, Novartis Pharmaceuticals and the Christopher Reeve
Paralysis Foundation.

The two proteins, GAP-43 and CAP-23, are known to reside in
the growing tip of the axon, known as the growth cone. The
proteins appear to play a poorly understood role in integrating
and modulating biochemical signals in the growing axon. It also
had long been known that the genes for such proteins were
switched on during development to foster growth of axons in the
brain and spinal cord, but that they were turned off in adults.
The genes for GAP-43 and CAP-23 were known to be re-activated
after damage to peripheral nerves, which regenerate
effectively, but not after spinal cord injury. Neuroscientists
had debated whether activation of these genes is needed for
regeneration of spinal cord axons, and which of the many genes
induced by peripheral nerve injury are critical for regrowth,
said Skene.

"Although we had known for some time that GAP-43 and CAP-23
are not ordinarily expressed after spinal cord injury, it was
not clear what role that lack of expression played in
preventing axon regeneration," he said. "This paper offers the
best evidence so far that expression of these genes is one of
the key factors determining the success or failure of
regeneration."

In their experiments, Skene and his colleagues used cultures
of dorsal root ganglion (DRG) nerve cells taken from adult
mice. The axons of DRG neurons carry sensory information from
the body up the spinal cord to the brain and form one of the
principal fiber tracts damaged by spinal cord injuries. But,
because the cell bodies of these neurons are located just
outside the spinal cord, they are more easily isolated for cell
culture than other adult neurons, Skene said.

"This in vitro system has two major advantages," he
explained. "First, it is much faster and more straightforward
than doing a complete study in intact animals, which can take
years. So, we can study many genes and combinations that appear
likely to support regenerative axon growth. And secondly, we
can study the whole cell in isolation and in a well-controlled
environment. By contrast, attempting to trace axon growth in
the intact animal is more difficult."

Using this in vitro assay, Bomze studied the response to
axon injury of DRG neurons taken from mice that had been
engineered to express genes for GAP-43 or CAP-23, or both.

"He found that cells expressing the genes for either GAP-43
or CAP-23 alone produced cell growth, but of the highly
branched type characteristic of local remodeling," Skene said.
"But when cells expressed the genes for both proteins, they
switched to a long, unbranched axon growth that resembles nerve
regeneration," he said. "We were very impressed that the
combination of these two genes produced a qualitatively
different kind of growth than either gene alone."

The scientists next sought to detect whether the combination
of genes produced the same growth affect in adult mice.

They produced spinal cord lesions in both normal wild-type
mice and transgenic mice engineered to produce both proteins as
adults. To give any potentially regenerating axons a support on
which to grow, the scientists grafted a segment of peripheral
nerve into the spinal cord lesion site.

After several weeks, they used a fluorescent axon tracer to
label any spinal cord axons that had been able to regenerate.
These staining measurements revealed that the transgenic mice
were 60 times as likely to regenerate their spinal cord axons
as the wild-type mice. According to Skene, further research
will include using the in vitro assay to explore the effects of
introducing growth-inducing genes after an injury.

"In these experiments, we used transgenic animals that
expressed the genes throughout life, whereas normally they turn
off after the spinal cord is completed," Skene said. "But
that's not what happens in a person who has an accident that
severs the spinal cord. So, we need to understand the effects
of expressing these genes in adults - after an injury has
occurred - and for how long they need to be expressed to get an
effect. Also, we need to develop techniques for inserting these
genes into neurons after an injury."

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