Jan. 5, 2001 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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