By David N. Leff
The terrible accident that befell movie actor Christopher Reeve nearly two years ago has refocused public attention on spinal cord injury. A fall from his horse in May 1995 left Reeve with a broken neck and severely damaged spinal cord, unable to move his arms or legs, and only limited ability to breathe.
There are a quarter million patients with permanent spinal injury in the U.S. alone. Their number increases by 10,000 to 12,000 annually, many of whom will live out long lives with their immobilizing disability. (See BioWorld Today, July 29, 1996, p. 1.)
When a limb suffers severe trauma, its cut nerve endings usually regenerate. These belong to the peripheral nervous system, which can sprout new axons to replace those damaged or destroyed. In contrast, axons of the central nervous system (CNS), which serves brain and spinal cord, have, in effect, forgotten how to reinvent themselves.
Forgotten, because mammalian embryos, during the gestational process of creating the future body's nervous system, manufacture vast networks of neurons, each of which sprouts forests and trees of long, branching axons.
Like a skilled arborist, the growing embryo prunes and discards excess neurons, as development proceeds. One of the genes responsible for this programmed pruning now has a new assignment -- teaching CNS neurons in adults to "remember" their embryonic origins, and turn on regrowth of axons lost to trauma.
That human gene is bcl-2. After planting the forest of neuronal trees and axonal branches in the growing embryo, it shuts down for good, at about the end of the first trimester in human gestation. Without the molecular tutelage of bcl-2's protein product, Bcl-2, CNS axons have no memory of how to regenerate, explained neuroscientist Gerald Schneider, at the Massachusetts Institute of Technology, in Cambridge, Mass.
He is co-senior author of an article in today's Nature, dated Jan. 30, 1996. Its title: "Bcl-2 promotes regeneration of severed axons in mammalian CNS."
Extra Gene Makes All The Difference
The mammals in question are transgenic mice, possessed of an extra copy of the human bcl-2 gene. While "shopping for genes that can explain the failure of CNS axons to regenerate," Schneider found them in the adjacent MIT laboratory of Nobel immunologist Susumu Tonegawa.
Swiss scientists had constructed these bcl-2 transgenics, Schneider told BioWorld Today "because of the gene's known function in controlling apoptosis -- programmed cell death. But because the failure of axonal regeneration occurs right at the age when excess embryonic axons start to die off, it seemed to me that perhaps the gene could have a role in axonal growth as well."
Schneider continued: "Susumu [Tonegawa] of course became interested in my project. His methods were very powerful; he had the lab facilities to do this, and is a co-senior author of our Nature paper."
They quickly discovered the difference between the transgenic mice and normal animals. "The main difference," Schneider went on, "is that they just don't show the drastic fall in regenerative capacity at the normal age. In the normals, gene transcription is down-regulated early in embryogenesis"
To soup up the animals' ability to express the bcl-2 gene product, Bcl-2, the Swiss had created transgenic mice containing an extra copy of the human gene, powered by a different promoter, specific for neurons.
To verify in vivo the ability of these animals to regrow severed CNS axons, the MIT team turned to nerves connecting their retinas to the optic lobe of the midbrain. "The retina," Schneider pointed out, "is a part of the CNS that is more readily accessible for study than the spinal cord proper."
The team selected young mice, about a week past regenerative failure in normal, non-transgenic, animals.
"We did neurosurgery," Schneider recounted, "exposed the surface of the midbrain, and made a knife cut clear across the optic tract's bundle of axons. That's when we found the big contrast between the transgenic and normal animals. The normals' axons failed to grow back; they stopped right at the side of the cut. But in the transgenics, they regenerated either through the cut, or, because they had enough growth vigor, they just regrew around it, in an end-run.
"This experiment demonstrated," Schneider said, "that we had found a gene, which, when turned off, results in regenerative failure. When we restore its function, we can overcome that initial large failure in this CNS axonal tract."
He added: "We wouldn't have discovered this if we had worked, as many labs do, on the adult. It's because we had this developmental model that we were able to make this finding."
Gene Therapy In MIT Group's Cards
But this promising result is only a beginning, he pointed out. "Other things happen later that we still have to work on. There are other molecules in this bcl-2 gene family that have to be found and tested. And, in collaboration with various molecular biologists, I'm looking for other genes that may play additional roles in improving the vigor of axonal growth."
He is also working with other academic collaborators on "genetic engineering to put the genes into adults, the next major step toward therapeutic treatments of spinal trauma."
Once developed, Schneider went on, this will take the form of "a package of chemical ingredients that will be used for tissue engineering after a CNS injury. As we now envision it, the major ingredient will be the bcl-2. DNA, for transfection into the neuronal cell."
A major conceptual spin-off of the MIT finding, Schneider concluded, is that it contradicted the dominant view that the place to seek axonal regeneration is at the site of the wounded axon itself, rather than its origin in the neuronal cell body.
"We discovered," he observed, "that the source of regeneration is not in the local tissue, but in the retina, site of the axons' neuronal origin, where the change was occurring that led to the big failure early in postnatal life." *