Rewiring damaged circuits
by Julie Kinney
Hundreds of millions of years ago, the remote ancestors of humans could regenerate nerve fibers to repair injuries that damaged perception, movement and memory, among other crucial functions regulated by our brains. We humans do, in fact, still have some ability to regenerate nerve fibers, but it disappears as we emerge from infancy into childhood.
"The lower vertebrates, such as zebrafish or newts, can regenerate nerve fibers after injury," says Larry Benowitz, PhD, director of the Laboratories for Neuroscience Research in the Department of Neurosurgery at Children's Hospital Boston. "It's the higher vertebrates that do not. One hypothesis is that somewhere during the course of evolution we lost this ability, probably because it is more important to maintain the stability of brain circuits than to be able to re-grow new pathways after injury. The wiring of the central nervous system represents the biological basis of how we understand the world, how we represent knowledge, process information and store memories. If nerve fibers were able to grow and change, the net effect could be harmful."
But what happens when the very wiring upon which all of this rests is disrupted? The numbers of such interruptions of wiring are staggering. Between one-quarter- and a half-million people in the United States are living with spinal cord injury. And there are approximately 500,000 new victims of stroke every year in the U.S. Damage caused by these injuries can permanently disconnect the communication between different parts of the central nervous system and permanently impair perception, thinking and movement, among other functions. Probably the most widely reported case is that of Christopher Reeve, the actor known for his role as Superman, whose tragic horseback riding accident fractured the uppermost vertebra in his spine and paralyzed him from the neck down.
Benowitz is among a handful of neuroscientists giving new hope to spinal cord injury and stroke victims. With funding from the National Institutes of Health, as well as the Christopher Reeve Paralysis Foundation, Boston Life Sciences Inc., and the Boston Neurosurgical Foundation, Benowitz and members of his laboratory are closing in on a molecular pathway in nerve cells that regulates axon growth. Axons are the branch-like extensions of the neuron that conduct impulses away from the cell body; axons typically end in synapses, where neurons initiate communication with other nerve cells or with muscles, enabling us to walk, talk and perform other functions.
It was mostly through chance that Benowitz, who has dedicated more than 20 years to understanding the brain's circuitry, stumbled onto inosine, a molecule that closely resembles two of the four building blocks of DNA and can play a crucial role in stimulating axon growth. His lab had previously found that the glial cells that surround nerve fibers release a small molecule that causes nerve cells of the goldfish retina to regenerate their axons. While trying to figure out the structure of this molecule, which they called AF-1, they tested a number of molecules with the right molecular size. One of the molecules they tested was adenosine, one of the building blocks of DNA. "I had read papers about some of the effects of adenosine on nerve cells, and thought, maybe it induces axon growth. It's the right size. We tried it and were amazed to see our nerve cells' response to it." Through additional experiments, they discovered that it is not adenosine itself, but inosine, a molecule that cells form by converting adenosine through an enzymatic reaction, that causes nerve cells to extend long axons.
Benowitz's team, including a senior research associate, a neurosurgical resident, two medical students, four post-doctoral fellows and three research assistants, treated nerve cells growing in petri dishes with inosine. Instead of succumbing to the potent suppressive agents that normally block nerve growth later in life, inosine flipped a switch of sorts in the nerve cells and turned on an entire constellation of genes required for axon growth. "I had never experienced a series of experiments where every experiment gave such a clear answer, and where everything came together and crystallized so beautifully," says Benowitz.
The next step was to repeat these results in vivo—or in live models. In rats, his group interrupted the major nerve pathway that controls voluntary movement of the paws and digits on one side of the body, replicating the injury caused by certain strokes or spinal cord trauma in humans. A catheter carried inosine into the brain's cerebral cortex, delivering it to the nerve cells that send their axons down to the spinal cord. Two weeks later, they examined whether new connections had formed by injecting a tracer dye into the brain. They found that inosine had stimulated up to several thousand new axon branches to extend from the remaining intact corticospinal tract across the midline into the part of the spinal cord that had lost its normal inputs, where they seemed to form new connections. The findings showed that inosine had induced remarkable levels of axon growth just where it was needed. Their most recent studies suggest that in addition to causing intact axons to sprout new branches that replace those that were lost, inosine may also stimulate damaged neurons to regenerate their axons.
It was the excitement generated in the late 1960s by the new approaches to the brain and to biology in general that swept Benowitz into the field of neuroscience. He was studying math and engineering at CalTech when the lab of Roger Sperry—who later won the Nobel Prize for his visionary understanding of the functions of the cerebral hemispheres in humans’Äîwelcomed him into its ranks. Benowitz studied the functional organization in the brain, intrigued by how the brain is organized in different species and how it forms memories. He peered into areas of the brains involved in memory formation in newborn chicks, and investigated the cognitive functions of the human right cerebral hemisphere before turning his attention to the molecules of the nervous system.
Since then, a number of findings led him further down a path that may soon provide medical therapies for physically devastating conditions caused by spinal cord injury and stroke. In the late 1970s, Benowitz discovered the protein GAP-43 and showed that it is involved in the regeneration of the optic nerve in goldfish. He then went on to study how this protein contributes to the formation of connections throughout the brain in most species, including humans, and how it may allow synapses to continue changing in certain parts of the adult brain when we learn. Upon coming to Children's in 1990, he turned his attention to the broader question of what stimulates nerve cells to form connections and to make GAP-43 in the first place. This research led to the discovery of the small growth factor AF-1, and then to inosine.
"We think we may have come across a final common pathway that controls the growth of axons in nerve cells," says Benowitz. "Inosine enables damaged neurons to at least partially regrow their nerve fibers, and can stimulate neurons whose axons were not damaged to sprout new branches and take the place of others that have been lost. We need to do more work and conduct longer treatments to see how far it will take us. As basic scientists, we're also deeply interested in understanding the precise molecular pathways that underlie axon growth."
In the meantime, Boston Life Sciences Inc., a company developing novel diagnostics and therapeutics for various diseases and disorders, plans to begin Phase I clinical trials using inosine with stroke patients this fall. Phase I trials target safety levels and toxicity of drugs. Since inosine is a naturally occurring molecule, and no toxicity has been detected in animal models, Benowitz and Boston Life Sciences have high hopes for its potential clinical uses.
As is the case in science, the Benowitz lab continues to look into alternatives that, in conjunction with inosine or on their own, may lead to even greater levels of nerve regeneration. Ongoing investigations by the lab entail cloning the gene for the enzyme they believe to be the target of inosine's actions and the "master switch" that controls axon growth; exploring methods to regenerate axons in the optic nerve of mammals; studying whether inosine and AF-1 cause a reorganization of connections and improve functional outcome after stroke; and obtaining a fuller understanding of the molecular changes that underlie axon growth.
"We're very excited and hopeful about what inosine might hold," says Benowitz. "These experiments have been among the most rewarding I've ever undertaken."