December/January 2001

Searching for the Key

By Moses V. Chao, PhD

New research could help unlock the door to repairing the damaged spinal cord.

How do we begin repairing the damaged spinal cord? More than 250,000 Americans suffer from spinal cord injury (SCI), and 10,000 new injuries are reported each year. However, a number of recent reports indicate damaged nerves in the spinal cord are able to recover, find their targets, and make functional connections that can lead to partial recovery.

The Possibility of Curing SCI

The possibility of overcoming the effects of serious injury to the spinal cord has dramatically improved over the past few years due to new research. Here are just a few of the findings that may hold a cure for paralysis from spinal injury:

• Injection of a monoclonal antibody (IN-1) into the lesioned spinal cord of rodents leads to significant functional recovery of limb movements.¹
• Mice immunized with purified myelin use their immune systems to facilitate regeneration with increased nerve growth.²
• Growth factors, such as neurotrophin-3 (NT-3) and glial derived neurotrophic factor (GDNF), are very effective in stimulating nerve extension into regions in the spinal cord that are not hospitable to regeneration.³ Delivery of these proteins in damaged regions restores the ability of animals to sense heat and pressure.
• Placement of stimulated macrophages into a transected rat spinal cord results in partial recovery of motor activity.⁴
• Transplantation of embryonic stem cells into rats whose hind limbs were incapacitated by spinal cord injury results in increases in leg movement.⁵
• Transplantation of ensheathing cells from the olfactory bulb into animals with a complete spinal cord transection facilitates motor axon regeneration and recovery of some locomotor and sensory functions.⁶

These results raise many questions. Why do some nerves regenerate poorly or not at all? What are the molecules that limit and promote nerve regeneration? What role will the emerging use of stem cells play in spinal cord repair? Finally, what are the mechanisms that account for axonal regeneration and functional recovery in the central nervous system?

One of the dogmas in neuroscience is that the adult mammalian central nervous system (CNS) cannot regenerate or recover from serious injury. This idea was overturned in the 1980s by two important realizations. First, grafting peripheral nerve segments into the spinal cord allowed damaged spinal nerve fibers to extend long distances. This finding, made by Albert Aguayo 20 years ago, changed the field, since the results clearly indicated that nerves in the spinal cord have an intrinsic ability to grow. Second, the environment of the spinal cord is very important. It contains many powerful inhibitory factors that prevent nerve growth. Myelin produced by oligodendroglial cells expresses proteins that block regeneration. Inhibitory proteins in myelin have been identified recently. Likewise, growth-inducing factors, such as neurotrophic factors and adhesion and guidance molecules, can overcome the inhibitory environment of the spinal cord.

Many of the advances have occurred as a result of cell and molecular biological approaches. These findings will soon be translated into clinical trials in human patients. Together, modulation of both inhibitory and growth-promoting molecules promises to provide many new strategies for turning an inhospitable CNS environment into one that will support regeneration.

Improving the Environment

Much of the progress made is based on attempts to improve the poor environment in the spinal cord. There have been two basic strategies. One way to coax more nerve cell growth in the spinal cord is by blocking or removing the inhibitory molecules that are present in the spinal cord. This approach has been successful with the use of myelin blocking antibodies, such as IN-1, or even more elaborate approaches of immunization. Adult mice injected with myelin proteins have been found to mount an immune response to block axon growth inhibitors. Remarkably, these immunized mice undergo functional recovery of the hind limbs after spinal cord lesion.

A second approach is to provide a better environment by the use of growth factors or cell-based therapies. Trophic factors, such as GDNF, neurotrophins (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], and NT-3), and ciliary neurotrophic factor (CNTF) have been studied for their ability to keep cells alive during the development of the nervous system. More recently, neurotrophins have been found to possess other effects on neurotransmitter release, synaptic strength, and axonal and dendritic branching. Many of these actions are activity-dependent events based on remodeling of nerve terminals that are important during functional connectivity of lesioned nerves.

Another way to provide a more favorable milieu is to place cells that are permissive for regeneration in the spinal cord. A variety of cell types have been used. Glial cells from peripheral nerve, such as Schwann cells, have shown that this approach is feasible. Transplantation of an ensheathing cell from the olfactory system and macrophage cells from the immune system have drawn considerable attention, as they can improve recovery of lesioned nerves. In another exciting development, embryonic stem cells capable of giving rise to new neurons and astrocytes have been effectively used in transplantation studies to improve recovery after spinal cord injury.

These new cells can act as guideposts to direct axons to the right targets and speed up the repair process. The idea that a bridge for the cut spinal cord can facilitate recovery has been verified in many animal studies. Grafted cells can survive, provide trophic support, restore electrical conductance across a lesion, and contribute to regeneration in the spinal cord. Although the exact effects are unknown, transplanted cells may be effective in producing growth factors and cytokines or removing inhibitory effects during scar formation.

Identifying Proteins

While the list of advances is impressive, we still do not know in many cases how recovery is achieved and what signals are necessary to tell a nerve to stop and start growing. The identification of proteins involved in promoting or preventing nerve growth is critical, as they will provide novel therapeutic targets to promote nerve regeneration.

One protein that accounts for the inhibitory nature of myelin is nogo, recently purified and cloned by Martin Schwab and others.⁷ The nogo protein, found in oligodendrocytes and myelin, is the target for the IN-1 antibody, which is effective in promoting long-distance regeneration of damaged nerves in the corticospinal tract. Proteins like nogo may be released from oligodendrocytes during nerve injury and thereby prevent nerves from regrowing. But nogo is only the tip of the iceberg, as there are many other proteins that can influence regenerating axons. These include guidance molecules, ie, netrins, semaphorins, and slits, and membrane-attached proteins, such as myelin-associated glycoprotein (MAG), ephrins, and proteoglycans. Netrins, semaphorins, and slits are proteins that are generally secreted from the cell, whereas the others span the cell membrane. The existence of many inhibitory proteins in myelin explains why stimulating an animal’s own immune system to generate antibodies against myelin membranes may serve as a future vaccination approach.

Regeneration depends on the balance of opposing inhibitory and stimulatory signals encountered by growing axons. Guidance proteins can act as attractants or repellents, instructing nerves to run in a linear direction, stop, or extend in another direction. A common property is that each protein acts either as a ligand or a receptor to generate signals. Growth factors and components of the extracellular matrix generally provide a favorable environment that allows neurite elongation. Inhibitory proteins serve as potent stop signals. Identifying the signals that tell damaged nerves to respond to guidance proteins is a major challenge for the future.

Future Prospects

In spite of the progress made in identifying molecules involved in neuronal rewiring, there are major obstacles for repairing the damaged spinal cord. Inflammation and cellular damage frequently occur. Besides motor, sensory, and muscular damage, local scar formation is a significant physical barrier for regenerating nerves. Glial scars contain high levels of repulsive molecules, such as tensacin and proteoglycans, that impede axon extension. Another important question is how appropriate targets are found and proper connections are made by regenerating fibers. Additionally, maintenance of these connections is often influenced by nerve activity, a process that would be enhanced by rehabilitation programs that could promote the reestablishment of neural connections and function.

Another promising approach to repair damage is the use of neural stem cells or genetically engineered cells, a topic that has sparked considerable debate. The advantage of stem cells is that they can turn into specific types of cells capable of producing chemicals and nutrients to facilitate regeneration. However, there remain many technical and ethical problems in the use of stem cells. The longevity and ultimate fates of these cells cannot be easily controlled in the spinal cord, not to mention the availability and feasibility of using large numbers of stem cells taken from human embryos. An alternative is to take advantage of stem cells already in the adult CNS. There are small groups of stem cells in the adult brain and spinal cord that can divide, migrate long distances, and replenish the nervous system. While we do not know if these cells can participate in CNS regeneration, learning more about what controls their proliferation and identity will point to new ways of coaxing cells in the damaged spinal cord to go through self-repair. For this to be achieved requires much more basic research.

Information from the Human Genome Project will reveal what genes are induced or repressed in the injured spinal cord. For each cell type, it will be possible to determine which genes or batteries of genes are actively being expressed or altered during injury and recovery. Some genes will be related to the action of trophic factors, which have been proposed as therapeutic agents for nerve injury. Another approach is to use structural information from guidance molecules to design small molecule agonists. The use of small molecules may obviate difficulties of delivery and pharmacokinetics in the central nervous system.

The steps forward must be placed in perspective by the enormous difficulties that lie ahead in overcoming paralysis. The ability to regenerate will come from finding ways to combine the additive effects of multiple growth factors with decreases in growth inhibitory substances. Clearly, an understanding of the basic molecular mechanisms of cell-cell communication in the spinal cord will provide new insights into how functional recovery will be achieved.

Moses V. Chao, PhD, is professor of cell biology, physiology, and neuroscience in the Molecular Neurobiology Program at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York. He is a member of the New York State Spinal Cord Injury Research Board and the Scientific Advisory Board of the Christopher Reeve Paralysis Foundation.

References

1. Thallmair M, Metz GAS, Z’Graggen WJ, Raineteau O, Kartje GL, Schwab ME. Neurite growth inhibitors restrict plasticity and functional recovery following corticospinal tract lesions. Nature Neuroscience. 1998;1:24-130.
2. Huang DW, McKerracher L, Braun PE, David S. A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron. 1999;24:639-647.
3. Ramer MS, Priestley JV, McMahon SB. Functional regeneration of sensory axons into the adult spinal cord. Nature. 2000;403:312-316.
4. Raplino O, Lazarov-Spiegler O, Agranov E, et al. Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nature Medicine. 1998;4:814-821.
5. McDonald JW, Liu XZ, Qu Y, et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nature Medicine. 1999;5:1410-1412.
6. Ramon-Cueto A, Cordero MI, Santos-Benito FF, Avila J. Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia. Neuron. 2000;25:425-435.
7. Goldberg JL, Barres BA. Nogo in nerve regeneration. Nature. 2000;403:369-370.

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