NINDS Workshop on Re-establishing Connectivity in the Damaged Spinal Cord

NINDS Workshop on Re-establishing Connectivity in the Damaged Spinal Cord
January 18-19, 2001
Pooks Hill Marriott, Bethesda, MD
Chaired by Albert Aguayo, Arlene Chiu and Josh Sanes

Table of Contents
Introduction
Inducing adult neurons to grow axons again
Changing the environment around regenerating axons
Change response of neurons to non-permissive environment
Reforming connections - Interactions between the neuron and the target
Making the most of what remains after injury
Recommendations from the participants
Collaborations between groups and investigators
Development of needed research tools for the community
Imaging
Injury models and outcome measures

Introduction
Injury to the spinal cord initiates a complex series of events that has devastating consequences such as partial or complete paralysis, muscle atrophy, compromised ability to breathe, lack of bladder and bowel control, sexual dysfunction and chronic pain. The quarter of a million patients with spinal cord injury (SCI) in the United States require specialized care to deal with these problems every day for the rest of their lives. A major cause of dysfunction arises from damage to nerve fibers passing through the injury site or lesion. We are dependent on long fibers that traverse the length of the cord to coordinate messages between the brain and other parts of the body. Interruption of these fibers can disrupt the voluntary control of movement and the experience of sensation. In developing treatments for SCI, a central question is how to restore connectivity by promoting the regeneration of axons in the environment of the damaged nervous system and the establishment of new and appropriate synaptic contacts within the spinal cord.

To address this question, this workshop focused on "bridging" damaged tissue, encouraging re-growth of axons across the lesion, and facilitating reconnection to synaptic targets beyond the site of injury. However, it was recognized that complementary efforts to limit cell death, reduce inflammation-related damage, optimize the function of surviving neurons, and train intact local circuits would all contribute to functional recovery from SCI.

Presentations and discussions revolved around the following topics:

1. Spurring adult neurons to grow axons again
2. Changing the environment encountered by regenerating axons
3. Altering the response of neurons to a non-permissive environment
4. Reforming connections - interactions between neurons and their targets
5. Making the most of what remains after injury

Finally, a number of recommendations arose from the discussion that could move the field of SCI research forward.
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Inducing adult neurons to grow axons again
Damaged neurons in the adult mammalian central nervous system (CNS) typically do not regenerate axons in vivo. In experimental situations, it has been observed that injuring a nerve more than once leads to more robust regeneration after the second injury. However impractical it may be in a clinical setting, the phenomenon of a pre-injury, termed a "conditioning lesion", seems to prime the nervous system, and suggests that intracellular conditions can be changed so that neurons are more in a growth state. Several growth-associated molecules are "switched on" following a conditioning lesion. GAP43 and CAP23 are growth cone membrane proteins that are up-regulated by peripheral nerve injury and whose expression is correlated with regenerative potential. Although over-expression of either protein alone does not result in axonal regeneration, both proteins together in transgenic mice mimic the effects of a conditioning lesion and lead to higher numbers of regenerating axons in the spinal cord. DNA microarrays have also been used to identify several thousand genes that are regulated in response to injury. The challenge now is to elucidate the function of these genes, and to identify the ones that are crucial for spinal cord repair.
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Changing the environment around regenerating axons
Getting axons to grow across the cavity or "cyst" that often develops at the site of injury is a major challenge in SCI, and many strategies have been proposed to bridge this gap. Cells and substances that have been tested as bridge material include Schwann cells, olfactory ensheathing glia, peripheral nerve segments, collagen embedded with neurotrophic or growth factors, and a variety of stem/progenitor cells. Although many of these bridges and scaffolds have supported axon growth, the axons tended to stay within the bridge and do not extend beyond it to innervate targets on the other side of the lesion. To entice axons out of the bridge, it may be possible to introduce or genetically engineer in factors such as neurotrophins into the distal side of the host cord to lead axons onward.

Scar tissue surrounding the injury site is well known to inhibit axonal growth. Some of this activity is caused by accumulations of extracellular matrix such as chondroitin sulfate proteoglycans. When growth cones contact this inhibitory barrier, they remain motile and can persist for months, but make no further progress along the cord. However, the long-term persistence of these dystrophic endings raises the possibility that axons might resume growing should conditions around them improve and become more permissive, even at times long after the initial injury.

One direct approach to optimize the environment was to replace the injured tissue with transplants of fetal rat spinal cord. When these were grafted into newborn rats, axons regenerated through the transplant and into the host cord. When similar studies were conducted in older rats, axon regeneration through the transplant diminished, even though the same transplant material (fetal cord) was used, suggesting that the age of the host was responsible for the difference in regeneration. The numbers of axons growing into the transplant increased however, if exogenous neurotrophic factors such as BDNF, CNTF, NT-3, NT-4 were supplied to the older rats. Interestingly, if transplantation was delayed for 2 weeks after the initial injury to the cord, descending axons now were able to traverse completely through the graft and into the host cord at the caudal end. The effects of the delayed transplantation may have mimicked that of a conditioning lesion. In addition, the developing glial scar may have been removed during the surgery to insert the graft. The best behavioral recovery was seen when transplantation of fetal cord was delayed for 2 weeks, and with exogenous neurotrophins. Under this regimen, animals had near normal weight support and stepping behavior, although coordinated quadrupedal movement was not sustained. Top

Change response of neurons to non-permissive environment
Understanding the neuronal response to inhibitory molecules may provide hints on how to manipulate the intracellular signals and pathways that regulate this response. We may then develop strategies and design molecules to override the "no-growth" signals. Over the last decade, numerous molecules have been shown to have inhibitory, or growth-stopping effects on neuronal growth cones. Some are molecules that are present during development where they act to facilitate correct guidance when pathways are first laid down. Others appear to be components of myelin and prominent in white matter tracts in the adult nervous system

The semaphorins are a family of proteins, some of which are secreted and others membrane bound. They interact with a receptor complex made up of neuropilins and plexins. Because family members can act as antagonists by competing for binding to the same receptor, the activity of semaphorins can be modulated from inhibitory for growth to positive for outgrowth. Intracellular signaling pathways activated by semaphorins involve rac-1 and rho, small GTPases that control actin polymerization in the cytoskeleton.

The ephrins, a second family of proteins with inhibitory activity, play an important role in organizing the retinotopic projection to the tectum. Ephrin receptors are tyrosine kinases and activate downstream signaling cascades that are also linked to rac and rho pathways. Reverse signaling has been documented for ephrins where the ligand-bearing cell responds as well as the receptor-bearing cell.

The netrins and slits are two other classes of guidance molecules. In the developing spinal cord, netrin acts as an attractant cue guiding commissural axons to the floor plate. However, netrin activity can be modulated from being an attractant to being a repellent. In nematodes, netrin functions as a repellent when associated with another protein such as UNC5. Like netrin, Slit is found at the midline where it acts to keep axons that have crossed over from returning. Its receptor, Robo, is present on axons.

While these molecules play importance guidance roles in the developing nervous system, they may have a deleterious function during regeneration. The expression of molecules such as semaphorins, ephrins, netrins, and slits after SCI needs to be explored, as well as the expression of their respective receptors. If any are expressed after injury, their actions could be inactivated by selective use of antagonists and thus allow regeneration of certain axonal tracts or pathways in the cord.

One consequence of trauma is the disruption of oligodendrocytes with the concomitant release of myelin proteins. Many of these proteins exert a highly inhibitory effect on axonal growth. Some of myelin's inhibitory activity may reside in a recently identified protein, NOGO that is expressed only by mature oligodendrocytes. Different fragments of NOGO cause growth cone collapse in a classic in vitro test of motility inhibiting effects. MAG is a second inhibitory molecule present in myelin. The response of neurons to MAG is modulated by a cAMP dependent pathway. If cAMP levels are raised, protein kinase A is activated and inhibition by MAG is blocked. Intracellular levels of cAMP can be increased by exposure to neurotrophins. Other agents include the nonhydrolyzable cAMP analog dibutyrl-cAMP, and rollipram, an inhibitor of the phosphodiesterase that inactivates cAMP; all overcome inhibition by MAG and myelin.

How does elevated cAMP promote neurite outgrowth even when the extracellular environment is hostile to growth? One enzyme activated by cAMP is arginase-1, which converts arginine to ornithine which in turn is converted by ornithine decarboxylase to putrescine. Treating neurons with putrescine alone overcame inhibition by MAG.

Rho, another downstream target of the cAMP signaling cascade, is a GTPase that regulates actin polymerization. Neurite outgrowth is reduced when Rho is activated. Rho activity can be blocked by C3, a bacterial toxin from clostridium botulinum. Treatment of neurons in tissue culture with C3 blocks inhibition by MAG. The application of C3 to the site of a dorsal hemisection in adult mice resulted in good regeneration and behavioral recovery. Although these results seem encouraging, it was puzzling that recovery seemed too rapid (measurable after 24 hours) to be due to regeneration.

In summary, manipulating different stages of the cAMP pathway appears effective in overcoming the inhibitory actions of myelin components and may provide an entrée into intracellular mechanisms to change the response of the neuron. Top

Reforming connections - Interactions between the neuron and the target
Re-establishing connections with the appropriate targets is the next critical hurdle to functional recovery. Unfortunately, the rules governing synapse formation are not well understood, and those for synapse re-formation after injury are even less clear. What is clear is that a great deal of communication occurs between potential pre- and post-synaptic partners when synapses first form during development. Some studies suggest that the initial contact may come from the postsynaptic cell. When Mauthner neurons extend the first axons down the zebrafish spinal cord, they grow past their target motor neurons in each segment without a pause or change in their rate of extension. Target motor neurons, however, actively explore the environment with their dendritic protrusions. Dendrites contacting a Mauthner axon becomes stabilized, and synaptic specializations can form within 30 minutes of the initial contact. Complex interactions between the presynaptic and postsynaptic cells have also been observed in Drosophila development.
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Making the most of what remains after injury
While the focus of the workshop was on regeneration of axons and reformation of synapses, awareness of the need to make the most of remaining neurons and circuits after injury was high. One approach is to identify ways to facilitate endogenous mechanisms for recovery.

Unlike central nerves, peripheral nerves face fewer barriers, and because they do regenerate in the adult nervous system, investigators have turned to this model system to explore ways that encourage appropriate regrowth following injury. Brief electrical stimulation of the cut femoral nerve greatly enhanced the speed of axonal regeneration. With electrical stimulation, all motor neurons regenerated axons after 3 weeks, rather than requiring 10 weeks.

In the nervous system, how well a circuit functions depends on the levels of synaptic communication between the contiguous neurons making up the circuit. Synaptic function can be enhanced by regulating the presence of neurotransmitter receptors as well as their function. For example, AMPA receptors mediate excitatory synaptic transmission in the spinal cord, and recent studies find that phosphorylation potentiates their activity by increasing open channel probability. Receptor function can also be regulated by mobilization and insertion of receptors into active zones. As the intracellular pathways involved in receptor trafficking start to be understood, it may be possible to change and optimize synaptic function. For example, reduction of these receptors after injury may prevent excitotoxicity. Conversely their upregulation may enhance residual synaptic function.
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Recommendations from the participants

1. Collaborations between groups and investigators
   Research in SCI now requires laboratory methods that range from molecular genetics to animal behavior - essentially the entire range of modern biomedical research techniques. Workshop participants discussed the daunting task faced by individual investigators in acquiring the expertise in their own laboratories to conduct molecular biological, cell biological, and animal behavioral studies. Ways to share expertise, and ways for investigators with one specialty to learn new techniques were frequently discussed and generally felt to be crucial for the field to move forward. The specific recommendations were:
   1. Resurrect linked RO1's, so that investigators at different institutions can collaborate on a formal basis. This would help small laboratories with limited resources to broaden their research goals. Since these are like PPGs without walls, they should be reviewed by a review panel that routinely reviews PPGs rather than having the individual proposals go to different study sections, as they do in the current review mechanism. Encourage or popularize the R21 mechanism and bioengineering research grants. These types of mechanisms can be vehicles to fund "descriptive" research, exploratory science that may generate important reagents or models but may not be highly hypothesis-driven and therefore are doomed in regular study sections.
   2. Provide supplemental funding to reimburse shipping costs, as well as the costs of producing extra animals. Animals that were generated or produced through NIH funding should be required to be generally available. However, this entails extra costs. Laboratories that are willing to share resources are often hindered from doing so for purely financial reasons. Shipping of special animals (rare and precious transgenics, experimentally injured animal models such as cats etc.) and international shipping are is an additional problems and courier services such as those used by NASA are very expensive.
   3. Support a public repository for animals or reagents and spinal cord tissue. Animals that were engineered with NIH funding could be deposited at such a facility that would handle breeding, shipping, reagent production, and quality control. A tissue bank that stores spinal cord tissue with different types of injury could be a resource for future testing of genes and proteins.
   4. Establish a central core or a national laboratory to facilitate SCI research. Participants were interested in a central site where new techniques could be learned, new models/molecules/genes could be tested, old and new injury models could be standardized, and experimental results compared. The exact format/organization was not defined but examples such as the Woods Hole Marine Laboratories and the Brookhaven National Laboratory were brought us.
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2. Development of needed research tools for the community. These include:
   1. bridges/polymers
   2. GFP mice with specific tracts labeled
   3. high through-put screens
   4. exogenous delivery systems
   5. source(s) of growth factors and recombinant proteins
   
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3. Imaging
   The discussion on imaging combined aspects of both of the items above. New techniques needed to be developed, and new facilities needed to be available to make imaging studies available and useful for SCI research. These include:
   1. Developing microPET, fMRI and optical imaging for viewing different aspects of the spinal cord
   2. Developing 2-photon microscopy for viewing and re-constructing damaged spinal cord
   3. Developing new, noninvasive methods to look deep into an intact animal
   4. Increase funding to buy and operate equipment. Currently, the ceiling for shared instrumentation grants is low ($450,000), and do not cover operating costs and support personnel. NCI currently funds mall animal imaging centers.
   5. Establish a National Laboratory (such as Brookhaven) to image recovery from spinal cord damage.
   
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4. Injury models and outcome measures The participants agreed that new animal models and functional outcome measures (for example for pain) needed to be developed and standardized. A manual for SCI that "catalogues" the pros and cons of each injury model would be useful for the field and for new researchers.
   
5. Partnerships with private organizations and foundations NINDS can "flag" promising but unfounded SCI grants for consideration by private organizations. The participants also encouraged the use of administrative supplements to enable NINDS grantees who are not currently working in SCI to expand their funded project into the SCI field.

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