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Spinal Cord Repair: Strategies to Promote Axon Regeneration
Neurobiology of Disease 8, 11–18 (2001) doi:10.1006/nbdi.2000.0359, available online at http://www.idealibrary.com on
REVIEW Spinal Cord Repair: Strategies to Promote Axon Regeneration Lisa McKerracher De´partement de Pathologie et biologie cellulaire, Universite´ de Montre´al, C.P. 6128, succursale Centre-ville, Montre´al, Que´bec H3C 3J7, Canada Received June 26, 2000; revised September 20, 2000; accepted for publication October 5, 2000; published online December 20, 2000
Neurons in the central nervous system have a remarkable capacity to regenerate their transected axons when provided with an appropriate growth environment. Advances in our understanding of axon regeneration have allowed the development of different experimental strategies to stimulate axon regeneration in animal models of spinal cord injury. Growth inhibitory proteins block axon regeneration in the CNS, and many of these proteins have been identified. Various methods that are now used to stimulate regeneration in the injured spinal cord are directed at overcoming the growth inhibitory environment of the CNS. Three general approaches tested in vivo stimulate regeneration in the spinal cord. First, antibodies that bind inhibitory proteins in myelin allow axon regeneration in the CNS. Second, methods that modulate neuronal intracellular signaling allow axons to grow directly on the inhibitory substrate of the CNS. Third, transplantation of cells to the lesioned spinal cord promotes repair. In this paper we review current advances in each of these research domains. © 2001 Academic Press
INTRODUCTION
tory reaction. This drug, however, does not restore functions that are lost when axons are cut. Several major advances in our understanding of axon regeneration have led to the ability to stimulate some axon regeneration and functional repair in animal models of spinal cord injury. In the 1980s experiments by Aguayo and colleagues to use peripheral nerve grafts that were inserted into the brain or spinal cord showed that CNS neurons have the capacity to regrow, and these studies highlighted that diverse classes of CNS neurons have the potential to regenerate when given a permissive growth environment (Aguayo et al., 1981; Bray et al., 1987a). Another major advance in our understanding of axon regeneration in the central nervous system was the discovery by Schwab and colleagues that the CNS environment did not simply lack growth promoting molecules, but that growth inhibitory molecules existed to block axon growth (Caroni & Schwab, 1988b; Schwab et al., 1993; Schwab & Thoenen, 1985). Experiments aimed at removing myelin in the injured region to promote re-
Traumatic injury of the spinal cord that transects axon processes results in permanent functional impairment, even when the neuronal cell bodies that are located away from the injury site remain alive. At present there are no clinical treatments available to stimulate regeneration of cut axons, but with the pace of research in the field of central nervous system (CNS) axon regeneration now increasing, the application of new discoveries may not be far away. Numerous studies now show that anatomical regeneration and functional recovery are possible in rodent models of spinal cord injury. Clinical treatments that foster regeneration would be a significant improvement over present treatments that serve only to limit the extent of secondary damage caused by non-neuronal cells that invade the injury site. Spinal cord injured patients receive high doses of the steroid methylprednisolone immediately following injury to suppress an unfavorable inflamma0969-9961/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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12 generation have conclusively demonstrated that inhibitory proteins in myelin are an important barrier to axon growth (Dyer et al., 1998; Keirstead et al., 1992; Savio & Schwab, 1997). Related to these discoveries is the important finding that the glial scar that forms after injury is a formidable barrier to axon regrowth (McKeon et al., 1991; Reier, 1989). A more recent advance is the demonstration that increasing the intrinsic growth capacity of neurons is sufficient to allow axon regeneration in the CNS and that neurons primed for regeneration with neurotrophins or a conditioning lesion have a better chance to grow on inhibitory substrates (Cai et al., 1999; Neumann, 1999). The inhibitory barriers present in the CNS are not entirely understood, and it is likely that not all CNS growth inhibitory molecules have been identified. To better overcome the growth inhibitory environment of the CNS, there has been a focused effort to identify the proteins that have growth inhibitory activity.
THE GROWTH INHIBITORY ENVIRONMENT OF THE CNS Growth inhibitory activity has been demonstrated to be associated with myelin (Caroni & Schwab, 1988a, 1988b). The first myelin-derived growth inhibitory protein to be identified was myelin-associated glycoprotein (MAG), a known myelin protein. The inhibitory activity of MAG was discovered independently by us (McKerracher et al., 1994) and by Filbin and collaborators (Mukhopadhyay et al., 1994). Next, several inhibitory proteoglycans that associate with myelin were identified (Niederost, 1999; Xiao et al., 1997b), although their role in myelin biology is less understood. Another growth inhibitory protein, called nogo, whose activity was first detected as a potent high molecular weight inhibitor in myelin (Caroni & Schwab, 1988a,b) was identified this year (Chen, 2000; GrandPre´, 2000; Prinjha, 2000). Different splice varients of nogo exist and a controversy concerning the inhibitory domains of nogo is relevant to whether the inhibitory forms of nogo exhibit oligodendrocyte-specific expression (Tessier-Lavigne, 2000). Function blocking monoclonal antibodies generated against nogo by the Schwab group bind to a unique portion of nogo expressed by oligodendrocytes (Chen, 2000), yet the unique domain is predicted to be intracellular by the Strittmatter group (GrandPre´, 2000). However, now that the sequence of nogo is known, we can expect that the relationship between structure, function, and inhibitory activity will be resolved in the near future. Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Lisa McKerracher
The inhibitory activity of MAG has been characterized both in vitro and in vivo (Li et al., 1996; McKerracher et al., 1994; Mukhopadhyay et al., 1994; Schafer et al., 1996). The extent to which this inhibitor limits outgrowth in vivo has been controversial because of studies performed with MAG null mutant mice: two groups were able to show a small improvement in regeneration on MAG ⫺/⫺ myelin (Li et al., 1996; Shen et al., 1998), whereas no change was observed in a third study (Bartsch et al., 1995). Regardless of the differences between these studies, regeneration in vivo in MAG⫺/⫺ mice was poor, likely because of the presence of other inhibitors, such as the inhibitory myelin proteoglycans and nogo. The inhibitory activity of nogo and MAG have been compared, and when tested in vitro these two proteins have equivalent growth inhibitory activity (Prinjha, 2000). If nogo knock out mice are generated and are viable, the MAG⫺/⫺ and nogo ⫺/⫺ could be compared with the same techniques. Perhaps the most interesting experiment will be when double mutant MAG⫺/⫺ and nogo⫺/⫺ mice will be generated to test the contribution of other inhibitors to block regeneration in the CNS. Other potential inhibitors are chemorepulsive guidance molecules such as netrins, semaphorins and ephrins. Developmental guidance molecules with chemorepulsive activity are expressed in the adult CNS (Miranda et al., 1999; Zhou, 1998). Growth inhibitory proteins present at the glial scar near an injury site are another important barrier of axon growth. Reactive astrocytes that form the glial scar express many different types of chondroitin sulfate proteoglycans (CSPG). CSPG core proteins that inhibit axon growth include versican (Asher et al., 1995; Bode-Lesniewska et al., 1996), phosphocan (Meyer-Puttlitz et al., 1996; Xiao et al., 1997a), NG2 (Levine, 1998), and neurocan (Meyer-Puttlitz et al., 1996; Rauch et al., 1991). The full complement of proteoglycans expressed at the glial scar, and their time course of expression, has not been thoroughly explored. However, in tissue culture, reactive astrocytes block axon growth and express inhibitory proteoglycans (Fawcett, 1999; McKeon & Silver, 1995). Other cell types also contribute to scar formation. For example, meningeal cells express growth inhibitory proteins on their cell surfaces and invade the scar region to line the lesion cavity (Fawcett, 1999). Oligodendrocyte precursor cells proliferate after CNS demyelinating injury and express the NG2 proteoglycan that is known to inhibit neurite growth (Dou & Levine, 1998; Fawcett, 1999; Keirstead, 1998). Invasion of blood vessels occurs after injury of the spinal cord (Imperato-Kalmar, 1997; Selle´s-Navarro, 2000) and recent experiments
Spinal Cord Axon Regeneration
suggest that anti-angiogenic agents given after spinal cord injury may reduce the area of damage (Wamil, 1998). Therefore, many different cell types contribute to the formation of a scar that blocks axon regeneration.
LONG DISTANCE AXON REGENERATION Success in achieving long distance regeneration in the CNS of adult animals was demonstrated with peripheral nerve grafts used to replace the inhibitory CNS environment (Aguayo et al., 1991; Bray et al., 1987b; Cheng et al., 1996). Peripheral nerve grafts are living nerve grafts that contain Schwann cells which provide a favorable substrate for growth as well as trophic support. In experiments where peripheral nerve grafts are used to promote regeneration, axons grow the full length of the graft. When retinal ganglion cells regenerate in grafts, axonal transport rates increase (McKerracher et al., 1990), and the regenerating neurons reexpress early developmental forms of tubulin (Fournier & McKerracher, 1997; McKerracher et al., 1993), indicating that the cell bodies revert to a growth mode similar to that observed in development. When peripheral nerve grafts used to replace the cut optic nerve were inserted into target regions, the formation of functional synapses was clearly demonstrated by both electron microscopy and recording of post-synaptic responses following stimulation by light (Keirstead et al., 1989; Vidal-Sanz et al., 1991; VidalSanz et al., 1987). These studies demonstrate the potential for long distance regeneration and reconnection in the CNS. While peripheral nerve grafts are an excellent experimental model to study regeneration, they are not likely to be useful to rewire the complex circuitry of the spinal cord after injury. The ideal strategy to stimulate regeneration will allow axons to regrow directly on their native terrain of the CNS, and we discuss below several different ways to achieve this.
BLOCKING MYELIN-DERIVED GROWTH INHIBITION WITH ANTIBODIES Long distance regeneration in the CNS by blocking growth inhibitory molecules with antibodies was first achieved in juvenile rats by neutralization of inhibitory protein activity with the IN-1 antibody in spinal cord (Schnell & Schwab, 1990) and optic nerve (Weibel
13 et al., 1994). The IN-1 antibody was raised to a high molecular weight growth inhibitory protein, and has been shown to block the inhibitory property of myelin (Caroni & Schwab, 1988a). To test the ability of IN-1 to promote regeneration in spinal cord, hybridoma cells secreting IN-1 antibody were implanted in the brain to give a continuous supply of antibody for the postlesion period. By this approach long-distance growth of a few corticospinal axons was observed, although the number of axons that regenerate long distance is quite small (Schnell & Schwab, 1990). Nonetheless, anatomical axon regeneration in the spinal cord following IN-1 treatment has been correlated with improvements in functional recovery (Bregman et al., 1995). These experiments highlight the remarkable potential for functional recovery when even small numbers of axons regenerate. As with other experimental strategies that stimulate recovery of function after spinal cord lesion (see below), it is not clear to what extent the functional recovery after IN-1 treatment results only from regrowth and reconnection of projection axons or is also stimulated by rearrangement of the local circuitry of the spinal cord. The application of IN-1 antibody can induce sprouting of uninjured axons (Buffo, 2000) suggesting it may affect local rearrangements in the spinal cord as well. The IN-1 antibody has been applied to a wide variety of injured CNS neurons with good success, further demonstrating the widespread potential for CNS axon regeneration. More recently, we have tested a novel variation of this antibody method to block growth inhibitory molecules by a therapeutic vaccine approach (Huang, 1999). The idea of the therapeutic vaccine is simple: use the animal’s own immune system to produce antibodies against all of the spinal cord-derived growth inhibitory proteins. We used an unconventional immunization approach to avoid the type of autoimmune reactions that led to demyelination, such as with experimental autoimmune encephalitis. This approach was based on immunization with spinal cord homogenate to generate polyreactive antibodies (IgM class) that stimulated remyelination in models of demyelinating disease (Rodriguez, 1990). The IN-1 antibody used by the Schwab group recognizes multiple proteins on Western blots and is also an IgM (Caroni & Schwab, 1988a), suggesting that it may be polyreactive rather than specific for a single inhibitor. For the therapeutic vaccine experiments, animals were immunized before the injury to ensure a high antibody titre at the time of spinal cord lesion. Tested in adult mice, the therapeutic vaccine led to a remarkable long distance regeneration of corticospinal tract fibres (Huang, Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
14 1999). We also have preliminary evidence that the therapeutic vaccine promotes the regeneration of adult rat retinal ganglion cells after optic nerve injury (Ellezam and McKerracher, unpublished). A future direction for this research will be to give antibodies against CNS inhibitors by passive immunization at the time of lesion, as has been successfully done for treatment of demyelinating lesion in the CNS (Pavelko, 1998; Warrington et al., 2000). This would allow antibodies to enter the CNS at the lesion site immediately after injury, so that axons could grow before the glial scar is well-formed.
STIMULATION OF AXON REGENERATION BY MODULATING THE NEURONAL SIGNALING RESPONSES There have been many investigations on the use of growth factors to promote regeneration in the CNS, some with notable success (Blesch et al., 1999; Liu et al., 1999; Ramer et al., 2000). Typically infusion pumps or gene therapy techniques are used to deliver growth factors to injured neurons. In general, trophic factors do not stimulate long distance regeneration, but stimulate more of a local sprouting response (MansourRobaey et al., 1994; Schnell et al., 1994). Recently, the creation of mice that are double null for BAX and nerve growth factor (NGF) allowed NGF-dependent dorsal root ganglion neurons to survive and grow axons. These studies showed that axons grew normally in the absence of NGF, but that target field innervation and neuropeptide expression were deficient (Patel et al., 2000). Similarly, regeneration of sensory nerves is successful when NGF is blocked by anti-NGF antibodies, but collateral sprouting was prevented (Diamond, 1992). Therefore, the potential for treatment with neurotrophic factors may reside in their ability to promote sprouting, reinnervation, and remodeling, rather than long-distance growth. We have used a totally different approach in developing a new strategy to stimulate regeneration after injury in the CNS. In recent experiments we have manipulated the intracellular neuronal signaling mechanisms to allow growth cones to ignore growth inhibitory proteins and regrow directly on inhibitory substrates. Growth inhibitory proteins cause growth cone collapse (Fan et al., 1993; Li et al., 1996) and it has become clear that GTPases of the Rho family (Rho, Rac, and Cdc42) are intracellular regulators of growth cone collapse (Jin & Strittmatter, 1997; Kuhn et al., 1999; Lehmann et al., 1999; Tigyi et al., 1996). These small GTPases exist in inactive (GDP) and active Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Lisa McKerracher
(GTP) forms, and the cycling between active GTPbound and inactive GDP-bound states regulates the cytoskeleton and cell motility (Hall, 1998). We have found that when Rho is inactivated by C3 toxin from C. botulinum, PC12 cells and retinal ganglion cells grow directly on myelin substrates (Lehmann et al., 1999). Moreover, PC12 cells transfected with dominant negative Rho were able to grow on inhibitory MAG substrates (Lehmann et al., 1999). Results on the critical role of Rho in allowing growth on inhibitory substrates are in agreement with the recent findings on the role of cAMP in growth inhibitory signaling. Treating neurons with neurotrophins to increase intracellular cAMP before plating on MAG substrates, or increasing intracellular cAMP through the use of cAMP analogues, allows neurons to grow on MAG substrates and ignore repulsive signaling by MAG (Cai et al., 1999; Song et al., 1998). It is likely that cAMP inactivates the Rho pathway through protein kinase A phosphorylation of Rho, as shown for nonneuronal cells (Dong et al., 1998; Lang et al., 1996). Therefore, increases in cAMP or direct inactivation of Rho allows neurons to grow on inhibitory MAG substrates in tissue culture. The ability of compounds that modulate cAMP to promote axon regeneration in vivo needs to be explored. With the in vitro evidence that Rho was a key protein to regulate the response to growth inhibitory molecules, we tested the ability of C3 to stimulate regeneration in vivo. We crushed adult rat optic nerves and applied C3 at the same time, directly at the lesion site (Lehmann et al., 1999). We found that large numbers of axons traversed the lesion to grow in the distal optic nerve. More recently we have tested this technique in injured adult mouse spinal cord, and found that axons could regenerate for long distances after a single treatment with C3 (Dergham and McKerracher, unpublished). The simplicity of application of C3 and the encouraging results in vivo suggest this strategy has great potential for further testing and development. Another method tested to promote regeneration is application of inosine, a purine nucleotide that is a metabolite present in all cells. Benowitz and colleagues showed that adenosine promoted neurite growth in tissue culture, and that the adenosine needed to be metabolized to inosine to be effective (Benowitz et al., 1998). Next they were able to demonstrate that after unilateral lesion of the corticospinal tract of adult rats, inosine stimulated uninjured neurons to sprout collaterals into denervated areas (Benowitz et al., 1999). The mechanism of action is unknown, and one limitation of this approach is the requirement for infusion with transplanted minipumps. Total hemisec-
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Spinal Cord Axon Regeneration
tions and complete spinal cord lesions need to be tested with this method to more directly compare it with other methods that stimulate long distance regeneration.
CELL TRANSPLANTATION AND AXON REGENERATION Cellular grafts transplanted in the injured CNS have enormous potential to serve as bridges across lesion cavities. Many different cell transplants have been extensively studied for their potential to promote regeneration and repair. These include Schwann cells (Guest, 1997; Tuszynski et al., 1998; Xu et al., 1996), fibroblasts modified to express trophic factors (Blesch et al., 1999; Liu et al., 1999; Nakahara et al., 1996; Tuszynski et al., 1994), fetal spinal cord transplants (Bregman, 1993; Diener & Bregman, 1998), macrophages (Lazarov-Spiegler et al., 1996), embryonic stem cells (McDonald et al., 1999), and olfactory ensheathing glia (Li et al., 1997; Ramon-Cueto et al., 2000; Ramon-Cueto et al., 1998). Transplants containing neurons, such as fetal spinal cord transplants or embryonic stem cells may act as relay stations between the host and transplant to help remodel the local circuitry near the injury site. The limitation transplants have for regeneration, however, is that the growth environment within the transplant is so favorable, that most axons do not grow out into the spinal cord toward their natural target regions. The expression of trophic factors by transplanted cells greatly helps this situation, perhaps because of their priming effect (Cai et al., 1999), allowing neurons to ignore growth inhibitory proteins. One case where axons extend long distances out of the initial transplant region is when olfactory ensheathing glial cells (OEGs) are used because these cells are themselves highly migratory (Li et al., 1997; Ramon-Cueto et al., 2000, 1998). The Ramon-Cueto et al. have studied long term effectiveness of OEG transplanted into adult rat spinal cord after a complete spinal cord transection that destroys all supraspinal projections (Ramon-Cueto et al., 2000). They demonstrated that eight months after OEG transplantation in injured spinal cord, long distance regeneration occurred along tracts of OEGs that migrated distally in the spinal cord. They were able to demonstrate regeneration of corticospinal, noradrenergic and serotonergic axons. Moreover, a careful positive correlation was shown between the extent of anatomical regeneration and the amount of functional recovery for each animal. Yet, as they point out, it still
remains to be determined if the regenerated axons are functional. Several studies have demonstrated that embryonic and adult neurons transplanted into the CNS have a robust capacity for growth in white matter (Davies et al., 1994; Davies et al., 1997; Wictorin & Bjorklund, 1992; Wictorin et al., 1992). However, the reason why transplanted neurons but not neurons injured in situ exhibit this response is not known. One clue to these differences may reside with the observation that a conditioning lesion can improve the success of regeneration. A conditioning lesion occurs when a nerve is injured twice; if an axon is cut a second time closer to the cell body about one week after the first lesion, the response to the second lesion is much more vigorous. Conditioning lesions increase the synthesis of cytoskeletal proteins and the rate of slow axonal transport, resulting in a faster rate of regeneration (Jacob, 1998; McQuarrie, 1984). Recently, a remarkably robust, long distance regeneration in the spinal cord of ascending dorsal column fibres was induced when a conditioning lesion was made prior to dorsal column injury (Neumann, 1999). Not only did the lesioned fibers regenerate long distances, but a substantial number of fibers grew past the lesion in the spinal cord. This important result demonstrates that the intrinsic growth state of an injured neuron is a critical determinant for axon regeneration in a typically inhibitory environment. Possibly, the explanation for vigorous growth of transplanted neurons in the CNS is that something about transplantation primes them for growth, as if they had received a conditioning lesion. The challenge in the future will be to discover the signals responsible for converting a neuron into a highly responsive growth state. Such discoveries may provide a clue to learn how to make old neurons regrow long after the injury occurred.
CONCLUSIONS Experiments in animal models of spinal cord injury now suggest the potential for three conceptually different approaches to stimulate long-distance axon regeneration and recovery of motor function. (1) Blocking myelin-derived inhibitors with antibodies can foster axon growth over long distances in white matter. The successful methods include treatment with IN-1 antibody, and stimulating an immune response to inhibitory proteins with a therapeutic vaccine. (2) Axon sprouting and regeneration can follow the modulation neuronal intracellular signaling. Methods that have been tested include the application of neurotrophins, Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
16 Rho antagonists, and inosine. The most successful of these to stimulate long-distance regeneration is the use of C3 as a Rho antagonist. (3) The transplantation of cells into the lesion site can stimulate axon growth into the transplant and behavioral recovery. When olfactory ensheathing glia are used as transplants, longdistance regeneration occurs because the transplanted cells migrate into the distal spinal cord. In the future, we can look forward to ongoing progress in our understanding of axon regeneration in injured spinal cord. It will be especially interesting when combination therapies begin to be explored—we might expect synergistic outcomes when some of the conceptually different approaches are combined. The current research trends demonstrate that axon regeneration and functional improvement is possible after injury in the CNS. The next challenge will be to bring some of these new techniques towards clinical trials.
ACKNOWLEDGMENTS The work reported in this review was supported by funds from MRC (Canada). I thank Drs. Adriana DiPolo, Guy Doucet, and Benjamin Ellezam for their comments on the manuscript.
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Lisa McKerracher Song, H.-J., Ming, G.-I., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne, M., & Poo, M.-M. (1998) Conversion of growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518. Tessier-Lavigne, M., & Goodman, C. S. (2000) Regeneration in the nogo zone. Science 287, 813– 814. Tigyi, G., Fischer, D. J., Sebok, A., Yang, C., Dyer, D. L., & Miledi, R. (1996) Lysophosphatidic acid-induced neurite retraction in PC12 cells: Control by phosphoinositide—Ca ⫹2 signaling and Rho. J. Neurochem. 66, 537–548. Tuszynski, M. H., Peterson, D. A., Ray, J., Baird, A., Nakahara, Y., & Gage, F. H. (1994) Fibroblasts genetically modified to produce nerve growth factor induce robust neuritic ingrowth after grafting to the spinal cord. Exp. Neurol. 126, 1–14. Tuszynski, M. H., Weidner, N., McCormack, M., Miller, I., Powell, H., & Conner, J. (1998) Grafts of genetically modified Schwann cells to the spinal cord: Survival, axon growth, and myelination. Cell Transplant 7, 187–196. Vidal-Sanz, M., Bray, G. M., & Aguayo, A. J. (1991) Regenerated synapses persist in the superior colliculus after the regrowth of retinal ganglion cell axons [published erratum appears in J. Neurocytol. (1992) 21(3):234]. J. Neurocytol. 20, 940 –952. Vidal-Sanz, M., Bray, G. M., Villegas-Perez, M. P., Thanos, S., & Aguayo, A. J. (1987) Axonal regeneration and synapse formation in the superior colliculus by retinal ganglion cells in the adult retina. J. Neurosci. 7, 2894 –2909. Wamil, A. W., Wamil, B. D., & Hellerqvist, C. G. (1998) CM101mediated recovery of walking abiity in adult mice paralyzed by spinal cord injury. Proc. Natl. Acad. Sci. USA 95, 13188 –13193. Warrington, A. E., Asakura, K., Bieber, A. J., Ciric, B., Van Keulen, V., Kaveri, S. V., Kyle, R. A., Pease, L. R., & Rodriguez, M. (2000) Human monoclonal antibodies reactive to oligodendrocytes promote remyelination in a model of multiple sclerosis. Proc. Natl. Acad. Sci. USA 97, 6820 – 6825. Weibel, D., Cadelli, D., & Schwab, M. E. (1994) Regeneration of lesioned rat optic nerve fibers is improved after neurtralization of myelin-associated neurite growth inhibitors. Brain Res. 642, 259 – 266. Wictorin, K., & Bjorklund, A. (1992) Axon outgrowth from grafts of human embryonic spinal cord in the lesioned adult rat spinal cord. Neuroreport 3, 1045–1048. Wictorin, K., Brundin, P., Sauer, H., Lindvall, O., & Bjorklund, A. (1992) Long distance directed axonal growth from human dopaminergic mesencephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats. J. Comp. Neurol. 323, 475– 494. Xiao, Z.-C., Bartsch, U., Margolis, R. K., Rougon, G., Montag, D., & Schachner, M. (1997a) Isolation of a tenascin-R binding protein from mouse brain membranes. J. Biol. Chem. 272, 32092– 32101. Xiao, Z.-C., David, S., Braun, P. E., & McKerracher, L. (1997b) Characterization of a new myelin-derived grrowth inhibitory activity. Soc. Neurosci. Absts. 23, 1994. Xu, X. M., Guenard, V., Kleitman, N., Aebischer, P., & Bunge, M. B. (1996) A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord. Exp. Neurol. 134, 261–272. Zhou, R. (1998) The Eph family receptors and ligands. Pharmacol. Ther. 77, 151–181.
REVIEW Spinal Cord Repair: Strategies to Promote Axon Regeneration Lisa McKerracher De´partement de Pathologie et biologie cellulaire, Universite´ de Montre´al, C.P. 6128, succursale Centre-ville, Montre´al, Que´bec H3C 3J7, Canada Received June 26, 2000; revised September 20, 2000; accepted for publication October 5, 2000; published online December 20, 2000
Neurons in the central nervous system have a remarkable capacity to regenerate their transected axons when provided with an appropriate growth environment. Advances in our understanding of axon regeneration have allowed the development of different experimental strategies to stimulate axon regeneration in animal models of spinal cord injury. Growth inhibitory proteins block axon regeneration in the CNS, and many of these proteins have been identified. Various methods that are now used to stimulate regeneration in the injured spinal cord are directed at overcoming the growth inhibitory environment of the CNS. Three general approaches tested in vivo stimulate regeneration in the spinal cord. First, antibodies that bind inhibitory proteins in myelin allow axon regeneration in the CNS. Second, methods that modulate neuronal intracellular signaling allow axons to grow directly on the inhibitory substrate of the CNS. Third, transplantation of cells to the lesioned spinal cord promotes repair. In this paper we review current advances in each of these research domains. © 2001 Academic Press
INTRODUCTION
tory reaction. This drug, however, does not restore functions that are lost when axons are cut. Several major advances in our understanding of axon regeneration have led to the ability to stimulate some axon regeneration and functional repair in animal models of spinal cord injury. In the 1980s experiments by Aguayo and colleagues to use peripheral nerve grafts that were inserted into the brain or spinal cord showed that CNS neurons have the capacity to regrow, and these studies highlighted that diverse classes of CNS neurons have the potential to regenerate when given a permissive growth environment (Aguayo et al., 1981; Bray et al., 1987a). Another major advance in our understanding of axon regeneration in the central nervous system was the discovery by Schwab and colleagues that the CNS environment did not simply lack growth promoting molecules, but that growth inhibitory molecules existed to block axon growth (Caroni & Schwab, 1988b; Schwab et al., 1993; Schwab & Thoenen, 1985). Experiments aimed at removing myelin in the injured region to promote re-
Traumatic injury of the spinal cord that transects axon processes results in permanent functional impairment, even when the neuronal cell bodies that are located away from the injury site remain alive. At present there are no clinical treatments available to stimulate regeneration of cut axons, but with the pace of research in the field of central nervous system (CNS) axon regeneration now increasing, the application of new discoveries may not be far away. Numerous studies now show that anatomical regeneration and functional recovery are possible in rodent models of spinal cord injury. Clinical treatments that foster regeneration would be a significant improvement over present treatments that serve only to limit the extent of secondary damage caused by non-neuronal cells that invade the injury site. Spinal cord injured patients receive high doses of the steroid methylprednisolone immediately following injury to suppress an unfavorable inflamma0969-9961/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
11
12 generation have conclusively demonstrated that inhibitory proteins in myelin are an important barrier to axon growth (Dyer et al., 1998; Keirstead et al., 1992; Savio & Schwab, 1997). Related to these discoveries is the important finding that the glial scar that forms after injury is a formidable barrier to axon regrowth (McKeon et al., 1991; Reier, 1989). A more recent advance is the demonstration that increasing the intrinsic growth capacity of neurons is sufficient to allow axon regeneration in the CNS and that neurons primed for regeneration with neurotrophins or a conditioning lesion have a better chance to grow on inhibitory substrates (Cai et al., 1999; Neumann, 1999). The inhibitory barriers present in the CNS are not entirely understood, and it is likely that not all CNS growth inhibitory molecules have been identified. To better overcome the growth inhibitory environment of the CNS, there has been a focused effort to identify the proteins that have growth inhibitory activity.
THE GROWTH INHIBITORY ENVIRONMENT OF THE CNS Growth inhibitory activity has been demonstrated to be associated with myelin (Caroni & Schwab, 1988a, 1988b). The first myelin-derived growth inhibitory protein to be identified was myelin-associated glycoprotein (MAG), a known myelin protein. The inhibitory activity of MAG was discovered independently by us (McKerracher et al., 1994) and by Filbin and collaborators (Mukhopadhyay et al., 1994). Next, several inhibitory proteoglycans that associate with myelin were identified (Niederost, 1999; Xiao et al., 1997b), although their role in myelin biology is less understood. Another growth inhibitory protein, called nogo, whose activity was first detected as a potent high molecular weight inhibitor in myelin (Caroni & Schwab, 1988a,b) was identified this year (Chen, 2000; GrandPre´, 2000; Prinjha, 2000). Different splice varients of nogo exist and a controversy concerning the inhibitory domains of nogo is relevant to whether the inhibitory forms of nogo exhibit oligodendrocyte-specific expression (Tessier-Lavigne, 2000). Function blocking monoclonal antibodies generated against nogo by the Schwab group bind to a unique portion of nogo expressed by oligodendrocytes (Chen, 2000), yet the unique domain is predicted to be intracellular by the Strittmatter group (GrandPre´, 2000). However, now that the sequence of nogo is known, we can expect that the relationship between structure, function, and inhibitory activity will be resolved in the near future. Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Lisa McKerracher
The inhibitory activity of MAG has been characterized both in vitro and in vivo (Li et al., 1996; McKerracher et al., 1994; Mukhopadhyay et al., 1994; Schafer et al., 1996). The extent to which this inhibitor limits outgrowth in vivo has been controversial because of studies performed with MAG null mutant mice: two groups were able to show a small improvement in regeneration on MAG ⫺/⫺ myelin (Li et al., 1996; Shen et al., 1998), whereas no change was observed in a third study (Bartsch et al., 1995). Regardless of the differences between these studies, regeneration in vivo in MAG⫺/⫺ mice was poor, likely because of the presence of other inhibitors, such as the inhibitory myelin proteoglycans and nogo. The inhibitory activity of nogo and MAG have been compared, and when tested in vitro these two proteins have equivalent growth inhibitory activity (Prinjha, 2000). If nogo knock out mice are generated and are viable, the MAG⫺/⫺ and nogo ⫺/⫺ could be compared with the same techniques. Perhaps the most interesting experiment will be when double mutant MAG⫺/⫺ and nogo⫺/⫺ mice will be generated to test the contribution of other inhibitors to block regeneration in the CNS. Other potential inhibitors are chemorepulsive guidance molecules such as netrins, semaphorins and ephrins. Developmental guidance molecules with chemorepulsive activity are expressed in the adult CNS (Miranda et al., 1999; Zhou, 1998). Growth inhibitory proteins present at the glial scar near an injury site are another important barrier of axon growth. Reactive astrocytes that form the glial scar express many different types of chondroitin sulfate proteoglycans (CSPG). CSPG core proteins that inhibit axon growth include versican (Asher et al., 1995; Bode-Lesniewska et al., 1996), phosphocan (Meyer-Puttlitz et al., 1996; Xiao et al., 1997a), NG2 (Levine, 1998), and neurocan (Meyer-Puttlitz et al., 1996; Rauch et al., 1991). The full complement of proteoglycans expressed at the glial scar, and their time course of expression, has not been thoroughly explored. However, in tissue culture, reactive astrocytes block axon growth and express inhibitory proteoglycans (Fawcett, 1999; McKeon & Silver, 1995). Other cell types also contribute to scar formation. For example, meningeal cells express growth inhibitory proteins on their cell surfaces and invade the scar region to line the lesion cavity (Fawcett, 1999). Oligodendrocyte precursor cells proliferate after CNS demyelinating injury and express the NG2 proteoglycan that is known to inhibit neurite growth (Dou & Levine, 1998; Fawcett, 1999; Keirstead, 1998). Invasion of blood vessels occurs after injury of the spinal cord (Imperato-Kalmar, 1997; Selle´s-Navarro, 2000) and recent experiments
Spinal Cord Axon Regeneration
suggest that anti-angiogenic agents given after spinal cord injury may reduce the area of damage (Wamil, 1998). Therefore, many different cell types contribute to the formation of a scar that blocks axon regeneration.
LONG DISTANCE AXON REGENERATION Success in achieving long distance regeneration in the CNS of adult animals was demonstrated with peripheral nerve grafts used to replace the inhibitory CNS environment (Aguayo et al., 1991; Bray et al., 1987b; Cheng et al., 1996). Peripheral nerve grafts are living nerve grafts that contain Schwann cells which provide a favorable substrate for growth as well as trophic support. In experiments where peripheral nerve grafts are used to promote regeneration, axons grow the full length of the graft. When retinal ganglion cells regenerate in grafts, axonal transport rates increase (McKerracher et al., 1990), and the regenerating neurons reexpress early developmental forms of tubulin (Fournier & McKerracher, 1997; McKerracher et al., 1993), indicating that the cell bodies revert to a growth mode similar to that observed in development. When peripheral nerve grafts used to replace the cut optic nerve were inserted into target regions, the formation of functional synapses was clearly demonstrated by both electron microscopy and recording of post-synaptic responses following stimulation by light (Keirstead et al., 1989; Vidal-Sanz et al., 1991; VidalSanz et al., 1987). These studies demonstrate the potential for long distance regeneration and reconnection in the CNS. While peripheral nerve grafts are an excellent experimental model to study regeneration, they are not likely to be useful to rewire the complex circuitry of the spinal cord after injury. The ideal strategy to stimulate regeneration will allow axons to regrow directly on their native terrain of the CNS, and we discuss below several different ways to achieve this.
BLOCKING MYELIN-DERIVED GROWTH INHIBITION WITH ANTIBODIES Long distance regeneration in the CNS by blocking growth inhibitory molecules with antibodies was first achieved in juvenile rats by neutralization of inhibitory protein activity with the IN-1 antibody in spinal cord (Schnell & Schwab, 1990) and optic nerve (Weibel
13 et al., 1994). The IN-1 antibody was raised to a high molecular weight growth inhibitory protein, and has been shown to block the inhibitory property of myelin (Caroni & Schwab, 1988a). To test the ability of IN-1 to promote regeneration in spinal cord, hybridoma cells secreting IN-1 antibody were implanted in the brain to give a continuous supply of antibody for the postlesion period. By this approach long-distance growth of a few corticospinal axons was observed, although the number of axons that regenerate long distance is quite small (Schnell & Schwab, 1990). Nonetheless, anatomical axon regeneration in the spinal cord following IN-1 treatment has been correlated with improvements in functional recovery (Bregman et al., 1995). These experiments highlight the remarkable potential for functional recovery when even small numbers of axons regenerate. As with other experimental strategies that stimulate recovery of function after spinal cord lesion (see below), it is not clear to what extent the functional recovery after IN-1 treatment results only from regrowth and reconnection of projection axons or is also stimulated by rearrangement of the local circuitry of the spinal cord. The application of IN-1 antibody can induce sprouting of uninjured axons (Buffo, 2000) suggesting it may affect local rearrangements in the spinal cord as well. The IN-1 antibody has been applied to a wide variety of injured CNS neurons with good success, further demonstrating the widespread potential for CNS axon regeneration. More recently, we have tested a novel variation of this antibody method to block growth inhibitory molecules by a therapeutic vaccine approach (Huang, 1999). The idea of the therapeutic vaccine is simple: use the animal’s own immune system to produce antibodies against all of the spinal cord-derived growth inhibitory proteins. We used an unconventional immunization approach to avoid the type of autoimmune reactions that led to demyelination, such as with experimental autoimmune encephalitis. This approach was based on immunization with spinal cord homogenate to generate polyreactive antibodies (IgM class) that stimulated remyelination in models of demyelinating disease (Rodriguez, 1990). The IN-1 antibody used by the Schwab group recognizes multiple proteins on Western blots and is also an IgM (Caroni & Schwab, 1988a), suggesting that it may be polyreactive rather than specific for a single inhibitor. For the therapeutic vaccine experiments, animals were immunized before the injury to ensure a high antibody titre at the time of spinal cord lesion. Tested in adult mice, the therapeutic vaccine led to a remarkable long distance regeneration of corticospinal tract fibres (Huang, Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
14 1999). We also have preliminary evidence that the therapeutic vaccine promotes the regeneration of adult rat retinal ganglion cells after optic nerve injury (Ellezam and McKerracher, unpublished). A future direction for this research will be to give antibodies against CNS inhibitors by passive immunization at the time of lesion, as has been successfully done for treatment of demyelinating lesion in the CNS (Pavelko, 1998; Warrington et al., 2000). This would allow antibodies to enter the CNS at the lesion site immediately after injury, so that axons could grow before the glial scar is well-formed.
STIMULATION OF AXON REGENERATION BY MODULATING THE NEURONAL SIGNALING RESPONSES There have been many investigations on the use of growth factors to promote regeneration in the CNS, some with notable success (Blesch et al., 1999; Liu et al., 1999; Ramer et al., 2000). Typically infusion pumps or gene therapy techniques are used to deliver growth factors to injured neurons. In general, trophic factors do not stimulate long distance regeneration, but stimulate more of a local sprouting response (MansourRobaey et al., 1994; Schnell et al., 1994). Recently, the creation of mice that are double null for BAX and nerve growth factor (NGF) allowed NGF-dependent dorsal root ganglion neurons to survive and grow axons. These studies showed that axons grew normally in the absence of NGF, but that target field innervation and neuropeptide expression were deficient (Patel et al., 2000). Similarly, regeneration of sensory nerves is successful when NGF is blocked by anti-NGF antibodies, but collateral sprouting was prevented (Diamond, 1992). Therefore, the potential for treatment with neurotrophic factors may reside in their ability to promote sprouting, reinnervation, and remodeling, rather than long-distance growth. We have used a totally different approach in developing a new strategy to stimulate regeneration after injury in the CNS. In recent experiments we have manipulated the intracellular neuronal signaling mechanisms to allow growth cones to ignore growth inhibitory proteins and regrow directly on inhibitory substrates. Growth inhibitory proteins cause growth cone collapse (Fan et al., 1993; Li et al., 1996) and it has become clear that GTPases of the Rho family (Rho, Rac, and Cdc42) are intracellular regulators of growth cone collapse (Jin & Strittmatter, 1997; Kuhn et al., 1999; Lehmann et al., 1999; Tigyi et al., 1996). These small GTPases exist in inactive (GDP) and active Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Lisa McKerracher
(GTP) forms, and the cycling between active GTPbound and inactive GDP-bound states regulates the cytoskeleton and cell motility (Hall, 1998). We have found that when Rho is inactivated by C3 toxin from C. botulinum, PC12 cells and retinal ganglion cells grow directly on myelin substrates (Lehmann et al., 1999). Moreover, PC12 cells transfected with dominant negative Rho were able to grow on inhibitory MAG substrates (Lehmann et al., 1999). Results on the critical role of Rho in allowing growth on inhibitory substrates are in agreement with the recent findings on the role of cAMP in growth inhibitory signaling. Treating neurons with neurotrophins to increase intracellular cAMP before plating on MAG substrates, or increasing intracellular cAMP through the use of cAMP analogues, allows neurons to grow on MAG substrates and ignore repulsive signaling by MAG (Cai et al., 1999; Song et al., 1998). It is likely that cAMP inactivates the Rho pathway through protein kinase A phosphorylation of Rho, as shown for nonneuronal cells (Dong et al., 1998; Lang et al., 1996). Therefore, increases in cAMP or direct inactivation of Rho allows neurons to grow on inhibitory MAG substrates in tissue culture. The ability of compounds that modulate cAMP to promote axon regeneration in vivo needs to be explored. With the in vitro evidence that Rho was a key protein to regulate the response to growth inhibitory molecules, we tested the ability of C3 to stimulate regeneration in vivo. We crushed adult rat optic nerves and applied C3 at the same time, directly at the lesion site (Lehmann et al., 1999). We found that large numbers of axons traversed the lesion to grow in the distal optic nerve. More recently we have tested this technique in injured adult mouse spinal cord, and found that axons could regenerate for long distances after a single treatment with C3 (Dergham and McKerracher, unpublished). The simplicity of application of C3 and the encouraging results in vivo suggest this strategy has great potential for further testing and development. Another method tested to promote regeneration is application of inosine, a purine nucleotide that is a metabolite present in all cells. Benowitz and colleagues showed that adenosine promoted neurite growth in tissue culture, and that the adenosine needed to be metabolized to inosine to be effective (Benowitz et al., 1998). Next they were able to demonstrate that after unilateral lesion of the corticospinal tract of adult rats, inosine stimulated uninjured neurons to sprout collaterals into denervated areas (Benowitz et al., 1999). The mechanism of action is unknown, and one limitation of this approach is the requirement for infusion with transplanted minipumps. Total hemisec-
15
Spinal Cord Axon Regeneration
tions and complete spinal cord lesions need to be tested with this method to more directly compare it with other methods that stimulate long distance regeneration.
CELL TRANSPLANTATION AND AXON REGENERATION Cellular grafts transplanted in the injured CNS have enormous potential to serve as bridges across lesion cavities. Many different cell transplants have been extensively studied for their potential to promote regeneration and repair. These include Schwann cells (Guest, 1997; Tuszynski et al., 1998; Xu et al., 1996), fibroblasts modified to express trophic factors (Blesch et al., 1999; Liu et al., 1999; Nakahara et al., 1996; Tuszynski et al., 1994), fetal spinal cord transplants (Bregman, 1993; Diener & Bregman, 1998), macrophages (Lazarov-Spiegler et al., 1996), embryonic stem cells (McDonald et al., 1999), and olfactory ensheathing glia (Li et al., 1997; Ramon-Cueto et al., 2000; Ramon-Cueto et al., 1998). Transplants containing neurons, such as fetal spinal cord transplants or embryonic stem cells may act as relay stations between the host and transplant to help remodel the local circuitry near the injury site. The limitation transplants have for regeneration, however, is that the growth environment within the transplant is so favorable, that most axons do not grow out into the spinal cord toward their natural target regions. The expression of trophic factors by transplanted cells greatly helps this situation, perhaps because of their priming effect (Cai et al., 1999), allowing neurons to ignore growth inhibitory proteins. One case where axons extend long distances out of the initial transplant region is when olfactory ensheathing glial cells (OEGs) are used because these cells are themselves highly migratory (Li et al., 1997; Ramon-Cueto et al., 2000, 1998). The Ramon-Cueto et al. have studied long term effectiveness of OEG transplanted into adult rat spinal cord after a complete spinal cord transection that destroys all supraspinal projections (Ramon-Cueto et al., 2000). They demonstrated that eight months after OEG transplantation in injured spinal cord, long distance regeneration occurred along tracts of OEGs that migrated distally in the spinal cord. They were able to demonstrate regeneration of corticospinal, noradrenergic and serotonergic axons. Moreover, a careful positive correlation was shown between the extent of anatomical regeneration and the amount of functional recovery for each animal. Yet, as they point out, it still
remains to be determined if the regenerated axons are functional. Several studies have demonstrated that embryonic and adult neurons transplanted into the CNS have a robust capacity for growth in white matter (Davies et al., 1994; Davies et al., 1997; Wictorin & Bjorklund, 1992; Wictorin et al., 1992). However, the reason why transplanted neurons but not neurons injured in situ exhibit this response is not known. One clue to these differences may reside with the observation that a conditioning lesion can improve the success of regeneration. A conditioning lesion occurs when a nerve is injured twice; if an axon is cut a second time closer to the cell body about one week after the first lesion, the response to the second lesion is much more vigorous. Conditioning lesions increase the synthesis of cytoskeletal proteins and the rate of slow axonal transport, resulting in a faster rate of regeneration (Jacob, 1998; McQuarrie, 1984). Recently, a remarkably robust, long distance regeneration in the spinal cord of ascending dorsal column fibres was induced when a conditioning lesion was made prior to dorsal column injury (Neumann, 1999). Not only did the lesioned fibers regenerate long distances, but a substantial number of fibers grew past the lesion in the spinal cord. This important result demonstrates that the intrinsic growth state of an injured neuron is a critical determinant for axon regeneration in a typically inhibitory environment. Possibly, the explanation for vigorous growth of transplanted neurons in the CNS is that something about transplantation primes them for growth, as if they had received a conditioning lesion. The challenge in the future will be to discover the signals responsible for converting a neuron into a highly responsive growth state. Such discoveries may provide a clue to learn how to make old neurons regrow long after the injury occurred.
CONCLUSIONS Experiments in animal models of spinal cord injury now suggest the potential for three conceptually different approaches to stimulate long-distance axon regeneration and recovery of motor function. (1) Blocking myelin-derived inhibitors with antibodies can foster axon growth over long distances in white matter. The successful methods include treatment with IN-1 antibody, and stimulating an immune response to inhibitory proteins with a therapeutic vaccine. (2) Axon sprouting and regeneration can follow the modulation neuronal intracellular signaling. Methods that have been tested include the application of neurotrophins, Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
16 Rho antagonists, and inosine. The most successful of these to stimulate long-distance regeneration is the use of C3 as a Rho antagonist. (3) The transplantation of cells into the lesion site can stimulate axon growth into the transplant and behavioral recovery. When olfactory ensheathing glia are used as transplants, longdistance regeneration occurs because the transplanted cells migrate into the distal spinal cord. In the future, we can look forward to ongoing progress in our understanding of axon regeneration in injured spinal cord. It will be especially interesting when combination therapies begin to be explored—we might expect synergistic outcomes when some of the conceptually different approaches are combined. The current research trends demonstrate that axon regeneration and functional improvement is possible after injury in the CNS. The next challenge will be to bring some of these new techniques towards clinical trials.
ACKNOWLEDGMENTS The work reported in this review was supported by funds from MRC (Canada). I thank Drs. Adriana DiPolo, Guy Doucet, and Benjamin Ellezam for their comments on the manuscript.
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