* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Mammalian Models of CNS Regeneration - Wiley-VCH
Single-unit recording wikipedia , lookup
Multielectrode array wikipedia , lookup
Neurotransmitter wikipedia , lookup
Premovement neuronal activity wikipedia , lookup
Synaptic gating wikipedia , lookup
Endocannabinoid system wikipedia , lookup
Nervous system network models wikipedia , lookup
Central pattern generator wikipedia , lookup
Molecular neuroscience wikipedia , lookup
Signal transduction wikipedia , lookup
Feature detection (nervous system) wikipedia , lookup
Stimulus (physiology) wikipedia , lookup
Neural engineering wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Optogenetics wikipedia , lookup
Node of Ranvier wikipedia , lookup
Development of the nervous system wikipedia , lookup
Channelrhodopsin wikipedia , lookup
Synaptogenesis wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Neuroanatomy wikipedia , lookup
Spinal cord wikipedia , lookup
Part I Mammalian Models of CNS Regeneration Model Organisms in Spinal Cord Regeneration Edited by Catherina G. Becker and Thomas Becker Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31504-8 3 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord Patrick N. Anderson, Jez Fabes, and David Hunt 1.1 Introduction In adult mammals axonal regeneration is vigorous following peripheral nerve injury, but meager after injury to the central nervous system (CNS). Several theories seek to explain this situation. First, the cell body response to axotomy may be inadequate in intrinsic CNS neurons. Second, there may be inadequate levels of support in terms of neurotrophic factors and cell adhesion molecules in the CNS. Third, the regeneration of axons in the CNS may be prevented by molecules which inhibit neurite outgrowth in vitro. In addition, the absence of a normal woundhealing response in mammalian CNS tissue may limit regeneration; whereas a lesion site in a peripheral nerve is rapidly repopulated by Schwann cells migrating from the two stumps, lesion sites in the CNS expand by secondary degeneration during the first week after injury. None of these hypotheses explains all of the data, but the idea that inhibitory molecules play a major role in preventing axonal regeneration in the CNS has dominated thought in this area for almost two decades. However, there remains much contradictory evidence concerning the roles inhibitory molecules and conflicting views as to their importance in limiting axonal regeneration in vivo (e.g., Raisman, 2004; Schwab, 2004). 1.1.1 CNS Neurons Have Widely Differing Phenotypes The heterogeneity of CNS neurons and their responses to injury greatly complicates the evaluation of hypotheses on CNS regeneration. This is best illustrated by the results of grafting peripheral nerves into the CNS. Richardson et al. (1980) showed that many adult mammalian CNS neurons could regenerate axons through a suitable environment in the form of a peripheral nerve graft. However, subsequent studies showed that many, perhaps most, neurons in the brain are very poor at regenerating axons, even into nerve grafts (Anderson et al., 1998; Anderson and Lieberman, 1999). This may be because CNS neurons differ dramatically in Model Organisms in Spinal Cord Regeneration Edited by Catherina G. Becker and Thomas Becker Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31504-8 4 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord their sensitivity to neurotrophic factors, the strength of their cell body responses to axotomy, and in their expression of receptors for inhibitory molecules (Hunt et al., 2002a; Josephson et al., 2002; Lauren et al., 2003; Pignot et al., 2003). 1.2 Difficulties in Assessing Axonal Regeneration in the Mammalian Spinal Cord A characteristic of scientific progress is that novel techniques that initially appear difficult become commonplace within a few years. This has not been the case with experimental studies of axonal regeneration in the mammalian spinal cord. There have been many claims of treatments resulting in successful axonal regeneration in the mammalian CNS, but there is a paucity of cases where those claims have been replicated in other laboratories, or have even developed into a series of confirmatory observations from the same laboratory. This may be because of the ease with which some axons can be left intact when lesioning tracts in the CNS; spared axons can be misinterpreted as regenerated axons. 1.2.1 Experimental Lesions and Problems of Interpretation Probably the best model for producing reliable complete lesions of a CNS tract is provided by the optic nerve, which can be completely sectioned or crushed by an experienced operator, with little chance of axonal sparing. This has allowed major discoveries to be made on the influence of neurotrophic stimuli (Berry et al., 1996; Logan et al., 2006) and inflammation (Leon et al., 2000; Lorber et al., 2005) on the vigor of axonal regeneration within CNS tissue. Yet even in the optic nerve, reports of remarkable axonal regeneration (Eitan et al., 1994) have sometimes gone without apparent replication or further development. The mammalian spinal cord can be transected, contused or compressed to produce a lesion. Transection or partial transection lesions (Fig. 1.1) have the advantage that the site of initial injury can be accurately estimated. The lesion sites are filled with blood and macrophages and then invaded by meningeal cells, endothelial cells and Schwann cells, together with axons, some of which are of peripheral origin (Zhang et al., 1997). Meningeal cells are the source of several molecules that can inhibit or repel regenerating axons (Zhang et al., 1997; Pasterkamp et al., 2001; Niclou et al., 2003). Astrocyte processes extend into the lesion sites, but few astrocyte or oligodendrocyte cell bodies are present. A region of reactive gliosis characterized by hypertrophic astrocytes develops rostral and caudal to the lesion site where CSPGs are up-regulated (Davies et al., 1999; Tang et al., 2003), and there may be cavitation, particularly if the lesion involves the central canal. Complete transection of the mammalian spinal cord should allow axonal regeneration to be studied without the complication of spared fibers. However, the animals require considerable care after surgery, including regular manual emptying of the bladder, and permission to perform such experiments can be difficult to obtain in Europe. 1.2 Difficulties in Assessing Axonal Regeneration in the Mammalian Spinal Cord GFAP immunohistochemistry identifying astrocytes in a horizontal section of the cervical spinal cord of an adult rat one week following dorsal column transection. The wound has enlarged since the initial injury and is characterized by the absence of CNS glia. The GFAP-negative ‘‘space’’ at the center is occupied by macrophages, other invading non-glial cells, some axons, and fluid-filled cysts. Reactive astrocytes are present bordering the lesion. Fig. 1.1. It is worth noting that even with attempts at complete transection, spared fibers can be left at the ventral surface of the cord (You et al., 2003). Contusion and compression lesions are good models of common types of spinal injury in the western world, but the lesion size and position are more difficult to control. In such lesions it is not possible to be precise about the position of the axotomy, and the possibility of spared fibers is more difficult to eliminate than with transection injuries. Contusion/compression lesions develop from the center of the spinal cord where there is extensive cell death and an invasion of hematogeneous cells (Popovich et al., 1997). Subsequently, large injury sites with cavitation around the central canal, spreading several millimeters rostral and caudal to the site of impact, develop in rats (Bresnahan et al., 1991). Fibrotic tissue instead of cavities is found in most strains of mice (Ma et al., 2001; Stokes and Jakeman, 2002). Typically, the dorsal corticospinal tracts are destroyed in all but the mildest contusion injuries with loss of much gray matter and sparing of a variable amount of peripheral white matter. The axons in the lesioned tracts often terminate well short of the region of primary impact. Although contusion and compression injuries are often less than ideal for studying axonal regeneration, they are excellent models in which to study effects of treatments on behavioral recovery. In all but the most severe lesions, functional recovery occurs to some extent. A number of behavioral tests have been developed for such purposes, including the BBB score (Basso et al., 1995) of open field motor function, grid and rope walking (for a re- 5 6 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord cent use of such tests, see Hendriks et al., 2006). Many more treatments including steroid treatment (Young, 1991) and environmental enrichment (Lankhorst et al., 2001) have been found to enhance behavioral recovery than to promote axonal regeneration. Such treatments presumably act through neuroprotection and/or enhancing plasticity in surviving connections between the rostral and caudal parts of the spinal cord. As the histopathological features differ from those of transection injuies, it would be reasonable to suggest that all potential therapies should be tested on contusion lesions prior to clinical use. However, whether the lesions are produced by transection, contusion or compression, and despite the structural differences between these lesions, regeneration of intrinsic CNS axons across the lesion site is always very poor. In summary, complete transection lesions are the best for proving axonal regeneration has taken place, but compression or contusion injuries are excellent for studying functional recovery. 1.2.2 Tracing Regenerating Axons Anterograde tracing of axons provides the ‘‘gold standard’’ for assessing the extent of regeneration following injury because it allows the course of regenerating axons to be followed around or through a lesion site. Retrograde tracing has the disadvantage that cell bodies may become labeled by spread of tracer through the tissues, necessitating careful analysis of the injection site. Anterograde tracing of descending axons is usually performed using biotinylated dextran amine (BDA; Fig. 1.2) (Li et al., 1997) or sometimes cholera toxin subunit B (CTB) (Hagg et al., 2005), injected near the cell bodies of the injured neurons. Enhanced green fluorescent protein (EGFP) delivered by lentiviral vectors is also useful for tracing axons from brainstem nuclei, and labels only those axons that arise from neurons in the region where the vector is applied (Fabes et al., 2006; Fig. 1.1). Ascending dorsal column axons may be labeled with CTB or CTB-HRP (Chong et al., 1996, 1999), injected into peripheral nerves. Regeneration of Corticospinal Axons is Difficult to Assess Corticospinal tract axons are widely distributed through a transverse section of the spinal cord of rodents (Fig. 1.2). Although most corticospinal tract axons passing through a segment of cord are found in the dorsal funiculus, others are present in the lateral and ventral funiculi of the white matter, and these fibers – if spared – will also send branches into the gray matter below a lesion. Hence, it is difficult to eliminate all corticospinal tract projections without a complete lesion, and sprouting of surviving axons caudal to a lesion – an interesting neurobiological phenomenon in its own right – may be confused with axonal regeneration. As corticospinal tract axons are present in much of the gray matter, it is particularly difficult to distinguish any regenerating axons that might grow through the gray matter around a partial lesion, from axons that were undamaged. Rubrospinal tracts (Fig. 1.2) are located entirely within the dorsal part of the lateral funiculus in ro1.2.2.1 1.2 Difficulties in Assessing Axonal Regeneration in the Mammalian Spinal Cord Corticospinal (upper panel) and rubrospinal (lower panel) axons in transverse sections of the cervical spinal cord of adult rats. The corticospinal axons were labeled by BDA injection into the motor cortex and detected with a streptavidin-conjugated fluorophore. The rubrospinal axons were labeled by the injection of a viral vector carrying EGFP (provided by R. Yáñez and A. Thrasher, UCL) into the red nucleus. Most corticospinal axons are present in the dorsal corticospinal tract (arrow) in the dorsal columns, but smaller numbers are also found in the dorsal part of the lateral white column (*), and in the ventral corticospinal tract Fig. 1.2. (arrowhead). The latter is on the same side as the BDA injection into cortex. The midline is indicated by the dashed line. Many corticospinal fibers are found throughout the dorsal horn (DH) of gray matter. Unless all of these are cut, there will always be some corticospinal axons caudal to a lesion. In contrast, rubro-spinal tracts (arrow) are confined to the dorsal part of the lateral white column and extend axons into the deep dorsal horn. A cut through the dorsal horn and dorsal part of the lateral white column will eliminate rubrospinal axons below a lesion. It is simple to completely cut the rubrospinal tracts. 7 8 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord dents, and can be completely transected by a lateral lesion, with less cavitation than occurs after a dorsal corticospinal tract injury, making them an excellent model for studying regeneration of descending tracts. Regeneration of Ascending Dorsal Column Axons Can Be Measured Simply and Accurately The ascending dorsal columns include axons of dorsal root ganglion neurons ascending to the dorsal column nuclei in the medulla. Because these ascending axons are confined to the dorsal columns and end in the dorsal column nuclei of the medulla, they are particularly useful for assessing axonal regeneration. Transection of the ascending dorsal columns results in meager axonal regeneration, but sprouting into the lesion site can be dramatically enhanced by performing a conditioning lesion on a peripheral nerve containing the peripheral processes of the injured neurons (Bavetta et al., 1999; Neumann and Woolf, 1999). A great advantage of the ascending dorsal column system is that it is possible to check for spared axons. Labeled axons can only be found in the dorsal column nuclei in the medulla if regeneration has been completely successful, which is highly unlikely if the lesion is many millimeters from the medulla, or if they were spared by the lesion. Thus, if fibers are found in these nuclei in individual animals it must be assumed that the lesion was incomplete in these particular animals. In summary, although the regeneration of many different tracts in the mammalian spinal cord can be followed by anterograde tracing, of these the corticospinal tract is the most difficult to assess, except following complete transection of the cord. 1.2.2.2 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration Molecules capable of inhibiting axonal growth in vitro have been found both in the intact CNS (Caroni et al., 1988; Schwab, 1990) and at sites of injury (Fawcett and Asher, 1999; Pasterkamp et al., 2001). All appear to act by activating the RhoA GTPase. Berry (1982) first postulated that CNS myelin was involved in the prevention of axonal regeneration. He drew attention to the observation that nonmyelinated axons in the CNS (from monoaminergic neurons that we now know to strongly express growth-associated genes) would regenerate after chemical axotomy, but not after mechanical axotomy. Since chemical axotomy did not involve injury to adjacent myelinated axons (unlike mechanical axotomy, which always damaged some myelinated axons), Berry hypothesized that degeneration products of CNS myelin were inhibitory to axonal growth. Subsequently, it was demonstrated that CNS myelin inhibited neurite growth in vitro (Caroni and Schwab, 1988a; Schwab and Caroni, 1988) and that two proteins extracted from CNS myelin had most of the inhibitory activity (Caroni and Schwab, 1988a). The generation of a function-blocking antibody, IN-1, against one of these proteins (Caroni and 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration Schwab, 1988b) eventually led to cloning of the Nogo gene (Chen et al., 2000; GrandPre et al., 2000; Prinjha et al., 2000). The roles of myelin-derived inhibitors of axonal regeneration have been most intensively studied, and will be the main focus of this chapter. Nogo, MAG and OMgp are the best known myelin-derived inhibitors. EphrinB3 and Sema 4D also contribute to the inhibitory effects of CNS myelin (Moreau-Fauvarque et al., 2003; Benson et al., 2005), but these will be considered under the heading of inhibitors at the lesion site. 1.3.1 Nogo Nogo is a member of the Reticulon family, and is otherwise known as Reticulon 4. Three isoforms – Nogo-A (the largest), Nogo-B and Nogo-C (the smallest) – are generated in the nervous system by the Nogo gene. A 66-residue extracellular domain sequence (Nogo-66), common to all three isoforms, inhibits axonal extension and induces growth cone collapse (GrandPre et al., 2002), but Nogo-A also has inhibitory domains in its unique N-terminal sequence (Amino-Nogo) (Prinjha et al., 2002; Oertle et al., 2003). Nogo-A is found in CNS myelin, and is highly expressed by oligodendrocytes. However, it has been reported that only 1–2% of total Nogo66/A in oligodendrocytes is actually expressed at the cell surface. This may be sufficient to exert its inhibitory influences on axonal elongation, but the quantity of Nogo-A that is retained in other subcellular compartments would be consistent with the molecule having other functions. 1.3.2 OMgp Oligodendrocyte myelin glycoprotein (OMgp) is a GPI-linked cell-surface protein first identified as arretin (McKerracher et al., 1994), an inhibitory extract from CNS myelin, and subsequently characterized as a growth-cone-collapsing factor and an inhibitor of axonal regeneration (Kottis et al., 2002; Wang et al., 2002a). Despite its name, OMgp has in fact been found to be strongly expressed by neurons, both by in-situ hybridization and immunohistochemistry (Habib et al., 1998). Furthermore, recent evidence suggests that OMgp is also expressed by glial cells that contact axons at nodes of Ranvier, probably NG2þcells (Huang et al., 2005), where it has a role in suppressing collateral formation. 1.3.3 MAG Myelin-associated glycoprotein (MAG; Siglec-4) is a member of the Siglec family of sialic acid-binding Ig-family member lectins (Crocker et al., 1998). Siglecs bind to sialic acid-bearing glycoconjugates, of which gangliosides are the most abundant in the brain (Yang et al., 1996). MAG is an oligodendrocyte protein which binds with 9 10 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord high affinity and specificity to two major brain gangliosides, GD1a and GT1b, that are expressed prominently on axons (Calderon et al., 1995). MAG was originally shown to be a promoter of neurite outgrowth from immature dorsal root ganglion (DRG) neurons (Johnson et al., 1989), but subsequently it was found to inhibit axonal growth from adult DRG cells, cerebellar granule cells and many other neurons (McKerracher et al., 1994; Mukhopadhyay et al., 1994). 1.3.4 The Nogo-66 Receptor, NgR1, (RTN4R), and Related Molecules Although Nogo-66, MAG and OMgp lack sequence homology, a single cell-surface protein, NgR1 acts as a functional receptor for all three molecules (Fournier et al., 2001; Liu et al., 2002; Wang et al., 2002a). NgR1 is a GPI-linked cell-surface protein and requires LINGO-1 and either p75 or TROY for signal transduction. There are two further homologues of NgR1: NgR2 (RTN4RL2; NgRL3; NgRH1) and NgR3 (RTN4RL1; NgRL2; NgRH2) (Barton et al., 2003; Lauren et al., 2003; Pignot et al., 2003). NgR2 binds MAG in a sialic acid-dependent manner and acts as a functional receptor for that protein (Venkatesh et al., 2005), although this was not reported in earlier studies (Pignot et al., 2003). It is not yet clear which co-receptors are required for NgR2 activity. Gangliosides play a poorly defined role in signaling from MAG (Yang et al., 1996; Vinson et al., 2001). Thus, NgR1 mediates growth cone collapse in response to three myelin proteins and NgR2 is a receptor for MAG, but many neurons in vivo lack these receptors or their co-receptors. 1.3.5 Co-Receptors: LINGO-1, p75 and TROY (TAJ) LINGO-1, is a transmembrane leucine-rich repeat-containing protein, which coimmunoprecipitates with NgR1 and p75; the expression of all three proteins is necessary for the downstream activation of RhoA in COS cells in response to Nogo-66, MAG or OMgp (Mi et al., 2004). Lingo-1 is a negative regulator of myelination (Mi et al., 2005). p75 is a transmembrane protein belonging to the tumor necrosis factor receptor (TNFR) superfamily (Roux and Barker, 2002). p75 interacts with several cell membrane proteins other than NgR1, including Trk family members and sortilin, to perform complex functions in neurons, including cell death (Bronfman and Fainzilber, 2004). p75 is subject to regulated intramembrane proteolysis by a number of metalloproteases and secretases, which in some cases is necessary for its functions (Jung et al., 2003; Kanning et al., 2003). TROY is another receptor in the TNFR family and binds to NgR1. It can replace p75 in the p75/NgR1/LINGO-1 complex to activate Rho-A in the presence of myelin-derived inhibitory molecules (Park et al., 2005; Shao et al., 2005). In summary, NOGO, MAG and OMgp are proteins found in CNS white matter and can cause growth cone collapse via a receptor complex comprising NgR1, LINGO-1 and p75 or TROY, but can also inhibit axonal regeneration through other mechanisms. 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration 1.3.6 Signal Transduction from Myelin-Derived Inhibitory Molecules The signaling pathways from the unknown receptor for Amino-Nogo to Rho-A are unknown, as are those from NgR2. In contrast, important steps on the signaling pathways between ligand binding to NgR1 and growth cone collapse or inhibition of neurite extension have been identified (Fig. 1.3). Nogo-66, MAG, or OMgp binding to the tripartite receptor comprising NgR1, LINGO-1 and p75 or TROY induces activation of RhoA, leading to rearrangement of the actin cytoskeleton and hence growth cone collapse. The role of p75 in the response to inhibitory ligands is likely to be complex, not least because of the interaction of p75 with other membraneassociated proteins, such as Trk tyrosine kinases and the ganglioside GT1b (Fujitani et al., 2005), and multiple intracellular signaling molecules (Dechant and Barde, 2002). The pathway appears to involve regulated intramembrane proteolysis of p75 (Domeniconi et al., 2005) (but see also Logan et al., 2006; Ahmed et al., 2006), and activation of PKC (Sivasankaran et al., 2004) and activation of EGFR (Koprivica et al., 2005). The link between p75 and activation of Rho-A is still obscure, but may involve p75 (or a cytoplasmic fragment thereof ) releasing Rho-A from its inhibitor Rho-GDI (Yamashita and Tohyama, 2003). Calcium and EGFR activation are essential for growth cone collapse in response to NgR ligands or other inhibitory molecules. The pathway from Rho-A activation to growth cone collapse was more obscure, until recent studies implicated ROCK and the antagonistic effects of LIM kinase and a phosphatase, Slingshot, on cofilin (Hsieh et al., 2006). 1.3.7 The Role of Nogo-A in Axonal Regeneration in the Spinal Cord Variations in the Extent of Axonal Regeneration in Different Strains of Nogo Knockout Mice Different strains of nogo-a/b= mice have been reported to exhibit either no corticospinal tract axon regeneration following dorsal cord hemisection at T7–T8 in 6to 14-week-old animals (Zheng et al., 2003), or significant regeneration and functional recovery following dorsal cord hemisection at T6 in 7- to 14-week-old mice (Kim et al., 2003). Selective nogo-a= mice are reported to exhibit a slight degree of corticospinal tract sprouting after dorsal column and dorsal horn transection at T8 in 8- to 17-week-old animals (Simonen et al., 2003). It is not clear how the disparity between these findings can be resolved, except by invoking the unknown effects of genetic background, or compensatory changes in the expression of other molecules during development in some strains. In either case, it would appear that factors other than the level of Nogo-A expression can play a potent role in preventing axonal regeneration in the spinal cord. None of the mutants used in regeneration experiments was in homogenous genetic backgrounds (i.e., backcrossed into the desired strain for 10 generations). One possible compensatory change in gene expression was identified in the nogo-a= animals: Nogo-B was up-regulated 1.3.7.1 11 12 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord Signal transduction pathways from NgR1. The encircled numbers in the diagram refer to the following publications (see References): 1. Fournier et al. (2001); 2. Wang et al. (2002a); 3. Liu et al. (2002); 4. Yang et al. (1996), Vinson et al. (2001); 5. Fujitani et al. (2005); 6. Yamashita et al. (2002); 7. Hasegawa et al. (2004); 8. Domeniconi et al. (2005), but see also Ahmed et al. (2006); Fig. 1.3. 9. Koprivica et al. (2005); 10. Matsui et al. (1996); 11. Hsieh et al. (2006); 12. Cai et al. (1999). * MAG-bound GT1b/GD1a induces translocation of p75 into lipid rafts. ! necessary but not sufficient for growth cone collapse/neurite outgrowth inhibition. ‘‘P’’ in a yellow circle indicates a phosphate group. 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration tenfold in the CNS of the mutants, and could have exerted an inhibitory effect on the injured corticospinal tract axons. This change could not, of course, explain the lack of regeneration in one strain of nogo-a/b= animals (Zheng et al., 2003). 1.3.7.2 Effects of Antibodies Against Nogo on Axonal Regeneration in Spinal Cord 1.3.7.2.1 Exogenous Antibodies Against Nogo Produce Some Regeneration of Corticospinal Tract Axons and Substantial Behavioral Recovery Mediated by Enhanced Plasticity Experiments with function-blocking antibodies, particularly IN-1 and its derivatives (Caroni and Schwab, 1988b; Rubin et al., 1994), have provided (until recently) the best evidence that Nogo is a significant player in controlling axonal sprouting and regeneration and behavioral recovery following experimental spinal cord injuries. The experiments in which the evidence was most conclusive involve corticospinal tract neurons, whose cell bodies contain very high levels of mRNAs for NgR1, Nogo-66 and Nogo-A. However, it is worth noting that there is some confusion as to which molecules contain epitopes recognized by IN-1 (Chen et al., 2000; Fournier et al., 2002) and that, in most studies, antibodies may have had access to the cell bodies as well as the growth cones of the corticospinal tract neurons. Schnell and Schwab (1990) used young rats in which the dorsal and dorsolateral parts of the midthoracic spinal cord were severed. The small ventral tract was presumably intact. Regenerating corticospinal tract axons were surprisingly found up to 2.6 mm distal to the lesion in controls, but in IN-1-treated animals they were identified even further (up to 11 mm) beyond the lesion. The regenerating corticospinal tract axons were mainly in the position of the former corticospinal tract, in contrast to the results of later studies, and there must be some question as to whether some of these axons were fibers spared from the lesion. The effects of IN-1 were enhanced by the implantation of fetal spinal cord at the lesion site (Schnell and Schwab, 1993). Treatment with NT-3 in conjunction with IN-1 (Schnell et al., 1994) improved regeneration further, with axons apparently regenerating up to 20 mm beyond the lesion. IN-1 treatment was subsequently shown to improve functional recovery after such lesions (Bregman et al., 1995), but both the effects on regeneration and on functional recovery were reduced if treatment was delayed until eight weeks after lesioning (von Meyenburg et al., 1998). The most robust evidence that IN-1 produces regeneration of corticospinal tract axons comes, however, from pyramidotomy experiments (Raineteau et al., 1999) which allowed the entire corticospinal tract to be severed: axons regenerated more than 2 mm. In the later studies the regenerating axons were often found in the ascending dorsal columns, lateral columns and gray matter, rather than the former corticospinal tract. It is significant that IN-1 also enhanced corticospinal tract sprouting and produced increased numbers of labeled corticospinal tract axons caudal to a lateral corticospinal tract injury in marmosets (Fouad et al., 2004), thereby demonstrating its efficacy in primates as well as rodents. Subsequently, other antibodies to Nogo-A have been developed for use in spinal injury experiments. A recombinant, partially humanized Fab fragment derived 13 14 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord from IN-1 was infused through a minipump in rats with a thoracic dorsal and dorsolateral spinal cord lesion, and produced enhanced corticospinal tract sprouting with labeled axons up to 9 mm beyond the lesion (Brosamle et al., 2000). Monoclonal antibodies against the active sites of the Nogo-A-specific region were produced and shown to enhance functional recovery following intrathecal infusion in rats (Liebscher et al., 2005). Although the use of partial lesion models allows the possibility that axons beyond the lesion were spared fibers or their sprouts, the weight of evidence strongly suggests that both IN-1 and antibodies against Nogo-A peptides can stimulate the regeneration of some corticospinal axons, mainly through intact tissue around transection lesions and through gray matter distal to the lesion. The numbers of regenerating axons are small compared with those in the intact corticospinal tracts, and they are often reported to be absent in animals with large lesions, extensive scarring or cysts (Brosamle et al., 2000; Liebscher et al., 2005). Perhaps this should not be surprising as Nogo-A is virtually absent from spinal cord lesion sites (Hunt et al., 2003), where many other inhibitors are strongly expressed (Pasterkamp et al., 2001; Tang et al., 2003; Zhang et al., 1997). Perhaps the most significant observations on the effects of IN-1 in vivo is that it increases sprouting from axons, including corticobulbar and corticospinal axons rostral to a unilateral pyramidotomy, corticospinal axons caudal to a unilateral pyamidotomy (Bareyre et al., 2002), and corticostriate axons (Kartje et al., 1999). Sprouting of corticospinal axons into abnormal territories was even observed in animals without axotomy (Bareyre et al., 2002). Following unilateral pyramidectomy, the behavioral recovery resulting from IN-1 treatment was not abolished by relesioning the pyramid rostral to the original injury (Z’Graggen et al., 1998), thus showing that plasticity rather than regeneration was responsible for the improvement. The improvement of motor behavior in injured animals treated with IN-1 may be the result of enhanced plasticity of corticofugal axons or of the descending serotonergic system (Bregman et al., 1995; Bareyre et al., 2002). 1.3.7.2.2 The Absence of Effects of IN-1 and Antibodies Raised Against Nogo-A on Regeneration of Ascending Axons Although knowledge of IN-1 has existed for almost two decades, there has been only one published morphological study of its effects on ascending axons within the injured spinal cord, and this concluded that the antibody does not enhance regeneration of the central processes of primary afferent (DRG) neurons in vivo (Oudega et al., 2000). The experimental model used was complex. Peripheral nerve grafts were implanted into the thoracic spinal cord of adult rats and the ascending dorsal column axons encouraged to grow into the grafts through the use of a conditioning lesion of the sciatic nerve. The issue addressed was whether IN-1 could promote regeneration of the sensory axons from the graft into the rostral spinal cord. This did not occur. IN-1 does, however, promote the regeneration of neonatal DRG neurons into CNS tissue (optic nerves) in vitro (Chen et al., 2000). Furthermore, a functional MRI study of rats with a spinal cord injury and treated with antibody 11C7 (raised against an 18-amino acid Nogo-A peptide corresponding to the rat Nogo-A amino acids 623 to 640), showed activation of somatosensory cortex 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration following hind paw stimulation in treated animals, but not in controls (Liebscher et al., 2005). Presumably, axonal sprouting or possibly regeneration of an ascending system other than the dorsal columns was stimulated by the antibody. There are a few other tracts where IN-1 is reported to enhance axonal regeneration. IN-1 treatment increased the number of regenerating septohippocampal axons following removal of the fimbria/fornix (Cadelli and Schwab, 1991). The axons in IN-1-treated animals grew for about 1.5 mm in the hippocampus, significantly further than in control animals. However, the regenerating axons were identified by cholinesterase staining, as opposed to anterograde tracing, which left some doubt as to their origin. Septal nuclei are rare examples of neurons thought to regenerate in response to IN-1 although expressing very low levels of NgR1 mRNA; however, being cholinergic neurons they probably have a higher propensity for regeneration than most other neurons in the brain (Anderson et al., 1998; Anderson and Lieberman, 1999). IN-1 together with brain-derived neurotrophic factor (BDNF) allowed about 1.5 mm regeneration of retinal ganglion cell axons after freeze/crush lesions of the intracranial optic nerve of young rats (Weibel et al., 1994). In mice, IN-1 also enhanced axonal regeneration after optic nerve crush (Bartsch et al., 1995a), but once again the furthest fibers were less than 2 mm beyond the lesion. Retinal ganglion cells in adult rodents show strong NgR1 mRNA expression (Fournier et al., 2001; Hunt et al., 2002a) and Nogo-A expression (Hunt et al., 2003), and are rare neurons in that they show some axonal regeneration in CNS tissue of the optic nerve or superior brachium, even without therapeutic intervention (Harvey and Tan, 1992; Campbell et al., 1999). In summary, IN-1 and related antibodies can stimulate considerable axonal sprouting and the regeneration of some types of intrinsic CNS axons for several millimeters. IN-1 also produces significant behavioral recovery in both rodents and primates in systems where enhanced plasticity can be effective. A major question still to be answered is whether regenerated axons are responsible for any part of the functional recovery achieved by IN-1 and other antibodies to Nogo-A. Axonal regeneration is only the first stage towards functional repair; after peripheral nerve injury, it has been estimated that only 50% of patients with repaired nerves experience a useful degree of functional recovery (Lee and Wolfe, 2000), even though regeneration is vigorous. Fiber misdirection, degeneration of glia and degeneration of targets during the period of regeneration are thought to be responsible for this unfortunate situation. It may be naive to expect CNS axons to regenerate long distances, to navigate to their original targets and to form appropriate synapses to produce functional recovery. Plasticity involving the formation of new pathways is likely to be an essential part of any recovery following spinal injury, but some regeneration will always be required for complete spinal lesions. Endogenous Antibodies Against Myelin Antibodies to CNS myelin and Nogo can also be generated directly within the animal with a spinal cord injury, by vaccination. Huang et al. (1999) reported that if mice were vaccinated with CNS myelin preparations, subsequent dorsal hemisection of the spinal cord was followed by extensive regeneration of corticospinal ax1.3.7.2.3 15 16 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord ons. The regenerating axons were present in the dorsal white matter, unlike those regenerating after treatment with IN-1. In a further study, Sicotte et al. (2003) reported that vaccination with a combination of MAG and Nogo-66 would also enhance corticospinal regeneration following dorsal hemisection at lower thoracic levels, although the effect was less than that of myelin. In this study the regenerating axons were less numerous and were no longer in a bundle in the dorsal white matter but rather spread out through the cord. A third study (Xu et al., 2004) reported the use of a DNA vaccine to deliver a construct containing the inhibitory epitopes of Amino-Nogo, Nogo-66, MAG and tenascin-R to adult rats. Appropriate antibodies were produced by the vaccinated animals, and regeneration of dorsal corticospinal axons claimed following a thoracic dorsal hemisection. However, unpublished studies in our laboratory (K. Rezajooi et al.) failed to show any enhancement of regeneration in rats vaccinated with CNS myelin using the same protocols as Huang et al. (1999). Differences in the immune responses of rats and mice might explain this negative result. However, since vaccination with myelin is a simple procedure, it is likely that other vaccination studies have been performed, but the results not published. Vaccination with a Nogo-A peptide can also have neuroprotective effects (Hauben et al., 2001); it remains to be seen whether increased axonal sparing can explain some of the apparent axonal regeneration in other vaccination experiments. Neuronal Nogo-A Antibodies against Nogo-A will not bind solely to myelin. Nogo-A is widely expressed in neurons (Josephson et al., 2001; Hunt et al., 2002b, 2003), where it reaches the cell surface (Dodd et al., 2005). During development, Nogo-A is expressed in all neuronal cell bodies and in growing axons (Tozaki et al., 2002). Nogo-A is down-regulated by most CNS neurons during the later stages of development and excluded from the axons of others. Some intrinsic CNS neurons – notably retinal ganglion cells, Purkinje cells, some hippocampal neurons and corticospinal tract neurons – retain high levels of Nogo-A in their perikarya (but not their axons) during adult life. In contrast to intrinsic CNS axons, axons within mature and regenerating peripheral nerves express Nogo-A strongly (Hunt et al., 2003). Hence axonal Nogo-A expression appears to be correlated with the ability of the axons to grow, form fascicles and regenerate. All neurons which are known to respond to IN-1 in adult animals in vivo strongly express Nogo-A mRNA and protein. The possibility exists that IN-1 acts in vivo by binding to neuronal Nogo and directly stimulating neurons, rather than by disinhibiting them. The evidence against such a possibility is the report of unpublished experiments showing that IN-1 does not enhance neurite outgrowth from DRG neurons grown on laminin, and immunohistochemical evidence that IN-1 binds mainly to white matter (Bartsch et al., 1995a; Huber et al., 2002). However, the environment surrounding neurons in vivo is complex and could influence the effects of IN-1 on them. IN-1 would be expected to bind neuronal Nogo. IN-1 promotes neurite outgrowth from hippocampal neurons on non-inhibitory substrates in vitro (Huber et al., 2002; Mingorance et al., 2004). The epitopes recog1.3.7.3 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration nized by IN-1 have never been published. It may be germane that Fournier et al. (2002) claimed that IN-1 recognizes other myelin proteins, but if this were the case it would be interesting to see if IN-1 binds other molecules expressed by cortical neurons. Finally, it is not clear why antibodies against Nogo-A should have profound effects on axonal sprouting and regeneration if they act through binding to myelin: CNS myelin fractions also contain MAG, OMgp and ephrin B3 (Benson et al., 2005) and Sema 4D (Moreau-Fauvarque et al., 2003) (see below), each of which is capable of inhibiting neurite growth in vitro. 1.3.8 The Role of NgR1, NgR2 and Their Co-Receptors in Axonal Regeneration Within the Spinal Cord The Distribution of NgR1 and NgR2 Does Not Suggest a General Regeneration-Inhibitory Function in the CNS NgR1 is differentially expressed by neurons in the adult mammalian nervous system: the highest levels of NgR1 mRNA are found in forebrain neurons (including pyramidal neurons of the motor cortex from which corticospinal tract axons originate), and retinal ganglion cells. There is no NgR1 mRNA in neostriatal neurons (Fig. 1.4). Neurons in the spinal cord have very low levels of NgR1 mRNA expression, and only 20–30% of neurons in DRG appear to express NgR1 mRNA (Hunt et al., 2002a; Josephson et al., 2002), although some studies have contained images showing wider expression (Park et al., 2005). There has been no systematic immunohistochemical study of NgR1 protein expression in the adult CNS, which proba1.3.8.1 In-situ hybridization for NgR1 on a section through an adult rat forebrain. Many neurons in the neocortex (C) strongly express NgR1 mRNA, but no such expression is seen in the neostriatum (St). NgR1 cannot explain the poor regenerative powers of striatal projection neurons. Fig. 1.4. 17 18 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord bly reflects the lability of the antigen. However, most DRG neurons are reported to express NgR1 protein in vitro (Ahmed et al., 2005). NgR2 is also differentially expressed, although there are some differences between the published accounts. Both, Pignot et al. (2003) and Lauren et al. (2003) reported a distribution broadly similar to that of NgR1 (strong in cortex, absent from striatum, weak in spinal cord), whereas Venkatesh et al. (2005) reported strong expression in the spinal cord and DRGs. Knockout Mice Do Not Provide a Clear Picture of the Role of NgR1 in Regeneration Two groups have reported mice with genetically inactivated NgR1. Kim et al. (2004) generated mice in which exon 2 of the mouse ngr gene was replaced with the neo R cassette. Later, Zheng et al. (2005) generated mice in which a 1.3-kb genomic fragment in exon 2 was replaced with an IRES-Tau-LacZ reporter gene. In both cases experimental animals were obtained by backcrossing onto a C57BL/6 strain, though it is not clear that a uniform genetic background was obtained. Both males and females were viable and fertile, showing that NgR1 is not required for normal development. Zheng et al. found no enhancement of corticospinal regeneration in the mutant animals. Furthermore, they showed that neurite outgrowth from P7 cerebellar granule cells and P10 DRG neurons derived from the mutant animals was still inhibited by CNS myelin, and that the outgrowth of neurites by granule cells was inhibited by Nogo-66. In contrast, Kim et al. reported P14 DRG neurons from their mutants to be less sensitive to Nogo-66, MAG and OMgp, and that rubrospinal and raphespinal axons showed enhanced regeneration after spinal injury, though corticospinal axons were unable to regenerate. Importantly, raphespinal axons even regenerated across complete lesions of the cord, which should have eliminated confusion of regenerated axons with spared fibers. Complete transection necessitates that the regenerating axons cross a lesion site, where the NgR1 ligands Nogo-A (Hunt et al., 2003) and MAG (Pasterkamp et al., 2001) are depleted, whereas it might have been expected that inactivating ngr would have its greatest effects in experiments where tissue bridges containing CNS myelin and oligodendrocytes remained. The only direct comparison possible between studies of the two mutants involves corticospinal axons in vivo, and neither study shows enhancement of regeneration of such fibers. This is a curious result because neocortical neurons express NgR1 more strongly than most other cells, including those in the red nucleus (Hunt et al., 2002a; Josephson et al., 2002; Lauren et al., 2003). Nonetheless, the differential regenerative abilities of various descending tracts in one strain of NgR1deficient mice, together with the lack of regeneration of corticospinal axons in both strains, confirms that both the NgR1 system and other factors contribute to regulating the degree of axon regrowth (Kim et al., 2004). The results of comparing Nogo-A-deficient and NgR1-deficient mice should elucidate the role of AminoNogo-A in the cellular responses to spinal injury. However, the comparison is somewhat confused by the different results obtained with the various strains of null mutation animals. 1.3.8.2 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration Pharmacological Blockade of NgR1 Enhances Axonal Sprouting and Regeneration It should be informative to compare the extent of axonal regeneration, the source of the regenerating axons and the extent of behavioral recovery following dorsal spinal hemisection in ngr= mice and animals treated with pharmacological agents to perturb the NgR1 signaling. Two NgR1 antagonists have been developed by Strittmatter’s group: NEP1-40 and NgRecto, the soluble ectodomain of NgR1. NEP1-40, which is a specific blocker of Nogo-66 signaling through NgR1, was reported to enhance corticospinal regeneration and raphespinal sprouting following dorsal spinal cord transection when administered intrathecally using a minipump (GrandPre et al., 2002). The behavioral results in NEP1-40-treated rats were generally similar to those in ngr= mice, and regeneration or sprouting of raphespinal axons was enhanced in both. However, small numbers of corticospinal axons were present, widely dispersed across the cross-section of the spinal cord caudal to the lesion in the NEP1-40-treated rats, including regions where corticospinal labeling is not normally found following dorsal hemisection. Whether these axons were the result of regeneration or the sprouting of ventral corticospinal tract fibers was not entirely clear. In subsequent experiments it was shown that subcutaneous administration of NEP1-40 to mice could enhance the regeneration of corticospinal, raphespinal and rubrospinal axons, even when there was a 7-day delay between injury and treatment (Li and Strittmatter, 2003). NgRecto should be able to block the effects of Nogo-66, MAG and OMgp on NgR1 signaling, and would therefore be expected to have greater effects on axonal regeneration. Intrathecal delivery of NgRecto was reported to be more effective at increasing sprouting of corticospinal fibers rostral to a lesion, but the effects on axonal regeneration and behavioral recovery were apparently similar to those of NEP1-40 (Li et al., 2004). Hence, both NEP1-40 and NgRecto enhance the regeneration of corticospinal tract axons, in contrast to the genetic inactivation of NgR1. The explanation for this discrepancy is not clear, but it may include compensatory changes in gene expression. For example, Nogo-A is up-regulated in mutant mice (at least in the early postnatal stages), and it is conceivable that Nogo-A signaling via its unidentified specific receptor could inhibit regeneration. Nonetheless, the observation that, among neurons with axons in the spinal cord, it is the cell type that normally expresses NgR1 most strongly that fails to regenerate axons in the NgR1-deficient mice which suggests that our understanding of the control of regeneration in the spinal cord is still poor. It is surprising that the regeneration of ascending dorsal column fibers has not been studied in ngr= mice or in animals treated with NgR1 antagonists. 1.3.8.3 The Pattern of Expression of LINGO-1 and p75 Does Not Suggest a General Role in Inhibiting Regeneration in Vivo Morphological investigations show that LINGO-1 is most strongly expressed by cerebral cortical neurons and perhaps DRG neurons, but is much more weakly expressed by neurons in the striatum, brainstem, cerebellum, and spinal cord. Carim-Todd et al. (2003) showed by in-situ hybridization, immunohistochemistry 1.3.8.4 19 20 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord and Northern blot of adult rat brain, that LINGO-1(LERN1) expression was largely restricted to neurons within the neocortex and limbic system, that very little expression was present in cerebellum, and that none was detectable in intact spinal cord. Mi et al. (2004) also reported that LINGO-1 mRNA expression was restricted to the nervous system. RT-PCR showed that LINGO-1 expression peaked at P1, and decreased thereafter. In-situ hybridization and immunohistochemistry showed LINGO-1 expression to be confined to neurons (but see below), and confirmed that there was a gradient in LINGO-1 expression, with the highest levels being present in the forebrain and much lower levels in the cerebellum and spinal cord. In contrast to Carim-Todd et al. (2003), Mi et al. found signal from at least some Purkinje cells, spinal motor neurons and DRG neurons. Shao et al. (2005) also found LINGO-1 mRNA and protein in Purkinje cells, motor neurons and many DRG neurons. Park et al. (2005) identified a strong in-situ hybridization signal for LINGO-1 in most DRG neurons, but only a very weak signal in Purkinje cells. Mi et al. (2004) briefly reported on studies of LINGO-1 expression following spinal cord injury; RT-PCR revealed a fivefold increase in LINGO-1 mRNA levels, 14 days after a dorsal hemisection injury. The protein was strongly up-regulated in the gray matter, and also to a lesser extent in white matter rostral to the lesion 10 days after the injury. LINGO-1 can also be detected by RT-PCR in cultured oligodendrocytes (Mi et al., 2005) in the CNS, where it negatively regulates myelination. How much of the elevated expression of LINGO-1 following spinal injury was produced by glia is unknown, however. The expression of p75 is widespread, both in the nervous system and beyond. In the nervous system it is differentially expressed on neurons and some glia. The strongest p75 expression by intrinsic CNS neurons is found in the cholinergic nuclei of the basal forebrain and septum, though lower levels have been reported in the hippocampus, cerebellum and frontal cortex (Buck et al., 1988). However, p75 is difficult to detect in many CNS neurons. DRG neurons have varied levels of p75 expression, but a general estimate is that about 40–50% are p75-positive (Wright and Snider, 1995; Bennett et al., 1996). Interestingly, the expression of p75 by retinal ganglion cells, often used in regeneration experiments, is disputed (Hu et al., 1999; Hirsch et al., 2000; Logan et al., 2006). As with NgR1, it is not always clear whether very low levels of signal represent the complete absence of protein. TROY is more widely expressed by intrinsic CNS and DRG neurons than is p75, including neocortical neurons, cerebellar granule cells, Purkinje cells and most retinal ganglion cells, most DRG neurons, and spinal motor neurons (Park et al., 2005; Shao et al., 2005). In summary, the patterns of expression of NgR1, NgR2, LINGO-1 and p75, do not suggest a uniform regeneration-inhibitory role throughout the CNS. LINGO-1, p75 and TROY Have Important Roles in Neurite Outgrowth in Vitro, But Their Significance for Axonal Regeneration in Vivo Has Not Yet Been Established Since LINGO-1 is apparently an obligatory co-receptor for NgR1, and either p75 or TROY is required to make up the functional receptor complex, genetic or pharmacological interference with these molecules might be expected to have considerable 1.3.8.5 1.3 Myelin Proteins as Inhibitors of Axonal Regeneration effects of axonal regeneration in the spinal cord. Transfection of embryonic cerebellar granule cells with a dominant-negative LINGO-1 construct attenuated the inhibitory effects of myelin, Nogo-66 and OMgp, as did addition of a LINGO-1-Fc fusion protein. These agents have not yet been used in the injured spinal cord. A LINGO-1 knockout mouse exists (Mi et al., 2005), but as yet there have been no reports of regeneration experiments in these animals. The absence of any obvious neurological abnormalities in this mouse suggests that the role of NgR1 signaling during development of the nervous system is minor. Soluble TROY-Fc also blocks the inhibitory effects of Nogo-66 and OMgp on neurite outgrowth from P7 cerebellar granule cells and DRG neurons (Park et al., 2005; Shao et al., 2005). Dominant-negative TROY transfected into P28 DRG neurons had similar effects (Park et al., 2005). These agents have not yet been tested for their ability to promote regeneration in vivo, but cerebellar granule cells and DRG neurons from TROY knockout mice show a greatly enhanced ability to elongate neurites on myelin-derived inhibitory substrates (Shao et al., 2005). TROY knockout mice are healthy and fertile, but no experiments on spinal cord injury using the mice have yet been reported. Neurons from mice lacking p75 show enhanced neurite outgrowth on myelin and myelin-derived inhibitory molecules (Wang et al., 2002b; Yamashita et al., 2002; Ahmed et al., 2005; Zheng et al., 2005). A possible role for p75 in preventing axonal regeneration in vivo was indicated by a study of sympathetic axonal sprouting into the CNS of mice. Elevated levels of nerve growth factor (NGF) in the brain, produced by cholinergic denervation, attract sprouts from NGF-sensitive sympathetic axons in the meninges and around the cerebral blood vessels (Crutcher et al., 1979; Crutcher, 1981; Crutcher and Davis, 1981; Crutcher and Marfurt, 1988). This phenomenon was also observed in transgenic mice overexpressing NGF in astrocytes (Kawaja and Crutcher, 1997), and was greatly enhanced in mice overexpressing NGF but lacking p75 (Walsh et al., 1999). More recently, it has been shown that sprouting within the spinal cord of serotonergic (raphespinal) axons and calcitonin gene-related peptide (CGRP)-containing primary afferents after dorsal rhizotomy is enhanced in p75-deficient mice (Scott et al., 2005). However, there was no evidence for corticospinal axonal regeneration in mice lacking p75 following dorsal spinal cord hemisection (Zheng et al., 2005). Sympathetic, serotonergic and primary afferent neurons have a much great propensity for axonal regeneration, at least into peripheral nervous tissue, than corticospinal neurons, and a reasonable interpretation of the evidence would suggest that p75 plays some role in inhibitory signaling in vivo but that such a function only becomes significant in regeneration-competent neurons. The role of p75 in peripheral nerve regeneration, where the axons are probably exposed to lower levels of inhibitory molecules than are present in the CNS, is perhaps more confusing. Although Ferri et al. (1998) reported that regeneration of facial nerve motor axons was accelerated in the absence of p75, Gschwendtner et al. (2003) found no effect on axonal regeneration or neuronal survival. In summary, it can be concluded that NgR1 signaling is involved in limiting the regeneration of some types of axon in the mammalian spinal cord, but its effects 21 22 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord may be sensitive to the genetic background of the animals investigated. The existence of other receptors for Amino-Nogo (unidentified) and MAG (NgR2), together with the presence of many other inhibitory molecules at CNS lesion sites, probably modulates the importance of NgR1. 1.3.9 Effects of MAG and OMgp on Axon Regeneration in the Mammalian CNS Mice lacking MAG are healthy and have been studied for more than a decade, but little evidence has been obtained that MAG is a significant factor preventing axonal regeneration in the CNS in vivo. Following either optic nerve crush or dorsal hemisection of the spinal cord at lower thoracic levels, there was no difference in the regenerative responses of axons in mice lacking MAG and controls, although IN-1 antibody did enhance regeneration (Bartsch et al., 1995b). However, in these experiments there was reported to be some corticospinal regeneration in both wildtype and transgenic animals. OMgp knockout mice are healthy and show increased axonal sprouting at nodes of Ranvier in the CNS (Huang et al., 2005) where OMgpexpressing glial processes are otherwise found. No regeneration experiments have been reported in these animals. 1.3.10 Strong Evidence That Myelin Proteins Are Not Always Effective Inhibitors of Axonal Regeneration in Vivo Although there is little doubt that CNS myelin is capable of inhibiting axonal growth in vitro, there is striking and unexplained evidence from transplantation studies that axons can regenerate within myelinated tracts in vivo. It was first demonstrated that fetal monoaminergic neurons and embryonic forebrain neurons could grow long axons when transplanted into the adult CNS (Bjorklund and Stenevi, 1979; Nornes et al., 1983; Wictorin et al., 1990; Davies et al., 1994). However, the most surprising data on the ability of neurons to regenerate axons in vivo came from experiments in which adult DRG neurons were transplanted into adult CNS. In landmark experiments, Davies et al. (1997) performed atraumatic microtransplantations of adult DRG neurons into the corpus callosum and fimbria, and showed that the neurons grew axons rapidly in the white matter at rates of about 1 mm per day. Subsequently, Davies et al. (1999) showed that microtransplanted adult DRG neurons also regenerated axons at up to 2 mm per day within spinal white matter. If the dorsal columns were first injured, caudal to the site of implantation, so that the neurons were placed into white matter undergoing Wallerian degeneration, regeneration of axons still occurred. However, the regenerating sensory axons could not grow through the lesion site, where chondroitin sulfate proteoglycans (CSPGs) were abundant, within the 10-day time course of the experiments. Thus, DRG neurons regrew their axons in intact or degenerating CNS white matter at approximately the same speed that they would regenerate in injured peripheral nerve; the extension of the regenerating axons was not prevented by the 1.4 Inhibitors at the Lesion Site undoubted presence of myelin proteins or their breakdown products. These important experiments call into question the importance of myelin-derived inhibitory molecules in the CNS. Possible explanations of the remarkable regenerative abilities of microtransplanted DRG neurons include the absence of parts of the receptor complexes for Nogo, etc., or the exceptional strength of the cell body response in the transplanted neurons. Most DRG neurons contain little or no mRNA for NgR1 (Hunt et al., 2002a; Josephson et al., 2002), and many do not express p75 (Wright and Snider, 1995; Bennett et al., 1996), although Troy and LINGO-1 are apparently widely expressed (Mi et al., 2004; Park et al., 2005), at least within ganglia. The degree of expression of these molecules in DRG neurons after isolation and transplantation is unknown. 1.4 Inhibitors at the Lesion Site (Fig. 1.5) A large variety of potentially inhibitory molecules are found in CNS lesion sites, including CSPGs (Tang et al., 2003; Carulli et al., 2005), tenascins (Zhang et al., 1997), semaphorins (Pasterkamp et al., 2001), and ephrins (Goldshmit et al., 2004), but little Nogo-A (Hunt et al., 2003). The evidence that any individual molecule in this region plays a significant role in blocking axonal regeneration is weak, but the evidence that the scar is a profoundly inhibitory region is strong. TenascinC knockout mice have been available for many years, and although some spinal injury experiments have been performed (Steindler et al., 1995), no reports of an effect on axonal regeneration have been published. Unpublished results from our laboratory showed that there was no enhancement of regeneration of corticospinal or ascending dorsal column axons in tenascin-R knockout mice following spinal injury. The molecules for which there is the best evidence of any significant role in axonal regeneration within the spinal cord include CSPGs, semaphorins, and ephrins. 1.4.1 CSPGs CSPGs are proteoglycans that comprise a core protein with variable numbers of glycosaminoglycan (GAG) side chains composed of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid. Aggrecan, versican, neurocan, brevican, neuroglycan D, NG2, the receptor-type protein tyrosine phosphatase PTPb and its splice variant phosphacan are among the CSPGs found in the CNS of mammals (Hartmann and Maurer, 2001; reviewed by Carulli et al., 2005). There have been many reports that CSPGs cause growth-cone collapse, inhibit neurite outgrowth, and are a non-conducive substrate for neural cell adhesion (Snow et al., 1990; McKeon et al., 1995; Hynds and Snow, 1999), although not all CSPGs have these effects (Davies et al., 2004) and some have neurotrophic activity (Junghans et al., 1995). The action of CSPGs has been reported to be a function of 23 24 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord Diagrammatic representation of the inhibitory molecules present in and around lesion sites in the mammalian spinal cord. Fig. 1.5. the GAG side chains (Yamada et al., 1997; Talts et al., 2000), the core proteins (Dou and Levine, 1994; Schmalfeldt et al., 2000), or both (Ughrin et al., 2003), depending on the CSPG studied and the type of investigation. The pattern of sulfation of the GAGs is also a factor in inhibitory activity (Gilbert et al., 2005). CSPGs are not evenly distributed at lesion sites in the CNS, and individual CSPGs have characteristic patterns of expression. Neurocan and NG2 are rapidly up-regulated in and around spinal injury sites, while phosphacan and brevican are more slowly up-regulated (an excellent description is given in Tang et al., 2003). Phosphacan, NG2 and tenascin-C are strongly expressed in the lesion by invading meningeal cells, whereas neurocan is strongly expressed by glia at the lesion margins. NG2 glia (synantocytes) accumulate around lesions and become rounded; it is difficult to ascertain whether rounded NG2-positive cells inside the lesion site are of glial or other origin. In contrast to the main myelin-associated in- 1.4 Inhibitors at the Lesion Site hibitors of axonal regeneration, little is known about signal transduction following CSPG exposure, although there is now evidence that activation of Rho-A through a mechanism involving EGFR and intracellular calcium is involved (Koprivica et al., 2005). The evidence that CSPGs play a role in inhibiting axonal regeneration in the spinal cord comes from two sources: first, they are most strongly expressed, unlike Nogo isoforms, in and around lesion sites where axons fail to regenerate; and, second, treatment with chondroitinase ABC to remove GAGs, is reported to enhance axonal regeneration in CNS tissue. Relationship Between the Distribution of CSPGs and Failure of Axonal Regeneration One of the best examples of the failure of axonal regeneration in regions of CSPG up-regulation was provided by Davies et al. (1999) (see above), who studied axonal growth by transplanted DRG neurons within the degenerating dorsal columns of rats with spinal injuries. The axons regenerated vigorously until they reached a region at the outskirts of the lesion site, showing high levels of chondroitin-6-sulfate, where growth abruptly stopped. It is undoubtedly the case that CSPG expression is up-regulated at all types of lesion site in the CNS, although expression levels are generally proportionate to the size of the lesion (Davies et al., 1997, 1999). However, not all investigators have interpreted the pattern of CSPG expression around lesions as indicating a role in suppressing regeneration (Lips et al., 1995). The dorsal root entry zone (DREZ) provides a particularly informative region for studying the relationship between CSPG expression and axonal regeneration. Following dorsal root injury, the central processes of DRG neurons regenerate up to the DREZ, but most then cease elongating or turn back to grow retrogradely towards the DRG. The DREZ therefore appears inhibitory for axonal regeneration, even though it has not been damaged by the dorsal root injury. Pindzola et al. (1993) reported that the adult DREZ contained high levels of tenascin-C and CSPGs, which could be correlated with its inhibitory properties. However, Zhang et al. (2001) found, using immunohistochemistry and in-situ hybridization, that overall CSPG and tenascin-C levels in the DREZ following dorsal root injury in adult rats were lower than those in the injured root where regeneration did occur. Of the putative inhibitory molecules tested, only NG2 and tenascin-R were more concentrated in the DREZ. Clearly, the localization of CSPGs following dorsal root injury does not provide strong evidence for their role in preventing regeneration. However, evidence from Steinmetz et al. (2005) suggests that chondroitin sulfate GAGs are significant players in preventing regeneration through the DREZ. The active molecules may be part of individual CSPGs, probably a minor part of the total population but located in critical areas. NG2 does not appear important in this respect because mice lacking NG2 show no enhancement of axonal regeneration from dorsal roots back into the cord (our unpublished observations). 1.4.1.2 Chondroitinase ABC and Axonal Regeneration The best evidence that chondroitin sulfate GAGs are important for preventing axonal regeneration comes from studies of the effects of chondroitinase ABC, a bacterial enzyme that can remove the GAG side chains from CSPGs, leaving only a dis1.4.1.3 25 26 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord accharide stub. Chondroitinase treatment eliminates the inhibitory effects of some CSPGs on neurite outgrowth in vitro, and it is not surprising that the enzyme has been used in several experiments on axonal regeneration in vivo. The results have varied from minor to remarkable enhancement of regeneration. Experiments with hyaluronidase, which has chondroitinase activity, showed limited enhancement of retinal ganglion cell axon regeneration in crushed optic nerves (Tona and Bignami, 1993) and some enhancement of sprouting by injured nigrostriatal axons (Moon et al., 2003). A single application of chondroitinase ABC was reported greatly to enhance the regeneration of dorsal spinocerebellar axons into a peripheral nerve graft in the thoracic spinal cord of adult rats (Yick et al., 2000). Chondroitinase ABC infusion into the brain was reported to allow injured nigrostriatal fibers to regenerate back to their targets (Moon et al., 2001). It may be germane that nigrostriatal neurons have a higher propensity to regenerate axons than most other CNS neurons (Woolhead et al., 1998; Anderson and Lieberman, 1999). In the spinal cord, chondroitinase was also reported to have considerable effects on regeneration (Bradbury et al., 2002). Following a partial crush injury to the cervical cord of adult rats, chondroitinase infusion produced regeneration of ascending dorsal column axons up to 4 mm rostral to the presumed boundaries of the lesion, and regeneration of corticospinal axons caudal to those boundaries. Some corticospinal axons were found at least 5 mm caudal to the lesion, though it should be noted that some axons were also present in vehicle-infused controls. Chondroitinase treatment was also correlated with a variety of functional improvements in the injured animals. Electrical stimulation of the motor cortex evoked large postsynaptic potentials up to 7 mm caudal to the lesion, which were largely absent in vehicle controls. A number of sensorimotor behavioral tests also showed functional improvement in the chondroitinase-treated animals. The only possible caveats to these experiments are that the type of lesion used has poorly defined boundaries, making the presence of spared axons difficult to rule out, and that the anatomical localization of apparently regenerating axons was difficult to determine. Chondroitinase also enhanced recovery of bladder and motor function in another study of forceps compression of the rat spinal cord (Caggiano et al., 2005). Yick et al. (2003) studied the regeneration of dorsal spinocerebellar axons following a lower thoracic lateral hemisection and chondroitinase application to the lesion site in gelfoam. Regeneration was assessed by retrograde labeling from the cervical cord. Following the single application of chondroitinase, up to 12% of the spinocerebellar neurons in the L1 segment (three segments caudal to the lesion) were retrogradely labeled, indicating that they had regenerated 30 mm within the cord. A similar application of chondroitinase was used in a study of rubrospinal regeneration following a lateral hemisection at C7 (Yick et al., 2004). Fluorogold was injected at T1 to retrogradely label regenerating neurons. Following chondroitinase treatment, 20% of rubrospinal axons apparently regenerated compared with none in controls. However, there must be concern about the short distance between the injection site and the lesion in these experiments. Recently, chondroitinase has been used in combination with an inflammatory stimulus in the DRG, to enhance the regeneration of sensory axons from dorsal roots into the spinal cord (Steinmetz 1.4 Inhibitors at the Lesion Site et al., 2005). Furthermore, chondroitinase has been used successfully to enhance axonal regeneration into and beyond grafts of olfactory ensheathing glia and/or Schwann cells in the spinal cord (Fouad et al., 2005). In these experiments the enzyme could be primarily affecting the axons or the grafted cells. In summary, there is good evidence that chondroitinase can enhance axonal sprouting in CNS gray matter and the functional plasticity that results from such sprouting (Pizzorusso et al., 2002; Tropea et al., 2003; Corvetti and Rossi, 2005; Massey et al., 2006). Its effects on functional recovery following experimental spinal injury may be mediated by such mechanisms. However, chondroitinase by itself has produced impressive results on axonal regeneration in a very limited number of studies, despite being readily available and easily administered. The inference is that the effects of chondroitinase on axonal regeneration are often minor, and that chondroitin sulfate GAGs, like other inhibitory molecules, have only a partial role in limiting axonal regeneration in the CNS. Scar-Reducing and Growth-Promoting Effects of Decorin It should not be assumed that all CSPGs are inhibitory to axonal regeneration. In particular, the small leucine-rich protoglycan decorin, which has a single chondroitin sulfate or dermatan sulfate side chain, reduces astrogliosis and extracellular matrix deposition following stab wounds to the cerebral cortex (Logan et al., 1999). Furthermore, recent very convincing studies have shown that decorin infused around partial spinal cord lesions in adult rats reduces scarring, downregulates the inhibitory CSPGs neurocan, NG2, phosphacan and brevican, and makes the lesion site more conducive to the regeneration of axons (Davies et al., 2004). Since decorin down-regulates EGFR on tumor cells and cell lines (Csordas et al., 2000; Santra et al., 2002; Zhu et al., 2005) and EGFR activation appears necessary for signaling from several axonal growth inhibitory molecules, decorin might also have a direct disinhibitory effect on regenerating axons. 1.4.1.4 1.4.2 Axonal Guidance Molecules Are Present in the Spinal Cord and Their Receptors Are Expressed by Specific Classes of Neuron Since the direction of axonal growth during development is regulated by a series of attractive and repulsive cues, which are still expressed in adult animals, it is not surprising that several of the repulsive guidance cues may be involved in regulating axonal regeneration in the injured spinal cord. Semaphorins The semaphorin family of proteins has been divided into eight classes based on membrane topology (transmembrane, secreted, GPI-linked) and conserved domains (for a review, see de Wit and Verhaagen, 2003). Classes 3 to 7 are expressed in vertebrates; class 3 contains secreted semaphorins and classes 4 to 7 transmembrane or membrane-anchored semaphorins. All semaphorins share a conserved, 500-amino acid motif, termed the sema domain. Sema 3a is the prototype sema1.4.2.1 27 28 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord phorin, and has been subject to the most investigation with regard to axonal growth and regeneration within the mammalian spinal cord. Semaphorin receptors include plexins and neuropilins; both are needed for class 3 semaphorin activity. Neuropilins bind the semaphorin and plexins transduce the signal. Other cellsurface molecules may form part of the receptor complex, including the adhesion molecule L1, which is necessary for the transduction of a repulsive signal from Sema 3A to corticospinal neurons. Neuropilins and plexins are expressed on specific classes of CNS neurons in adult mammals (Chen et al., 1997; Steup et al., 1999; Fujita et al., 2001; Murakami et al., 2001). DRG neurons from which ascending dorsal column axons originate express Neuropilin-1 (NP-1) and Plexin A1 (Pasterkamp et al., 2001). Neocortical pyramidal neurons express NP-1, NP-2 (Holtmaat et al., 2002; Barnes et al., 2003), and are sensitive to Sema 3A (Castellani et al., 2000). The signaling cascades that lead from receptor activation to growth cone collapse are complicated, and molecules including kinases, Rho family GTPases (Liu and Strittmatter, 2001), cyclic nucleotides, redox signaling (Terman et al., 2002) and eicosanoids (Mikule et al., 2003) have been implicated. Of considerable interest is the involvement of cyclic GMP. Elevating cyclic GMP levels alters the response of cultured Xenopus spinal neurons from repulsion to attraction towards a source of Sema 3A (Song et al., 1998). Activating the cyclic GMP pathway also blocked the growth cone-collapsing effects on mammalian DRG neurons. An interesting observation linking semaphorins and CSPGs is that CSPGs are required to convert the signal from Sema 5A from an attractive to a repulsive cue for some developing neurons (Kantor et al., 2004). Sema 3A is expressed in spinal cord transection lesion sites in adult rats by invading meningeal cells (Pasterkamp et al., 2001), but not in contusion lesion sites (De Winter et al., 2002) where such cells are uncommon (Pasterkamp et al., 1998, 2001; De Winter et al., 2002). Contusion lesion sites are nonetheless profoundly inhibitory to axonal regeneration. However, when ascending dorsal column axons were stimulated to regenerate into a transection lesion site, they avoided areas of Sema 3A expression (Pasterkamp et al., 2001), indicating that this secreted semaphorin may constitute one of the inhibitory influences present where the glia limitans around the CNS is breached. Sema 4D, a potent inhibitor of DRG and cerebellar granule cell neurite outgrowth, is expressed in myelin and strongly upregulated by oligodendrocytes around an injury (Moreau-Fauvarque et al., 2003). In summary, various semaphorins are present at lesion sites in the CNS, and in myelin and receptors are expressed by some axons in the spinal cord. However, strong evidence that they play a major role in limiting regeneration in the spinal cord is still lacking. Ephrins The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are important contact-dependent regulators of development, particularly neuronal pathfinding (Pasquale, 2005). The ligands are grouped into two subfamilies: the A-subclass (ephrins A1–A6) that are GPI-linked; and the B-subclass (ephrins B1–B3) that are integral membrane proteins with one transmembrane do1.4.2.2 1.4 Inhibitors at the Lesion Site main and a short cytoplasmic region. In total, 16 Ephs have been found in vertebrates, and are divided into A- and B-subclasses on the basis of ligand affinity and sequence similarity. Ephs and ephrins bind promiscuously with most members of the corresponding subclass, interacting in the nanomolar affinity range. Some promiscuity exists between subclasses; in particular EphA4 binds B-class ephrins with high affinity. Eph dimerization is required for activation; soluble monomeric ephrins do not induce signal transduction, but artificially clustered ephrins or membrane-bound ephrins are effective (Stein et al., 1998). Cell–cell contact is therefore probably required for ephrin signaling in vivo. The principal mediators of ephrin-induced repulsion are the Rho family of small GTPases, particularly RhoA (Kullander and Klein, 2002). Cell-surface ephrins can signal in the ‘‘reverse’’ direction to transduce signals into cells in response to Eph receptors acting as ligands (Kullander and Klein, 2002). For example, A-class ephrin signaling has been implicated in vomeronasal axon mapping to the accessory olfactory bulb (Knoll et al., 2001), while B-class signaling mediates commissural axon guidance (Henkemeyer et al., 1996). Ephrin signaling is complicated by emerging data that receptor/ligand coexpression can modulate receptor sensitivity, and increasing evidence for coexpression of ligands and receptors on neurons (Iwamasa et al., 1999; Eberhart et al., 2000) suggests that this may play a significant functional role. Ephrin and Eph Expression in the Adult Mammalian Nervous System Ephrin expression is down-regulated in most tissues postnatally, but substantial expression of ephrins and receptor ephs remains in adult brain and spinal cord of humans (Hafner et al., 2004; Sobel, 2005), other primates (Xiao et al., 2006) and 1.4.2.2.1 Horizontal section of the cervical spinal cord of an adult rat one week following a dorsal column transection injury. The inhibitory ligand EphrinB2 (red) is found on GFAP-positive astrocytes (green) around, and extending into, the lesion (*). Scale bar ¼ 50 mm. Fig. 1.6. 29 30 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord rodents (Liebl et al., 2003). EphrinB3 is well placed to play an important role in limiting axonal regeneration in the mammalian spinal cord. It is a midline guidance marker for growing EphA4-positive corticospinal tract axons (Kullander et al., 2001), but is also strongly expressed in adult myelin (Benson et al., 2005). Myelin ephrinB3 is potent: the contribution of the ephrinB3 in CNS myelin to the repulsion/retraction response of neocortical neurites was equivalent to that of Nogo, MAG and OMgp combined (Benson et al., 2005). A- and B-family receptor Ephs are expressed on various classes of neuron in adult mammals, and are up-regulated following contusion injuries of the thoracic spinal cord (Miranda et al., 1999; Willson et al., 2002). Of particular interest is the phylogenetically conserved expression of EphA4 in cortical neurons (Liebl et al., 2003) and prominent accumulation of EphA4 in lesioned corticospinal axons (Fabes et al., 2006), which would make these fibers sensitive to most ephrins. It is not yet possible to say if many of the other long tracts in the spinal cord also express receptor Ephs. Some mammalian DRG neurons express EphA4 during development, which seems to control their sprouting in response to skin lesions (Moss et al., 2005), but it is not clear whether expression in DRGs includes those neurons that contribute to the ascending dorsal columns or whether expression is retained into adulthood. Many Ephs are expressed in chick embryo DRGs (Munoz et al., 2005). Eph expression by astrocytes (Miranda et al., 1999) has been implicated in gliosis and scar formation. Bundesen et al. (2003) showed that, following partial spinal cord transection injuries in mice, reactive astrocytes up-regulated ephrinB2 and meningeal cells up-regulated EphA4. These authors suggested that bidirectional signaling between astrocytes and meningeal cells may limit meningeal ingrowth into the cord and initiate the development of a glia limitans at the injured surface of the cord. The presence of ephrinB2 in the astrocytic scar (Fig. 1.6) would also present a further barrier to the regeneration of Eph-positive axonal tracts. Regeneration in EphA4 Knockout Mice Exciting evidence for a role of ephrin signaling in axonal regeneration in the spinal cord was obtained by Goldshmit and colleagues, who studied EphA4 null mice (Goldshmit et al., 2004) with lateral hemisections of the cord at T12. These authors reported that astrogliosis was greatly reduced and CSPG expression at the lesion site reduced in EphA4= mice, which also showed enhanced motor recovery. Evidence of axonal regeneration was produced by injecting FluoroRuby into the cervical cord and Fast blue into the lumbar enlargement. Many anterogradely labeled axons crossed the presumptive lesion site, and retrogradely (Fast Blue) labeled neuronal perikarya were found in the cerebral cortex (corticospinal axons), red nucleus (rubrospinal axons) and other brainstem nuclei. Although the Fast Blue injection was relatively close to the lesion site and spread of the tracer may have produced some false-positive results, the anterogradely labeled axons in the lesion site must have taken up tracer in the cervical cord. The results on regeneration and behavioral recovery were attributed to the attenuated astroglial reaction and meningeal scarring. If these findings could be replicated, it would suggest that signaling via EphA4 is a major contributor to the failure of axonal regeneration in the spinal cord. 1.4.2.2.2 1.4 Inhibitors at the Lesion Site Finally, extraneuronal ephrin signaling is involved in controlling phenomena relevant to spinal injury including immune system functions such as T- and B-cell signaling, chemotaxis, immunoregulation and co-stimulation (Sharfe et al., 2002; Freywald et al., 2003; Yu et al., 2003), and integrin signaling (Huynh-Do et al., 2002). A likely role for ephrin signaling in the inflammatory responses of the injured cord is indicated by the expression by perivascular mononuclear cells of numerous A-class ligands and receptors (Sobel, 2005). In summary, ephrins are present in and around lesion sites in the spinal cord and in myelin. Receptors are present on axons in the spinal cord, including those in the corticospinal tracts. A single study in the EphA4 knockout mouse reported extensive axonal regeneration in the spinal cord. Ephrin/Eph signaling is probably involved in the responses of neurons, glia and immune cells to spinal cord injury, but the significance of each of these effects is yet to be established. Slits and Netrins in the Mammalian Spinal Cord Slits and Netrins are potent axon guidance molecules during development. Netrins are expressed in the midline of all bilaterally symmetrical animals (Barallobre et al., 2005). Most members of the Netrin family are secreted proteins that act as bifunctional signals, chemoattractive for some neurons and chemorepellent for others, depending on the receptor types that are expressed and the levels of cAMP within the growth cone (Hopker et al., 1999). Netrins bind to members of the UNC5 and DCC receptor families. Netrin-1 acting on DCC receptors attracts dorsal commissural interneurons, but acting via UNC5 receptors it repels certain classes of motor neurons (Chisholm and Tessier-Lavigne, 1999). Netrin-1, Netrin-3/NTL2, Netrin-4/b and G-Netrins have been cloned (Barallobre et al., 2005). In adult rodent spinal cord Netrin-1 is expressed by many neurons and by oligodendrocytes, but not astrocytes (Manitt et al., 2001), and is present as a membrane- or extracellular matrix (ECM)-bound form rather than as a diffusible molecule. Netrin-1 is strongly expressed by cells within lesion sites in mouse spinal cord (Wehrle et al., 2005). The prototypic function of slit proteins is as a midline chemorepulsive signal in Drosophila. Three mammalian slit genes (slit1–slit3), all of which encode large ECM glycoproteins of about 200 kDa, have been cloned. Vertebrate Slits are repulsive factors in vitro for axons from developing spinal cord (Brose et al., 1999), dentate gyrus (Nguyen Ba-Charvet et al., 1999) and retina (Erskine et al., 2000; Plump et al., 2002). Slit2 can, however, stimulate DRG neurite elongation and branch formation (Wang et al., 1999; Brose and Tessier-Lavigne, 2000). Slits bind to receptors called roundabouts (Robo). Robo-1, -2, -3 and -4, are known in mammals, though the ability of the divergent family member, Robo-4, to bind Slits is controversial. Slit-1 and Slit-3 – but not Slit-2 – are strongly expressed in the center of partial transection injuries of the mouse spinal cord (Wehrle et al., 2005), mainly by macrophages and/or fibroblasts (presumably of meningeal origin). An antibody to Robo-1 and -2 produced strong staining of the corticospinal tract in adult mouse spinal cord (Sundaresan et al., 2004). In summary, slits and netrins are present at lesion sites in the spinal cord that are profoundly inhibitory for regenerating axons, and at least some axons in 1.4.2.3 31 32 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord adult mammals express suitable receptors. However, experimental evidence that they play a major role in preventing axonal regeneration in spinal cord is still awaited. 1.5 The Most Consistent Effects of Interfering with Inhibitory Molecules or Their Signaling Are on Raphespinal Axons When an overall view is taken of the effects of interfering with Nogo-A or NgR1 in vivo, the most obvious conclusion is that serotoninergic raphespinal axons are the most responsive fibers in terms of sprouting and regeneration. Serotoninergic axons have been reported to sprout and regenerate after spinal lesions treated with NEP1-40, NgRecto, IN-1, antibodies raised against Nogo-A, and antibodies against NgR1. They show marked regenerative responses in some Nogo-A knockout mice and in some NgR1 knockout mice. In view of the presence of the potent neurite outgrowth inhibitors ephrin B3 and Sema 4D in CNS myelin (Moreau-Fauvarque et al., 2003; Benson et al., 2005), and numerous inhibitors at the lesion site (whose signal transduction does not involve NgR1, LINGO-1, TROY or p75), it might be speculated that raphespinal axons may lack suitable receptor Ephs, Robos and mechanisms for transducing signals form CSPGs. However, such neurons also express high levels of growth-associated proteins and may have a special propensity for regeneration (Kruger et al., 1993; Cheng and Olson, 1995; McNamara and Lenox, 1997; Vinit et al., 2005). Serotonin has been shown to have important functions in motor control (Gerin et al., 1995; Nishimaru et al., 2000; Slawinska et al., 2000; Hains et al., 2001) which forms the basis of most tests used to show improvements in behavior after spinal injury; raphe spinal sprouting and regeneration may provide the anatomical substrate of much of the reported improvements in behavior. 1.6 Interfering with Downstream Effectors of Inhibitory Signaling Several strategies have been tried to enhance axonal regeneration in the mammalian CNS by targeting molecules in the signaling cascades from receptors for inhibitory molecules (see Fig. 1.3). 1.6.1 Cyclic AMP Can Modulate the Responses of Neurons to Inhibitory Molecules in Vitro But is Only a Weak Promoter of Axonal Regeneration in the Spinal Cord Neuronal cyclic nucleotide levels have powerful modulatory effects on signaling from inhibitory molecules in vitro, for example altering the effects of MAG on 1.6 Interfering with Downstream Effectors of Inhibitory Signaling Xenopus motor axons from repulsion to attraction (Song et al., 1998). Polyamine synthesis is downstream of cAMP in the pathway, reducing the effects of myelinderived inhibitors (Cai et al., 2002). Arginase-1 is a key enzyme in polyamine synthesis and is up-regulated after axotomy in peripheral neurons capable of vigorous regeneration (Costigan et al., 2002; Boeshore et al., 2004). Injection of cAMP into the sciatic DRGs has been reported to increase sprouting of ascending dorsal column axons into lesion sites in the thoracic cord (Qiu et al., 2002). Similarly, the phosphodiesterase inhibitor, rolipram, increased the sprouting of serotonergic axons into fetal cord grafts in cervical cord lesions in adult rats (Nikulina et al., 2004). Transfection of DRG neurons with a constitutively active cAMP response element binding protein (CREB), mediating the transcriptional effects of cAMP, also increased sprouting of dorsal column axons into spinal lesions (Gao et al., 2004). Finally, cAMP injections into DRGs combined with NT-3 treatment and implants of bone marrow stromal cells allowed dorsal column axons to regenerate at least 2 mm beyond cervical lesion sites (Lu et al., 2004). Little regeneration was found with cAMP treatment alone. The latter experimental results are likely to prove particularly robust because the absence of dorsal column nucleus labeling showed that the axons rostral to the lesion were regenerating, rather than spared, fibers. 1.6.2 Rho-A Inhibition The activation of RhoA is a common mechanism leading to growth cone collapse in response to signaling from NgR1 (Niederost et al., 2002), Ephs (Wahl et al., 2000), semaphorin receptors (Driessens et al., 2001; Swiercz et al., 2002) and CSPGs (Jain et al., 2004; Schweigreiter et al., 2004). Decreasing RhoA expression or inhibiting its activity should, therefore, be an interesting prospect for enhancing axonal regeneration in an environment containing myelin-derived and/or other inhibitory molecules. Treating retinal ganglion cells with C3 transferase, a potent inhibitor of Rho-A, has only a modest effect on optic axon regeneration, unless the cells also receive a pro-regenerative stimulus (Fischer et al., 2004; Bertrand et al., 2005). However, C3 transferase-mediated inactivation of Rho-A together with inhibition of ROCK (a downstream target) using Y27632 in mice with a thoracic dorsal hemisection was reported to result in regenerated corticospinal axons as far as 12 mm caudal to the lesion, as well as enhanced behavioral recovery (Dergham et al., 2002). However, C3 delivered via a minipump to a partial spinal cord transection site in adult rats failed to promote corticospinal regeneration, although Y27632 increased corticospinal spouting into the lesion (Fournier et al., 2003). Two possible explanations of this discrepancy are that Dergham et al. (2002) used a single large dose of C3, whereas Fournier et al. (2003) used continuous infusion at a lower dose; the large initial dose may have been necessary to allow regeneration. Alternatively, since C3 has been shown to exert a potent neuroprotective effect (Dubreuil et al., 2003), the treated animals in the experiments by Dergham et al. (2002) could have had more spared fibers. 33 34 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord In summary, since Rho-A is downstream of several receptors for inhibitory molecules, blocking Rho-A activation would be expected to produce more substantial disinhibition of regenerating axons in the spinal cord than blocking individual receptors. However, in general the effects on axonal regeneration in the CNS have not been as impressive as might have been expected. Either there are signaling pathways that do not involve Rho-A, or disinhibition is insufficient to produce extensive regeneration in the adult mammalian spinal cord. 1.6.3 Interfering with Other Targets on Signaling Pathways Inhibiting PKC can cause MAG or Nogo to stimulate neurite outgrowth from cerebellar granule cells in vitro (Hasegawa et al., 2004). Intrathecal infusion of a PKC inhibitor is reported to allow ascending dorsal column axons to regenerate up to 6 mm beyond a cervical dorsal hemisection of the spinal cord in adult rats (Sivasankaran et al., 2004). A screen for small molecules that could block the inhibition of neurite outgrowth on CNS myelin showed that EGFR antagonists could also prevent inhibitory signaling. An EGFR antagonist was able to promote the regeneration of large numbers of retinal ganglion cells axons for up to 2 mm following optic nerve crush in adult mice (Koprivica et al., 2005), but no experiments on spinal cord regeneration using the antagonist have yet been reported. 1.7 Inhibitory Molecules and the Control of Neuronal Growth-Associated Genes The most obvious effects of growth-inhibitory molecules on neurons in vitro are to produce growth cone collapse and/or repulsion of neurites. There are, however, intriguing in-vivo data that inhibitory molecules block aspects of the neuronal cell body response to axotomy, and that some of the treatments designed to interfere with signaling from inhibitory molecules may produce enhanced expression of neuronal growth-associated molecules. The IN-1 antibody against Nogo-A has been shown to produce up-regulation of some neuronal growth-associated proteins in both the spinal cord and cerebellum. Delivery of the IN-1 antibody to intact or pyramidotomized rats by an intracerebral hybridoma graft produced up-regulation in the spinal cord of genes associated with axonal growth including actin, myosin, and GAP-43 (Bareyre et al., 2002), together with aberrant sprouting of corticospinal axons. It was not clear in which neurons gene expression was altered, although corticospinal neurons with their cell bodies in the forebrain would not have been sampled. Spinal cord neurons express NgR1 weakly or not at all, so that it is unlikely that IN-1 blocked interactions between NgR1 on such neurons and Nogo-A. Many spinal neurons express Nogo-66 mRNA (Hunt et al., 2002a) and Nogo-A protein (Hunt et al., 2003). Other data derives from studies of the cerebellum where Purkinje cells are conspicuously unresponsive to axotomy (Vaudano et al., 1998; Chaisuksunt et al., 1.7 Inhibitory Molecules and the Control of Neuronal Growth-Associated Genes 2000; Rossi et al., 2001). Axotomized Purkinje cells up-regulate very few of the genes that are expressed in neurons that successfully regenerate axons (Zagrebelsky et al., 1998; Chaisuksunt et al., 2000). Application of IN-1 brought about upregulation of some growth-associated genes in both intact and axotomized Purkinje cells, including the transcription factors c-jun, P-Jun and JunD, and NADPH diaphorase (NOS) (Zagrebelsky et al., 1998). It may be significant that granule cells were unaffected by IN-1, although they express NgR1 (but not Nogo) at least as strongly as Purkinje cells. If IN-1 acted solely by blocking NgR1/Nogo interactions it would be expected that growth-associated proteins would also be up-regulated by granule cells. Overall, IN-1 has so far been reported to induce the up-regulation of some growth-associated neuronal genes, but not the strong up-regulation of dozens of such genes that are induced in regeneration-competent neurons following peripheral nerve injury. NgR1 knockout mice also showed increases in another growth-associated gene, small proline-rich repeat protein 1A (SPRR1A), in neuronal cell bodies and axons above and below a spinal cord lesion (Kim et al., 2004), but not in corticospinal or brainstem neurons. Presumably, NgR1 signaling represses SPRR1A up-regulation by axotomized neurons near the lesion site – those cells which would be expected to show the greatest response to axotomy. A similar result was obtained in rats with a thoracic cord lesion following infusion of the NgR1 antagonist NEP1-40 (Li and Strittmatter, 2003). Rubrospinal and corticospinal neurons do not up-regulate growth-associated genes in response to a thoracic axotomy (Fernandes et al., 1999; Mason et al., 2003). A possibly related observation is that an inhibitor of RhoA (downstream of NgR1; see below) applied to a thoracic cord lesion site both enhanced corticospinal regeneration and increased expression of the prototypic growth-associated neuronal gene, GAP-43, in neurons in the motor cortex (Dergham et al., 2002). Chondroitinase infusion into the subarachnoid space has been shown to produce up-regulation of GAP-43 in cervical DRG neurons (Bradbury et al., 2002). The most likely explanation is that GAGs in the spinal cord suppress the expression of GAP-43 in DRG neurons. Expression of other growth-associated genes was not examined and, as with IN-1, it is possible that the enzyme had a direct effect on the neurons in addition to modifying the environment around the injured axons. It is not yet clear whether the increased expression of neuronal growth-associated genes produced by treatments directed at blocking signaling from inhibitory molecules can explain any of their influence on axonal sprouting and regeneration. Is their most important action in vivo to prevent growth cone collapse, or to produce signals that allow a growth response in the neurons? Another, possibly related, insight into the complex nature of the responses to injury in the CNS is provided by evidence that neurotrophic stimulation (Cai et al., 1999; Logan et al., 2006) or a vigorous cell body response to axotomy (Leon et al., 2000) may decrease the sensitivity to inhibitory molecules. The control of axonal regeneration involves a dynamic interaction between many influences that are predominantly arranged for regeneration in peripheral nerves and against regeneration in the spinal cord. 35 36 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord 1.8 Conclusions A large number and variety of neurite outgrowth inhibitory molecules have been identified in the adult spinal cord, both in myelinated tracts and at lesion sites (see Fig. 1.5). In some cases (the myelin-derived inhibitors, ephrins, semaphorins and other axonal guidance molecules), specific receptors are known for the inhibitory molecules, yet none is expressed by all neurons. This leads to the perhaps surprising conclusion that different classes of axon may be prevented from regenerating by different inhibitory molecules. Jerry Silver has compared the injured CNS to a shark (inhibitory molecule)-infested sea; it appears that the sharks are of many species, preying on different sets of axons. This diversity may explain the limited range of axons that can be stimulated to regenerate by interfering with Nogo or its receptors, for example. However, corticospinal tract neurons seem to express receptors for a number of inhibitors including Sema 3A, ephrins and myelin-derived inhibitors, and most axons are sensitive to inhibitory CSPGs. In general, it is serotonergic axons with a known propensity for regeneration, which have been most successfully stimulated to regenerate by disinhibitory treatment or genetic manipulation of inhibitory signaling. These axons have been claimed to regenerate across complete spinal cord transections with purely surgical treatment (Cheng and Olson, 1995). One of the greatest disappointments in the study of myelin-derived inhibitors has been the failure of conventional knockout mice to provide a clear picture of the roles of such inhibitors or their receptors. Although analysis of such mice in more homogeneous genetic backgrounds may clarify matters, it seems likely that conditional knockouts – for example, inactivating Nogo-A in oligodendrocytes or neurons separately in adult animals – may be required for an accurate analysis of the significance of some inhibitors. NgR1 and its ligands clearly play a part in limiting the regeneration of some types of axon. Nonetheless, since some neurons do not express NgR1 and since its inactivation has failed to produce significant regeneration of one class of axon (the corticospinal tracts) that express the receptor strongly, it is difficult to conclude that NgR1 signaling is the major cause of the failure of regeneration in the spinal cord. Surprisingly, inactivating NgR1 has been shown to allow rubrospinal and raphespinal axons to regenerate across complete transections of the spinal cord – that is, through regions lacking the myelinderived inhibitors. This may be explained by invoking the possibility that overcoming the inhibitory effects of some myelin-derived factors may up-regulate neuronal growth-associated genes, altering the growth state of the neurons, so that vigorously regenerating axons can overcome other inhibitory influences. Such considerations may also explain the strong evidence that some neurons can regenerate axons through CNS white matter. Finally, it is not clear why data indicating that myelin possesses inhibitory molecules that do not signal through NgR1 (AminoNogo, Sema 4D, ephrin B3) are largely ignored. With regard to inhibitors at the lesion site, remarkable effects have been claimed for interfering with various molecules, but in each case by a very limited number References of studies. The single report showing the dramatic effects on axonal regeneration of knocking out EphA4 suggests that interfering with EphA4 signaling may be a tool to allow both manipulation of the lesion site and disinhibition of some axons. The relatively few published experiments using chondroitinase to digest GAGs were aimed directly at manipulating the environment in and around the lesion site. Both an enhanced neuronal cell body response to axotomy and impressive axonal regeneration in the spinal cord have been reported in such experiments. The use of decorin to down-regulate inhibitory CSPGs at lesion sites also appears promising. However, the publication of attempts at repetition and development of these experiments is urgently needed, even if the experiments produce negative results. It is, therefore, too early to be confident that the most significant inhibitory molecules at lesion sites have been identified and their effects overcome. Furthermore, the outcome of the numerous studies that have attempted to elicit regeneration by blocking inhibitory signaling suggests that disinhibition may be only one stage in that process. Direct stimulation of the feeble cell body response to axotomy shown by many intrinsic CNS neurons, together with the implantation of regeneration-conducive cells into the lesion site may also be required if axons are to regenerate vigorously in the adult mammalian spinal cord. References Ahmed Z, Dent RG, Suggate EL, Barrett LB, Seabright RJ, Berry M, Logan A (2005) Disinhibition of neurotrophininduced dorsal root ganglion cell neurite outgrowth on CNS myelin by siRNAmediated knockdown of NgR, p75NTR and Rho-A. Mol. Cell. Neurosci. 28: 509–523. Ahmed Z, Suggate EL, Brown ER, Dent RG, Armstrong SJ, Barrett LB, Berry M, Logan A (2006) Schwann cell-derived factor-induced modulation of the NgR/ p75NTR/EGFR axis disinhibits axon growth through CNS myelin in vivo and in vitro. Brain 129: 1517–1533. Anderson PN, Lieberman AR (1999) Intrinsic determinants of differential axonal regeneration by adult mammalian CNS neurons. In: Saunders NR, Dziegielewska KM (Eds.), Degeneration and regeneration in the nervous system. Harwood Academic Press, pp. 53–75. Anderson PN, Campbell G, Zhang Y, Lieberman AR (1998) Cellular and molecular correlates of the regeneration of adult mammalian CNS axons into peripheral nerve grafts. Prog. Brain Res. 117: 211–232. Barallobre MJ, Pascual M, del Rio JA, Soriano E (2005) The Netrin family of guidance factors: emphasis on Netrin-1 signalling. Brain Res. Brain Res. Rev. 49: 22–47. Bareyre FM, Haudenschild B, Schwab ME (2002) Long-lasting sprouting and gene expression changes induced by the monoclonal antibody IN-1 in the adult spinal cord. J. Neurosci. 22: 7097– 7110. Barnes G, Puranam RS, Luo Y, McNamara JO (2003) Temporal specific patterns of semaphorin gene expression in rat brain after kainic acid-induced status epilepticus. Hippocampus 13: 1–20. Barton WA, Liu BP, Tzvetkova D, Jeffrey PD, Fournier AE, Sah D, Cate R, Strittmatter SM, Nikolov DB (2003) Structure and axon outgrowth inhibitor binding of the Nogo-66 receptor and related proteins. EMBO J. 22: 3291–3302. Bartsch U, Bandtlow CE, Schnell L, Bartsch S, Spillmann AA, Rubin BP, Hillenbrand R, Montag D, Schwab ME, Schachner M (1995a) Lack of evidence that myelin-associated glycoprotein is a 37 38 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord major inhibitor of axonal regeneration in the CNS. Neuron 15: 1375–1381. Bartsch U, Bandtlow CE, Schnell L, Bartsch S, Spillmann AA, Rubin BP, Hillenbrand R, Montag D, Schwab ME, Schachner M (1995b) Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS. Neuron 15: 1375–1381. Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12: 1–21. Bavetta S, Hamlyn PJ, Burnstock G, Lieberman AR, Anderson PN (1999) The effects of FK506 on dorsal column axons following spinal cord injury in adult rats: neuroprotection and local regeneration. Exp. Neurol. 158: 382–393. Bennett DLH, Averill S, Priestley JV, McMahon SB (1996) Postnatal changes in the expression of trkA high affinity NGF receptor in primary sensory neurones. Eur. J. Neurosci. 8: 2204–2208. Benson MD, Romero MI, Lush ME, Lu QR, Henkemeyer M, Parada LF (2005) EphrinB3 is a myelin-based inhibitor of neurite outgrowth. Proc. Natl. Acad. Sci. USA 102: 10694–10699. Berry M (1982) Post-injury myelin-breakdown products inhibit axonal growth: an hypothesis to explain the failure of axonal regeneration in the mammalian central nervous system. Bibl. Anat. 23: 1–11. Berry M, Carlile J, Hunter A (1996) Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J. Neurocytol. 25: 147–170. Bertrand J, Winton MJ, RodriguezHernandez N, Campenot RB, McKerracher L (2005) Application of Rho antagonist to neuronal cell bodies promotes neurite growth in compartmented cultures and regeneration of retinal ganglion cell axons in the optic nerve of adult rats. J. Neurosci. 25: 1113–1121. Bjorklund A, Stenevi U (1979) Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system. Physiol. Rev. 59: 62–100. Boeshore KL, Schreiber RC, Vaccariello SA, Sachs HH, Salazar R, Lee J, Ratan RR, Leahy P, Zigmond RE (2004) Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis. J. Neurobiol. 59: 216–235. Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, McMahon SB (2002) Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416: 636–640. Bregman BS, Kunkel-Bagden E, Schnell L, Ning Dai H, Gao D, Schwab ME (1995) Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature 378: 498–501. Bresnahan JC, Beattie MS, Stokes BT, Conway KM (1991) Three-dimensional computer-assisted analysis of graded contusion lesions in the spinal cord of the rat. J. Neurotrauma 8: 91–101. Bronfman FC, Fainzilber M (2004) Multitasking by the p75 neurotrophin receptor: sortilin things out? EMBO Rep. 5: 867–871. Brosamle C, Huber AB, Fiedler M, Skerra A, Schwab ME (2000) Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J. Neurosci. 20: 8061–8068. Brose K, Tessier-Lavigne M (2000) Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr. Opin. Neurobiol. 10: 95–102. Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, Tessier-Lavigne M, Kidd T (1999) Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96: 795–806. Buck CR, Martinez HJ, Chao MV, Black IB (1988) Differential expression of the nerve growth factor receptor gene in multiple brain areas. Brain Res. Dev. Brain Res. 44: 259–268. Bundesen LQ, Scheel TA, Bregman BS, Kromer LF (2003) Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats. J. Neurosci. 23: 7789–7800. Cadelli D, Schwab ME (1991) Regeneration of lesioned septohippocampal acetylcholinesterase-positive axons is improved by antibodies against the myelin-associated References neurite growth inhibitors NI-35/250. Eur. J. Neurosci. 3: 825–832. Caggiano AO, Zimber MP, Ganguly A, Blight AR, Gruskin EA (2005) Chondroitinase ABCI improves locomotion and bladder function following contusion injury of the rat spinal cord. J. Neurotrauma 22: 226–239. Cai D, Shen Y, De Bellard M, Tang S, Filbin MT (1999) Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron 22: 89–101. Cai D, Deng K, Mellado W, Lee J, Ratan RR, Filbin MT (2002) Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron 35: 711–719. Calderon RO, Attema B, DeVries GH (1995) Lipid composition of neuronal cell bodies and neurites from cultured dorsal root ganglia. J. Neurochem. 64: 424–429. Campbell G, Holt JKL, Shotton HR, Anderson PN, Bavetta S, Lieberman AR (1999) Spontaneous regeneration after optic nerve injury in adult rat. NeuroReport 10: 3955–3960. Carim-Todd L, Escarceller M, Estivill X, Sumoy L (2003) LRRN6A/LERN1 (leucinerich repeat neuronal protein 1), a novel gene with enriched expression in limbic system and neocortex. Eur. J. Neurosci. 18: 3167–3182. Caroni P, Schwab ME (1988a) Two membrane proteins fractions from rat central myelin with inhibitory properties for neurite outgrowth. J. Cell Biol. 106: 1281–1288. Caroni P, Schwab ME (1988b) Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron 1: 85–96. Caroni P, Savio T, Schwab ME (1988) Central nervous system regeneration: oligodendrocytes and myelin as nonpermissive substrates for neurite growth. Prog. Brain Res. 78: 363–370. Carulli D, Laabs T, Geller HM, Fawcett JW (2005) Chondroitin sulfate proteoglycans in neural development and regeneration. Curr. Opin. Neurobiol. 15: 116–120. Castellani V, Chedotal A, Schachner M, Faivre-Sarrailh C, Rougon G (2000) Analysis of the L1-deficient mouse phenotype reveals cross-talk between Sema3A and L1 signaling pathways in axonal guidance. Neuron 27: 237–249. Chaisuksunt V, Zhang Y, Anderson PN, Campbell G, Vaudano E, Schachner M, Lieberman AR (2000) Patterns of expression and distribution of mRNAs for L1, CHL1, c-jun and GAP-43 in identified regenerating neurons of the cerebellum and brainstem of the adult rat. Neuroscience 100: 87–108. Chen H, Chedotal A, He Z, Goodman CS, Tessier-Lavigne M (1997) Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 19: 547–559. Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F, Schwab ME (2000) Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403: 434–439. Cheng H, Olson L (1995) A new surgical technique that allows proximodistal regeneration of 5-HT fibers after complete transection of the rat spinal cord. Exp. Neurol. 136: 149–161. Chisholm A, Tessier-Lavigne M (1999) Conservation and divergence of axon guidance mechanisms. Curr. Opin. Neurobiol. 9: 603–615. Chong M-S, Woolf CJ, Turmaine M, Emson PC, Anderson PN (1996) Intrinsic vs extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurons: the influence of a peripheral nerve graft. J. Comp. Neurol. 370: 97–104. Chong M-S, Woolf CJ, Haque NSK, Anderson PN (1999) Regeneration of axons from injured dorsal roots into the spinal cord in adult rats. J. Comp. Neurol. 410: 42–54. Corvetti L, Rossi F (2005) Degradation of chondroitin sulfate proteoglycans induces sprouting of intact purkinje axons in the cerebellum of the adult rat. J. Neurosci. 25: 7150–7158. Costigan M, Befort K, Karchewski L, Griffin RS, D’Urso D, Allchorne A, 39 40 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord Sitarski J, Mannion JW, Pratt RE, Woolf CJ (2002) Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci. 3: 16. Crocker PR, Clark EA, Filbin M, Gordon S, Jones Y, Kehrl JH, Kelm S, Le Douarin N, Powell L, Roder J, Schnaar RL, Sgroi DC, Stamenkovic K, Schauer R, Schachner M, van den Berg TK, van der Merwe PA, Watt SM, Varki A (1998) Siglecs: a family of sialic-acid binding lectins. Glycobiology 8: v–vi. Crutcher KA (1981) Cholinergic denervation of rat neocortex results in sympathetic innervation. Exp. Neurol. 74: 324–329. Crutcher KA, Davis JN (1981) Sympathohippocampal sprouting is directed by a target tropic factor. Brain Res. 204: 410–414. Crutcher KA, Marfurt CF (1988) Nonregenerative axonal growth within the mature mammalian brain: ultrastructural identification of sympathohippocampal sprouts. J. Neurosci. 8: 2289–2302. Crutcher KA, Brothers L, Davis JN (1979) Sprouting of sympathetic nerves in the absence of afferent input. Exp. Neurol. 66: 778–783. Csordas G, Santra M, Reed CC, Eichstetter I, McQuillan DJ, Gross D, Nugent MA, Hajnoczky G, Iozzo RV (2000) Sustained down-regulation of the epidermal growth factor receptor by decorin. A mechanism for controlling tumor growth in vivo. J. Biol. Chem. 275: 32879–328872. Davies JE, Tang X, Denning JW, Archibald SJ, Davies SJ (2004) Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries. Eur. J. Neurosci. 19: 1226–1242. Davies SJ, Field PM, Raisman G (1994) Long interfascicular axon growth from embryonic neurons transplanted into adult myelinated tracts. J. Neurosci. 14: 1596–1612. Davies SJ, Fitch MT, Memberg SP, Hall AK, Raisman G, Silver J (1997) Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390: 680– 683. Davies SJ, Goucher DR, Doller C, Silver J (1999) Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19: 5810–5822. Dechant G, Barde YA (2002) The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat. Neurosci. 5: 1131–1136. Dergham P, Ellezam B, Essagian C, Avedissian H, Lubell WD, McKerracher L (2002) Rho signaling pathway targeted to promote spinal cord repair. J. Neurosci. 22: 6570–6577. De Winter F, Oudega M, Lankhorst AJ, Hamers FP, Blits B, Ruitenberg MJ, Pasterkamp RJ, Gispen WH, Verhaagen J (2002) Injury-induced class 3 semaphorin expression in the rat spinal cord. Exp. Neurol. 175: 61–75. de Wit J, Verhaagen J (2003) Role of semaphorins in the adult nervous system. Prog. Neurobiol. 71: 249–267. Dodd DA, Niederoest B, Bloechlinger S, Dupuis L, Loeffler JP, Schwab ME (2005) Nogo-A, -B, and -C are found on the cell surface and interact together in many different cell types. J. Biol. Chem. 280: 12494–12502. Domeniconi M, Zampieri N, Spencer T, Hilaire M, Mellado W, Chao MV, Filbin MT (2005) MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth. Neuron 46: 849–855. Dou CL, Levine JM (1994) Inhibition of neurite growth by the NG2 chondroitin sulfate proteoglycan. J. Neurosci. 14: 7616–7628. Driessens MH, Hu H, Nobes CD, Self A, Jordens I, Goodman CS, Hall A (2001) Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr. Biol. 11: 339–344. Dubreuil CI, Winton MJ, McKerracher L (2003) Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system. J. Cell Biol. 162: 233–243. Eberhart J, Swartz M, Koblar SA, Pasquale EB, Tanaka H, Krull CE (2000) Expression of EphA4, ephrin-A2 and ephrin-A5 during axon outgrowth to the hindlimb indicates potential roles in pathfinding. Dev. Neurosci. 22: 237–250. References Eitan S, Solomon A, Lavie V, Yoles E, Hirschberg DL, Belkin M, Schwartz M (1994) Recovery of visual response of injured adult rat optic nerves treated with transglutaminase. Science 264: 1764–1768. Erskine L, Williams SE, Brose K, Kidd T, Rachel RA, Goodman CS, TessierLavigne M, Mason CA (2000) Retinal ganglion cell axon guidance in the mouse optic chiasm: expression and function of robos and slits. J. Neurosci. 20: 4975–4982. Fabes J, Anderson PN, Yáñez-Muñoz R, Thrasher A, Brennan C, Bolsover S (2006) Accumulation of the inhibitory receptor EphA4 may prevent regeneration of corticospinal tract axons following lesion. Eur. J. Neurosci. 23(7): 1721–1730. Fawcett JW, Asher RA (1999) The glial scar and central nervous system repair. Brain Res. Bull. 49: 377–391. Fernandes KJ, Fan DP, Tsui BJ, Cassar SL, Tetzlaff W (1999) Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M. J. Comp. Neurol. 414: 495–510. Ferri CC, Moore FA, Bisby MA (1998) Effects of facial nerve injury on mouse motor neurons lacking the p75 low-affinity neurotrophin receptor. J. Neurobiol. 34: 1–9. Fischer D, Petkova V, Thanos S, Benowitz LI (2004) Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J. Neurosci. 24: 8726–8740. Fouad K, Klusman I, Schwab ME (2004) Regenerating corticospinal fibers in the Marmoset (Callitrix jacchus) after spinal cord lesion and treatment with the antiNogo-A antibody IN-1. Eur. J. Neurosci. 20: 2479–2482. Fouad K, Schnell L, Bunge MB, Schwab ME, Liebscher T, Pearse DD (2005) Combining Schwann cell bridges and olfactory-ensheathing glia grafts with chondroitinase promotes locomotor recovery after complete transection of the spinal cord. J. Neurosci. 25: 1169–1178. Fournier AE, GrandPre T, Strittmatter SM (2001) Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 409: 341–346. Fournier AE, Gould GC, Liu BP, Strittmatter SM (2002) Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J. Neurosci. 22: 8876–8883. Fournier AE, Takizawa BT, Strittmatter SM (2003) Rho kinase inhibition enhances axonal regeneration in the injured CNS. J. Neurosci. 23: 1416–1423. Freywald A, Sharfe N, Rashotte C, Grunberger T, Roifman CM (2003) The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptormediated responses. J. Biol. Chem. 278: 10150–10156. Fujita H, Zhang B, Sato K, Tanaka J, Sakanaka M (2001) Expressions of neuropilin-1, neuropilin-2 and semaphorin 3A mRNA in the rat brain after middle cerebral artery occlusion. Brain Res. 914: 1–14. Fujitani M, Kawai H, Proia RL, Kashiwagi A, Yasuda H, Yamashita T (2005) Binding of soluble myelin-associated glycoprotein to specific gangliosides induces the association of p75NTR to lipid rafts and signal transduction. J. Neurochem. 94: 15–21. Gao Y, Deng K, Hou J, Bryson JB, Barco A, Nikulina E, Spencer T, Mellado W, Kandel ER, Filbin MT (2004) Activated CREB is sufficient to overcome inhibitors in myelin and promote spinal axon regeneration in vivo. Neuron 44: 609–621. Gerin C, Becquet D, Privat A (1995) Direct evidence for the link between monoaminergic descending pathways and motor activity. I. A study with microdialysis probes implanted in the ventral funiculus of the spinal cord. Brain Res. 704: 191–201. Gilbert RJ, McKeon RJ, Darr A, Calabro A, Hascall VC, Bellamkonda RV (2005) CS-4,6 is differentially upregulated in glial scar and is a potent inhibitor of neurite extension. Mol. Cell. Neurosci. 29: 545–558. Goldshmit Y, Galea MP, Wise G, Bartlett PF, Turnley AM (2004) Axonal regeneration and lack of astrocytic gliosis in EphA4deficient mice. J. Neurosci. 24: 10064–10073. GrandPre T, Nakamura F, Vartanian T, Strittmatter SM (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403: 439–444. GrandPre T, Li S, Strittmatter SM (2002) Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 417: 547–551. 41 42 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord Gschwendtner A, Liu Z, Hucho T, Bohatschek M, Kalla R, Dechant G, Raivich G (2003) Regulation, cellular localization, and function of the p75 neurotrophin receptor (p75NTR) during the regeneration of facial motoneurons. Mol. Cell. Neurosci. 24: 307–322. Habib AA, Marton LS, Allwardt B, Gulcher JR, Mikol DD, Hognason T, Chattopadhyay N, Stefansson K (1998) Expression of the oligodendrocyte-myelin glycoprotein by neurons in the mouse central nervous system. J. Neurochem. 70: 1704–1711. Hafner C, Schmitz G, Meyer S, Bataille F, Hau P, Langmann T, Dietmaier W, Landthaler M, Vogt T (2004) Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers. Clin. Chem. 50: 490–499. Hagg T, Baker KA, Emsley JG, Tetzlaff W (2005) Prolonged local neurotrophin-3 infusion reduces ipsilateral collateral sprouting of spared corticospinal axons in adult rats. NeuroReport 130: 875–887. Hains BC, Johnson KM, McAdoo DJ, Eaton MJ, Hulsebosch CE (2001) Engraftment of serotonergic precursors enhances locomotor function and attenuates chronic central pain behavior following spinal hemisection injury in the rat. Exp. Neurol. 171: 361–378. Hartmann U, Maurer P (2001) Proteoglycans in the nervous system – the quest for functional roles in vivo. Matrix Biol. 20: 23–35. Harvey AR, Tan MM (1992) Spontaneous regeneration of adult rat retinal ganglion cell axons in vivo. NeuroReport 3: 239–242. Hasegawa Y, Fujitani M, Hata K, Tohyama M, Yamagishi S, Yamashita T (2004) Promotion of axon regeneration by myelinassociated glycoprotein and Nogo through divergent signals downstream of Gi/G. J. Neurosci. 24: 6826–6832. Hauben E, Ibarra A, Mizrahi T, Barouch R, Agranov E, Schwartz M (2001) Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury promotes recovery via a T-cell-mediated neuroprotective response: comparison with other myelin antigens. Proc. Natl. Acad. Sci. USA 98: 15173–15178. Hendriks WT, Eggers R, Ruitenberg MJ, Blits B, Hamers FP, Verhaagen J, Boe GJ (2006) Profound differences in spontaneous long-term functional recovery after defined spinal tract lesions in the rat. J. Neurotrauma 23: 18–35. Henkemeyer M, Orioli D, Henderson JT, Saxton TM, Roder J, Pawson T, Klein R (1996) Nuk controls pathfinding of commissural axons in the mammalian central nervous system. Cell 86: 35–46. Hirsch S, Labes M, Bahr M (2000) Changes in BDNF and neurotrophin receptor expression in degenerating and regenerating rat retinal ganglion cells. Restor. Neurol. Neurosci. 17: 125–134. Holtmaat AJ, De Winter F, de Wit J, Gorter JA, da Silva FH, Verhaagen J (2002) Semaphorins: contributors to structural stability of hippocampal networks? Prog. Brain Res. 138: 17–38. Hopker VH, Shewan D, Tessier-Lavigne M, Poo M, Holt C (1999) Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature 401: 69–73. Hsieh SH, Ferraro GB, Fournier AE (2006) Myelin-associated inhibitors regulate cofilin phosphorylation and neuronal inhibition through LIM kinase and Slingshot phosphatase. J. Neurosci. 26: 1006–1015. Hu B, Yip HK, So KF (1999) Expression of p75 neurotrophin receptor in the injured and regenerating rat retina. NeuroReport 10: 1293–1297. Huang DW, McKerracher L, Braun PE, David S (1999) A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron 24: 639–647. Huang JK, Phillips GR, Roth AD, Pedraza L, Shan W, Belkaid W, Mi S, FexSvenningsen A, Florens L, Yates JR, III, Colman DR (2005) Glial membranes at the node of Ranvier prevent neurite outgrowth. Science 310: 1813–1817. Huber AB, Weinmann O, Brosamle C, Oertle T, Schwab ME (2002) Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions. J. Neurosci. 22: 3553–3567. Hunt D, Mason MRJ, Campbell G, Coffin R, Anderson PN (2002a) Nogo receptor mRNA expression in intact and regenerating CNS neurons. Mol. Cell. Neurosci. 20: 537–552. Hunt D, Coffin R, Anderson PN (2002b) The Nogo receptor, its ligands and axonal References regeneration in the spinal cord: a review. J. Neurocytol. 31: 93–120. Hunt D, Coffin RS, Prinjha RK, Campbell G, Anderson PN (2003) Nogo-A expression in the intact and injured nervous system. Mol. Cell. Neurosci. 24: 1083– 10102. Huynh-Do U, Vindis C, Liu H, Cerretti DP, McGrew JT, Enriquez M, Chen J, Daniel TO (2002) Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis. J. Cell Sci. 115: 3073–3081. Hynds DL, Snow DM (1999) Neurite outgrowth inhibition by chondroitin sulfate proteoglycan: stalling/stopping exceeds turning in human neuroblastoma growth cones. Exp. Neurol. 160: 244–255. Iwamasa H, Ohta K, Yamada T, Ushijima K, Terasaki H, Tanaka H (1999) Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles. Dev. Growth Differ. 41: 685–698. Jain A, Brady-Kalnay SM, Bellamkonda RV (2004) Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycandependent inhibition of neurite extension. J. Neurosci. Res. 77: 299–307. Johnson PW, Abramow-Newerly W, Seilheimer B, Sadoul R, Tropak MB, Arquint M, Dunn RJ, Schachner M, Roder JC (1989) Recombinant myelinassociated glycoprotein confers neural adhesion and neurite outgrowth function. Neuron 3: 377–385. Josephson A, Widenfalk J, Widmer HW, Olson L, Spenger C (2001) NOGO mRNA expression in adult and fetal human and rat nervous tissue and in weight drop injury. Exp. Neurol. 169: 319–328. Josephson A, Trifunovski A, Widmer HR, Widenfalk J, Olson L, Spenger C (2002) Nogo-receptor gene activity: Cellular localization and developmental regulation of mRNA in mice and humans. J. Comp. Neurol. 453: 292–304. Jung KM, Tan S, Landman N, Petrova K, Murray S, Lewis R, Kim PK, Kim DS, Ryu SH, Chao MV, Kim TW (2003) Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor. J. Biol. Chem. 278: 42161–42169. Junghans U, Koops A, Westmeyer A, Kappler J, Meyer HE, Muller HW (1995) Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan. Eur. J. Neurosci. 7: 2341–2350. Kanning KC, Hudson M, Amieux PS, Wiley JC, Bothwell M, Schecterson LC (2003) Proteolytic processing of the p75 neurotrophin receptor and two homologs generates C-terminal fragments with signaling capability. J. Neurosci. 23: 5425–5436. Kantor DB, Chivatakarn O, Peer KL, Oster SF, Inatani M, Hansen MJ, Flanagan JG, Yamaguchi Y, Sretavan DW, Giger RJ, Kolodkin AL (2004) Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans. Neuron 44: 961–975. Kartje GL, Schulz MK, Lopez-Yunez A, Schnell L, Schwab ME (1999) Corticostriatal plasticity is restricted by myelinassociated neurite growth inhibitors in the adult rat. Ann. Neurol. 45: 778–786. Kawaja MD, Crutcher KA (1997) Sympathetic axons invade the brains of mice overexpressing nerve growth factor. J. Comp. Neurol. 383: 60–72. Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM (2003) Axon regeneration in young adult mice lacking nogo-a/b. Neuron 38: 187–199. Kim JE, Liu BP, Park JH, Strittmatter SM (2004) Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron 44: 439–451. Knoll B, Zarbalis K, Wurst W, Drescher U (2001) A role for the EphA family in the topographic targeting of vomeronasal axons. Development 128: 895–906. Koprivica V, Cho KS, Park JB, Yiu G, Atwal J, Gore B, Kim JA, Lin E, TessierLavigne M, Chen DF, He Z (2005) EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science 310: 106–110. Kottis V, Thibault P, Mikol D, Xiao ZC, Zhang R, Dergham P, Braun PE (2002) Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth. J. Neurochem. 82: 1566–1569. 43 44 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord Kruger L, Bendotti C, Rivolta R, Samanin R (1993) Distribution of GAP-43 mRNA in the adult rat brain. J. Comp. Neurol. 333: 417–434. Kullander K, Klein R (2002) Mechanisms and functions of Eph and ephrin signalling. Nat. Rev. Mol. Cell. Biol. 3: 475–486. Kullander K, Croll SD, Zimmer M, Pan L, McClain J, Hughes V, Zabski S, DeChiara TM, Klein R, Yancopoulos GD, Gale NW (2001) Ephrin-B3 is the midline barrier that prevents corticospinal tract axons from recrossing, allowing for unilateral motor control. Genes Dev. 15: 877– 888. Lankhorst AJ, ter Laak MP, van Laar TJ, Van Meeteren NL, de Groot JC, Schrama LH, Hamers FP, Gispen WH (2001) Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat. J. Neurotrauma 18: 203–215. Lauren J, Airaksinen MS, Saarma M, Timmusk T (2003) Two novel mammalian Nogo receptor homologs differentially expressed in the central and peripheral nervous systems. Mol. Cell. Neurosci. 24: 581–594. Lee SK, Wolfe SW (2000) Peripheral nerve injury and repair. J. Am. Acad. Orthoped. Surg. 8: 243–252. Leon S, Yin Y, Nguyen J, Irwin N, Benowitz LI (2000) Lens injury stimulates axon regeneration in the mature rat optic nerve. J. Neurosci. 20: 4615–4626. Li S, Strittmatter SM (2003) Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J. Neurosci. 23: 4219–4227. Li S, Liu BP, Budel S, Li M, Ji B, Walus L, Li W, Jirik A, Rabacchi S, Choi E, Worley D, Sah DW, Pepinsky B, Lee D, Relton J, Strittmatter SM (2004) Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J. Neurosci. 24: 10511–10520. Li Y, Field PM, Raisman G (1997) Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells. Science 277: 2000–2002. Liebl DJ, Morris CJ, Henkemeyer M, Parada LF (2003) mRNA expression of ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse central nervous system. J. Neurosci. Res. 71: 7–22. Liebscher T, Schnell L, Schnell D, Scholl J, Schneider R, Gullo M, Fouad K, Mir A, Rausch M, Kindler D, Hamers FP, Schwab ME (2005) Nogo-A antibody improves regeneration and locomotion of spinal cord-injured rats. Ann. Neurol. 58: 706–719. Lips K, Stichel CC, Muller HW (1995) Restricted appearance of tenascin and chondroitin sulphate proteoglycans after transection and sprouting of adult rat postcommissural fornix. J. Neurocytol. 24: 449–464. Liu BP, Strittmatter SM (2001) Semaphorinmediated axonal guidance via Rho-related G proteins. Curr. Opin. Cell Biol. 13: 619–626. Liu BP, Fournier A, GrandPre T, Strittmatter SM (2002) Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science 297: 1190–1193. Logan A, Baird A, Berry M (1999) Decorin attenuates gliotic scar formation in the rat cerebral hemisphere. Exp. Neurol. 159: 504–510. Logan A, Ahmed Z, Baird A, Gonzalez AM, Berry M (2006) Neurotrophic factor synergy is required for neuronal survival and disinhibited axon regeneration after CNS injury. Brain 129: 490–502. Lorber B, Berry M, Logan A (2005) Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors. Eur. J. Neurosci. 21: 2029–2034. Lu P, Yang H, Jones LL, Filbin MT, Tuszynski MH (2004) Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury. J. Neurosci. 24: 6402–6409. Ma M, Basso DM, Walters P, Stokes BT, Jakeman LB (2001) Behavioral and histological outcomes following graded spinal cord contusion injury in the C57Bl/6 mouse. Exp. Neurol. 169: 239–254. Massey JM, Hubscher CH, Wagoner MR, Decker JA, Amps J, Silver J, Onifer SM (2006) Chondroitinase ABC digestion of the perineuronal net promotes functional collateral sprouting in the cuneate nucleus References after cervical spinal cord injury. J. Neurosci. 26: 4406–4414. Manitt C, Colicos MA, Thompson KM, Rousselle E, Peterson AC, Kennedy TE (2001) Widespread expression of netrin-1 by neurons and oligodendrocytes in the adult mammalian spinal cord. J. Neurosci. 21: 3911–3922. Mason MRJ, Lieberman AR, Anderson PN (2003) Corticospinal neurons upregulate a range of growth-associated genes following intracortical, but not spinal, axotomy. Eur. J. Neurosci. 18: 789–802. Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K (1996) Rhoassociated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15: 2208– 2216. McKeon RJ, Hoke A, Silver J (1995) Injuryinduced proteoglycans inhibit the potential for laminin-mediated axon growth on astrocytic scars. Exp. Neurol. 136: 32–43. McKerracher L, David S, Jackson DL, Kottis V, Dunn RJ, Braun PE (1994) Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron 13: 805–811. McNamara RK, Lenox RH (1997) Comparative distribution of myristoylated alanine-rich C kinase substrate (MARCKS) and F1/GAP-43 gene expression in the adult rat brain. J. Comp. Neurol. 379: 48–71. Mi S, Lee X, Shao Z, Thill G, Ji B, Relton J, Levesque M, Allaire N, Perrin S, Sands B, Crowell T, Cate RL, McCoy JM, Pepinsky RB (2004) LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat. Neurosci. 7(3): 221–228. Mi S, Miller RH, Lee X, Scott ML, ShulagMorskaya S, Shao Z, Chang J, Thill G, Levesque M, Zhang M, Hession C, Sah D, Trapp B, He Z, Jung V, McCoy JM, Pepinsky RB (2005) LINGO-1 negatively regulates myelination by oligodendrocytes. Nat. Neurosci. 8: 745–751. Mikule K, Sunpaweravong S, Gatlin JC, Pfenninger KH (2003) Eicosanoid activation of protein kinase C epsilon: involvement in growth cone repellent signaling. J. Biol. Chem. 278: 21168– 21177. Mingorance A, Fontana X, Sole M, Burgaya F, Urena JM, Teng FY, Tang BL, Hunt D, Anderson PN, Bethea JR, Schwab ME, Soriano E, del Rio JA (2004) Regulation of Nogo and Nogo receptor during the development of the entorhinohippocampal pathway and after adult hippocampal lesions. Mol. Cell. Neurosci. 26: 34–49. Miranda JD, White LA, Marcillo AE, Willson CA, Jagid J, Whittemore SR (1999) Induction of Eph B3 after spinal cord injury. Exp. Neurol. 156: 218–222. Moon LD, Asher RA, Rhodes KE, Fawcett JW (2001) Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC. Nat. Neurosci. 4: 465–466. Moon LD, Asher RA, Fawcett JW (2003) Limited growth of severed CNS axons after treatment of adult rat brain with hyaluronidase. J. Neurosci. Res. 71: 23–37. Moreau-Fauvarque C, Kumanogoh A, Camand E, Jaillard C, Barbin G, Boquet I, Love C, Jones EY, Kikutani H, Lubetzki C, Dusart I, Chedotal A (2003) The transmembrane semaphorin Sema4D/ CD100, an inhibitor of axonal growth, is expressed on oligodendrocytes and upregulated after CNS lesion. J. Neurosci. 23: 9229–9239. Moss A, Alvares D, Meredith-Middleton J, Robinson M, Slater R, Hunt SP, Fitzgerald M (2005) Ephrin-A4 inhibits sensory neurite outgrowth and is regulated by neonatal skin wounding. Eur. J. Neurosci. 22: 2413–2421. Mukhopadhyay G, Doherty P, Walsh FS, Crocker PR, Filbin MT (1994) A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron 13: 757–767. Munoz LM, Zayachkivsky A, Kunz RB, Hunt JM, Wang G, Scott SA (2005) Ephrin-A5 inhibits growth of embryonic sensory neurons. Dev. Biol. 283: 397–408. Murakami Y, Suto F, Shimizu M, Shinoda T, Kameyama T, Fujisawa H (2001) Differential expression of plexin-A subfamily members in the mouse nervous system. Dev. Dyn. 220: 246–258. Neumann S, Woolf CJ (1999) Regeneration of dorsal column fibres into and beyond the 45 46 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord lesion site following adult spinal cord injury. Neuron 23: 83–91. Nguyen Ba-Charvet KT, Brose K, Marillat V, Kidd T, Goodman CS, Tessier-Lavigne M, Sotelo C, Chedotal A (1999) Slit2Mediated chemorepulsion and collapse of developing forebrain axons. Neuron 22: 463–473. Niclou SP, Franssen EH, Ehlert EM, Taniguchi M, Verhaagen J (2003) Meningeal cell-derived semaphorin 3A inhibits neurite outgrowth. Mol. Cell. Neurosci. 24: 902–912. Niederost B, Oertle T, Fritsche J, McKinney RA, Bandtlow CE (2002) Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1. J. Neurosci. 22: 10368–10376. Nikulina E, Tidwell JL, Dai HN, Bregman BS, Filbin MT (2004) The phosphodiesterase inhibitor rolipram delivered after a spinal cord lesion promotes axonal regeneration and functional recovery. Proc. Natl. Acad. Sci. USA 101: 8786– 8790. Nishimaru H, Takizawa H, Kudo N (2000) 5-Hydroxytryptamine-induced locomotor rhythm in the neonatal mouse spinal cord in vitro. Neurosci. Lett. 280: 187–190. Nornes H, Bjorklund A, Stenevi U (1983) Reinnervation of the denervated adult spinal cord of rats by intraspinal transplants of embryonic brain stem neurons. Cell Tissue Res. 230: 15–35. Oertle T, van der Haar ME, Bandtlow CE, Robeva A, Burfeind P, Buss A, Huber AB, Simonen M, Schnell L, Brosamle C, Kaupmann K, Vallon R, Schwab ME (2003) Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions. J. Neurosci. 23: 5393–5406. Oudega M, Rosano C, Sadi D, Wood PM, Schwab ME, Hagg T (2000) Neutralizing antibodies against neurite growth inhibitor NI-35/250 do not promote regeneration of sensory axons in the adult rat spinal cord. NeuroReport 100: 873–883. Park JB, Yiu G, Kaneko S, Wang J, Chang J, He XL, Garcia KC, He Z (2005) A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron 45: 345–351. Pasquale EB (2005) Eph receptor signalling casts a wide net on cell behaviour. Nat. Rev. Mol. Cell. Biol. 6: 462–475. Pasterkamp RJ, De Winter F, Giger RJ, Verhaagen J (1998) Role for semaphorin III and its receptor neuropilin-1 in neuronal regeneration and scar formation? Prog. Brain Res. 117: 151–170. Pasterkamp RJ, Anderson PN, Verhaagen J (2001) Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A. Eur. J. Neurosci. 13: 457–471. Pignot V, Hein AE, Barske C, Wiessner C, Walmsley AR, Kaupmann K, Mayeur H, Sommer B, Mir AK, Frentzel S (2003) Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor. J. Neurochem. 85: 717–728. Pindzola RR, Doller C, Silver J (1993) Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during development and after root and sciatic nerve lesions. Dev. Biol. 156: 34–48. Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei L (2002) Reactivation of ocular dominance plasticity in the adult visual cortex. Science 298: 1248–1251. Plump AS, Erskine L, Sabatier C, Brose K, Epstein CJ, Goodman CS, Mason CA, Tessier-Lavigne M (2002) Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33: 219–232. Popovich PG, Wei P, Stokes BT (1997) Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats. J. Comp. Neurol. 377: 443–464. Prinjha RK, Moore SE, Vinson M, Blake S, Morrow R, Christie G, Michalovich D, Simmons DL, Walsh FS (2000) Inhibitor of neurite outgrowth in humans. Nature 403: 383–384. Prinjha RK, Hill C, Irving E, Roberts J, Campbell C, Parsons A, Morrow R, Woodhams PL, Philpott KL, Pangalos M, Walsh FS. Mapping the functional inhibitory sites of Nogo-A. Discovery of regulated expression following neuronal injury. SFN Itinerary planner CD-ROM References Program No. 333.12. Society for Neuroscience, Washington, DC, 2002. Qiu J, Cai D, Dai H, McAtee M, Hoffman PN, Bregman BS, Filbin MT (2002) Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34: 895–903. Raineteau O, Z’Graggen WJ, Thallmair M, Schwab ME (1999) Sprouting and regeneration after pyramidotomy and blockade of the myelin-associated neurite growth inhibitors NI 35/250 in adult rats. Eur. J. Neurosci. 11: 1486–1490. Raisman G (2004) Myelin inhibitors: does NO mean GO? Nat. Rev. Neurosci. 5: 157–161. Richardson PM, McGuinness UM, Aguayo AJ (1980) Axons from CNS neurons regenerate into PNS grafts. Nature 284: 264–265. Rossi F, Buffo A, Strata P (2001) Regulation of intrinsic regenerative properties and axonal plasticity in cerebellar Purkinje cells. Restor. Neurol. Neurosci. 19: 85–94. Roux PP, Barker PA (2002) Neurotrophin signaling through the p75 neurotrophin receptor. Prog. Neurobiol. 67: 203–233. Rubin BP, Dusart I, Schwab ME (1994) A monoclonal antibody (IN-1) which neutralizes neurite growth inhibitory proteins in the rat CNS recognizes antigens localized in CNS myelin. J. Neurocytol. 23: 209–217. Santra M, Reed CC, Iozzo RV (2002) Decorin binds to a narrow region of the epidermal growth factor (EGF) receptor, partially overlapping but distinct from the EGF-binding epitope. J. Biol. Chem. 277: 35671–35681. Schmalfeldt M, Bandtlow CE, DoursZimmermann MT, Winterhalter KH, Zimmermann DR (2000) Brain derived versican V2 is a potent inhibitor of axonal growth. J. Cell Sci. 113 (Pt 5): 807–816. Schnell L, Schwab ME (1990) Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature 343: 269–272. Schnell L, Schwab ME (1993) Sprouting and regeneration of lesioned corticospinal tract fibres in the adult rat spinal cord. Eur. J. Neurosci. 5: 1156–1171. Schnell L, Schneider R, Kolbeck R, Barde Y-A, Schwab ME (1994) Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion. Nature 367: 170–173. Schwab ME (1990) Myelin-associated inhibitors of neurite growth and regeneration in the CNS. Trends Neurosci. 13: 452–456. Schwab ME (2004) Nogo and axon regeneration. Curr. Opin. Neurobiol. 14: 118–124. Schwab ME, Caroni P (1988) Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in vitro. J. Neurosci. 8: 2381–2393. Schweigreiter R, Walmsley AR, Niederost B, Zimmermann DR, Oertle T, Casademunt E, Frentzel S, Dechant G, Mir A, Bandtlow CE (2004) Versican V2 and the central inhibitory domain of NogoA inhibit neurite growth via p75NTR/ NgR-independent pathways that converge at RhoA. Mol. Cell. Neurosci. 27: 163–174. Scott AL, Borisoff JF, Ramer MS (2005) Deafferentiation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. Eur. J. Neurosci. 21: 81–92. Shao Z, Browning JL, Lee X, Scott ML, Shulga-Morskaya S, Allaire N, Thill G, Levesque M, Sah D, McCoy JM, Murray B, Jung V, Pepinsky RB, Mi S (2005) TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron 45: 353–359. Sharfe N, Freywald A, Toro A, Dadi H, Roifman C (2002) Ephrin stimulation modulates T cell chemotaxis. Eur. J. Immunol. 32: 3745–3755. Sicotte M, Tsatas O, Jeong SY, Cai CQ, He Z, David S (2003) Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord. Mol. Cell. Neurosci. 23: 251–263. Simonen M, Pedersen V, Weinmann O, Schnell L, Buss A, Ledermann B, Christ F, Sansig G, van der PH, Schwab ME (2003) Systemic deletion of the myelinassociated outgrowth inhibitor nogo-a improves regenerative and plastic responses after spinal cord injury. Neuron 38: 201–211. Sivasankaran R, Pei J, Wang KC, Zhang YP, Shields CB, Xu XM, He Z (2004) PKC mediates inhibitory effects of myelin and 47 48 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord chondroitin sulfate proteoglycans on axonal regeneration. Nat. Neurosci. 7: 261–268. Slawinska U, Majczynski H, Djavadian R (2000) Recovery of hindlimb motor functions after spinal cord transection is enhanced by grafts of the embryonic raphe nuclei. Exp. Brain Res. 132: 27–38. Snow DM, Lemmon V, Carrino DA, Caplan AI, Silver J (1990) Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp. Neurol. 109: 111–130. Sobel RA (2005) Ephrin A receptors and ligands in lesions and normal-appearing white matter in multiple sclerosis. Brain Pathol. 15: 35–45. Song H, Ming G, He Z, Lehmann M, McKerracher L, Tessier-Lavigne M, Poo M (1998) Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281: 1515– 1518. Stein E, Lane AA, Cerretti DP, Schoecklmann HO, Schroff AD, Van Etten RL, Daniel TO (1998) Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses. Genes Dev. 12: 667–678. Steindler DA, Settles D, Erickson HP, Laywell ED, Yoshiki A, Faissner A, Kusakabe M (1995) Tenascin knockout mice: barrels, boundary molecules, and glial scars. J. Neurosci. 15: 1971–1983. Steinmetz MP, Horn KP, Tom VJ, Miller JH, Busch SA, Nair D, Silver DJ, Silver J (2005) Chronic enhancement of the intrinsic growth capacity of sensory neurons combined with the degradation of inhibitory proteoglycans allows functional regeneration of sensory axons through the dorsal root entry zone in the mammalian spinal cord. J. Neurosci. 25: 8066–8076. Steup A, Ninnemann O, Savaskan NE, Nitsch R, Puschel AW, Skutella T (1999) Semaphorin D acts as a repulsive factor for entorhinal and hippocampal neurons. Eur. J. Neurosci. 11: 729–734. Stokes BT, Jakeman LB (2002) Experimental modelling of human spinal cord injury: a model that crosses the species barrier and mimics the spectrum of human cytopathology. Spinal Cord 40: 101–109. Sundaresan V, Mambetisaeva E, Andrews W, Annan A, Knoll B, Tear G, Bannister L (2004) Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system. J. Comp. Neurol. 468: 467–481. Swiercz JM, Kuner R, Behrens J, Offermanns S (2002) Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology. Neuron 35: 51–63. Talts U, Kuhn U, Roos G, Rauch U (2000) Modulation of extracellular matrix adhesiveness by neurocan and identification of its molecular basis. Exp. Cell Res. 259: 378–388. Tang X, Davies JE, Davies SJ (2003) Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue. J. Neurosci. Res. 71: 427–444. Terman JR, Mao T, Pasterkamp RJ, Yu HH, Kolodkin AL (2002) MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion. Cell 109: 887–900. Tona A, Bignami A (1993) Effect of hyaluronidase on brain extracellular matrix in vivo and optic nerve regeneration. J. Neurosci. Res. 36: 191–199. Tozaki H, Kawasaki T, Takagi Y, Hirata T (2002) Expression of Nogo protein by growing axons in the developing nervous system. Brain Res. Mol. Brain Res. 104: 111–119. Tropea D, Caleo M, Maffei L (2003) Synergistic effects of brain-derived neurotrophic factor and chondroitinase ABC on retinal fiber sprouting after denervation of the superior colliculus in adult rats. J. Neurosci. 23: 7034–7044. Ughrin YM, Chen ZJ, Levine JM (2003) Multiple regions of the NG2 proteoglycan inhibit neurite growth and induce growth cone collapse. J. Neurosci. 23: 175–186. Vaudano E, Campbell G, Hunt SP, Lieberman AR (1998) Axonal injury and peripheral nerve grafting in the thalamus and cerebellum of the adult rat: upregulation of c-jun and correlation with regenerative potential. Eur. J. Neurosci. 10: 2644– 2656. Venkatesh K, Chivatakarn O, Lee H, Joshi PS, Kantor DB, Newman BA, Mage R, Rader C, Giger RJ (2005) The Nogo-66 References receptor homolog NgR2 is a sialic aciddependent receptor selective for myelinassociated glycoprotein. J. Neurosci. 25: 808–822. Vinit S, Boulenguez P, Efthimiadi L, Stamegna JC, Gauthier P, Kastner A (2005) Axotomized bulbospinal neurons express c-Jun after cervical spinal cord injury. NeuroReport 16: 1535–1539. Vinson M, Strijbos PJ, Rowles A, Facci L, Moore SE, Simmons DL, Walsh FS (2001) Myelin-associated glycoprotein interacts with ganglioside gt1b. a mechanism for neurite outgrowth inhibition. J. Biol. Chem. 276: 20280–20285. von Meyenburg J, Brosamle C, Metz GA, Schwab ME (1998) Regeneration and sprouting of chronically injured corticospinal tract fibers in adult rats promoted by NT-3 and the mAb IN-1, which neutralizes myelin-associated neurite growth inhibitors. Exp. Neurol. 154: 583–594. Wahl S, Barth H, Ciossek T, Aktories K, Mueller BK (2000) Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase. J. Cell Biol. 149: 263–270. Walsh GS, Krol KM, Crutcher KA, Kawaja MD (1999) Enhanced neurotrophin-induced axon growth in myelinated portions of the CNS in mice lacking the p75 neurotrophin receptor. J. Neurosci. 19: 4155–4168. Wang KC, Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL, He Z (2002a) Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417: 941–944. Wang KC, Kim JA, Sivasankaran R, Segal R, He Z (2002b) p75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature 420: 74–78. Wang KH, Brose K, Arnott D, Kidd T, Goodman CS, Henzel W, Tessier-Lavigne M (1999) Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96: 771–784. Wehrle R, Camand E, Chedotal A, Sotelo C, Dusart I (2005) Expression of netrin-1, slit-1 and slit-3 but not of slit-2 after cerebellar and spinal cord lesions. Eur. J. Neurosci. 22: 2134–2144. Weibel D, Cadelli D, Schwab ME (1994) Regeneration of lesioned rat optic nerve fibers is improved after neutralization of myelin-associated neurite growth inhibitors. Brain Res. 642: 259–266. Wictorin K, Brundin P, Gustavii B, Lindvall O, Bjorklund A (1990) Reformation of long axon pathways in adult rat central nervous system by human forebrain neuroblasts. Nature 347: 556–558. Willson CA, Irizarry-Ramirez M, Gaskins HE, Cruz-Orengo L, Figueroa JD, Whittemore SR, Miranda JD (2002) Upregulation of EphA receptor expression in the injured adult rat spinal cord. Cell Transplant. 11: 229–239. Woolhead CL, Zhang Y, Lieberman AR, Schachner M, Emson PC, Anderson PN (1998) Differential effects of autologous peripheral nerve grafts to the corpus striatum of adult rats on the regeneration of axons of striatal and nigral neurons and on the expression of GAP-43 and the cell adhesion molecules N-CAM and L1. J. Comp. Neurol. 391: 259–273. Wright DE, Snider WD (1995) Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia. J. Comp. Neurol. 351: 329–338. Xiao D, Miller GM, Jassen A, Westmoreland SV, Pauley D, Madras BK (2006) Ephrin/Eph receptor expression in brain of adult nonhuman primates: Implications for neuroadaptation. Brain Res. 1067: 67–77. Xu G, Nie DY, Chen JT, Wang CY, Yu FG, Sun L, Luo XG, Ahmed S, David S, Xiao ZC (2004) Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: a potential strategy for axonal regeneration. J. Neurochem. 91: 1018–1023. Yamada H, Fredette B, Shitara K, Hagihara K, Miura R, Ranscht B, Stallcup WB, Yamaguchi Y (1997) The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons. J. Neurosci. 17: 7784–7795. Yamashita T, Tohyama M (2003) The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI. Nat. Neurosci. 6: 461–467. Yamashita T, Higuchi H, Tohyama M (2002) The p75 receptor transduces the 49 50 1 The Role of Inhibitory Molecules in Limiting Axonal Regeneration in the Mammalian Spinal Cord signal from myelin-associated glycoprotein to Rho. J. Cell Biol. 157: 565–570. Yang LJ, Zeller CB, Shaper NL, Kiso M, Hasegawa A, Shapiro RE, Schnaar RL (1996) Gangliosides are neuronal ligands for myelin-associated glycoprotein. Proc. Natl. Acad. Sci. USA 93: 814–818. Yick LW, Wu W, So KF, Yip HK, Shum DK (2000) Chondroitinase ABC promotes axonal regeneration of Clarke’s neurons after spinal cord injury. NeuroReport 11: 1063–1067. Yick LW, Cheung PT, So KF, Wu W (2003) Axonal regeneration of Clarke’s neurons beyond the spinal cord injury scar after treatment with chondroitinase ABC. Exp. Neurol. 182: 160–168. Yick LW, So KF, Cheung PT, Wu WT (2004) Lithium chloride reinforces the regeneration-promoting effect of chondroitinase ABC on rubrospinal neurons after spinal cord injury. J. Neurotrauma 21: 932–943. You SW, Chen BY, Liu HL, Lang B, Xia JL, Jiao XY, Ju G (2003) Spontaneous recovery of locomotion induced by remaining fibers after spinal cord transection in adult rats. Restor. Neurol. Neurosci. 21: 39–45. Young W (1991) Methylprednisolone treatment of acute spinal cord injury: an introduction. J. Neurotrauma 8 Suppl 1: S43–S46. Yu G, Luo H, Wu Y, Wu J (2003) Mouse ephrinB3 augments T-cell signaling and responses to T-cell receptor ligation. J. Biol. Chem. 278: 47209–47216. Z’Graggen WJ, Metz GA, Kartje GL, Thallmair M, Schwab ME (1998) Functional recovery and enhanced corticofugal plasticity after unilateral pyramidal tract lesion and blockade of myelin-associated neurite growth inhibitors in adult rats. J. Neurosci. 18: 4744–4757. Zagrebelsky M, Buffo A, Skerra A, Schwab ME, Strata P, Rossi F (1998) Retrograde regulation of growth-associated gene expression in adult rat Purkinje cells by myelin-associated neurite growth inhibitory proteins. J. Neurosci. 18: 7912–7929. Zhang Y, Winterbottom JK, Schachner M, Lieberman AR, Anderson PN (1997) Tenascin-C expression and axonal sprouting following injury to the spinal dorsal columns in the adult rat. J. Neurosci. Res. 49: 433–450. Zhang Y, Tohyama K, Winterbottom JK, Haque NS, Schachner M, Lieberman AR, Anderson PN (2001) Correlation between putative inhibitory molecules at the dorsal root entry zone and failure of dorsal root axonal regeneration. Mol. Cell. Neurosci. 17: 444–459. Zheng B, Ho C, Li S, Keirstead H, Steward O, Tessier-Lavigne M (2003) Lack of enhanced spinal regeneration in nogodeficient mice. Neuron 38: 213–224. Zheng B, Atwal J, Ho C, Case L, He XL, Garcia KC, Steward O, Tessier-Lavigne M (2005) Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo. Proc. Natl. Acad. Sci. USA 102: 1205–1210. Zhu JX, Goldoni S, Bix G, Owens RT, McQuillan DJ, Reed CC, Iozzo RV (2005) Decorin evokes protracted internalization and degradation of the epidermal growth factor receptor via caveolar endocytosis. J. Biol. Chem. 280: 32468–32479.