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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
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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-
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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.
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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.
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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.
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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
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