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n an effort to develop therapies for promoting neurological recovery after spinal cord injury,
I
much work has been done to identify the cellular and molecular factors that control axonal
regeneration within the injured central nervous system. This review summarizes the current
understanding of a number of the elements within the spinal cord environment that inhibit
axonal growth and outlines the factors that influence the neuron’s ability to regenerate its axon
after injury.
Recent insights in these areas have identified important molecular pathways that
are potential targets for therapeutic intervention, raising hope for victims of spinal cord injury.
Brian K. Kwon1,2, Jaimie F. Borisoff1, and Wolfram Tetzlaff1,3
1
International Collaboration on Repair Discoveries (ICORD)
2
Department of Orthopaedics, 3Department of Surgery,
University of British Columbia
Vancouver, British Columbia, Canada V6T 1Z4
244
Therapeutic Intervention in Spinal Chord Injury
INTRODUCTION
Few survivable injuries compare with the devastating physical and
psychological consequences of the paralysis that results from
trauma to the spinal cord. Each year, over 10,000 North
Americans, most below the age of thirty, sustain such spinal cord
injury (1). Whereas the loss suffered by each of these individuals is
incalculable, societal costs, in terms of the medical, surgical, and
rehabilitative care for patients with acute and chronic spinal cord
injuries, come to four billion dollars per annum (estimated from
decade-old data) (2). Clearly, therapies to enhance neurological
function after spinal cord injury are urgently needed. This need
has sparked great interest in the neuroscience community, and
many exciting experimental strategies have emerged. The purpose
of this review is to summarize some of these advances, with a
particular focus on the molecular elements deemed to play an
important role in preventing axonal regeneration after spinal cord
injury, and how these elements can be targeted by therapeutic
interventions.
INJURY CONSIDERATIONS: PRIMARY AND
SECONDARY DAMAGE
Typical injury to the human spinal cord occurs during blunt, nonpenetrating trauma (e.g., motor vehicle and diving accidents),
whereby the bony and ligamentous components of the spinal
column are displaced and impart sudden mechanical forces to the
spinal cord. A number of animal (predominantly rodent) injury
models have been developed in an attempt to reproduce such
injuries (3). The primary, mechanical disruption of tissue is
quickly followed by a complex cascade of secondary injury
processes that include regional blood flow alterations, electrolyte
homeostasis perturbations, free radical generation, excitotoxicity,
inflammation, and apoptotic cell death (4, 5). These secondary
mechanisms damage spinal cord tissue that was otherwise spared
during the initial insult and thus represent a potential target for
neuroprotective strategies. In current clinical practice, the
mainstay of neuroprotective therapy is limited to the judicious
medical resuscitation and stabilization of the acutely traumatized
patient (6) and high-dose administration of the steroid
methylprednisolone (7–10). A number of other pharmacological
means of influencing the course of secondary injury are currently
in various stages of animal and human evaluation (5, 11). Novel
neuroprotective agents will undoubtedly play an important role in
the future for minimizing neurological damage in the acutely
injured patient.
As secondary injury processes subside during the weeks and
months following the trauma, the site of spinal cord injury
becomes typically characterized by disrupted axons, a cystic cavity
encased within a gliotic scar, and variable amounts of intact tissue
surrounding the lesion (12, 13). In this peripheral rim of intact
tissue reside axons that are either uninjured or that have lost part
of their myelin sheaths. The clinical challenge at this stage is to
promote compensatory sprouting of the remaining intact axons or
the re-growth of severed axons across the injury site (Figure1).
Permanent paralysis after spinal cord injury testifies to the fact that
this challenge has gone largely unanswered. It has become
apparent that axonal regeneration after spinal cord injury fails
both because of elements within the injury environment that
inhibit axonal growth, and because the central nervous system
(CNS) neurons themselves demonstrate a relatively weak intrinsic
ability to regenerate axons after injury. Although many exciting
experimental approaches have been developed to overcome these
obstacles individually, it is generally agreed that an optimal
therapeutic strategy for human patients will require combinatorial
treatments that address both extrinsic and intrinsic barriers to
regeneration.
INHIBITORY ELEMENTS WITHIN THE
ENVIRONMENT OF THE INJURED CNS
Although the inhibitory environment of the CNS has been
recognized for nearly a century (14), not until the past two
decades have CNS myelin and the glial scar become appreciated as
primary blockades to CNS regeneration after injury (15–17).
Much has been done to identify the specific elements of CNS
myelin and the glial scar that inhibit axonal growth, and recent
advances in our understanding of the intracellular signalling
pathways that mediate these inhibitory effects may offer potential
therapeutic targets.
MYELIN INHIBITORS
OF
AXONAL REGENERATION
In the late 1980s, pioneering work in the laboratory of Martin
Schwab demonstrated that oligodendrocytes and their myelin
membranes are major inhibitors of axonal growth within the CNS
(18, 19). Corroborating this finding, John Steeves and colleagues
demonstrated that the failure of axonal regeneration within chick
spinal cord coincide with the onset of spinal myelination; in fact,
the experimental delay of myelination acts to extend the
permissive period for axonal regrowth (20). Subsequently, at least
five separate inhibitory elements have been attributed to myelin:
arretin (21); a chondroitin sulfate proteoglycan (22); the relatively
well-characterized Nogo-A protein (23–25); myelin associated
glycoprotein (MAG) (26, 27); and the recently identified
oligodendrocyte myelin glycoprotein (OMgp) (28).
Nogo-A
Schwab and colleagues provided some of the earliest evidence that
axonal growth could be promoted by targeting specific aspects of
CNS myelin (18). They biochemically separated 35- and 250-kDa
inhibitory fractions from CNS myelin and developed a monoclonal
murine antibody known as IN-1 that could block their inhibitory
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A
Cell body treatment
Lesion site treatment
B
Corticospinal tract
Figure 1. Experimental treatment of rat spinal
cord injuries. A. Treatments are applied
typically at the lesion site but can also be applied
to the cell body. B. Cellular events typify a
chronic spinal cord injury. Primary cellular
damage spreads the insult to surrounding cells
and creates secondary damage that can supersede
the original injury. Secondary damage includes a
central acellular fluid-filled cyst surrounded by a
glial scar. In a typical crush injury, axons per se
are often left uninjured, intact but partially
demyelinated, or severed. Degeneration of axons
and myelin occurs distal to the injury site.
Treatment paradigms may promote regeneration of
severed axons into or around the injury site,
possibly into the distal spinal cord. In addition,
treatments often promote the sprouting of
uninjured axons into areas of deafferentation.
membrane proteins (Nogo-A, -B, and -C) and
has been assigned (on the basis of sequence
homology to three previously sequenced
proteins) to the reticulon family of proteins.
Consistent with its role as an inhibitor within
CNS myelin, Nogo-A is highly expressed by
oligodendrocytes but not by Schwann cells
(23). The original descriptions of the Nogo
protein have led to some uncertainty
Cyst
Uninjured axon
Activated glial cells
regarding the actual components responsible
Sprouting axon
Myelin sheath
Glial scar
for axonal inhibition (37). Whereas the
Regenerating axon
Degenerating myelin
Original injury
Schwab and Walsh laboratories ascribe the
Degenerating axons
Demyelinated axon
inhibitory effect to the N-terminal domain of
Intact axons
Nogo-A (24, 25), the Strittmatter group has
assigned
inhibitory
functionality
to an extracellular loop of sixtyproperties in vitro (29). Subsequent in vivo application of the
six amino acids (Nogo-66) found in all three Nogo isoforms (23).
monoclonal antibody resulted in substantial axonal sprouting and
Fortunately, significant progress has been made in the recent
some long-distance corticospinal axonal regeneration within the
identification and cloning of a receptor for the Nogo-66 protein
adult mammalian CNS (30). Importantly, this antibody treatment
(NgR) (38). This landmark study demonstrates that the NgR is
was associated with improved performance in a variety of
anchored to the cell membrane through a glycophosphatidylfunctional tests, including open-field locomotion, rope climbing,
inositol linkage, suggesting that NgR is in fact part of a receptor
and food pellet reaching (31, 32). Although the partial spinal cord
complex. The role of the NgR in axonal growth inhibition is
injury models used in these early antibody studies could
illustrated by the positive correlation between its expression level
conceivably have permitted functional recovery due either to
in chick neurons of the dorsal root ganglion (DRG) and
regeneration of injured axons or to sprouting and compensation
responsiveness to myelin (i.e., Nogo) inhibition. Furthermore,
from uninjured axons (Figure 1), subsequent work has suggested
transfection techniques that cause (normally unresponsive) E7
that much of the benefit derived from the antibody treatment is in
retinal ganglion cells to express NgR confer sensitivity to Nogo-66.
fact mediated by sprouting and functional plasticity of spared
Interestingly, although Nogo-66 inhibits neurite outgrowth of E12
axons (33). With the hopes of translating this experimental
chick DRG neurons, the soluble form of the N-terminal domain is
approach to the clinical setting, a humanized version of the
not inhibitory, lending further credence that the sixty-six–residue
monoclonal IN-1 antibody has been generated and shown to be
extracellular domain possesses “Nogo” activity (38).
beneficial in animal models of partial spinal cord injury (34) and
In experiments to delineate the residues of Nogo-66 that are
stroke (35).
specifically responsible for inhibitory activity, Strittmatter and
The IN-1 antibody recognizing several components of myelin
colleagues found that a truncated peptide comprising amino acids
was also instrumental in the characterization and cloning of the
1 to 40 in the Nogo extracellular polypeptide loop (NEP1–40) fails
target antigen (23–25, 36), the neurite outgrowth inhibitor now
to inhibit neurite growth, and in fact acts as a competitive
known as Nogo. The cloned gene encodes three integral
246
Therapeutic Intervention in Spinal Chord Injury
antagonist of Nogo-66 binding to the NgR (39). After dorsal
hemisection of the rat thoracic spinal cord, local application of
NEP1-40 significantly increases the number of corticospinal and
serotonergic axon profiles distal to the site of injury, presumably
indicative of antagonism of the NgR and resultant neuronal
sprouting/regeneration. However, models of partial injury, such as
dorsal hemisection, leave room for uncertainty as to whether the
newly sprouted axons arise from injured axons in the dorsal
column versus ininjured ventral axons. Nevertheless, many
corticospinal axon sprouts occur in the dorsal spinal cord,
suggesting origination from the injured dorsal corticospinal tract.
Perhaps most excitingly, the injured animals treated with NEP1-40
show significant and sustained (i.e., at twenty-eight days postinjury) improvement in open field locomotor testing, which the
authors attribute to both early sprouting and subsequent longdistance regeneration (39). Clearly, NEP1-40 raises the exciting
potential for specifically targeting myelin inhibition of axonal
regeneration in the context of human spinal cord injury, and may
find relevance in the treatment of neurotraumatic and
neurodegenerative disorders.
Myelin Associated Glycoprotein (MAG)
Although NEP1–40 is clearly effective as an antagonist of Nogo66, its ability to promote neuronal regeneration is, even at high
concentration in vitro, incomplete (39). Elements other than Nogo
that reside within CNS myelin and inhibit neuronal regeneration
must thus be considered. One such element is myelin-associated
glycoprotein (MAG), which inhibits neurite outgrowth from adult
CNS neurons in vitro (26, 27). The role of MAG as an inhibitor of
regeneration in the peripheral nervous system is clear from the
increased axonal growth of peripheral nerves manifested in certain
mice that are deficient in MAG (40). Within the CNS, however,
the importance of MAG as an impediment to axonal regeneration
has been difficult to establish, most likely because of the presence
of the other inhibitors of axonal growth (41).
Our understanding of the function of MAG would be
significantly enhanced by the identification of its receptor, and
quite recently, several molecules have emerged as candidate
receptors for MAG: the gangliosides GD1a and GT1b (43); the p75
neurotrophin receptor (44); and interestingly, the Nogo-66
receptor, NgR (45, 46) (Figure 2). The indication that the NgR
might mediate the inhibitory effect of both Nogo and MAG (and
OMgp, as discussed below) is particularly intriguing, because such
a receptor would represent a point of convergence for numerous
therapeutic strategies (Figure 2). The Filbin group (45) has
recently demonstrated that the removal of GPI-linked proteins
such as the NgR from the surface of cerebellar neurons precludes
their interaction with an experimental variant of MAG (i.e.,
MAG–Fc), and as a result, neurite outgrowth is no longer
inhibited. The interaction between MAG and NgR is more directly
demonstrated by the binding of MAG to NgR-expressing CHO
MAG
Nogo
OMgp
Myelin
membrane
Nogo-66
domain
NgR
?
Growth cone
membrane
lgG domains
Signal peptide
Leucine rich repeats
Serine/threonine repeats
P75 death domain
Cysteine rich repeats
Oligosaccharide
Leucine rich repeat
C terminal domain
Ganglioside
GT1b
p75
??
Rho
Figure 2. Inhibitory myelin components and their putative receptors.
Three myelin membrane proteins that inhibit axonal growth are Nogo,
OMgp, and MAG. All three may bind to the Nogo receptor (NgR) in the
axonal growth cone, although downstream signaling events are less clear.
MAG has also been shown to bind both to ganglioside GT1b and to the
p75 low-affinity neurotrophin receptor. p75 may regulate the affects of
MAG through Rho-GTPase signaling to the actin cytoskeleton. (OMgp,
oligodendrocyte-myelin glycoprotein; MAG, myelin associated
glycoprotein; see text for details.).
cells, as well as by the binding of a soluble NgR to MAGexpressing CHO cells. Moreover, the in vitro inhibition of neurite
outgrowth by MAG can be overcome by three methods: with a
polyclonal antibody to NgR; with a soluble version of NgR to
compete for MAG binding with membrane-bound NgR; and with
a recombinant dominant-negative version of the NgR (N-NgR).
Interestingly, the binding of the experimental variant of MAG to
NgR-expressing CHO cells is inhibited by soluble form of Nogo66, implying that the two proteins compete for the same binding
site on the NgR (45). The Strittmatter group has provided similar
evidence regarding the relationship between MAG and the NgR,
but the authors state that competitive inhibition between MAG
and Nogo-66 was not observed (46). Also, the NEP1-40 peptide,
which competitively antagonizes the inhibitory effect of Nogo-66,
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had no effect on the inhibition of neurite growth by MAG,
suggesting that the Nogo-66 and MAG bind to NgR at distinct
sites, a proposition supported by the lack of homologies between
the two proteins (46).
Oligodendrocyte Myelin Glycoprotein (OMgp)
The list of identified inhibitory elements of CNS myelin was
recently lengthened to include oligodendrocyte-myelin
glycoprotein (OMgp) (28), a 105-kDa GPI-linked protein
described over a decade ago to be highly expressed in CNS myelin
(47). Quite remarkably, like Nogo-66 and MAG, OMgp also
appears to act through the NgR. Wang et al. identified OMgp as a
potential culprit in the growth cone–collapsing activity of the
proteins released by the treatment of myelin with
phosphatidylinositol-specific phospholipase C, and demonstrated
that the expression of recombinant OMgp in COS-7 cells induces
the collapse of E13 chick DRG growth cones (28). The screening
of a COS cell cDNA expression library with OMgp fused to
alkaline phosphatase revealed NgR as a possible receptor for
OMgp, findings that were subsequently confirmed in coprecipitation studies. Most compelling, NgR transfection of E7
DRG neurons (normally not yet expressing the NgR) rendered
these neurons sensitive to Omgp—a gain of function similarly
elicited with respect to Nogo-66 (38). Deletion constructs of the
NgR revealed that OMgp and Nogo-66 bind to overlapping
regions of the receptor, and consistent with this finding, no
additive effect was observed with regard to growth cone collapse
when OMgp and Nogo-66 were administered coincidentally as
opposed to single administration of either factor.
Hence, NgR has recently emerged as a single receptor for the
three major myelin proteins known to inhibit axonal outgrowth:
Nogo-A, MAG, and OMgp (Figure 2). These exciting findings
point strongly to the convergence of numerous inhibitory elements
onto the same signaling pathway (see below), which may represent
a specific molecular target for interventions to overcome the failure
of CNS regeneration.
GLIAL SCAR INHIBITORS
OF
AXONAL REGENERATION
At the site of CNS injury, a complex cellular reaction represented
by the activation of astrocytes, microglial cells, and the invasion of
macrophages, fibroblasts, and meningeal cells gives rise to what is
commonly referred to as the “glial scar” (15, 48–51). Although
most spinal cord injuries are caused by non-penetrating trauma,
potentially important differences in the nature of glial scarring
exist after blunt versus penetrating injury, with a connective tissue
basal laminar component and meningeal cell invasion being more
prominent in the latter. The glial scar is generally accepted to
represent an impediment to axonal regeneration after CNS injury,
but the relative significance of its molecular and cellular
constituents in inhibiting axonal growth is not entirely clear.
248
Nevertheless, due to their expression of inhibitory molecules (see
below) and the potentially impenetrable physical barrier created
by the dense intertwining of their processes, reactive astrocytes are
widely believed to play an important role in inhibiting nerve
regeneration (49). The extent to which this inhibition is due to the
physical environment created by the tightly interwoven astrocytic
processes is uncertain, although there is some evidence that the
three dimensional structure erected by these astrocytes has some
implications for axonal growth. For example, whereas astrocyte
monolayers support DRG neurite outgrowth in vitro, outgrowth is
not observed within astrocytes packed into a three dimensional
tube structure (52).
The importance of astrocytes as in vivo inhibitors of CNS
axonal growth is implied in a transgenic mouse model where the
ablation of astrocytes results in pronounced axonal sprouting
penetrating across the CNS injury (53). Perhaps the most
convincing demonstration that glial scars inhibit axonal
regeneration comes from Silver and colleagues, who show that
embryonic or adult DRG cells, microinjected into the spinal cords
of young adult rats without trauma, can extend axons for long
distances along myelinated white matter tracts until they
encounter the glial scar (16). The long-distance regeneration is
prevented if the microinjection inadvertently induces local glial
scarring at the transplant site, thus entrapping the DRG axons.
Reactive astrocytes (50, 54–56) and surrounding inflammatory
cells and oligodendrocyte progenitors (57, 58) express inhibitory
chondroitin sulfate proteoglycans (CSPGs), which have lately
achieved some notoriety as possible targets for therapeutic
intervention. Two recent studies have demonstrated the use of
chondroitinase ABC (Ch-ABC) to degrade CSPG in vivo and
thereby induce axonal growth (59, 60). In fact, intrathecal bolus
infusions of Ch-ABC, at the time of dorsal column crush lesion
and every other day for ten days thereafter, results in a rostrocaudal 4-mm span of spinal cord that is free of
glycosaminoglycan side chains (59). Anterograde axonal tracing
of sensory fibers, furthermore, reveals some growth along the wall
of the injury cavity, and corticospinal fibers 1 and 2 mm
proximal, as well as 1 mm distal, to the lesion epicenter show
significant growth. Ch-ABC treated animals also manifest, upon
electrophysiological stimulation of the cortex, a small short
latency wave 2 mm caudal to the lesion (indicating the
propagation of postsynaptic potentials across the injury site) that
can be abolished by a subsequent re-lesion to the dorsal column.
Finally, the Ch-ABC–treated animals show significant behavioral
improvements (albeit transient in some cases) in tape removal,
beam walking, and footprint analyses.
Although the results of Ch-ABC–treated animals are all very
promising results, the partial nature of the dorsal column injury
used in these studies most likely spares ventral and dorsolateral
corticospinal tracts, making it difficult to determine whether the
increase in corticospinal profiles observed distal to the injury site
represents growth from axotomized neurons or collateral sprouts
Therapeutic Intervention in Spinal Chord Injury
from spared corticospinal fibers (Figure1). Increased terminal
arborization from such sprouts could also increase postsynaptic
field potentials and account for the observed electrophysiological
findings. The inhibitory properties of CSPG are sometimes
associated primarily with its core protein (22, 61), so that some
inhibitory activity would be resistant to Ch-ABC treatment. As
the researchers of the study demonstrate, CSPGs are also
expressed by oligodendrocytes (62), and thus, Ch-ABC treatment
may promote corticospinal plasticity normally suppressed by
oligodendrocytes/myelin. Even so, the promotion of axonal
sprouting of spared fibers and subsequent functional recovery
clearly has significant clinical relevance, as the majority of
human spinal cord injuries (even with functionally “complete”
paralysis) are in fact anatomically partial injuries. The
distinction between strategies that promote axonal regeneration
versus sprouting would, however, be important for those with
anatomically complete lesions (for example, full spinal cord
transections from penetrating trauma), in which axonal sprouting
could be crucial. Despite many unanswered questions, the
promising results from the use of Ch-ABC suggest that the
approach of targeting inhibitory elements within the glial scar
holds promise for clinically appreciable treatments of spinal cord
injury.
Although much attention has been placed on CSPGs, it
should be mentioned that several other putative axonal growth
inhibitors have been ascribed to the glial scar, or to its
extracellular connective tissue components, such as collagen-IV
(63), tenascin (64, 65), class-3-semaphorins (66, 67), and Eph3B
(68). The relative importance of these molecules as inhibitors of
axonal growth is still a matter of some debate, awaiting
conclusive loss-of-function verification in vivo. The digestion of
basal laminar collagen is reported to promote some regeneration
in the retrospenial fornix (63), but the inability to reproduce
these findings in the spinal cord setting (69) leaves uncertainty
around the candidacy of collagen as a target of molecular
intervention.
INTRINSIC REGENERATIVE ABILITY OF
INJURED CNS NEURONS
That the permissive environment of peripheral nerves allows
neurons from the CNS to extend their axons after injury was
demonstrated by Tello and Ramon y Cajal almost a century ago
(14), and has been reiterated with more contemporary methods in
landmark work performed by Richardson, Aguayo, and colleagues
(70). The regenerative limits of mature CNS neurons, however, lie
in stark contrast to the fairly robust regenerative abilities of
neurons projecting into the peripheral nervous system. This
contrast is certainly explained in part by the different
environments encountered by neurons of the central as opposed to
the peripheral nervous system (the latter environment being far
more conducive), but it is also clear that CNS neurons intrinsically
fail to mount or to maintain some of the gene expression programs
necessary for a regenerative response to injury (71, 72). The
regenerative response is thought to involve the expression of genes
that encode a wide spectrum of proteins, including transcription
factors, cytoskeletal proteins, growth cone proteins, and cell
adhesion molecules (73). The identification and clinical
manipulation of the expression of these proteins represents a
potential therapeutic strategy for neuron regeneration and spinal
cord repair (74). It is important to recognize that our current
appreciation of the full battery of genes that are regulated to
produce axonal outgrowth is fairly rudimentary, although
microarray technology will undoubtedly accelerate our
understanding of such regulation in the future.
LESSONS FROM THE DORSAL ROOT GANGLION: GAP-43
CONDITIONING LESIONS
AND
The sensory neurons of the DRG, which possess axons that
project into the central and peripheral nervous systems, have
provided an extremely useful model for studying intrinsic
neuronal regenerative propensity. Richardson and Issa made the
intriguing observation in 1984 that injured central sensory axons
regenerate into permissive peripheral nerve transplants inserted
into the dorsal columns much more readily if a “conditioning”
injury to the peripheral sensory axon is implemented beforehand
(75). These observations suggested that the peripheral axotomy
initiated a molecular response within the cell body that was
necessary for the regeneration of the subsequently injured central
axon. It was later shown that such a conditioning peripheral
lesion could augment the intrinsic regenerative response
sufficiently to drive injured central axons across the otherwise
nonpermissive spinal cord injury environment (76). One of the
components that may play an important role in driving this
regenerative response is GAP-43, a protein localized to the
growth cone where, by virtue of its interactions with calmodulin,
PI(4,5)P2, G proteins, and the actin cytoskeleton, it influences
axonal growth and synaptic plasticity (77, 78). The correlation
between increased GAP-43 expression and axonal growth and
sprouting (79, 80) has made GAP-43 a commonly used indicator
of neuronal growth propensity, although one should be aware
that there do exist examples in which GAP-43 is neither
sufficient (81) nor necessary (82) for axonal growth. Bomze at al.
have demonstrated in vivo that the concomitant overexpression
of both GAP-43 and CAP-23 (a related growth cone protein)
results in significant regeneration of the central DRG axons into
peripheral nerve transplants, whereas overexpression of either
protein alone failed to promote such axonal regeneration (83).
Still, the most extensive regeneration was observed after a
conditioning peripheral axotomy, indicating that the two genes
(i.e., those encoding GAP-43 and CAP-23) do not account
completely for the conditioning effect of the peripheral nerve
lesion.
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CYCLIC AMP
PROPENSITY
AS AN INTRINSIC
DETERMINANT
OF
GROWTH
The DRG model has also been extremely valuable for the
identification of cAMP as an important intrinsic determinant of
axonal growth. Two very recent studies, in particular, have
strongly implicated cAMP in the promotion of DRG central axon
regeneration (84, 85), much like the increased growth propensity
observed with a conditioning peripheral axotomy (76). In studies
by Neumann et al. and Qiu et al., a single injection of a cAMP
analog into DRG neurons in vivo significantly enhanced their
neurite outgrowth in vitro from short, highly branched neurites to
an elongated pattern with few branches. The cAMP injection was
also associated with regenerative sprouting in vivo of ascending
injured sensory axons after a dorsal column spinal cord lesion,
with axonal growth occurring mainly along the wall of the injury
site cavity (84, 85). The extent to which the cAMP-activated
regenerating sensory axons can extend beyond the injury site and
make synaptic contact with target neurons is difficult to
determine. Consistent with the enhanced regenerative propensity
achieved with a conditioning lesion to the peripheral branch of the
DRG neuron, cAMP levels were also found to be significantly
increased by peripheral axotomy.
Marie Filbin’s group has presented data that may explain some
of the mechanisms by which cAMP mediates these growth-inducing
effects. In particular, the cAMP-enhanced growth that allows DRG
neurons to extend neurites on inhibitory substrates (86) is dependent
on activation of cAMP-responsive binding protein (CREB), which
results in the upregulation of arginase I and subsequent downstream
activation of polyamine synthesis (87, 88). The implication of
polyamine synthesis in DRG regeneration is consistent with the
increase in ornithine decarboxylase activity (a rate-limiting enzyme in
polyamine synthesis) observed in regenerating facial motoneurons
after axotomy (89); indeed, exogenous application of polyamines
stimulates such regeneration (90).
It is worth noting that in the recent demonstrations of in vivo
regeneration of DRG axons within the spinal cord, the cAMP was
administered to the DRGs two (85) or seven days (84) before the
spinal cord injury was actually induced. Obviously, within the
context of translating these important findings into a clinically
relevant strategy for spinal cord repair, the beneficial effects of
manipulating cAMP levels would have to be demonstrated after
injury. Nevertheless, these and other studies have provided
important insights into the role of cAMP in neuronal regenerative
capacity.
NEUROTROPHIC FACTOR ENHANCEMENT
COMPETENCE
OF
REGENERATIVE
Neurotrophic factors are small proteins that play diverse roles in
cell survival, axonal regeneration, and synaptic plasticity, and may
thus find therapeutic application in spinal cord injury and a host
250
of other neurological disorders (91). The implication of cAMP
(and downstream PKA activation) in neuronal regenerative
capacity suggests that DRG neurons, “primed” with neurotrophic
factors that may function via cAMP could extend neurites even on
nonpermissive substrates such as MAG and myelin (86). Indeed,
the in vivo treatment with brain-derived neurotrophic factor
(BDNF) or glia-derived neurotrophic factor (GDNF) promotes
regeneration of sensory neurons across the dorsal root entry zone
and into the spinal cord, with demonstrable functional recovery
(92). We have similarly found that BDNF, applied to the cell
bodies of acutely and chronically injured rubrospinal neurons, can
enhance regenerative capacity as evidenced by the upregulation
GAP-43 and T1 tubulin, as well as axonal growth into peripheral
nerve transplants (93, 94). Also, application of BDNF to
corticospinal neurons promotes the sprouting response within the
spinal cord (95). Although a direct link between neurotrophic
factors and cyclic nucleotides was not evaluated in our studies of
the rubrospinal and corticospinal system, it is certainly
conceivable that the application BDNF to the neuronal cell body
stimulates the activation of the MAPK/ERK pathway, with
subsequent activation of CREB, as has been previously
demonstrated for hippocampal neurons in vitro (96) and in vivo
(97). Hence, the activation of CREB may represent a point of
convergence for the intracellular effects of neurotrophic factors
and cAMP, mediating common downstream changes in the
expression of relevant genes such as arginase I and possibly many
others that have not yet been identified.
Increasing intracellular levels of cAMP with
phosphodiesterase inhibitors such as rolipram represents a
potentially useful strategy for promoting axonal growth, and may
be pharmacologically easier to achieve than the application of
neurotrophic factors, although overcoming undesirable side effect
(e.g., nausea) may be important considerations in both cases.
Although the growth-inducing effects of neurotrophins and cAMP
discussed herein involve molecular alterations at the level of the
cell bodies, we should point out that cAMP has also been shown
to play an important role at the level of the growth cone,
influencing the growth cone’s response to axonal guidance cues
(discussed below).
MANIPULATING GROWTH CONE SIGNALING
TO PROMOTE REGENERATION—THE ROAD
TO RHO
The actual extension of an axon occurs at its distal tip, where the
growth cone plays a key role in axonal regeneration by negotiating
the environment and integrating signals from an array of guidance
molecules. The growth cone cytoskeleton is primarily composed
of filamentous actin (F-actin) in its peripheral and central
domains, and microtubules extending from the central domain
along the entire shaft of the axon (reviewed in 98–100). The
peripheral domain comprises two dynamic F-actin–based
Therapeutic Intervention in Spinal Chord Injury
A
The
Growth
Cone
AXON
Microtubules
Filop
IInhibitory
hibi
Ligands
and
Receptors
B
RhoGDI
Rho
cAMP/PKA
GDP
Rho
GAP
GTP
Rho
GEF
C3
Y-27632
MYOSIN
ROCK
MLC
Phosphatase
LIM Kinase
Actin Filaments
Cofilin
Figure 3. The growth cone: Features and inhibitory signaling
pathways. A. A growth cone has distinct actin filament-based features,
including distal, finger-like filopodia and central, fan-shaped
lamellipodia. B. Schematic diagram of the Rho-signaling pathway in the
growth cone, a possible pathway of convergence for numerous
extracellular stimuli. Receptor activation somehow alters the balance of
Rho-GEF and Rho-GAP activity so as to favor an increase in activated,
GTP-bound Rho. Conversely, Rho activity can be down-regulated by
RhoGDI sequestration to the cytoplasm, an effect promoted by
intracellular cAMP/PKA elevation. Rho-GTP activates its downstream
effector kinases, of which ROCK is depicted here. ROCK in turn has
effects on several substrates that affect actin events that lead to growth
cone advance or retraction. (GAP, GTPase activating proteins; GEF,
guanosine nucleotide exchange factors; RhoGDI, Rho guanosine
nucleotide dissociation inhibitor; ROCK, Rho-kinase; PKA, protein
kinase A.)
structures: filopodia, made from polarized bundles of F-actin; and
lamellipodia, made from a meshwork of F-actin (Figure 3A).
These actin-rich structures contribute to the force necessary for the
forward extension of the growth cone (reviewed in 101, 102); thus,
a better understanding of how actin dynamics are regulated could
provide key insights into how axonal regeneration might be
promoted. It turns out that the members of the Rho family of
small guanosine triphosphatases (GTPases), in particular RhoA,
Rac1, and Cdc42 (reviewed in 103), appear to play a critical role
in the control and regulation of actin dynamics in the growth cone
and thus have emerged as potentially important molecular targets
for inducing axonal regeneration.
In neuronal cell lines, Rac and Cdc42 promote the formation
of lamellipodia and filopodia, whereas Rho activity causes neurite
retraction and growth cone collapse (104–107). [In primary
neuron cultures, however, the effects of Rho GTPases are not so
straightforward, indicating that an optimal level of Rac and Cdc42
GTPase activity, or optimal cycling between the GTP- and GDPbound forms, is needed for effective neurite outgrowth (108–110).]
Activated (i.e., GTP-bound) Rho modulates the activities of
numerous proteins, among which Rho-kinase/ROK/ROCK-2
(ROCK) is the best characterized. ROCK mediates Rho signaling
to the actin cytoskeleton (111) by phosphorylating myosin light
chain (MLC) (112, 113); ROCK also phosphorylates LIM-kinase
(114, 115), which is thereby activated so that it, in turn,
phosphorylates cofilin (116), which thereby contributes, along
with phosphorylation of myelin light chain, to growth cone
retraction (Figure 3B).
A number of studies have demonstrated a prominent role of
the Rho-ROCK pathway in mediating the growth cone collapse
and/or retraction effects of several inhibitory molecules found
within the injured CNS. Inhibition of Rho by C3 exoenzyme
promotes axonal elongation from chick DRG cells that are
otherwise inhibited by Semaphorin 3A (117). In addition, C3
exoenzyme–treatment (or the inhibition of ROCK with the specific
ROCK inhibitor Y-27632) promotes axonal elongation of retinal
ganglion cell axons that are otherwise inhibited by EphA receptor
signaling (118). Rho inhibition by C3 exoenzyme–treatment thus
overcomes the inhibitory effects of myelin, as well as of purified
MAG on the neurite (117, 119). Importantly, the in vivo
application of C3 exoenzyme promotes axonal regeneration of
crushed optic nerves in adult rats by as much as 500 m from the
crush site (119). This C3–exoenzyme treatment has been reported
to promote axonal regeneration of the corticospinal tract after a
full spinal cord transection injury (120), and we eagerly await the
full-length publication of these results. Consistent with these
findings, our laboratory has observed that the extension of DRG
axons grown on aggrecan, an inhibitory CSPG substrate, could be
stimulated by up to tenfold when ROCK was inhibited by Y-27632
(J. Borisoff et al., unpublished results).
As an interesting adjunct to the discussion of cAMP and its
role in neuronal regenerative propensity, elevating cAMP levels
July 2002
Volume 2, Issue 4
251
Review
may be yet another method of inhibiting Rho, in perhaps a
somewhat less specific manner than the C3 exotoxin. Rho can
undergo phosphorylation in a PKA-dependent manner (121),
resulting in its inactivation and the prevention of stress fiber
formation in non-neuronal cells. Experiments using rat brain
membrane fractions indicate that this PKA-dependent
phosphorylation inhibits Rho by enhancing its interactions with
the Rho-GDP-dissociation inhibitor (122). Interestingly, cAMP and
PKA activation have been shown in vitro to be critical for the
directional growth responses of Xenopus spinal neurons to
chemoattractive and chemorepulsive agents such as BDNF and
MAG (123, 124).
If Rho and its subsequent activation of ROCK represent a key
pathway that mediates axonal inhibition, and myelin and its
elements are prominent effectors of similar axonal inhibition, what
are the molecular means by which these intra- and extracellular
components interact? Very recent work has provided significant
insight into this question. Inhibition of neurite growth due to
MAG is mediated by the binding of MAG to the p75 neurotrophin
receptor, with subsequent activation of Rho (44). The relationship
among MAG, p75, and Rho is further supported by observations
that MAG colocalizes with and binds to p75, activates RhoA, and
fails to inhibit adult DRG neurons that express nonfunctional p75
receptors (44). Because MAG also binds to the NgR (46), one
could also speculate that the p75 receptor interacts with the GPIlinked NgR to form part of the elusive signal transducing NgR
receptor complex (Figure 2).
In summary, it is well established that members of the Rho
family of small GTPases plays a central role in mediating signaling
from permissive as well as inhibitory extracellular guidance
molecules. With regard to the numerous inhibitory molecules that
have been identified as potential obstacles to axonal regeneration,
there is accumulating evidence that their intracellular signaling
pathways converge by means of Rho, making the Rho pathway a
potentially important target for repair strategies (e.g., ROCK
inhibition). Although the mechanism by which cAMP influences
extension and directional decisions of the growth cone have not
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Many significant advances have been made recently in our
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Acknowledgments
The authors gratefully acknowledge the funding of the
Neuroscience Canada Foundation (NCF), Canadian Institutes for
Health Research (CIHR), Rick Hansen Institute, and the National
Science and Engineering Research Council of Canada and the
Christopher Reeve Paralysis Foundation. BKK is the Gowan and
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July 2002
Volume 2, Issue 4
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Review
Brian K. Kwon, MD, (center), completed clinical training in orthopedic surgery at the University of British Columbia before arriving at
ICORD as a Gowan and Michele Guest Fellow, where he is in his final year of studies for his PhD in Neuroscience. Jaime F. Borisoff
(right) is presently completing his PhD studies in Neuroscience at the University of British Columbia, where he is supported by a Rick
Hansen Studentship. Wolfram Tetzlaff, MD, PhD, (left) obtained his medical degree in Germany, trained at the Max-Planck-Insitute for
Psychiatry in Munich, and obtained his PhD in Neuroscience at the University of Calgary. Subsequent to faculty positions at the
University of Calgary and Ottawa he now holds the Rick Hansen Man in Motion Chair in Spinal Cord Research at UBC and is Associate
Director for Discovery Science for ICORD.
258