Download molecular mechanisms of axonal regeneration in the central

PROCEEDINGS
MOLECULAR MECHANISMS OF AXONAL REGENERATION
IN THE CENTRAL NERVOUS SYSTEM*
—
Marion Murray, PhD†
ABSTRACT
The regeneration of injured axons in the adult
central nervous system (CNS) is limited by a number of obstacles, including the loss of growth factors
that promote neuron survival, expression of nerve
growth inhibitors, activation of programmed cell
death pathways, and physical obstruction caused
by scar formation at the site of injury. Evidence
from several animal models of CNS injury suggests
it is possible to improve neuronal survival and support axon regeneration. Direct application of nerve
growth factors to the site of injury, either by
gelfoam, osmotic pump, or transplantation of
genetically modified cells, has been shown to
increase the number of neurons that survive axotomy and that can regenerate axons into or through
the lesion site. Inhibitors of growth-suppressing or
cell death pathways also enhance neuron survival.
Combination treatments may be required to produce clinically meaningful improvements in patients
with severe CNS injury.
(Adv Stud Med. 2004;4(4B):S335-S338)
lthough neurons of the adult central
nervous system (CNS) are known for
their inability to repair themselves following injury, it has long been recognized that these cells possess limited
regenerative capacity.1 Three distinct types of axonal
sprouting have been described as a result of axonal
injury in the CNS. Axon transection is followed by a
very short and abortive period of regenerative sprouting,
A
*Based on a presentation given by Dr Murray at an educational symposium held in New York City, December 2003.
†Professor of Neurobiology and Anatomy, Drexel
University College of Medicine, Philadelphia, Pennsylvania.
Address correspondence to: Marion Murray, PhD, Drexel
University College of Medicine, 3200 Henry Ave,
Philadelphia, PA 19129. E-mail: [email protected].
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during which the severed axons extend briefly then
retract. Compensatory sprouting occurs when the
transected axon also emits collateral sprouts to targets
proximal to the injury. Collateral sprouting is the
growth by undamaged neurons. When an axon is transected, neighboring undamaged axons can extend
sprouts to occupy the synaptic space formerly occupied by the transected axon.
These observations suggest neurons can initiate regenerative growth following CNS injury but that obstacles
prevent significant elongation. Adult neurons may fail to
express growth-promoting genes that are normally active
during CNS development. Axonal injury triggers programmed cell death (apoptosis) pathways that result in
neuronal death. The formation of a cyst or scar at the
injury site may act as a physical barrier preventing axon
regrowth, and scar-forming cells may also secrete substances that suppress axon regeneration. Other targetderived chemical mediators that normally support
neuronal survival may become unavailable to the neuron
after axonal transection. Inflammation at the site of
injury may cause the release of other chemical mediators
that suppress axon growth.
Although there are many obstacles to neuronal survival
or regeneration following CNS injury, solutions have been
devised to overcome many of them. Considerable experimental evidence suggests nerve growth factors can be
delivered to injured neurons either by direct injection or
transplantation of cells that secrete them. These growth
factors improve neuronal survival and promote axonal
regeneration. Within the lesion, the formation of a cyst or
scar can be prevented by enzymatic digestion, and a
“bridge” for regenerating axons can be formed using transplanted cells. Anti-inflammatory agents can be used to
suppress activation of the immune system.
RESCUING AXOTOMIZED NEURONS
One model system that is particularly well suited for
studies of neurodegeneration and regeneration is the
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transection of axons that originate from neurons in
Clarke’s nucleus of the spinal cord. Clarke’s nucleus is
anatomically well defined and projects entirely ipsilaterally to targets within the spinal cord and the cerebellum.2
Injury of the spinal cord along this pathway results in the
axotomy of all of the axons that project from Clarke’s
nucleus on that side of the spinal cord. After several
weeks, extensive cell death is observed in the Clarke’s
nucleus on the hemisected side, whereas on the contralateral side, the nucleus remains intact. Calculating
the ratio of neurons remaining on the axotomized side to
the number on the intact side provides an index of neurodegeneration that can be used to test various strategies
to improve neuron survival following axotomy.
This model was used to demonstrate that transplanting tissue grafts of fetal neural tissue into the lesion site
protected many of the Clarke’s nucleus axons from
death.2 In hemisected animals that did not receive fetal
tissue grafts, the number of cells surviving in Clarke’s
nucleus decreased by 30% over a 2-month period compared with control animals. Transplantation with tissue
grafts obtained from fetal spinal cord, cerebellum, or
neocortex within the first few days after axotomy resulted in nearly complete rescue of Clarke’s nucleus neurons.
Embryonic striatal tissue transplants, in contrast, did not
improve survival of the axotomized neurons (Figure 1).
Animals treated with the growth factor neurotrophin-3
(NT-3) into the site of spinal hemisection exhibited significant improvement in survival of Clarke’s nucleus
neurons compared with untreated animals, whereas
administration of several other growth factors (ie, nerve
growth factor, brain-derived neurotrophic factor, ciliary
neurotrophic factor) did not improve survival.3 In a subsequent study in which animals received grafts of fibroblasts that were genetically modified to secrete growth
factors, fibroblasts that secreted NT-3 appeared to completely protect Clarke’s nucleus neurons from death, and
nerve growth factor provided partial protection.4 It has
also been shown that transfer of the human BCL-2 gene
(which is believed to prevent apoptotic cell death) to cells
in the spinal cord above and below the lesion site significantly improved the survival of neurons in Clarke’s
nucleus following spinal hemisection.5
Similar outcomes were observed in studies that
examined the effects of transection on neurons of the
rubrospinal tract. In this case, axons from cell bodies that
originate in the red nucleus of the brainstem project
almost entirely to the contralateral spinal cord. Using
this model system, fibroblasts that were genetically mod-
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ified to secrete brain-derived neurotrophic factor
(BDNF)—a growth factor that is of particular importance for the survival or rubrospinal neurons—were
transplanted into the site of hemisection. In the red
nucleus contralateral to the spinal cord hemisection,
about 65% of cells survived in untreated animals, whereas approximately 90% of neurons survived in animals
that received the genetically modified fibroblasts. In a
second study, administration of apoptosis-inhibiting
BCL-2 rescued many (but not all) of the axotomized
neurons in the rubrospinal tract.6 These studies confirm
that cell death in axotomized neurons can be prevented
if the cells are provided appropriate neurotrophic factors.
AXON REGENERATION
As described previously, transected axons often
exhibit a brief transitory period of spontaneous regeneration. Two general approaches have been used to
increase axonal regeneration following CNS injury:
Figure 1. Survival of Axotomized Neurons in
Clarke’s Nucleus
*Significantly fewer neurons vs other groups.
Cell survival at the L1 level of Clarke’s nucleus of adult rats following hemisection of the ipsilateral spinal cord, expressed as the ratio of the number
of cells on the operated side to the number on the control side. Cell survival was significantly reduced (indicated by asterisks) in animals that underwent spinal hemisection alone or spinal hemisection followed by a graft of
fetal striatal tissue. Grafts of tissue from fetal spinal cord, cerebellum, or cortex prevented the loss of cells in the hemisected Clarke’s nucleus.
HX = hemisection only; HX-SC = hemisection with spinal cord transplant;
HX-CB = hemisection with cerebellum transplant; HX-CTX = hemisection
with neocortex transplant; HX-STR = hemisection with striatum transplant.
Adapted with permission from John Wiley & Sons, Inc. Himes et al. Grafts
of fetal central nervous system tissue rescue axotomized Clarke’s nucleus
neurons in adult and neonatal operates. J Comp Neurol. 1994;339(1):
117-131.2 Copyright © 2000, Wiley-Liss, Inc, A Wiley Company.
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PROCEEDINGS
providing the regenerating cells with growth factors or
other substances that stimulate growth or preventing
the development of injury processes that suppress
growth. Both approaches have been shown to improve
axon regeneration in animal injury models.
The use of genetically modified cells (eg, fibroblasts)
that secrete neurotrophic factors is of particular interest in
studies that examine axon regeneration. The growth factors may stimulate axonal sprouting and regrowth while
the fibroblasts form a physical bridge for the growing
axons. Axonal regeneration following the transplantation
of genetically modified fibroblasts has been studied in the
rubrospinal tract model system. Fibroblasts modified to
secrete BDNF were transplanted into the lesion site, and
axonal regeneration was assessed using 2 methods.7 The
regenerating axons were examined using an anterograde
staining procedure in which a dye (biotinylated dextran
amine, or BDA) was injected into the red nucleus. BDA
is taken up by the cell bodies and transported down the
length of the axons. Retrograde staining was performed
by injecting a different dye (fluorogold) into the spinal
cord below the level of the injury. Fluorogold is taken up
by axons that extend into the dye-injected region and
transported back along the axon to the cell bodies of neurons whose axons have regenerated beyond the lesion.
In transplanted animals, BDA-labeled axons were
observed extending through the grafted fibroblasts and
into the caudal portion of the spinal cord. Some of these
axons could be seen leaving the spinal cord white matter
and projecting into gray matter. Although it could not be
determined whether these axons established functional
synapses, they did project to the appropriate spinal laminae for rubrospinal neurons. With the retrograde tracing
technique, no red nucleus neurons were stained in animals that underwent transection without receiving the
modified fibroblasts. In treated animals, the number of
stained neurons was about 7% to 10% of the number
observed in normal animals that did not undergo spinal
hemisection. Although this is a relatively small fraction of
the total number of red nucleus axons projecting to the
normal spinal cord, even a few axons forming synapses in
the spinal cord could produce clinically significant
improvement in patients with severe spinal cord damage.
the injured spinal cord. These findings suggest it may
be possible to significantly improve recovery following
some types of CNS injury. However, they also illustrate that providing growth-promoting molecules may
not be sufficient to produce extensive axonal regrowth.
A significant obstacle to axon regeneration is the
development of a scar at the site of injury. Scarring within the CNS creates a physical barrier to axon regrowth
and is associated with the release of many chemical mediators that interfere with axonal regeneration. Several
inhibitory molecules have been identified in CNS lesions,
and strategies to suppress the growth-inhibiting effects of
these mediators are in development. Myelin-associated
inhibitors may be the principal impediment to axon
regeneration immediately after injury.8 Recent research
suggests that there are at least 3 of these inhibitors, all of
which appear to bind to a common neuronal receptor.
These include Nogo, a protein found on the cell surface
of oligodendrocytes and on the myelin membrane;
myelin-associated glycoprotein; and the recently identified oligodendrocyte myelin glycoprotein, which is
expressed by oligodendrocytes and neurons in the CNS
and in the peripheral nervous system. All of these ligands
activate a common receptor (the Nogo receptor, or Ngr),
and recent studies suggest that infusion of a peptide
antagonist to this receptor into the site of CNS injury
improves axon regeneration following transection.9
Semaphorins are a group of molecules that are
important in the developing nervous system, where
they provide inhibitory guidance by causing growth
cone collapse and thus prevent inappropriate growth.
In the adult they may act to prevent regenerative
growth. Development and administration of new
broad-spectrum protein kinase inhibitors can block
these effects (G. Gallo, oral communication).
Several inhibitory molecules have also been identified
that are related to the scar that forms after CNS injury.
These include chondroitin sulfate proteoglycans (CSPGs)
and semaphorins. Chondroitinase ABC, an enzyme that
cleaves CSPG and dissolves the scar, improved histologic
and functional measures of axonal regeneration after cervical dorsal column crush injury (Figure 2).10
SUMMARY AND CONCLUSIONS
THE HOSTILE ENVIRONMENT
Providing injured axons with appropriate growth
factors supports neuronal survival and allows some
axons to regenerate for relatively long distances within
Advanced Studies in Medicine
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Several techniques have been developed to improve
neuronal survival and enhance axonal regeneration
after CNS injury. Similar techniques may be applicable in MS; it has recently been noted that embryonic
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PROCEEDINGS
Figure 2. ChAB Promotes Regeneration of
Corticospinal Tract Neurons
spinal cord injury is that the distal portion of the spinal
cord often remains largely intact despite its physical separation from the more rostral regions of the CNS. It may
therefore be possible to develop neurologic prostheses
that can exploit the surviving circuitry and supplement
repair processes.
REFERENCES
ChAB promoted regeneration of corticospinal tract neurons following
crush injury in adult rats. Corticospinal tract neurons were counted in white
matter tracts extending 4 mm rostral and caudal to the site of injury.
Compared with sham-injury animals, crush injury resulted in the significant
loss of axons in the spinal cord rostral to and projecting through the lesion
in vehicle-treated animals. Treatment with ChAB significantly increased the
number of axons projecting to and through the lesion site.
*P <.05 between vehicle and ChAB treatment groups.
ChAB = chondroitinase ABC.
Adapted with permission from Bradbury et al. Chondroitinase ABC promotes
functional recovery after spinal cord injury. Nature. 2002;416(6881):636-640.10
stem cells implanted into injured spinal cord are capable
of differentiating into oligodendrocytes and thus of
myelinating axons.11 Many techniques have produced at
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date, however, have been relatively limited. Due to the
large number of molecular mediators of axonal growth
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to substantially improve functional outcomes. This may
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problems that must be overcome to develop new therapies for clinical use. Developing interventions for chronic spinal cord injury is further complicated because many
of the cells that survive injury may exhibit markedly
reduced activity and may never regenerate. Established
chronic lesions also have extensive permanent scarring,
making it difficult for axons to grow. However, it has
been shown that implanting fibroblasts engineered to
secrete neurotrophic factors can improve neuronal survival even when implantation is delayed for several weeks
after spinal cord hemisection.12 A significant feature of
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