mini disseration kabir nigam
TRANSCRIPT
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A Survey of the Neuronal Mechanisms
Underlying CNS Injury and the Barriers
Preventing Successful Recovery
By Kabir Nigam
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TABLE OF CONTENTS
Abstract…….……………………………………..…………………………..…………………………..…..pg. 3
Introduction……………………………………..…………………………..…………………………..…..pg. 4
Astrocytes……………………………………..…………………………..…………………………..…...…pg. 9
Developmental Molecules…………………………………………………………………….………..pg. 13
Oligodendrocytes……………………………………..…………………………..……………………….pg. 15
Regeneration……………………………………..…………………………..…………………………..…pg. 19
Conclusion……………………………………..…………………………..…………………………..…….pg. 24
References……………………………………..…………………………..……………………….…..……pg. 27
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ABSTRACT
Injuries to the central nervous system can be caused by either physical impact or by
restriction of blood flow to its constituents. These injuries often have very serious
consequences, in which full rehabilitation is rare. The main reason for this is that
upon injury, the affected area turns into an environment where axonal regeneration
is not possible. This includes the formation of a dense glial scar, the secretion of
repulsive growth factors, and upregulation of glycoproteins that induce actin
depolymerization. Strategies to overcome such inhibition include studying the
intercellular mechanisms by which these molecules act, and targeting either the
molecule itself or downstream secondary molecules that these inhibitory
components affect, with the greater goal of blocking the transduction of their signals
and creating an environment that is permissible to neuronal growth.
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INTRODUCTION
The central nervous system is perhaps the most complex system known to the
human race, with over 100 billion nerve cells making hundreds of trillions of
synaptic connections (Koch & Laurent, 1999). It governs the vital functions that
sustain life, and is thus a priority in terms of biomedical study. Given its complexity,
the CNS is still relatively a black box, a massive uncharted territory of current
exploration. Damage to the cells that encompass the CNS often has devastating
results that severely impair the affected individual, if not resulting in death. Thus, it
is important to study how the CNS responds to such insult, as through
understanding the natural mechanisms that mediate recovery, we can use medical
technology and research to facilitate this process.
There are three main types of CNS injury: traumatic brain injury, spinal cord injury
and ischemia. The first is defined as sudden physical damage to the brain, either
through impact to the intact skull or physical penetration of actual brain tissue
(Finnie & Blumbergs, 2002). Spinal cord injury is physical damage to the spine,
again through impact or penetration that damages the axons that encompass the
spinal cord, which carry information from the brain to the body and vice versa
(McDonald & Sadowsky, 2002). Ischemia involves the restriction of blood to
neurons, often as a result of a stroke, restricting them of oxygen and glucose that is
necessary for proper cellular function (Aarts & Tymianski, 2005).
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“Approximately 200,000 people die each year in the United States from brain
injuries, with an additional 500,000 hospitalized for treatment. About 10% of
surviving individuals have continuing disabilities that may impair their
ability to live independently… In the US, approximately 8,000 new cases of
spinal cord injury occur each year, and an estimated 450,000 people in the
country live with the condition.” (Physicians’ Desk Reference)
Neuroinflammation is a relatively recent term within neuroscience that defines the
CNS’s response process to injury. It has only been within the past decade that
scientists discovered that the brain exhibits immune activity unique to that of the
rest of the body. This response process is highly complex, and is not fully
understood, however the study of neuroinflammation has become a very “hot” topic
in modern neuroscience, with many labs dedicating their research to studying this
process.
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Figure adapted from Nature Reviews Neuroscience (Popovich & Longbrake, 2008)
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There are a multitude of different aspects of the neuroinflammatory process that
have been identified, and research shows these aspects, though acutely beneficial in
the healing process, can also be harmful (Nakajima & Kohsaka, 2001). Such aspects
are largely mediated by the activation of astrocytes and microglia, two types of glial
cells that play important roles in maintaining proper neuronal functionality.
Astrocytes help provide nutrients to nervous tissue and maintain extracellular ion
balances, while microglia are the macrophages of the CNS, constantly clearing
pathogens and extracellular debris through phagocytosis (Nakajima & Kohsaka,
2001; Kimelberg & Nedergaard, 2010). But upon CNS insult, these cells quickly
respond by producing inflammatory mediators. Microglia are activated by CNS
damage or infection, and are recruited rapidly to the area of injury. The presence of
damaged cells causes microglia to produce cytokines and chemokines that can
either damage or protect neighboring cells. Cytokines can cross the blood–brain
barrier to recruit mediators like leukocytes originating from the periphery (Lucas et
al., 2009). Astrocytes are most known for their ability to form scars that isolate
damaged tissue from healthy tissue, minimizing the spread of infection and cellular
damage while allowing for the damaged area to heal through the reorganization of
blood vessels that provide factors necessary for proper healing (Stichel & Müller,
1998).
However despite promoting the healing of damaged tissue, neuroinflammation,
otherwise known as gliosis, has effects that inhibit axonal regeneration, severing
proper communication between the affected region and the rest of the brain, thus
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resulting in a loss of function. The purpose of this paper will be to investigate what
factors inhibit axonal regeneration and to discuss therapeutic techniques that show
promise with regards to overcoming regenerative failure.
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ASTROCYTES
Upon CNS insult, astrocytes quickly react by forming what is known as a glial scar.
This process involves the proliferation as well as morphological and functional
changes within astrocytes. Such cells are known as reactive astrocytes (Fawcett &
Asher, 1999; Sofroniew, 2005). Proliferation has been marked by a strong increase
in the synthesis of Glial Fibrillary Acidic Protein and an increase in extracellular
matrix molecules (Wilhelmsson et al., 2006). The proliferation of astrocytes serves
to form a dense web of interconnected tissue that surrounds and fills the vacant
spaces caused by degenerating and dead neuronal tissue (Stichel & Müller, 1998).
This layer of cells functions to protect surrounding healthy tissue from being
damaged by potential microbial agents, while maintaining a homeostatic
environment and protecting the damaged are from other proinflammatory
molecules, growth factors, and free radicals (Rolls et al., 2009). Additionally,
astrocytes mediate the revascularization of the damaged tissue that promotes repair
by providing the affected area with nutritional and metabolic support. However,
reactive astrocytes also produce molecules that chemically inhibit neuronal growth,
preventing full recovery of the damaged area in the long-‐term (Huang et al., 2014).
The most obvious explanation for this inhibitory mechanism is that the density of
the glial scar is so strong that it provides a mechanical barrier that prevents
anything from getting into the area it surrounds (Windle & Chambers, 1950).
However what seems to play a bigger role are the inhibitory molecules that are
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upregulated upon reactive astrogliosis, as it has been shown that axonal
regenerative failure will still occur in vitro when the glial scar is removed from the
neuroinflammatory environment (Rudge and Silver, 1990). Such molecules include
Semaphorin 3 (Pasterkamp et al., 2001), ephrin-‐B2 (Bundesen et al., 2003) and
chondroitin sulfate proteoglycans (Jones et al., 2003). Evidence that supports the
environment as a factor in axonal regenerative failure includes micro-‐
transplantation of adult dorsal root ganglion cells into either intact host glial
environment or a damaged one. Though “these sensory axons regenerate rapidly
over long distances within adult white matter tracts of the brain and spinal cord, as
the growing adult neurons reach an area of CNS damage, with associated
inflammatory infiltrates and inhibitory molecules, the axons convert into a
dystrophic state and are unable to continue” (Fitch & Silver, 2008).
Chondroitin sulfate proteoglycans are strongly upregulated following CNS injury.
There are various types of CPSG’s that are differentially expressed that have been
shown to inhibit neuronal growth (Snow et al., 1990; Jones et al., 2003). Degradation
of CPSG’s following injury is shown to partially enhance axonal growth in animals
(Lee et al., 2010). Cleavage of the glycosaminoglycan side chains by Chondroitinase
ABC also makes the environment substantially more permissive to neuronal
outgrowth, suggesting that the inhibitory effect on axonal regeneration is primarily
dependent on the “sulfation pattern of GAG chains, since preventing GAG sulfation
eliminates much of the inhibitory activity on axon growth in vitro” (Sharma et al.,
2012).
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Figure adapted from The University of Leipzig (Schnabelrauch et al.)
The mechanisms through which CPSGs mediate their inhibitory effects on axonal
regeneration are complex and not fully understood. The first and most obvious
reason is that the diverse network of GAG side chains produces a dense barrier
prohibiting growth-‐promoting molecules from interacting with the cell surface
(Silver & Miller, 2004). Recent studies have shed light on the intercellular
mechanisms through which CPSGs may act. The Epidermal Growth Factor Receptor
is one such target, and addition of CPSGs to cerebral granular neurons was shown to
elicit EGFR phosphorylation. Furthermore, EGFR inhibitors were found to neutralize
the growth inhibitory effects of CPSGs (Koprivica et al., 2005). Another molecule
identified that binds to CPSGs was the transmembrane protein tyrosine
phosphatase, PTPsigma, a receptor for heparin sulfate proteoglycans that serves to
guide axons during development (Aricescu et al., 2002; Johnson & Van Vactor,
2003). Because of the structural homology shared between HPSGs and CPSGs
CPSG
CPSG
GAG
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(Kjellén & Lindahl, 1991), scientists tested whether CPSGs bind to PTPsigma
receptors, and found that binding occurs with high affinity. Dorsal root ganglion
cells from PTPsigma knockout mice were then probed with CPSGs, with results
showing that the knockout PTPsigma DRG cells exhibited substantially less
inhibition of growth than the wild-‐type DRG cells (Shen et al., 2009). Last, it has
been shown that when CPSGs inhibit axon growth, there is activation of the
Rho/ROCK pathway, a pathway that will be described later when discussing myelin
inhibitors. Use of a ROCK inhibitor was able to sufficiently suppress the inhibition
induced by CPSGs in outgrowth assays (Monnier et al., 2003).
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DEVELOPMENTAL MOLECULES
Figure adapted from Riken Brain Science Institute (Kamiguchi)
Studying molecules that regulate the development of the nervous system has been
useful in identifying other factors that play a role in inhibiting neuronal outgrowth
after CNS injury. One such molecule is ephrin-‐B3, which is shown to be upregulated
after spinal cord injury (Miranda et al., 1999). During development, expression of
this molecule prevents crossing of neurons in the coritospinal tract across the
midline, as these neurons possess the receptor for this protein, EphA4 receptor
tyrosine kinase, and knocking out of this receptor results in undesired crossing
(Yokoyama et al., 2001). It has been noted that ephrin-‐B3 expression is limited to
myelinating oligodendrocytes, and that “postnatal EphA4-‐positive cortical neurons
retain their sensitivity to ephrin-‐B3 in myelin and that this ligand accounts for a
fraction of the inhibitory activity in CNS myelin” (Benson et al., 2005). Similarly,
ephrin-‐B2 seems to be expressed by astrocytes, and upregulation of its expression is
Semaphorins, ephrins
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observed to occur in later time points following spinal cord injury. Though this is
thought to account for the segregation of EphB2 receptor -‐bearing fibroblasts from
ephrin-‐B2 expressing astrocytes in the glial scar, it is possible that ephrin-‐B2
expressing astrocytes may repulse other EphB2 receptor-‐containing axons from the
injured area (Bundesen et al., 2003).
Another factor in this group of developmental molecules is semaphorins, a family of
secreted and membrane-‐bound proteins that serve as repulsive guidance cues
during development (Bagnard et al., 1998). In the injured CNS, semaphorins are
expressed by both oligodendrocytes and by meningeal fibroblasts that occupy a
portion of the glial scar (De Winter et al., 2002; Moreau-‐Fauvarque et al., 2003). This
is supported by the fact that that injured CNS neurons express semaphorin receptor
components, potentially making them sensitive to the repulsive effects of
semaphorins (Pasterkamp & Verhaagen, 2001). Sema3s are the family of
semaphorins found in the glial scar, and it has been shown that “sprouting sensory
axons are responsive to Sema3-‐mediated axon repulsion in vitro and in vivo” and
that “conditioning peripheral nerve injuries that allow dorsal root ganglia axons to
regenerate centrally do not promote regenerative axon growth through Sema3-‐
expressing scar tissue” (De Winter et al., 2002; Pasterkamp & Verhaagen, 2006).
Sema4D and Sema5A are two other semaphorins that are expressed in
oligodendrocites (Goldberg et al., 2004; Yamaguchi et al., 2012). Both have also been
shown to inhibit axonal growth, with Sema4D being strongly upregulated following
CNS injury (Moreau-‐Fauvarque et al., 2003).
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OLIGODENDROCITES
Oligodendrocites are a type of neuroglia that provide support and insulation to
neurons in the CNS. These cells join together to form what is known as the myelin
sheath, which functions to enhance conduction of the action potential along the axon
by increasing the resistance and lowering the capacitance of the axonal membrane
(Edgar & Sibille, 2012). It has become known that myelin also has the ability to
block neuronal regeneration (Berry, 1982). This knowledge was furthered through
experiments by Martin Schwab using a monoclonal antibody IN-‐1, which when
“raised against a fraction of myelin that did not support neurite extension, allowed
axons to grow on myelin both in culture and in vivo” (Caroni & Schwab, 1988; Filbin,
2003).
It was later discovered that the molecule which was inhibited by this antibody was a
protein called Nogo-‐A which associates with the endoplasmic reticulum and on the
oligodendrocyte cell surface (Chen et al., 2000; Huber et al., 2002; Wang et al.,
2002). Nogo-‐A has two inhibitory domains where one is extracellular and the other
is in a cytoplasmic compartment. These domains are specifically a 66 amino acid
extracellular domain and the acidic amino terminus of Nogo-‐A (GrandPré et al.,
2000). Both domains have been shown to induce growth cone collapse regardless of
their location. Upon CNS injury, it is possible that both the Nogo-‐A located in the
endoplasmic reticulum as well as the Nogo-‐A located in the cytoplasmic
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compartment would become exposed to an extracellular environment that upon
interacting with axons attempting to regenerate, would inhibit them (Filbin, 2003).
Two additional glycoproteins have been identified that inhibit axonal regeneration:
Myelin-‐associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein
(Omgp). MAG is a member of the immunoglobulin super family, and appears to
exhibit bifunctionality, promoting growth during early stages of development while
inhibiting growth in older stages (Lai et al., 1987; McKerracher et al., 1994; Turnley
& Bartlett, 1998). MAG is located on the periaxonal membrane, allowing for easy
interaction with an axonal receptor (Trapp, 1988; Filbin, 2003). Omgp has also been
shown to induce growth cone collapse and inhibit neurite outgrowth (Wang et al.,
2002). In the spinal cord, Omgp is expressed at the Nodes of Ranvier, functioning to
maintain the structure of these areas by inhibiting axon growth (Nie et al., 2006).
This glycoprotein is linked to the cell membrane in myelinating oligodendrocytes,
and is localized to the glial–axonal interface of myelinated axons (Kottis et al., 2002).
Interestingly, although bearing no structural or sequence homology, Nogo, Mag and
Omgp all act on the same receptor, the glycosylphosphatidylinositol-‐linked Nogo
receptor, which is expressed on the surface of various neurons (Fournier et al.,
2001; Domeniconi et al., 2002; Wang et al., 2002). Because this receptor has no
transmembrane or cytoplasmic domains, it requires co-‐receptors to transduce the
inhibitory signal (Filbin, 2003). The first co-‐receptor identified was the p75
neurotrophin receptor (Yamashita et al., 2002). Similar to the mechanism
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underlying CPSG inhibition, “binding of myelin inhibitors to the NgR1-‐p75NTR
receptor complex activates protein kinase C (PKC) and causes the small GTPase Rho
to assume its active, GTP-‐bound state. Rho-‐mediated activation of downstream
effectors such as Rho-‐associated kinase (ROCK) then induces actin polymerization,
which leads to growth cone collapse and inhibition of neurite outgrowth” (Hannila &
Filbin, 2008). In order for p75 to activate Rho, it must be cleaved, as it is the
molecule’s intracellular domain that is functional in activating Rho (Domeniconi et
al., 2005).
Although inactivation of p75 is shown to promote neuronal outgrowth, co-‐
expression of p75 with NgR1 in non-‐neuronal cells led for an inability for Omgp to
activate Rho, suggesting the presence of another protein that mediates functionality
in this complex (Yamashita et al., 2002; Mi et al., 2004). Surveying CNS proteins for
their ability to interact with NgR1 led to the discovery of Lingo-‐1. The existence of a
ternary complex was supported by binding assays and experiments that showed
occurrence of Rho activity only when all three proteins were present, as opposed to
the lack of Rho activity in binary complexes expressing only two of the three
proteins. When analyzing the structural morphology of Lingo-‐1, the presence of an
EGFR-‐like tyrosine phosphorylation site was discovered. Deletion of this region
resulted in a diminished inhibitory effect on neuron outgrowth (Mi et al., 2004).
However, p75 is not expressed in all regions of the brain, suggesting the presence of
yet another protein that is associated with NgR1. This led to the discovery of Troy, a
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tumor necrosis factor receptor that is expressed on adult neurons. Similar to the
experiments with Lingo-‐1, binding assays and assessment of RhoA activity
supported the presence of another ternary complex consisting of NgR1, Lingo-‐1 and
Troy, with Troy functioning as a substitute for p75. Troy-‐knockout mice exhibit
substantially larger neuronal outgrowths as compared to wild-‐type mice, further
supporting its role in mediating the functional activity of NgR1. These results
indicate Troy as a functional homolog to p75 in areas of the brain where p75 is not
expressed (Park et al., 2005; Mandemakers & Barres, 2005).
Figure adapted from Experimental Neurology (Akbik et al., 2012)
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REGENERATION
The goals of attempts to promote axonal regeneration all involve trying to make the
damaged area permissive to growth. One way to do this is to transplant cells from
regions known to promote axonal regeneration to the areas of CNS injury. Data
supports the regeneration of axons in the olfactory bulb. “Normal and sectioned
olfactory axons spontaneously grow within the adult olfactory bulb, establishing
synaptic contacts with their targets. The major difference between a regenerating
system such as the olfactory bulb and the rest of the CNS is the presence of
ensheathing glia (EG) in the former” (Ramón-‐Cueto et al., 1998). To see if EGs have
the ability to regenerate axons in the CNS, pure EGs were transplanted to the dorsal
root entry zone of a transected region of one dorsal root at the cord entry point.
“Three weeks after transplantation, numerous regenerating dorsal root axons were
observed re-‐entering the spinal cord… Neither ensheathing cells nor regenerating
axons invaded those laminae they did not innervate under normal circumstances”
(Ramón-‐Cueto & Nieto-‐Sampedro, 1994). Further experimentation showed
successful recovery of forepaw function in mice after the region of the spinal cord
controlling this area was transected and EGs were transplanted to the area, allowing
for a permissive growth environment that restored functional connectivity (Li et al.,
1997).
An enzyme discussed earlier called Chondroitinase ABC (ChABC) has been shown to
promote functional recovery of locomotor behavior in mouse models of dorsal
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column spinal cord injury. When administered, ChABC functions to delete the
glycosaminoglycan side chains on the chondroitin sulfate proteoglycan, which is
thought to render the protein ineffective with regards to transducing the inhibitory
signal. In a rat model of spinal cord injury, electrophysiological and behavioral
testing showed functional recovery of synaptic connections through the area of
injury. Furthermore, upregulation of growth-‐associated protein 43 was observed, a
protein seen in PNS injury environments thought to mediate the regenerative state
(Bradbury et al., 2002).
Another approach to studying ways to promote axonal growth and block inhibition
involves studying the intracellular mechanisms by which myelin associated
inhibitors and CPSGs act. Based on the bifunctionality of MAG depending on the
stages of development, both its growth-‐promoting and growth-‐inhibiting actions
were studied. Comparing the structure of MAG during these two stages reveled no
changes, suggesting the role of intracellular mechanisms mediating the response to
MAG. It was found that during early stages when MAG is growth-‐promoting, levels of
cyclic AMP are high, while during later stages when MAG is inhibitory, levels of
cyclic AMP were low (Cai et al., 2001). This led to the idea of using pharmacological
agents to stimulate cAMP activity in cells in order to promote axonal growth.
Through administering a phosphodiesterase inhibitor that prevents the breakdown
of cAMP, scientists were able to overcome MAG-‐associated inhibition in a rat model
of spinal cord injury (Nikulina et al., 2004). Elevated levels of cAMP activate the
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transcription factor cAMP response element binding protein (CREB) through
phosphorylation of CREB by cAMP-‐activated protein kinase A. This leads to the
transcription of genes that are responsible for promoting axonal regeneration (Gao
et al., 2004; Hannila & Filbin, 2008).
In an attempt to identify other cellular mechanisms by which CNS myelin inhibition
works, one study screened for compounds with the ability to prevent axonal growth
inhibition and assessed each’s effect on neuronal outgrowth on an immobilized
myelin substrate. “Several EGFR kinase inhibitors showed a remarkable ability to
counter the effects of myelin inhibition, suggesting the involvement of EGFR kinase
activity in the inhibitory effects of myelin inhibitors” (Koprivica et al., 2005).
Further experiments showed that both Nogo-‐A and Omgp induce rapid EGFR
phosphorylation, however indirectly through events that occur downstream from
the NgR1 receptor, as coimmunoprecipitation experiments failed to detect the
presence of EGFR with the NgR1 receptor. Although how exactly myelin inhibitors
induce EGFR phosphorylation is unclear, treatment of CNS lesion sites with EGFR
inhibitors like Erlotinib, which is approved for the treatment of cancer, was able to
block the neurite outgrowth inhibition caused by myelin-‐associated inhibitors
(Koprivica et al., 2005).
Based on the Rho/ROCK pathway by which both myelin associated inhibitors and
CPSGs act, one study looked at the effect of using both Rho and ROCK inhibitors to
promote axonal regeneration on inhibitory substrates. Using C3, a Rho inhibitor, and
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Y27632, a ROCK inhibitor, scientists showed that both had an effect of substantially
increasing the degree of axonal growth when plated on substrates containing only
myelin associated inhibitors, only CPSGs, and both myelin associated inhibitors and
CPSGs. Inhibition of Rho showed significantly better effects on promoting
regeneration than did inhibition of ROCK, suggesting Rho may affect another
downstream secondary molecule that is separate from ROCK. The authors took this
a step forward and applied the Rho inhibitor to mouse spinal cord injury models to
test if inhibition of Rho allows for functional recovery of motor function. Use of C3
allowed for long-‐distance axonal generation extending past the lesion into distal
white matter and showed a remarkable recovery of motor functions, allowing mice
to walk with weight support as opposed to control mice who moved by pulling
themselves forward with their forelimbs (Dergham et al., 2002) .
Looking further into the intercellular signalling pathways initiated by myelin
associated inhibitors, one group found a molecule that when bound, promotes down
regulation of a key protein in the receptor complex that mediates myelin associated
inhibition. This molecule is retinoic acid, and binds to a receptor known as the
retinoic acid receptor beta. When cerebellar granule neurons were cultured with
both myelin substrates and retinoic acid, neurite outgrowth was promoted. With
knowledge of the myelin associated inhibition signalling pathway, the group
assessed the activity of Rho to see if retinoic acid was affecting this pathway, and
found that Rho activity was decreased when retinoic acid was bound to its receptor.
Further exploration of the molecular mechanisms mediating this decrease in activity
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found that retinoic acid binding resulted in the down regulation of the Lingo-‐1
protein, a protein part of the NgR complex that is necessary for myelin associated
inhibitors to transmit their inhibitory signal. This down regulation is mediated by
RA-‐bound RAR-‐β occupying “a specific RA response element (RARE) on the Lingo-‐1
promoter, transcriptionally repressing Lingo-‐1 myelin-‐dependent gene activation”
(Puttagunta et al., 2011).
This same group took an epigenetic approach to study the changes in gene
expression associated with recovery from PNS injury with hopes of being able to
induce the same changes after CNS injury. They found that “p300/CBP-‐associated
factor (PCAF)-‐dependent acetylation of histone 3 lysine 9 (H3K9ac), paralleled by a
reduction in methylation of H3K9 (H3K9me2), occurred at the promoters of select
genes only after PNS axonal injury” (Puttagunta et al., 2014). This histone
modification was promoted by extracellular signal-‐regulated kinase acting as a
retrograde signal to induce activation of PCAF that then causes increased expression
of regeneration associated genes like H3K9ac, Galanin and BDNF. The group then
tested whether PCAF overexpression in a spinal cord injury model would stimulate
axonal regeneration, finding a significant increase in the number of regenerating
fibres across the lesion (Puttagunta et al., 2014). This study represents a technique
unique to the other regeneration strategies presented as opposed to using inhibitors
or transplantation, this strategy employs overexpression, potentially the least
harmful method with regards to downstream negative effects.
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CONCLUSION
CNS injuries are a very serious matter, as they often lead to irreversible motor
defects due to the inability of neurons in the CNS to regenerate through the injury.
However, current neuroscience is targeting this area with hopes of both shedding
light on the mechanisms that make CNS injuries irreversible and how to manipulate
these mechanisms as to restore motor functionality by promoting CNS axonal
regeneration. A number of the mechanisms underlying the inhibition of axonal
regeneration come from the neuroinflammatory response to CNS injury. Reactive
astrogliosis is the process by which astrocytes respond to CNS injury by
proliferating and upregulating different molecules that function to block off the
injured site from being exposed to healthy tissue. Besides the formation of the glial
scar that acts as a physical barrier to axonal regeneration, there is a large increase in
the number of CPSGs that are found in the extracellular matrix. CPSGs exhibit their
inhibitory effects through diverse and complex signaling pathways that are not yet
fully understood. They also function to sequester growth-‐promoting molecules,
blocking them from accessing the damaged tissue to promote regeneration. Other
molecules that play important roles during development are shown to be
upregulated following CNS injury and are likely to play a role in inhibition. Such
molecules include ephrins, semaphorins, slit proteins, and tenascin.
The other major component of axonal inhibition after CNS inhibition are the
inhibitory glycoproteins associated with myelin. These are proteins that are always
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present on myelin, and though they exhibit no change in expression following CNS
injury, they still play a major role in inhibiting axonal regeneration. These proteins
often have a crucial role developmentally, promoting and directing growth during
development while inhibiting growth later on in life. These proteins are myelin-‐
associated glycoprotein and oligodendrocyte myelin glycoprotein. Both of these
molecules exhibit their inhibitory effects through acting on the same receptor, the
Nogo receptor. Thus, they all induce a similar cascade of intercellular events. This
pathway includes activation of protein kinase C that causes RhoA to activate Rho-‐
associated kinase (ROCK), which then induces actin polymerization, leading to
growth cone collapse and inhibition of neurite outgrowth.
Strategies to overcome the inhibition of axonal growth following CNS injury have
come from understanding the mechanisms by which the molecules that are
inhibiting axonal regeneration are acting. Strategies include inhibition of the
molecules directly involved or more effectively, of the downstream signaling
cascade that they induce, transplantation of growth-‐promoting regions to create a
growth-‐permissive environment, as well as upregulation or activation of different
molecules and genes that are shown to induce growth either during development or
in the PNS. Though many of these strategies have shown promise in restoring
functionality to CNS injury models in animals, clinical effectiveness of any of the
mentioned methods has yet to be established. One might conclude that due to the
multifaceted nature of CNS inhibition, a combination of many strategies would be
the most effective in promoting CNS recovery after injury. Because many of the
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molecules involved in axonal inhibition generally serve other functional roles in the
CNS, it is important to establish a methodology that takes into account many factors
such as the time course of administration of such inhibitors or activators, as failure
to do so may result in downstream negative effects due to ineffective signaling.
Another issue that would be important to account for is how to direct axonal
regeneration such that original connectivity is maintained, and so that new
connections that might functionally hinder the affected individual are not made.
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