Mini Disseration Kabir Nigam

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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

25

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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REFERENCES

1. "Brain and Spinal Cord Injury Causes, Symptoms, Diagnosis, Treatment." PDR

Health. Physcians Desk Reference, n.d. Web. 04 Nov. 2015.

2. Kamiguchi, Hiroyuki. "Neuronal Growth Mechanisms." Faculty. Riken Brain

Science Institute, n.d. Web. 4 Nov. 2014.

3. Schnabelrauch, Matthias. "Synthesis and Characterization of ECM-‐specific

Polysaccharides." Funktionelle Biomaterialien Zur Steuerung Von

Heilungsprozessen in Knochen-‐ Und Hautgewebe -‐ Vom Material Zur Klinik.

University of Leipzig, n.d. Web. 04 Nov. 2015.

4. Aarts, M. M. & Tymianski, M. TRPM7 and ischemic CNS injury. Neuroscientist

11, 116–23 (2005).

5. Aricescu, A., McKinnell, I., Halfter, W. & Stoker, A. Heparan sulfate

proteoglycans are ligands for receptor protein tyrosine phosphatase sigma.

Mol. Cell. Biol. 22, 1881-‐92 (2002).

6. Akbik, F., Cafferty, W. B. J. & Strittmatter, S. M. Myelin associated inhibitors: a

link between injury-‐induced and experience-‐dependent plasticity. Exp.

Neurol. 235, 43–52 (2012).

7. Bagnard, D., Lohrum, M., Uziel, D., Puschel, A. & Bolz, J. Semaphorins act as

attractive and repulsive guidance signals during the development of cortical

projections. Development 125, 5043–5053 (1998).

28

8. Benson, M., Romero, M., Lush, M., Lu, Q., Henkemeyer, M. & Parada, L. Ephrin-‐

B3 is a myelin-‐based inhibitor of neurite outgrowth. Proc. Natl. Acad. Sci. U. S.

A. 102, 10694–9 (2005).

9. Berry, M. Post-‐injury myelin-‐breakdown products inhibit axonal growth: an

hypothesis to explain the failure of axonal regeneration in the mammalian

central nervous system. Bibl. Anat. 23, 1–11 (1982).

10. Bradbury, E., Moon, L., Popat, R., King, V., Bennett, G., Patel, P., Fawcett, J. &

McMahon, S. Chondroitinase ABC promotes functional recovery after spinal

cord injury. Nature 416, 636–40 (2002).

11. Bundesen, L., Scheel, T., Bregman, B. & Kromer, L. Ephrin-‐B2 and EphB2

Regulation of Astrocyte-‐Meningeal Fibroblast Interactions in Response to

Spinal Cord Lesions in Adult Rats. J. Neurosci. 21, 7789-‐7800 (2003).

12. Cai, D., Qiu, J., Cao, Z., McAtee, M., Bregman, B. & Filbin, M. Neuronal Cyclic

AMP Controls the Developmental Loss in Ability of Axons to Regenerate. J.

Neurosci. 21, 4731–4739 (2001).

13. Caroni, P. & Schwab, M. E. Antibody against myelin-‐associated inhibitor of

neurite growth neutralizes nonpermissive substrate properties of CNS white

matter. Neuron 1, 85–96 (1988).

14. Chen, M., Huber, A., van der Haar, M., Frank, M., Schnell, L., Spillmann, A.,

Christ, F. & Schwab, M. Nogo-‐A is a myelin-‐associated neurite outgrowth

inhibitor and an antigen for monoclonal antibody IN-‐1. Nature. 403, 434-‐9

(2000).

29

15. Dergham, P., Ellezam, B., Essagian, C., Avedissian, H., Lubell, W. &

McKerracher, L. Rho Signaling Pathway Targeted to Promote Spinal Cord

Repair. J. Neurosci. 22, 6570–6577 (2002).

16. De Winter, F., Oudega, M., Lankhorst, A., Hamers, F., Blits, B., Ruitenberg, M.,

Pasterkamp, R., Gispen, W. & Verhaagen, J. Injury-‐induced class 3 semaphorin

expression in the rat spinal cord. Exp. Neurol. 175, 61–75 (2002).

17. Domeniconi, M., Cao, Z., Spencer, T., Sivasankaran, R., Wang, K., Nikulina, E.,

Kimura, N., Cai, H., Deng, K., Gao, Y., He, Z. & Filbin, M. Myelin-‐associated

glycoprotein interacts with the Nogo66 receptor to inhibit neurite

outgrowth. Neuron 35, 283–290 (2002).

18. Domeniconi, M., Zampieri, N., Spencer, T., Hilaire, M., Mellado, W., Chao, M. &

Filbin, M. MAG induces regulated intramembrane proteolysis of the p75

neurotrophin receptor to inhibit neurite outgrowth. Neuron 46, 849-‐855

(2005).

19. Edgar, N. & Sibille, E. A putative functional role for oligodendrocytes in mood

regulation. Transl. Psychiatry 2, e109 (2012).

20. Fawcett, J. & Asher, R. The glial scar and central nervous system repair. Brain

Res Bull. 49, 377-‐91 (1999).

21. Filbin, M. T. Myelin-‐associated inhibitors of axonal regeneration in the adult

mammalian CNS. Nat. Rev. Neurosci. 4, 703–13 (2003).

22. Finnie, J. W. & Blumbergs, P. C. Traumatic Brain Injury. Vet. Pathol. 39, 679–

689 (2002).

30

23. Fitch, M. T. & Silver, J. CNS injury, glial scars, and inflammation: Inhibitory

extracellular matrices and regeneration failure. Exp. Neurol. 209, 294–301

(2008).

24. Fournier, A., GrandPre, T. & Strittmatter, S. Identification of a receptor

mediating Nogo-‐66 inhibition of axonal regeneration. Nature 409, 341–346

(2001).

25. Gao, Y., Deng, K., Hou, J., Bryson, J., Barco, A., Nikulina, E., Spencer, T., Mellado,

W., Kandel, E. & Filbin, M. Activated CREB is sufficient to overcome inhibitors

in myelin and promote spinal axon regeneration in vivo. Neuron 44, 609–21

(2004).

26. Goldberg, J., Vargas, M., Wang, J., Mandemakers, W., Oster, S., Sretavan, D. &

Barres, B. An oligodendrocyte lineage-‐specific semaphorin, Sema5A, inhibits

axon growth by retinal ganglion cells. J. Neurosci. 24, 4989–99 (2004).

27. GrandPré, T., Nakamura, F., Vartanian, T. & Strittmatter, S. Identification of

the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature. 403,

439-‐44 (2000).

28. Hannila, S. S. & Filbin, M. T. The role of cyclic AMP signaling in promoting

axonal regeneration after spinal cord injury. Exp. Neurol. 209, 321–32 (2008).

29. Huang, L., Wu, Z., Zhuge, Q., Zheng, W., Shao, B., Wang, B., Sun, F. & Jin, K. Glial

scar formation occurs in the human brain after ischemic stroke. Int. J. Med.

Sci. 11, 344–8 (2014).

31

30. Huber, A., Weinmann, O., Brösamle, C., Oertle, T. & Schwab, M. Patterns of

Nogo mRNA and protein expression in the developing and adult rat and after

CNS lesions. J. Neurosci. 22, 3553-‐67 (2002).

31. Johnson, K. & Van Vactor, D. Receptor protein tyrosine phosphatases in

nervous system development. Physiol. Rev. 83, 1-‐24 (2003).

32. Jones, L. L., Margolis, R. U. & Tuszynski, M. H. The chondroitin sulfate

proteoglycans neurocan, brevican, phosphacan, and versican are

differentially regulated following spinal cord injury. Exp. Neurol. 182, 399–

411 (2003).

33. Kimelberg, H. K. & Nedergaard, M. Functions of astrocytes and their potential

as therapeutic targets. Neurotherapeutics 7, 338–53 (2010).

34. Kjellén, L. & Lindahl, U. Proteoglycans: structures and interactions. Annu. Rev.

Biochem. 60, 443-‐75 (1991).

35. Koch, C. & Laurent, G. Complexity and the nervous system. Science 284, 96–8

(1999).

36. Koprivica, V., Cho, K., Park, J., Yiu, G., Atwal, J., Gore, B., Kim, J., Lin, E., Tessier-‐

Lavigne, M., Chen, DF. & He, Z. EGFR activation mediates inhibition of axon

regeneration by myelin and chondroitin sulfate proteoglycans. Science 310,

106–10 (2005).

37. Kottis, V., Thibault, P., Mikol, D., Xiao, Z., Zhang, R., Dergham, P. & Braun, P.

Oligodendrocyte-‐myelin glycoprotein (OMgp) is an inhibitor of neurite

outgrowth. J. Neurochem. 82, 1566–1569 (2002).

32

38. Lai, C., Watson, J., Bloom, F., Sutcliffe, J. & Milner, R. Neural protein

1B236/myelin-‐associated glycoprotein (MAG) defines a subgroup of the

immunoglobulin superfamily. Immunol. Rev. 100, 129-‐51 (1987)

39. Lee, H., McKeon, R. J. & Bellamkonda, R. V. Sustained delivery of

thermostabilized chABC enhances axonal sprouting and functional recovery

after spinal cord injury. Proc. Natl. Acad. Sci. U. S. A. 107, 3340–5 (2010).

40. Li, Y., Field, P. & Raisman, G. Repair of Adult Rat Corticospinal Tract by

Transplants of Olfactory Ensheathing Cells. Science (80-‐. ). 277, 2000–2002

(1997).

41. Lucas, S.-‐M., Rothwell, N. J. & Gibson, R. M. The role of inflammation in CNS

injury and disease. Br. J. Pharmacol. 147 Suppl , S232–40 (2006).

42. Mandemakers, W. J. & Barres, B. A. Axon regeneration: it’s getting crowded at

the gates of TROY. Curr. Biol. 15, R302–5 (2005).

43. McKerracher, L., David, S., Jackson, D., Kottis, V., Dunn, R. & Braun,

P. Identification of myelin-‐associated glycoprotein as a major myelin-‐derived

inhibitor of neurite growth. Neuron 13, 805–811 (1994).

44. McDonald, J. W. & Sadowsky, C. Spinal-‐cord injury. Lancet 359, 417–25

(2002).

45. Mi, S., Lee, X., Shao, Z., Thill, G., Ji, B., Relton, J., Levesque, M., Allaire, N., Perrin,

S., Sands, B., Crowell, T., Cate, R., McCoy, J. & Pepinsky, R. LINGO-‐1 is a

component of the Nogo-‐66 receptor/p75 signaling complex. Nat. Neurosci. 7,

221–8 (2004).

33

46. Miranda, J., White, L., Marcillo, A., Wilson, C., Jagid, J. & Whittemore, S.

Induction of Eph B3 after Spinal Cord Injury. Exp. Neurology. 156, 218-‐22

(1999).

47. Monnier, P. P., Sierra, A., Schwab, J. M., Henke-‐Fahle, S. & Mueller, B. K. The

Rho/ROCK pathway mediates neurite growth-‐inhibitory activity associated

with the chondroitin sulfate proteoglycans of the CNS glial scar. Mol. Cell.

Neurosci. 22, 319–330 (2003).

48. Moreau-‐Fauvarque, C., Kumanogoh, A., Camand, E., Jaillard, C., Barbin, G.,

Boquet, I., Love, C., Jones, E., Kikutani, H., Lubetzki, C., Dusart, I. & Chédotal, A.

The Transmembrane Semaphorin Sema4D/CD100, an Inhibitor of Axonal

Growth, Is Expressed on Oligodendrocytes and Upregulated after CNS Lesion.

J. Neurosci. 23, 9229–9239 (2003).

49. Nakajima, K. & Kohsaka, S. Microglia: activation and their significance in the

central nervous system. J. Biochem. 130, 169–75 (2001).

50. Nie, D., Ma, Q., Law, J., Chia, C., Dhingra, N., Shimoda, Y., Yang, W., Gong, N.,

Chen, Q., Xu, G., Hu, Q., Chow, P., Ng, Y., Ling, E., Watanabe, K., Xu, T., Habib, A.,

Schachner, M. & Xiao, Z. Oligodendrocytes regulate formation of nodes of

Ranvier via the recognition molecule OMgp. Neuron Glia Biol. 2, 151–64

(2006).

51. Nikulina, E., Tidwell, J. L., Dai, H. N., Bregman, B. S. & Filbin, M. T. The

phosphodiesterase inhibitor rolipram delivered after a spinal cord lesion

promotes axonal regeneration and functional recovery. Proc. Natl. Acad. Sci.

U. S. A. 101, 8786–90 (2004).

34

52. Park, J., Yiu, G., Kaneko, S., Wang, J., Chang, J., He, X., Garcia, K. & He, Z. A TNF

receptor family member, TROY, is a coreceptor with Nogo receptor in

mediating the inhibitory activity of myelin inhibitors. Neuron 45, 345–51

(2005).

53. Pasterkamp, R. Emerging roles for semaphorins in neural regeneration. Brain

Res. Rev. 35, 36–54 (2001).

54. Pasterkamp, R., Anderson, P. & Verhaagen, J. Peripheral nerve injury fails to

induce growth of lesioned ascending dorsal column axons into spinal cord

scar tissue expressing the axon repellent Semaphorin3A. Eur. J. Neurosci. 13,

457-‐71 (2001).

55. Pasterkamp, R. J. & Verhaagen, J. Semaphorins in axon regeneration:

developmental guidance molecules gone wrong? Philos. Trans. R. Soc. Lond. B.

Biol. Sci. 361, 1499–511 (2006).

56. Popovich, P. G. & Longbrake, E. E. Can the immune system be harnessed to

repair the CNS? Nat. Rev. Neurosci. 9, 481–93 (2008).

57. Puttagunta, R., Schmandke, A., Floriddia, E., Gaub, P., Fomin, N., Ghyselinck, N.

& Di Giovanni, S. RA-‐RAR-‐β counteracts myelin-‐dependent inhibition of

neurite outgrowth via Lingo-‐1 repression. J. Cell Biol. 193, 1147–56 (2011).

58. Puttagunta, R., Tedeschi, A., Sória, M., Hervera, A., Lindner, R., Rathore, K.,

Gaub, P., Joshi, Y., Nguyen, T., Schmandke, A., Laskowski, C., Boutillier, A.,

Bradke, F. & Di Giovanni, S. PCAF-‐dependent epigenetic changes promote

axonal regeneration in the central nervous system. Nat. Commun. 5, 3527

(2014).

35

59. Ramón-‐Cueto, A. & Nieto-‐Sampedro, M. Regeneration into the spinal cord of

transected dorsal root axons is promoted by ensheathing glia transplants.

Exp. Neurol. 127, 232–44 (1994).

60. Ramón-‐Cueto, A., Plant, G. W., Avila, J. & Bunge, M. B. Long-‐Distance Axonal

Regeneration in the Transected Adult Rat Spinal Cord Is Promoted by

Olfactory Ensheathing Glia Transplants. J. Neurosci. 18, 3803–3815 (1998).

61. Rolls, A., Shechter, R. & Schwartz, M. The bright side of the glial scar in CNS

repair. Nat. Rev. Neurosci. 10, 235-‐41 (2009).

62. Rudge, J. & Silver, J. Inhibition of neurite outgrowth on astroglial scars in

vitro. J. Neurosci. 10, 3594–3603 (1990).

63. Sharma, K., Selzer, M. E. & Li, S. Scar-‐mediated inhibition and CSPG receptors

in the CNS. Exp. Neurol. 237, 370–8 (2012).

64. Shen, Y., Tenney, A., Busch, S., Horn, K., Cuascut, F., Liu, K., He, Z., Silver, J. &

Flanagan, J. PTPsigma is a receptor for chondroitin sulfate proteoglycan, an

inhibitor of neural regeneration. Science 326, 592–6 (2009).

65. Silver, J. & Miller, J. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5,

146–156 (2003)

66. Snow, D., Lemmon, V., Carrino, D., Caplan, A. & Silver, J. Sulfated

proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp.

Neurol. 109, 111-‐30 (1990).

67. Sofroniew, M. Reactive astrocytes in neural repair and protection.

Neuroscientist. 11, 400-‐7 (2005).

36

68. Stichel, C. C. & Müller, H. W. The CNS lesion scar: new vistas on an old

regeneration barrier. Cell Tissue Res. 294, 1–9 (1998).

69. Trapp, B. Distribution of the myelin-‐associated glycoprotein and P0 protein

during myelin compaction in quaking mouse peripheral nerve. J. Cell

Biol. 107, 675–685 (1988).

70. Turnley, A. & Bartlett, P. MAG and MOG enhance neurite outgrowth of

embryonic mouse spinal cord neurons. Neuroreport 9, 1987–1990 (1998).

71. Wang, X., Chun, S., Treloar, H., Vartanian, T., Greer, C. & Strittmatter, S.

Localization of Nogo-‐A and Nogo-‐66 receptor proteins at sites of axon-‐myelin

and synaptic contact. J. Neurosci. 22, 5505-‐15 (2002).

72. Wang, K., Koprivica, V., Kim, J., Sivasankaran, R., Guo, Y., Neve, R. & He, Z.

Oligodendrocyte-‐myelin glycoprotein is a Nogo receptor ligand that inhibits

neurite outgrowth. Nature 417, 941–944 (2002).

73. Wilhelmsson, U., Bushong, E., Price, D., Smarr, B., Phung, V., Terada, M.,

Ellisman, M. & Pekny, M. Redefining the concept of reactive astrocytes as cells

that remain within their unique domains upon reaction to injury. Proc. Natl.

Acad. Sci. 103, 17513–17518 (2006).

74. Yamaguchi, W., Tamai, R., Kageura, M., Furuyama, T. & Inagaki, S. Sema4D as

an inhibitory regulator in oligodendrocyte development. Mol. Cell. Neurosci.

49, 290-‐99 (2012).

75. Yamashita, T., Higuchi, H. & Tohyama, M. The p75 receptor transduces the

signal from myelin-‐associated glycoprotein to Rho. J. Cell Biol. 157, 565–70

(2002).

37

76. Yokoyama, N., Romero, M., Cowan, C., Galvan, P., Helmbacher, F., Charnay, P.,

Parada, L. & Henkemeyer, M. Forward signaling mediated by ephrin-‐B3

prevents contralateral corticospinal axons from recrossing the spinal cord

midline. Neuron. 29, 85-‐97 (2001).

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