Axonal Growth and Regeneration

Question 1

After the neurone has migrated to its final position what influences it’s connectivity?

Answer 1

Once they have migrated to their final positions, neurones begin to extend an axon towards an appropriate target tissue.

The pathway may be long and circuitous, requiring the axon to use molecular cues in the environment for navigation.

These molecular cues (trophic factors) can be short or long-range, repulsive or attractive, or promote survival . They are mainly released by glial cells.

Question 2

How is the axon adapted for axonal guidance and synaptogenesis?

Answer 2

The distal tip of the axon (the growth cone) is morphologically specialised and enriched in receptors that detect these cues, and stimulate intracellular signalling pathways, leading to cytoskeleton rearrangement and changes in gene transcription. The cytoskeletal changes (actin filaments and microtubules) underlie growth cone dynamics, and hence control axon growth.

Depending on the molecular cue, the growth cone responds by changing direction, collapsing, stalling, forming a pre-synaptic terminal, spreading, or growing faster.

Question 3

What is the main intracellular signalling pathway that controls growth cone formation and collapse?

Answer 3

Rho GTPases control actin assembly and disassembly. One example is RhoA, which causes growth-cone collapse
• Receptor activation leads to activation of Rho GTPases.
• This leads to controlling RhoA, which activates ROCK and LIM Kinases.
• LIMK phosphorylases to activate the Cofilin molecule, which is an essential protein for F-actin depolymerisation. Blocking LIMK prevents neurite outgrowth inhibition in embryonic chicks (Hsieh et al., 2006).

Question 4

How does the history of the neurone determine axonal growth response?

Answer 4

A growth cone can only respond to an extracellular signal if it contains a receptor for that ligand. Each neuronal population has a specific complement of growth cone receptors. Therefore this signalling depends on the history of the neurone.

Question 5

Describe the mechanism of activity-independent synaptogenesis

Answer 5

Axons respond to synaptogenic factors, which stimulate pre-synaptic differentiation by modulating microtubule dynamics in the growth cone.

Question 6

Give examples of positive regulators of axonal growth

Answer 6

Long-range:
• Growth factors such as the Neurotrophins stimulate axon growth and survival of selective groups of neurones. Neurotrophins include Nerve Growth Factor (NGF), Bone Derived Neurotrophic Factor (BDNF), and NT-3, NT4/5.
• Netrin which can attract (and repel) different classes of neurones.

Short-range (Cell-associated)
• The immunoglobulin superfamily (e.g. NCAM) or
• The cadherin superfamily (e.g. N-cadherin)

Short-range (ECM-associated) include laminin and collagen, which are secreted by cells to stimulate growth cone advance down specific guidance pathways.

Question 7

Give examples of negative regulators of axonal growth

Answer 7

Long-range:
• Netrins can attract and repel different classes of neurones
• Semaphorins such as semaphorin II (collapsin), which stimulates growth cone collapse
• Slit, which is a soluble repellent secreted by midline cells.

Short-Range (Cell-associated) are mainly ephrins which are membrane-associated repulsive ligands, which may be either GPI- anchored (A-type) or transmembrane (B-type).

Short-range (ECM-associated) such as S-laminin, tenascin, chondroitin sulphate proteoglycan (CSPGs).

Question 8

How do netrins influence axonal growth?

Answer 8

Netrins are released by cells during spinal cord development.

Commissural neurones express a transmembrane receptor called DCC (Depleted in Colorectal Cancer) which can bind to netrins.

This allows commissural neurones which are in the dorsal half to be attracted ventrally towards the floor plate by gradients of netrin 2 (gradual) and netrin 1 (steep).

Question 9

How do neurotrophins influence axonal growth?

Answer 9

The target for the neurotrophins are Trk tyrosine kinase receptors. Different neuronal populations have different Trk receptors - this provides a mechanism for target selection.
• Trk A has greater affinity to NGF
• Trk B has greater affinity to BDNF
• Trk C has greater affinity to NT-3/4/5

Dimerisation of the Trk receptors by the neurotrophins stimulates intracellular signalling pathways. These stimulate changes in gene expression (via CREB) that promote cell survival and axon outgrowth, and also local cytoskeleton changes via ?Ras/MAPK.

Question 10

How do immunoglobulins influence axonal growth?

Answer 10

There is only one NCAM (Neural cell adhesion molecule) gene. However, several different forms of NCAM are achieved by alternate splicing. It is expressed on the surface of neurons, oligodendrocytes, astrocytes and Schwann cells in the nervous system.

Numerous in vitro studies have established than NCAM is a stimulator of axonal growth.

Question 11

Why is alternate NCAM splicing important?

Answer 11

Alternative splicing is important: NCAM with PSA is abundant in early development, but decreases during later development. Itstimulates greater/faster neurite outgrowth than NCAM lacking PSA.
Whereas, VASE-NCAM is not present in early development. When it is present, stops from NCAM actin on regeneration and blocks responses to growth substratum.

This is very interesting, because nature here has used the same gene (just alternatively spliced) to change response during different times. PSA-NCAM to stimulate faster neurite outgrowth at the beginning of development, but VASE-NCAM to slow/stop this down during later development.

Question 12

How do ECM components influence axonal growth?

Answer 12

Components of the basal lamina such as laminin and collagen provide a permissive substratum for neurite growth. Laminin in fact guides axons during development by this mechanism.

The major growth cone receptor for extracellular matrix molecules are the beta 1 integrins. Integrin receptors consistent of heterodimers composed of alpha and beta sub-units. Different combinations of alpha and beta bind different ECM molecules
• The RGD [Arg-Gly-Asp] motif in ECM molecules is a common beta 1 integrin binding motif

Repulsive cues are also present in the ECM, such as S-laminin, tenascin, chondroitin sulphate proteoglycan.

Question 13

Give an example where ECM cues are important in development.

Answer 13

Retinal ganglion cells navigate from the retina to the optic tectum on a pathway of laminin laid down by astrocytic end-feet lining the path of the optic nerve. When they reach their target, they downregulate their integrin receptors for laminin. Essentially, astrocytes are leaving laminin bread-crumbs for the retinal ganglion cells.

Question 14

Give an example where Netrins cause repulsion instead of positive axonal growth

Answer 14

The trochlear motor neurons in the ventral half of the spinal cord, however, are repulsed by netrin and grow dorsally away from the floor plate. They express DCC but also Unc5, and the combination changes a chemoattractive response to chemorepulsion. (Involves modulation of cAMP levels)

This is a good example of the fact that different neuronal populations can respond differently to the same ligand depending on receptor expression.

Question 15

How do semaphorins influence axonal growth?

Answer 15

Semaphorins are soluble chemorepulsants that stimulate growth cone collapse. The receptors for semaphorins are plexins. Semaphorins are selectively repulsive and control patterning in the spinal cord. They stimulate growth-cone collapse through RhoA/ROCK/LIMK pathway.

Question 16

Give an example where Semaphorins influence axonal growth.

Answer 16

An example of how these are used in conjunction with positive regulators of axonal growth:
• Sema III is expressed in ventral half of spinal cord
• NT3 responsive sensory neurons terminate in the ventral part
• NGF responsive sensory neurons terminate in the dorsal part.
• In vitro studies using Sema III transfected fibroblasts show that NGF responsive neurons are repulsed by Sema III, but NT3 responsive neurons are not.
Therefore NGF-responsive neurons do not enter ventral part, while NT3-responsive neurons do.

Question 17

Give an example where Slit influences axonal growth.

Answer 17

Slit is produced by midline cells is a soluble (long-range) repulsive molecule. Neurons expressing Robo receptors for slit are repulsed.

However, they need to cross the midline, so their Robo receptors are removed from the growth cone with the help of Commissureless (Com). This allows them to cross the midline (attracted by netrin). After they have crossed, Com is down-regulated, Robo receptors are therefore again expressed in the growth cone. This prevents the neurons from re-crossing back again.

If confused look at notes ‘Axonal Growth and Guidance Lecture’.

Question 18

How do ephrins influence axonal growth?

Answer 18

Growth cone receptors for ephrins are the Eph Receptors. There are two classes of Eph receptor in vertebrates, EphA & EphB receptors. They are tyrosine kinase receptors that activate Rho GTPases to
stimulate growth cone collapse.

Question 19

Give an example where Ephrins influences axonal growth.

Answer 19

Gradients of ephrin expression in the optic tectum (e.g. xenopus), and differential expression levels of Eph Rs in retinal ganglion cells control the development of the topographic map of the retina in the visual cortex.

Question 20

Following spinal cord damage, what determines the level of functional impairment?

Answer 20

Functional impairment is linked to synaptic dysfunction as a consequence of axonal/dendritic damage or degeneration. In other words, functional impairment is linked to a loss of connectivity rather than a loss of neurones.

Question 21

Does the CNS regenerate spontaneously?

Answer 21

The central nervous system does not regenerate spontaneously. If you put a peripheral nerve graft into the CNS, it does not growth. However, it does facilitate growth of CNS neurones within the graft. This suggests the PNS has something that can facilitate growth, with the CNS can’t.

Question 22

Do CNS axons sprout spontaneously?

Answer 22

The CNS axons and dendrites are plastic and sprout spontaneously following injury. This occurs both spared and lesioned fibres within the CNS. This is the mechanism that underpins some level of functional recovery following a brain injury such as stroke. As there is more space in the brain than spinal chord, there is more room for functional recovery following a brain lesion rather than spinal cord lesion.

Question 23

Are neural progenitors involved after CNS injury?

Answer 23

Neural progenitor cells can proliferate, differentiate and migrate following CNS injury. This is another aspect of recovery. It’s contribution to recovery is still very unclear. Interesting research being done in it.

Question 24

What are the goals of regenerative neuroscience?

Answer 24

1. Inhibit the glial scar formation. Gliosis mainly involves astrocytes, which extend processes to the lesion. Also involves microglia which secrete cytokines etc.
2. Inhibit the axon regeneration inhibitory signalling.
3. Promote pro-regenerative pathways
4. Replace cell loss.

Question 25

What is the difference between regeneration and sprouting?

Answer 25

• A regenerated axon is one that grows back to it’s intended target from the stump/lesion. This does not happen in the CNS. (CNS Paradigm 1)
• Sprouting occurs from nearby healthy axons, which send collaterals to nearby targets. Whether this is from the same neuronal population or not will determine the extent of functional connectivity.

Question 26

What is the molecular response to axotomy in the PNS?

Answer 26

A: There is a lesion, which causes the membrane to ‘break’. There is a wave of depolarisation, as the membrane becomes permeable to Na+ ions. There is an immediate Ca2+ influx. Ca2+ activates PKA and ERK, which affect local translation (as mRNA is in the axon).

B: Removal of retrograde (transport from axon to cell body) negative growth cues. There are usually negative growth cues that transport retrograde. Axotomy/axonal damage stops these cues. In the PNS, inhibition of these cues can lead to partial recovery.

C: Induction of retrograde positive cues. Signals generated at the site of axonal damage, are transported by dyenin to the cell body. This leads to activation of gene transcription, to promote axonal growth.

Question 27

How do glial cells inhibit regeneration in the CNS?

Answer 27

Astrocytes secrete a number of molecules (importantly Chondroitin sulfate proteoglycans - CSPGs) which bind to receptors (PTP), which activate RhoA, and growth cone collapse. CSPGs include aggrecan, brevican, neurocan, versican, phos- phacan and NG2

After injury, myelin debris are exposed by oligodendrocytes. Proteins such as MAG (Myelin Associated Glycoprotein), Nogo, Omgp (have to remember). These proteins bind to a receptor complex called Nogo receptor, which then also activated RhoA inducing growth cone collapse.

Question 28

What is the role of Retinoic Acid in axonal growth?

Answer 28

Retinoic Acid (RA) is an important molecule during development. The receptor is intranuclear. Binding to the receptor allows it activate signalling pathways, in particular gene expression which blocks axonal collapse (inhibits Lingo-1 from the Nogo complex) as well as promoting growth.

Question 29

What is the role of mTOR in axonal growth?

Answer 29

mTOR is a powerful stimulus to promote growth of axons, as well as stimulating cell proliferation.

Question 30

What contributes to the difference in axonal regeneration/growth ability in development and in adults?

Answer 30

We have learned about the mechanisms that are present in the developing nervous system for axonal growth. E.g. Trks, Ephs, ROBOs and their respective neurotrophins, netrins, SLITs etc ec.

However, the environment of the mature CNS is different, and many of these mechanisms are lost.

Instead, astrocyte and oligodendrocyte (inhibitory) mechanisms are present. So when an axon is injured in the mature CNS, more of these molecules are available to impair axonal growth.

Question 31

Why may it be important to prevent axonal regeneration in adults?

Answer 31

As to prevent growth, and therefore to control tumour genesis. Furthermore, any growth in the mature CNS would have more trouble reaching its target to make an appropriate regenerative effort. Nature has chosen for regeneration to not happen instead of messy re-connections (which can lead to pain, dysregulation of heart rate, blood pressure etc.).

Question 32

How does CNS sprouting contribute towards functional recovery after spinal cord damage?

Answer 32

There is some level of spontaneous regeneration after spinal cord injury, although often incomplete, and not enough to make a functional difference.

Following a lesion, there is sprouting from:
• Intact fibre tracts to compensate for the lack of activity from injured axons
• Lesioned axons to other supraspinal projections.

Question 33

How can we track axonal regeneration?

Answer 33

One way is by taking advantage of the retrograde transport mechanisms (via dyenin and endosomes). Injecting the motor target cells with molecules which can show up in the dorsal root ganglia (cell bodies). Each bright spot is where a motor neurone is reconnected.

Another way is looking at the dorsal columns. Inject tracer in the motor cortex and follow the axons.

Question 34

Give an example which shows the role of epigenetics in the context of axonal regeneration

Answer 34

Following PNS injury, several factors are retrogradely transported, which leads to the activation of PCAF and therefore histone acetylation (epigenetics) and expression of a number of regeneration associated genes.

This does not happen for spinal cord injury (CNS). However, an experiment where PCAF was overexpressed in the spinal cord, showed that this is able to promote sensory axon growth.

p300 is another histone-associated acetyltransferase, which has been shown to help regeneration in the optic nerve.

Question 35

Describe the pathogenesis of spinal tissue damage

Answer 35

• Seconds to minutes: Immediately after the lesion, microglia and macrophages from the blood stream invade the site of injury. They release cytokines, chemokines, and trophic factors.
• Hours: disruption of myelin.
• Days to weeks: Proliferation of reactive astrocytes. They then release proteoglycans, fibronectin, etc. CSPGs in particular are key players in axonal growth inhibition.
• Weeks-months later: glial scar tissue has formed, which is essential for structure and stability. However, this scar tissue prevents electrical signals up and down. The scar tissue is formed by mainly astrocytes, but also oligodendrocytes and microglia/macrophages.

Question 36

What prevents collateral sprouting after a SCI?

Answer 36

There is some collateral sprouting that go around the lesion, but often it dies off because it doesn’t find a target (it gets pruned). A range of molecular factors are working against this sprouting, as well as the physical space gap created by the lesion or cyst. Furthermore, there is little or no neurogenesis in the injured CNS.

Question 37

How is Taxol thought to affect SCI?

Answer 37

Taxol enhances dorsal column (DC) axonal regeneration. Scar tissue has a number of molecules that inhibit regeneration - e.g. Nogo, MAG, etc. Taxol is originally an anti-cancer drug. It works by stabilises microtubules and as a result, interferes with the normal breakdown of microtubules during cell division. With taxol, there is less glial scar.

Question 38

How are Anti-Nogo Antibodies thought to affect SCI?

Answer 38

Anti-NogoA antibodies promotes CST [corticospinal tract] regeneration (in rats and non-human primates). Nogo is a myelin protein that binds to the Nogo receptor and causes growth cone collapse. With anti-Nogo antibodies, there is some axonal sprouting and in some trials, show some motor improvements.

Question 39

How is chondroitinase thought to affect SCI?

Answer 39

This is an enzyme which breaks CSPGs. Chondroitin Sulphate Proteoglycans can bind to PTP receptors and can also bind to Nogo and give inhibitory signals. Chondroitinase dissolves Chondroitin Sulphate Proteoglycans, so less inhibitory signals, so axons can grow past the scar site.

Question 40

Explain the role of Stem Cell Transplantation in SCI

Answer 40

Stem cells promotes some level of improvement of motion (as opposed to fibroblast controls). Also showed some synaptic reconnections. Anatomical regeneration was impressive, but functional regeneration not massive compared to other therapies. However, can have too much growth and some signs of early tumour growth.

Embryonic stem cell transplantation has shown small improvements in function. This is through remyelinating neurones and reconstituting circuits as well as increasing plasticity. More recently, researchers are using pre-differentiated progenitor cells that can differentiate into neurones and glia. This has shown better improvement in rats?

Adult stem cells such as haematopoietic stem cells from the bone marrow and bone marrow stromal cells can be used in SCI. Small scale trials have shown that BMSCs helped functional recovery, however no controls in that trial. These therapies would be good as it bypasses the ethical concerns of embryonic stem cells, and the issue of immune rejection.

Question 41

Explain the role of Tissue Transplantation in SCI

Answer 41

The main aims of tissue transplantation is to provide a physical bridge, to allow axonal growth over cysts or cavities. The bridge is hoped to also create a favourable environment for axonal regeneration.

Peripheral nerve transplants where tried. These were able to promote axonal regeneration, however did not translate to any functional benefit. Schwann cells and olfactory nervous system cells have also been transplanted into the SCI site. The latter showed promise in rats, with some functional recovery. This has only been tried in humans in small trials - not good enough to be considered a clinical trial by international standards.

Embryonic CNS tissue transplantation after SCI lead to functional improvement in rats and cats. It allows axons to grow into the transplant. However, the axons end where the transplant tissue ends. The authors suggest that functional improvement is still seen because the transplanted tissue act as relays for the circuits.

Question 42

What are some targets for gene therapy in the treatment of SCI?

Answer 42

• PTEN Deletion
• Overexpression of VP16-KLF7
• Sox11 overexpression
• MDM4 deletion
• Chondroitinase expression

Question 43

How could PTEN deletion be used in SCI treatment?

Answer 43

PTEN Deletion enhances CST [corticospinal tract] axonal regeneration. By deleting PTEN (proto oncogene – hence deleting it promotes growth), there is promotion of regeneration of corticospinal tract. (As cells atrophy after axon injury, this could be a good way of “re-activating” them).

PTEN is an inhibitor of mTOR which is a powerful signal to promote axonal growth.

Question 44

How could Chondroitinase be used in SCI treatment?

Answer 44

Chondroitinase ABC (ChABC) is a bacterial enzyme that removes the glycosaminoglycan chains from the CSPG molecules. As mentioned earlier, CSPG molecules are potent inhibitors of axonal growth, explaining why ChABC delivery to the injured spinal cord promotes axonal regeneration and functional recovery in rats. A mammalian-compatible ChABC delivered by gene therapy has also been successfully trialled in rats, boasting axonal regeneration as well as upper limb functional recovery following SCI.

Question 45

Explain the role of Neurorehabilitation and Robotics in SCI

Answer 45

In 2012, there was a nice example where a rat was attached to a robot, which helped it with neurorehabilitation. There was also an electrical signal generator, as well as injections of serotonin.

Training, electrical epidural and serotoninergic/dopamine stimulation enhance axonal regeneration and recovery. This is through strengthening of spared pathways and synapses, as well as limited injured axonal sprouting, and new functional synapses.

Another method involves using brain-machine interfaces to read activity in the motor cortex → motor states decoding → create implantable pulse generator spatially selective implant. This promotes some functional recovery in primates (but again very, very minimal).

Question 46

Explain the role of Epigenetic modification in SCI

Answer 46

Axonal outgrowth and target innervation are very active during neurodevelopment . The genetic and epigenetic programs are accordingly very plastic and dynamic. However, adult neurons in the CNS lose their plastic properties and may be locked in a dormant gene expression state due to epigenetic factors.

Signalling pathways that translate immediate modifications in the environment around injured axons into long-term gene reprogramming for phenotype modifying outcomes. Transcriptional and epigenetic-dependent gene reprogramming is at the core of these mechanisms

PCAF is required for conditioning dependent axonal regeneration. PCAF is a histone acetyltransferase. When acetyltransferase is missing, physiological regeneration that happens in dorsal columns doesn’t happen.

Hence using vectors and gene therapy research, we find that PCAF overexpression promotes spinal cord regeneration.

Question 47

Outline the mechanisms inhibiting/opposing regeneration of axons in CNS (essay structure)

Answer 47

• Presence of extrinsic mechanisms preventing growth. CNS has both astrocytes and oligodendrocytes.
  o CSPGs released by reactive astrocytes.
  oAstrocytes also contribute to the glial scar formation.
  oVASE-NCAM is a cell-adhesion molecule that is upregulated in reactive-astrocytes.
  oAfter axonal injury, oligodendrocyte and myelin proteins are exposed and present around the injury site.
  oOther negative modulators of axonal growth: ephrins and semaphorins are also upregulated after axonal injury.
• Absence of extrinsic mechanisms permitting growth. In the developmental CNS, neurotrophins and netrins enhance neurite growth. However, many of these mechanisms are lost in the mature CNS, contributing to the unfavourable
• Intrinsic Mechanisms
  o (Stam et al 2007) showed how differential gene expression in the central and peripheral neurites of the dorsal root ganglia neurones are responsible for their differential regenerative properties.
  o Central nervous system neurones have low expression of regeneration-associated genes (RAGs).
  o Epigenetics also plays an important role in the intrinsic regenerative ability of neurites.

Question 48

Why have the inhibiting mechanisms of axonal regeneration not been removed by evolution?

Answer 48

o Primitive vertebrates such as newts and salamanders can regenerate after SCI. However, this does not translate to higher vertebrates and mammals.

o Glial scar inhibition is evolutionarily more recent, and the presence of intrinsic mechanisms that would allow regeneration, such as those that allow PNS mechanisms, suggest a deliberate evolutionary pressure to inhibit regeneration in mammalian CNS. The glial scar is also important in revascularisation and maintenance/repair of the BBB, reducing the risk of infection.

o The CNS is more complicated than the PNS. Incorrect innervation could lead to disrupted complex neuronal networks, leading to unwanted symptoms and neuropathic pain. It may be evolutionarily advantageous for the mammal to lose function of their limbs, rather than incorrect innervation also leading to loss if limb function, but on top of other disruptive symptoms such intense neuropathic pain, which may prevent mating.