Module 1 - Axonal Regeneration

Question 1

What is a growth cone and what are the possible responses of it?

Answer 1

A growth cone is the distal tip of the axon, enriched in receptors that detect molecular cues and stimulate intracellular signalling pathways that lead to changes in direction, collapsing, stalling, pre-synaptic terminal formation, spreading or growing faster, via cytoskeletal changes and transcription factor regulation.

Question 2

Briefly describe what molecular process controls actin assembly and dissassembly:

Answer 2

Rho GTPases affect Rho, Rac and Cdc42 which affect factors such as ROCK and LIMK whic control cofilin and myosin and N-WASP with leads to the Arp2/3 complex which directs actin assembly. Enas affect profilin.

Question 3

Give some examples of synaptogenic factors:

Answer 3

Wnt - pre-synaptic differentiation by modulating microtubule dynamics in growth cone.
Target cells expressing neuroligin bind to neurexin on incoming cell to stimulate pre-synaptic differentiation.

Question 4

How do neurons stabilise their connections?

Answer 4

Activity-dependent remodelling and trophic factors.

Question 5

Give some examples of positive soluble guidance cues:

Answer 5

Growth factors eg. neurotrophins, eg. nerve growth factor. Cell bodies deprive of NGF survive as long as terminals are exposed to it.
Netrin

Question 6

Describe the molecular cues in the spinal cord:

Answer 6

Dorsal commisural neurons with DCC receptors are attracted to the ventral half by gradual netrin 2 gradient and steep netrin 1 gradient.
Meanwhile, ventral trochlear motor neurons are repulsed by netrin and grow away (dorsally). They express DCC and Unc5 (modulate of cAMP).
Sema III is expressed in the ventral half. NT3 responsive sensory neurons terminate in the ventral part whilst NGF responsive sensory neurons terminate in the dorsal part.

Question 7

Which receptors are for which neurotrophins?

Answer 7

Trk A for NGF (eg. nociceptive sensory neurons in the dorsal root ganglion)
Trk B for Brain Derived Neurotrophic Factor and Neurotrophin 4/5
Trk C for Neurotrophin 3
Dimerisation of Trk receptor tyrosine kinases stimulates intracellular signalling pathways which lead to changes in genen expression for cell survival and axon outgrowth and Rho GTPase controlled cytoskeletal changes.
p75 for all (inhibition)

Question 8

Describe a mouse model for Alzheimer’s disease using growth factors and their receptors:

Answer 8

Mice with antibodies for NGF (Capsoni 2000) - the neurons that died were cholinergic.
Or mice with TrkA because NGF-TrkA signalling complexes are endocytosed and transported in APP-containing vesicles retrogradely to the nucleus for neuronal survival.

Question 9

Give some examples of positive cell-associated cues:

Answer 9

Homophilic binding cell-cell adhesion molecules from immunoglobulin superfamily (eg. NCAM) or cadherin superfamily (eg. N-cadherin)
Extracellular matrix associated, eg laminin and collagen (secreted by cells too)

Question 10

What are the different types of NCAM?

Answer 10

NCAM can be spliced to give three major size classes with different modes of membrane attachment.
PSA-NCAM are made of high negatively charged poly sialic acid sugar chains attached to the 5th Ig-like domain. VASE-NCAM has a VASE exon aa attached to the 4th Ig domain.

Question 11

What is the function of PSA-NCAM?

Answer 11

Decreases during development because it’s used for neuronal migration and stimulates greater neurite growth. Prevents remyelination on oligodendrocytes and is expressed on adult neural stem cells too.

Question 12

What is the function of VASE-NCAM?

Answer 12

It increases during development because it increases NCAM homophilic interaction force between cells.

Question 13

How does NCAM homophilic binding stimulate neurite outgrowth?

Answer 13

non-receptor tyrosine kinase Fyn and activation of neuronal fibroblast growth factor receptor tyrosine kinase.

Question 14

Describe the integrin receptors:

Answer 14

Receptor for extracellular matrix molecules. Made up of heterodimers of alpha and beta sub units, depending on ECM molecule. RGD motif is a common beta 1 integrin binding motif.

Question 15

Give some examples of negative soluble guidance cues:

Answer 15

Semaphorins, eg. semaphorin III/collapsin for Plexin receptors.
Slit
Netrin

Question 16

Describe the role of guidance cues in commisural fibres.

Answer 16

Slit is produced by midline cells. Neurons expressing Robo are repulsed but Robo receptors are downregulated by Commissureless (Com). Com is then downregulated once they have crossed the midline so they don’t cross back.The complement of Robo receptors decides how sensitive it is to slit and which longitudinal path it will take.

Question 17

Give some examples of negative cell-associated cues:

Answer 17

Ephrins - A (GPI-anchored) or B (transmembrane) for EphA and EphB receptors (tyrosine kinase receptors that activate Rho GTPases to stimulate growth cone collapse).
Semaphorins
ECM-associated, eg. S-laminin, chondroitin sulphate and proteoglycan.

Question 18

Describe the role of molecular cues in the optic tectum:

Answer 18

Retinal ganglion cells from the nasal part of the retina project to the posterior part of the tectum whereas those from the temporal part of the retina project to the anterior part of the tectum.
This occurs because of an ephrinA gradient of low to high from the anterior to the posterior tectum. Temporal RGCs express high levels of EphA while nasal RGCs express lowl levels of EphA receptors.
Axons from retina to optic tectum via laminin on astrocytic end-feet lining path of optic nerve. Downregulate integrin receptors once they reach their target.

Question 19

What are some of the processes that occur after axotomy?

Answer 19

Loss of target-derived signals.
Retrograde signals to the cell body.
Gene transcription factors and RhoA is activated and cause cytoskeletal changes.

Question 20

What is the pathogenesis of spinal tissue damage?

Answer 20

Macrophages/microglia are the first to the site of injury and start inflammatory processes. Myelin surrounds the site (Nogo inhibits axonal regrowth). Astrocytes are the next cells at the site and form a barrier of scarring molecules. They fill the site using proteoglycans and fibronectin, collagen, tenascin, etc. The last cells at the site are oligodendrocytes.

Question 21

What is the function of taxol?

Answer 21

Taxol stabilises microtubules so stops their normal breakdown during cell division (anti-cancer drug).

Question 22

Give examples of molecules that appear to improve axonal regeneration when blocked?

Answer 22

Anti-NogoA antibody promotes CST regeneration (Liebsher 2005)
Inhibition of CSPG using chondroitinase-ABC (Bradbury 2002)
PTEN deletion (Liu 2010)
MDM4 deletion (Toledo 2006)
HDAC inhibition, especially HDAC3 (promoted outgrowth of primary DRG neurons) and HDAC5 (peripheral nerve axon regeneration)

Question 23

Give examples of improvements in axonal regeneration when molecules are overexpressed/something is added:

Answer 23

Stem cells (Lu 2012)
Overexpression of VP16-KLF7 (Murray 2012)
Sox11 overexpression reduces net retraction in DRG and promotes growth of injured CST axons (Zimei 2015)
Robots, electrical epidural and serotoninergic/dopamine stimulation (Van den Brand 2012) Electrical stimulation spine (Harkema 2011)
PCAF for conditional dependent axonal regeneration (Puttagunta 2014)

Question 24

Why is there regenerative failure after CNS injury?

Answer 24

CNS axons and dendrites are plastic and sprout spontaneously following injury
Neural progenitor cells can proliferate, differentiate and migrate following CNS injury

Poor neuronal intrinsic regenerative capacity
Presence of a growth inhibitory environment
Cell loss-imperfect generation of new cells

Therapies must: Inhibit the scar formation
Inhibit the axon regeneration inhibitory signalling
Promote pro-regenerative pathways
Replace cell loss
Promote functional recovery