Lecture 13

Question 1

Give an example of experimental evidence that implies a central role of cyclic nucleotides in axon guidance

Answer 1

Analogues of cyclic nucleotides such as dibutyryl cAMP are able to turn growth cones in cultures

Question 2

How can cyclic nucleotides influence the response of axons to guidance cues

Answer 2

Manipulation of intracellular cyclic nucleotide concentrations affects the response to a wide range of guidance cues. For example netrin dependence on cAMP signalling is shown by inhibiting the activity of cAMP-dependent PKA which results in the growth cone actually turning away rather than just not responding to the netrin. Hence the concentration of cAMP actually acts a switch for the response to netrin between attractive and repulsive

Question 3

Attractive guidance cues raise cAMP levels T or F

Answer 3

T

Question 4

Other than netrins give examples of attractive growth cues

Answer 4

BDNF NT-3 NGF and acetylcholine

Question 5

Repulsive cues only raise cGMP levels T or F

Answer 5

F – whilst they do raise cGMP levels repulsive cues such as MAG and Nogo also have their effect by inhibiting the production of cAMP

Question 6

What specifically is it about cyclic nucleotides that actually controls the response of neurons to cues

Answer 6

The ratio of cAMP to cGMP

Question 7

Describe the reciprocal regulation of cAMP and cGMP

Answer 7

When cGMP levels rise these increase the activation of PKGs which then in turn induce the activation of phosphodiesterases such as PDE-4D which increases the hydrolysis of cAMP. This hence leads to a reciprocal reduction in cAMP as a result of cGMP increase. Conversely if cAMP levels rise this results in an increased activation of PKA. One of the targets for phosphorylation by PKA is phosphodiesterase 5 (PDE5). Increased activation of the cGMP-specific PDE5 results in the increased hydrolysis of cGMP.

Question 8

Describe how the response to netrins can change

Answer 8

Netrin is a chemoattractive signal normally when its receptor DCC is expressed on neurons. However netrin is actually repulsive if DCC is accompanied by the co-receptor Unc5

Question 9

What is odd about the changes in the levels of Ca2+ as a result of encountering a growth/guidance cue

Answer 9

Once a guidance cue is encountered there is an elevation in Ca2+ levels irrespective of direction and regardless of whether the cue is attractive or repulsive. In fact the turning of axons is Ca2+ dependant and higher concentrations of Ca2+ are consistently seen in the side of the growth cone facing the source of the cue

Question 10

Outline the differences seen in Ca2+ signalling as a result of attractive and repulsive cues

Answer 10

Attractive cues – attractive cues elevate [cAMP] and also either activate TRPC channels at the cell surface to let in extracellular Ca2+ or elevate intracellular [IP3]. Both Ca2+ and IP3 trigger further release of Ca2+ from intracellular stores (CICR). This store release of Ca2+ feeds back to further elevate [cAMP] which in turn causes further increase in [Ca2+]. Its this positive feedback loop that then results in a high amplitude Ca2+ flux which activates extension of the growth cone. Repulsive Cues – repulsive cues elevate [cGMP] and also activate TRPC channels at the cell surface to let in extracellular Ca2+. cGMP however inhibits Ca2+ release from intracellular stores as well as from L-type Ca2+ channels at the cell surface. It also inhibits rises in [cAMP] through reciprocal feedback. Therefore the store release of Ca2+ (CICR) does not occur and [cAMP] levels do not get amplified. This results in low amplitude Ca2+ flux which somehow activates repulsion and growth cone collapse this blocking the positive feedback loop

Question 11

Give an example of another modulator of cNMPs

Answer 11

Integrins bind laminin that lowers cAMP levels. Laminin receptors are members of the integrin family which are known to suppress intracellular [cAMP] when they signal

Question 12

Describe the role of laminin in guidance of the retinal ganglion cells to the tectum

Answer 12

In the initial stage of their journey to the tectum retinal ganglion cells (RGCs) are attracted by netrin expressed by cells in the optic nerve head (ONH). However after contact with laminin in the optic nerve the response to netrin is reverses and may serve to direct axons away from the ONH.

Question 13

What is different in the central and peripheral nervous systems in mammals with regards to their ability to regenerate

Answer 13

The mammalian peripheral nervous system can regenerate to some extent but the central nervous system cannot

Question 14

What are the key players in inhibiting regeneration in the central nervous system and how do these molecules exert their effects

Answer 14

Myelin associated glycoprotein and nogo amongst others. Nogo and MAG and other inhibitory molecules actually affect cAMP levels by activating RhoA

Question 15

Describe the process of regrowth that occurs in the peripheral nervous system in response to injury

Answer 15

Following injury macrophages invade to remove myelin debris. The neuron then begins to express growth-related genes to begin forming a new growth cone. There is also a proliferation of Schwann cells that help to promote subsequent regeneration.

Question 16

In contrast describe what happens in the central nervous system as a result of injury and the features that prevent regeneration

Answer 16

Following injury to neurons in the central nervous system there is a prolonged clearage of myelin. This leaves behind some inhibitory factors such as MAG and Nogo. These inhibitory molecules disrupt axon extension. In addition the invasion of astrocytes and other glia results in the formation of a glial scar which ultimately impede nerve repair. Thus the failure of the CNS to regeneration is due to a number of factors including a failure to activate growth promoting program in injured neuron the presence of inhibitory factors in CNS myelin and the formation of Glial Scar that presents a physical barrier to axon growth

Question 17

It was later found that the central nervous system can regenerate under certain conditions what were these hypothesised to be

Answer 17

If they are given the appropriate substrate and if the correct cues are activated.

Question 18

Describe some experimental evidence to show that the central nervous system is capable of regeneration

Answer 18

A crush experiment was carried out on the optic nerve. This was then followed by the grafting of a peripheral nerve sheath (sciatic nerve) near to the optic nerve crush site. This provided an alternative route for axon regrowth and after some time axons did regrow in the grafted nerve sheath. This shows that the central nervous system is capable of re-growing if the substrate is right

Question 19

Describe some experimental evidence that shows the central nervous system is able to regenerate specifically if the right cues are activated

Answer 19

If a conditional lesion is made in the DRG axons some regeneration is seen. An initial cut is made in the peripheral branch of the sensory DRG axons the central branch cannot regrow normally. Then a few days later make another cut on the central branch and allow to repair. This results in the regeneration of the central branch which wouldn’t be seen if the peripheral branch was not cut prior. Hence the once the correct signals are activated the central nervous system can regenerate. Turning on the right genes can override inhibition of central nervous system regrowth

Question 20

Discuss the significance of RhoA in regeneration of neurons

Answer 20

The activation of the nogo receptor (NgR) activates RhoA. This leads to a change in the RhoA/Rac balance leading to growth cone collapse. Conversely elevating cAMP levels leads to an activation of PKA one of whose targets is RhoA. Phosphorylation of RhoA leads to its inactivation.

Question 21

What three different ways are there of stimulating regeneration in a neuron

Answer 21

Artificially raising cAMP levels blocking RhoA activation (through C3 glycosyl transferases ibuprofen and cethrin) or using Rho kinase (ROCK) inhibitors.

Question 22

Describe the significance of cAMP in regeneration of axons

Answer 22

db-cAMP promotes regrowth of central sensory axons. In addition a pre-conditioning peripheral lesion has been found to elevate cAMP levels which could explain why conditional lesions are capable of allowing repair of the central branch of the DRGs. Furthermore db-cAMP injection into the DRG prior to lesion can enhance regrowth through subsequent dorsal column lesions. In addition db-cAMP actually allows the growth of sensory fibres in vitro in the presence of inhibitory central nervous system myelin.

Question 23

cAMP stimulation alone is enough to result in functional recovery in central nervous system axon injury T or F

Answer 23

F - functional recovery after db-cAMP alone is poor

Question 24

The conditioning lesions (CL) alone are more effective in promoting regrowth than db-cAMP treatment in central nervous system axons why is this thought to be the case

Answer 24

Transcriptomics reveals large array of changes after conditional lesions such as the upregulation of regeneration-associated genes (RAGs). This hints that cAMP/db-cAMP is only playing a small role in regeneration. In addition conditioning lesions induce a global increase in trafficking of numerous intracellular components into the injured central branch that is not seen without CL or with cAMP alone. This includes mitochondria MAPs 14-3-3 proteins RhoGDIs and CRMP

Question 25

How can ROCK inhibitors be used to help regrowth

Answer 25

The Y27632 ROCK inhibitor when administered at the same time as the lesion can promote regrowth through the dorsal column lesion. This is accompanied by some recovery of motor control

Question 26

Discuss the case of ibuprofen in promoting central nervous system regeneration

Answer 26

Ibuprofen has been found to enhance DRG growth on myelin containing inhibitory molecules such as CSPGs. Ibuprofen actually inhibits the activation of RhoA at the injury site and can result in enhanced recovery of lesions and subsequent recovery of motor function in distal corticospinal tract sections

Question 27

Describe the mechanism of action of ibuprofen in stimulating central nervous system regeneration

Answer 27

Ibuprofen is the only NSAID to have this regenerative effect. Naproxen and other COX inhibitors do not work. Hence the normal NSAID pathway cannot be involved. Ibuprofen appears to act by activation of a transcription factor PPARγ. PPARγ upregulates SHP-2 phosphatase which may inhibit a RhoA GEF to suppress RhoA activation

Question 28

Describe the experimental setup that lead to the identification of the role of PTEN in central nervous system regeneration

Answer 28

A pool of mice carrying conditional knockouts for major growth control genes were tested for their ability to regrow optic nerve neurons following crush injuries. Mice were generated with a floxed allele for the growth control gene (flanked by loxP sites). A cre-recombinase construct downstream of a strong ubiquitous promoter was introduced into a lentivirus genome. These viral particles were then injected into the vitreous humour of the eye and allowed to infect the cells of the optic nerve etc. Meanwhile a tracer was also injected into the optic nerve to visualise the axons. After some time the optic nerve was then looked at again to see if any subsequent regeneration was seen.

Question 29

Only PTEN significantly enhanced neuronal survival and allowed retinal ganglion cell (RGC) axon regrowth in adult mice. What is the role of PTEN

Answer 29

PTEN is a phosphatase that dephosphorylates PIP3 to PIP2. Importantly it regulates the mTOR pathway which is active early in development. During normal development the mTOR (mammalian target of rapamycin) pathway is progressively inhibited. mTOR is a kinase that in-turn phosphorylates S6Kinase which is involved in promoting protein synthesis. Interestingly severed axons downregulate p-S6 which could indicate why they are unable to regenerate.

Question 30

What is the effect of PTEN deletion

Answer 30

Enhancement of spinal axon regrowth