Cue integration and regeneration strategies Flashcards
How are responses to cues modified?
1) Receptors for cues may interact to silence each other (DCC/Robo)
2) Modification to signals depending on the combination of cues received
What does the response to a cue depend on?
Example?
Depends on the CONTEXT
Eg. if a growth cone, depends if pre-crossing or post-crossing
What are cyclic nucleotides important in?
In determining the POLARITY of a growth cone’s response to chemotropic signals (ATTRACTED OR REPELLED)
What is cAMP activated by?
What does this do?
NGF activates cAMP
NGF turns growth cones in culture
What molecule of cAMP turns GC? Why?
db-cAMP (can cross the membrane)
cAMP cannot cross the membrane
Is higher [cAMP] attractive or repulsive?
Attractive
How can the polarity of response to a cue by reversed/negated? (2 ways)
1) By manipulating the concentrations of intracellular cyclic nucleotides
2) Reversed according to the combination of receptors present
What happens when block cAMP inside the cell
What does this show?
No activation of cAMP-dependant PKA
Reversal of the response to netrin
Shows:
- cAMP acts as a SWITCH, determining the polarity of the netrin response
How does [cAMP] and [cGMP] affect the polarity of response?
Binding to attractant (eg. netrin/NGF) - increases cAMP and decreases cGMP
Binding to repellant (eg. sema) - increases cGMP and decreases cAMP
When is DCC receptor involved in attractring/repelling?
Attractant - when bound to NETRIN
Repellant - when ACCOMPANIED to co-receptor Unc5
—-> causes netrin to become REPLLANT
When is netrin a repellant?
How does this act?
When its receptor DCC is accompanied by a coreceptor Unc5
Acts through the [cAMP]:[cGMP] balance
How do MAG and Nogo act as repellants?
How is this different to other repellants?
By INHIBITING cAMP
Other repellants (eg. sema, netrin (with unc5 coreceptor), ephrin) have their effect by INCREASING cGMP
How are cyclic nucleotide levels affected?
1) By extracellular cues
2) By the combination of receptors present
3) By signals from other receptors
What ligand binding to their receptor affect cAMP levels?
How?
Receptors binding to laminin or glutamate
Lowers cAMP levels
What ligand binding to their receptor affect cGMP levels?
Receptors binding to nitric oxide
What does how the GC behave depend upon?
COMBINATION of signals from the environment
How do specific environments alter guidance responses?
By modulating cyclic nucleotides
Describe the journey of retinal ganglion cells to/after the tectum
How are the neurons directed away from the ONH once they have crossed it?
- Initially attracted by NETRIN to cells in the optic nerve head
- After reach ONH - contact with LAMININ in the optic nerve –> REVERSES the response of the neurons to netrin (serves to direct neurons AWAY from the ONH and towards the tectum)
- Lamanin receptors are members of the integrin family - SUPRESS cAMP when they signal
(Lower cAMP –> REPULSION)
How does 14-3-3 alter the response of the cell to an attractive cue? (eg. shh)
14-3-3 INHIBITS PKA (which is normally activated by cAMP)
How does cAMP signal?
Through PKA
How does cGMP signal?
Through PKG
What are 2 inhibitory molecules that are implicated in the faliure of adult mammalian CNS to regenerate after nerve injury?
How do they do this?
1) MAG
2) Nogo
They LOWER/inhibit cAMP by activating RhoA
How can spinal cord injury be treated?
Manipulating cAMP and RhoA
Where does regeneration occur/not occur?
Occurs in LOWER vertebrates and the PERIPHERAL NS but not in the CNS
What happens in the periphery during regeneration?
- Injury
- MACROPHAGES move in and clear debris
- Expression in the injured nerve of RAG (regeneration associated genes)
- Proliferating SCHWANN cells –> promote axon regeneration
Why does the CNS fail to regenerate? (3 things)
1) Failure to activate growth promoting program in the injured neuron
2) Presence of INHIBITORY factors in the CNS myelin that disrupt axon extension
3) Formation of a ‘Glial scar’ - PHYSICAL barrier to for axon growth
When do CNS neurons have the capacity to regenerate? (2 things)
1) If given the APPROPRIATE SUBSTRATE
2) If the RIGHT GENES are activated
When can retinal ganglion cells regenerate?
When they bypass the optic nerve and go down the sciatic nerve (which they don’t normally do)
When can the neurons in the DRG regenerate/not regenerate?
In the PERIPHERAL branch but NOT in the CNS branch
What happens if cut the peripheral branch of the DRG axons a few days BEFORE cut the central branch?
What does this show?
Leads to regeneration in the central branch as well as the peripheral branch
Shows substrates and factors released from the periphery when cut can activate a regeneration program that can IGNORE CNS inhibition
What is the conditioning lesion?
What happens if cut the CNS branch without the conditioning lesion?
Cutting the peripheral branch of the DRG
NO regeneration
What do all the factors/receptors that inhibits CNS regeneration converge on?
RhoA
What ligands activate RhoA?
What does this cause? How?
MAG, OMgp and NOGO
All bind to NgR to change the RhoA/Rac balance
Activates RhoA –> growth cone collapse and the inhibition of nerve fibre regeneration
How is RhoA inhibited?
What does this allow?
By db-cAMP:
- Increases cAMP
- Activates PKA
- PKA phosphorylates RhoA –> inactivation of RhoA
How does a pre-conditioning lesion allow regeneration in the CNS?
By elevation of cAMP
What happens when db-cAMP is administered in the presence of inhibitory myelin?
REGROWTH of neurons
What does db-cAMP injection into the DRG INSTEAD of the pre-conditioning lesion cause?
Regrowth of the cut CNS neuron
What 2 points of intervention have been used to try and allow regeneration in the CNS?
1) db-cAMP to elevate cAMP
2) Activation of PKA pathway
What shows that cAMP alone is not enough to cause effective regeneration in the CNS?
- Treatment using db-cAMP has poor regeneration
- Pre-conditioning lesion alone is MORE EFFECTIVE in promoting regrowth that db-cAMP treatment
- Transcriptome following pre-conditioning lesion shows a LARGE ARRAY of changes/genes get switched on
- CL induces GLOBAL increase in trafficking of many intracellular components into the injured cell branch that is NOT SEEN without CL or with cAMP alone
What genes get switched on following the CL?
RAGs (Regeneration-associated genes)
What intracellular components get trafficked into the injureed cell branch following the CL?
What does this show?
- Mitochondria
- MAPs
- 14-3-3 proteins
- RhoGDI
- CRMP
Shows:
CL may affect the OVERALL POLARISATION in the cell
What does ROCK (Rho-kinase) inhibition cause?
Promotion of CST(corticospinal tract) regrowth
What is the CST?
Descending motor pathway
What is C3 transferase?
How does this affect CST regrowth?
How about Cethrin/VX-210?
INHIBITOR of RhoA
Allows regrowth of CST neurons IN VITRO
But NOT in vivo
Cethrin/VX-210 - cell permeable C3 transferase, some success in animals
What is Y27632?
How does this affect CST regrowth?
ROCK inhibitor
Promotes regrowth of of the DC column when administered at the same time as the lesion
What affects does ibuprofen (NSAID) have on neuron regeneration?
How?
Enhances DRG growth on myelin and CSPGs (a component of glial scars)
By inhibiting RhoA activation a
What are CSPGs?
Chondroitin sulphate proteoglycan
What does ibuprofen cause?
How?
Enhanced recovery DISTAL to the CST lesion (beyond lesion site)
AND mild recovery of motor function
By inhibiting RhoA activation in the injury site
How can see improvement of motor function in animals?
Increase stride length and reduce stride width
BBB increase
Does ibuprofen work through the NORMAL NSAID pathway to cause spinal tract regeneration?
How is this seen?
NO
Other NSAIDs (eg. Naproxen) doesn't have this affect Also other COX inhibitors do not work
How does ibuprofen cause regeneration of spinal cord tracts?
Activation of transcription factor (PPAR):
–> up regulates a PHOSPHATASE (SHP-2) which:
IHIBITS a RhoA GEF –> suppresses RhoA activation
What did Park et al suggest?
Pathways involved in regulating cell GROWTH may also regulate the ability of axons to grow (control regeneration)
What did Park et al do?
Why did they do this in the retina?
Tested a pool of mice containing CONDITIONAL KOs of growth control genes for ability to regrow OPTIC NERVE after injury
In the retina:
- Easy to get to
- RGCs project into the CNS - good paradigms for
What genes are involved in growth control?
P53
Smad4
LKB1
PTEN (antagonist for PI3K)
How are conditional KOs made?
- Mouse with floxed allele with loxP sites either side of gene to KO
- Adenovirus carrying cre under control of a strong promoter into this mouse
- Where ever virus goes - turn on cre and KO target genes (which are floxed)
How can follow the regeneration of a nerve?
Put GFP into the adenovirus
What did the conditional KOs of growth control genes that Park et al show?
What does this show?
Only PTEN KO significantly enhanced neuronal survival, allowing retinal ganglion cell axon regrowth adult mice
Shows PTEN normally REPRESSES axon growth
What pathway does PTEN regulate? How?
The mTOR pathway
Through Akt
What is rapamycin?
How does it work?
Immunosuppressant drug for organ transplantation
Anti-proliferative drug (for treatment of cancer)
Works by INHIBITING the mTOR function
What happens to mTOR pathway when development CEASES?
What does this cause?
PROGRESSIVELY inhibited
Causes levels of phospho-s6K to decrease (start high in development)
As mTOR phosphorylates S6K –> p-S6K to DRIVE GROWTH
What does ‘mTOR’ stand for?
mammalian Target Of Rapamycin
What does p-s6K do?
DRIVES growth
What does a axotomised axon do to p-s6K?
DOWNREGULATES it
What activates mTOR?
What does this cause?
Nutrients
Causes mTOR to phosphorylate s6 KINASE –> phospho-S6K
–> Growth
How does PTEN normally inhibit axon regrowth?
PTEN causes PIP3 –> PIP2
Less docking for Akt
Akt NORMALLY inhibits TSC1 which inhibits Rheb
Rheb NORMALLY activates mTOR
NO inhibition of TSC1 by Akt –> allows inhibition of Rheb
- -> lowers mTOR
- -> less p-S6 which drives growth
What happens in PTEN KO?
PIP2 –> PIP3
Akt docked at PIP3
Akt INHIBITS TSC1 which normally inhibits Rheb
Rheb no longer inhibited and can activate mTOR
AXON REGROWTH
How was it shown that the mTOR pathway is affected by PTEN?
KO of TSC1:
- Retinal axons allowed to grow
(mTOR functional - TSC1 normally inhibits Rheb–>mTOR)
What is bad about PTEN KO to regrow axon?
No functional recovery reported
Why is it difficult to develop drugs that INHIBT PTEN (in order to drive axon regrowth)?
Because most drugs act to ACTIVATE PTEN, due to its anti-proliferative action (in cancer treatment)
When has db-cAMP treatment only been successful?
When administered BEFORE injury
Are results using RhoA inhibitors promising?
Yes! now in phase 2/3 clinical trials
What are 3 problems still need to overcome for axon regeneration?
1) Other approaches needed (eg. anti-inflammatories) to block GLIAL SCAR formation (still major barrier for axon regrowth)
2) Axons still need to find appropriate targets after regeneration
What are 3 drugs being trialed for axon regeneration?
1) RhoA inhibitors
2) Inhibitors of PTEN
3) db-cAMP
