What is important for determining the polarity of a growth cones response?
levels of intracellular cyclic nucleotides
what is db-cAMP?
a membrane permeable version of cAMP
was shown to be attractive
how can you reverse an attractive response?
Kt5720 can be used to inhibit activity of cAMP-dependent protein kinase (PKA)
(cAMP signals through PKA)
What receptors signal to increase cAMP?
Acetyl Choline, Netrin, BDNF, NGF, NT-3
What receptors inhibit the increase in cAMP?
MAG, Nogo
(also integrin binding to laminin)
What receptors signal to increase cGMP?
Sema3A, EphrinB
netrin can be attractive or repulsive..
attractive w/ DCC
repulsive when DCC is accompanied by Unc5
Describe the change in axon response of retinal ganglion cells:
Retinal ganglion cells grow towards the tectum
initially retinal ganglion cell axons are attracted by cells in the optic nerve head
contact with laminin in the optic nerve reverses response to netrin
What 2 inhibitory mol are implicated in the failure of CNS regeneration?
Myelin associated glycoprotein (MAG)
Nogo
they affect cAMP levels by activating RhoA
(RhoA affects the RhoA/Rac balance)
Axon regeneration in the PNS:
- Injury to the peripheral nerve
- Macrophage recruitment - clear up debris (esp myelin)
- Activation of growth related genes
- proliferating Schwann cells promote axon regen and regrowth
Axon regeneration in the CNS:
- Injury to the central nerve
- Prolonged clearing of myelin debris
(the CNS is an immunologically priveliged area = macrophages don’t get in easily) - Debris from myelin contains cAMP inhibitory factors e.g. MAG and Nogo
- these interfere with axon extension - Astrocytes and glia from a glial scar - acts as a physical barrier
What 2 things allow the CNS axon to regenerate?
- if given the appropriate substrate
(retinal ganglion cell axons regrow if they go through sciatic nerve) - If the right genes are activated
(preconditioning lesion)
What were the preconditioning lesion experiments?
injuries to the peripheral branch of the DRG can regrow
Injury to CNS cannot regrow
however….
a conditioning lesion to the peripheral branch of the DRG
= lesion a few days later of central branch can regenerate
What ligands is the NogoR activated by?
MAG,OMgp, Nogo
How has this inhibitory pathway been targeted to prevent its inhibition of regeneration?
- Db-cAMP to elevate cAMP
increase in cAMP activates PKA which phosphorylates and inactivates RhoA
What does the conditioning lesion lead to?
a large range of transcriptional changes
- of regeneration associated genes (RAGs)
What is ROCK?
Rho- kinase
is downstream to RhoA
inhibition promotes regrowth through the DC lesion
What are 2 inhibitors of ROCK?
- Y27632
- C3 transferase (use a cell permeable version)
What effect does ibuprofen have on regrowth?
- enhances DRG growth on myelin and GSPG (component of glial scars)
- Inhibits RhoA activation in the spinal chord injury site
enhanced recovery distal to lesion + enhanced motor recovery
How does ibuprofen work?
acts by activating the TF PPARy
this upregulates phosphatase SHP-2
- inhibits RhoA GEF
= Suppresses RhoA activation!!!
What gene involved in growth control was also implicated in regeneration?
PTEN
What did KO of PTEN allow in the optic nerve after injury?
KO of PTEN =
enhanced neuronal survival
+
allows retinal ganglion cells axon regrowth in adult mice
= ALLOWS REGENERATION
What does PTEN regulate?
The MTOR pathway
What is the MTOR pathway involved in?
oxidative responses and is a key growth pathway
in normal development the mTOR pathway is progressively inhibited as development ceases
