B-RAF signaling promotes optic nerve regeneration

Abstract

 

Purpose

 

To investigate the role of intracellular B-RAF signaling in promoting regeneration in the optic nerve after injury.

 

Methods

 

We genetically engineered mice to study gain of B-RAF function in promoting axon regeneration. These mice bear in the endogenous B-RAF locus a construct that expresses a kinase-activated B-RAF (kaB-RAF) upon Cre recombination. Adult mice were injected with AAV2-Cre into the vitreous of the eye to activate B-RAF signaling in the RGCs, and 14 days later were subjected to proximal crush lesion of the optic nerve. Outgrowing axons were labeled by CTB-Alexa anterograde tracers. Optic nerves were dissected, sectioned and imaged two weeks after the injury. The same procedures were performed with mice lacking the canonical B-RAF effectors MEK1 and MEK2, as well as with mice homozygous for the conditional deletion of PTEN (Park et al., 2008).

 

Results

 

In mice expressing kaB-RAF in RGCs we observed robust axon regeneration up to 3.5 mm past the lesion site. Comparing to the PTEN loss-of-function model, the number of fibers growing past the lesion was almost 4fold higher in the kaB-RAF model. Average length of regeneration was similar in both models. Blocking of the canonical B-RAF signaling pathway by conditional elimination of MEK1 and MEK2 completely abolished axon regeneration induced by kaB-RAF. Concomitant activation of both B-RAF and the PI3 kinase-mTOR pathway (by removal of PTEN) drove axon growth beyond a simple additive effect.

 

Conclusions

 

We conclude that activation of B-RAF drives substantial axon regeneration in the crushed optic nerve via canonical B-RAF - MEK signaling.

 

 

Activation of B-RAF enables regenerative axon growth in the crush-lesioned optic nerve via the canonical effectors MEK1/MEK2. (A) Experimental time course. (B) Intravitreal injection of AAV-Cre induces expression of TdTomato reporter in RGCs. (C) Upper, whole mount crushed Bax -/- optic nerve. Crush site is indicated by a red asterisk. Bax null background was used to prevent RGC death after optic nerve crush. Lower, confocal fluorescence image of the same nerve. Green, axons anterogradely labeled with CTB-Alexa488. (D) Regenerative growth in lesioned kaB-RAF expressing optic nerve. Inset, axons at approx. 3.5 mm from the crush site. (E) Loss of MEK1 and MEK2 abolishes the regeneration driven by kaB-RAF. (F) Optic nerve regeneration in absence of PTEN. (G) Quantification. Scale bar = 0.5 mm. N=3 for each group.

 

Keywords: 687 regeneration • 629 optic nerve • 714 signal transduction