Abstract
Purpose: :
Our recent data demonstrates that Caspase-6 (CASP6) inhibition promotes Retinal Ganglion Cell (RGC) survival and axon regeneration; however, the downstream targets of CASP6 remain unclear. In the present study, we examined the role of a CASP6-specific substrate (Focal Adhesion Kinase- FAK) in RGC axon regeneration.
Methods: :
Optic nerve transection or optic nerve crush was performed in adult rats. FAK cleavage and phosphorylation after optic nerve transection was assayed by Western immunoblots of retinal lysates at 4 days after axotomy. After optic nerve crush, animals received intraocular injections of GM6001 (broad spectrum MMP inhibitor), Necrostatin-1 (Nec-1; RIP phosphorylation inhibitor), Y-27632 (ROCK inhibitor), or control (PBS) to determine if pathways that stimulate FAK activation promote axon regeneration in adult RGCs. Intraocular injections were delivered at 3 and 10 days after optic nerve crush. FAK phosphorylation in RGC growth cones (21 days after crush) was assessed by immunohistochemistry in longitudinal optic nerve sections, where RGC axons had been anterogradely labeled by intraocular injection of Cholera-Toxin B-FITC (CTB-FITC). RGC axon regeneration was quantified in fixed longitudinal sections of optic nerve at three different bins from the lesion site: 0-250 microns, 250-500 microns, and >500 microns.
Results: :
Western blots of axotomized retinas showed that FAK cleavage products were increased at 4 days postaxotomy, relative to normal retinas. Furthermore, FAK-phosphorylation (Y397) in the retina was reduced after axotomy, indicative of decreased FAK activation. In the crushed optic nerve, phospho-FAK was localized to regenerating RGC axons at 21 days after optic nerve crush, when Y-27632 was used to promote regeneration. Intraocular injections of the broad spectrum MMP inhibitor, GM6001, or the RIP phosphorylation inhibitor, Nec-1, significantly increased RGC axon regeneration through the lesion site at 21 days postaxotomy (p<0.01), resulting in a 3-fold increase in the mean number of growth cones in each of the three bins. Axons >500 microns in length were only observed after GM6001 or Necrostatin-1 treatment and not in controls.
Conclusions: :
Optic nerve transection causes the dephosphorylation and degradation of FAK. Activated phospho-FAK is an integral component of regenerating RGC growth cones, and enhancing FAK activation promotes RGC regeneration.
Keywords: ganglion cells • regeneration • lesion study