The role of focal adhesion kinase (FAK) in retinal ganglion cells regeneration

Abstract

Purpose: Extracellular matrix (ECM) remodeling in optic neuropathy results in RGC death and low regenerative capacity. We recently showed that laminin-integrin-FAK signaling is disrupted in RGC in optic neuropathy. FAK has a central role in neurons integrating growth factors and ECM signals through phosphorylation/dephosphorylation. The purpose of this study was to determine if gene therapy with activated FAK results in increased RGC survival and regeneration in purified RGCs in vitro and after optic nerve (ONC) injury in vivo.

Methods: Purified rat P8-9 RGCs were transfected by electroporation with constructs containing YFP fused to either wild type, or constitutively active, and non-phosphorylatable FAK mutants in the essential Tyr397 phosphorylation site and cultured on laminin. RGC survival was determined after immunostaining with β-III tubulin (TUJ1) and DAPI, by counting YFP/TUJ1 positive live cells out of total DAPI count, and neurite growth was determined by measuring neurites in Image J software after 48 hours in culture. C57/bl6 mice were treated with AAV2 caring GFP (AAV2-GFP) delivered by intravitreal injection. 2 weeks after injection the animals were subjected to ONC. RGC survival was determined by counting TUJ1 positive RGCs in flat-mounted retinas and quantitation of RGC regeneration was done by counting cholera toxin B labeled axons extending from the ONC site 2 weeks post-injury. Immunohistochemistry with antibodies anti-integrin β1, FAK, P-FAK was performed in retinal sections 4 days post-ONC.

Results: RGCs transfected with constitutively active FAK had 1.5 fold increased survival and 1.5 fold increased neurite growth, while transfection of FAK non-phosphorylatable mutant resulted in 1.7 fold decreased survival, and 1.4 fold decreased neurite growth when compared with wild type FAK after 48 hours in culture. We successfully injected intravitreally AAV2-GFP in the mouse resulting in more than 90% targeted GFP expression to RGC. ONC resulted in 80% RGC death and very limited axon regeneration 2 weeks post-injury. Immunostaining with specific antibodies showed decreased β1 integrin expression, and FAK activation, while FAK expression was unchanged in RGCs after ONC.

Conclusions: Our results suggest that transfecting RGCs with constitutively activated FAK promotes RGC survival and neurite growth in vitro. We are currently exploring the effect of gene therapy with activated FAK after ONC in vivo.

Keywords: 531 ganglion cells • 519 extracellular matrix • 615 neuroprotection