Axon Guidance Molecules Upregulated at the Optic Nerve Head of DBA/2J Glaucomatous Mice Alter RGC Intra–Axonal Calcium Levels in vitro

Abstract

Abstract: : Purpose: To study the expression of axon guidance molecules at the optic nerve head of DBA/2J mice and investigate their functional effects on adult RGC axons in vitro. Methods: The expression of axon guidance molecule genes was investigated in the optic nerve head region of adult DBA/2J mice, a strain of mice that develops pigmentary glaucoma resulting in RGC axon loss starting about 9–10 months of age. In situ hybridization was performed on sections of retina and optic nerves from 3, 8, 10 month old DBA/2J mice and in 10 month old C57/Bl6 mice. Sequence specific probes for the axon guidance molecules EphrinB1, EphrinB2, EphrinB3, EphB2, EphB3, EphB4, Slit1, Slit2, Slit3, Robo–1, Robo–2, Netrin–1, and Sema5A were used. To determine whether adult RGC axons were responsive to axon guidance molecules, adult retinal explants from C57/Bl6 mice that received an optic nerve crush 8–11 days beforehand were grown in culture. The RGC axons emanating from explants were then exposed to Fc–tagged recombinant axon guidance proteins and changes in the levels of intra–axonal calcium determined using Fluo–4AM fluorescence imaging. Axons were exposed to Fc protein alone in control experiments. Results: Of the axon guidance molecule genes examined, we found that mRNA encoding EphrinB1, EphrinB2, and EphB2 were elevated at the optic nerve head region of 10 month old DBA/2J mice compared to DBA/2J mice at 3 and 8 months of age. 10 month old C57/Bl 6 mice, which do not develop glaucoma, did not show a similar increase in the expression of these axon guidance molecules. Adult RGC axons exposed to EphB2 protein in culture exhibited an increase in the levels of intra–axonal Ca2⁺. Application of EphB2 caused Ca levels to rise over two–fold in cultured RGC axons within 10 minutes. Conclusions: Although previous work on glaucoma has implicated the optic nerve head as a critical site of axonal damage that subsequently leads to retrograde RGC cell death, the molecular triggers of RGC axon damage remain uncertain. Here, we found that the expression of EphrinB1, EphrinB2, and EphB2 axon guidance molecules was upregulated at the optic nerve head of DBA/2J glaucomatous mice. This increase in guidance molecule mRNA expression coincided with the known onset of RGC axon loss in DBA/2J mice. Furthermore, exposure of RGC axons to EphB2 protein in vitro caused an elevation in intra–axonal calcium, indicating that adult RGC axons were sensitive to axon guidance molecules. Together these findings are consistent with the possibility that axon guidance molecules may play a role in glaucoma pathogenesis or modulate the severity of this disease.

Keywords: neuro-ophthalmology: optic nerve • gene/expression • calcium