Elevated Retinal EphB/EphrinB Reverse Signaling Contributes To Retinal Ganglion Cell Injury In A Rat Experimental Glaucomatous Model

Glaucoma, the second leading cause of blindness, is a neurodegenerative disease that is characterized by optic nerve degeneration resulting from apoptotic death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is commonly regarded as a hallmark risk factor for the generation of glaucoma. Currently, strategy to treat glaucoma is combining neuronal protection with lowering IOP. However, like most of the other neurodegenerative diseases, the pathogenesis of RGC death following intraocular hypertension is still poorly understood.Erythropoietin-producing hepatocyte receptors (Eph receptors) are the largest family of tyrosine kinase receptors in vertebrates, consisting of 16 structurally related transmembrane tyrosine kinases. Eph receptors and their ligands ephrins have been found widely distribution in a variety of tissues, which play important roles in modulation of physiological functions, such as actin cytoskeleton regulation, adhesion, movement, survival, proliferation, differentiation and secretion, etc. Previous work has shown that ephrinB2/EphB2 signaling was upregulated in the optic nerve head of glaucomatous DBA/2J mice. Application of EphB2-Fc to the optic nerve heads of DBA/2J mice leads to elevation of Ca2+ concentration in single retina axon, indicating ephrinB2/EphB2 signaling invlovement in the pathology of glaucoma. However, the exact role of ephrinB2/EphB2 signaling in glaucoma and the underlying mechanisms are largely unknown.In the present work, we investigated the possible involvement of EphB/ephrinB signaling in the pathogenesis of RGCs death in a rat chronic ocular hypertension (COH) model, explored the underlying mechanisms and analyzed the relationship between elevation of EphB/ephrinB reverse signaling and downregulation of GluR2-containning AMPA receptor in rat retinas using immunohistochemistry, Western blot, Co-immunoprecipitation, and patch-clamp techniques. Results are listed as follows. (1) Rat COH model was reproduced by blocking episcleral veins. (2) In the COH rat retinas, EphB1 protein expression was shown a robust increase as early as the first day after the surgery (1d) and then maintained at a higher level for 6 weeks, which was localized in Muller cells. EphrinB2 protein expression was significantly elevated from the second week (2 w) after the surgery on Muller cells and RGCs. There are no significant changes in the expression of EphB2 and ephrinBI proteins. (3) Using a specific tyrosine phosphorylated antibody against ephrinB, we found that EphB/ephrinB reverse signaling was activated in the COH rat retinas. (4) To elucidate the role of elevation of EphB/ephrinB reverse signaling in the COH rats retinas, TUNEL assay in whole-mounted retinas was employed. A single intravitreal injection of EphB2-Fc (1μg/μl) to activate EphB/ephrinB reverse signaling in normal rats caused much more RGCs apoptosis as compared with control Fc injection. (5) To explore the underlying mechanisms of RGCs apoptosis caused by activation of EphB/ephrinB reverse signaling, we performed co-immunoprecipitation (co-IP) assay and found that the there were direct interactions between ephrinB2, GluR2 and phosphorylated src kinase, suggesting that EphB/ephrinB reverse signaling could regulate GluR2 phosphorylation through src tyrosine kinases. (6) We further confirmed by Western blot analysis that tyrosine phosphorylated src and GluR2 were significantly enhanced both in the COH rats and the EphB2-Fc intravitreally injected normal rats. (7) Intravitreal injection of EphB2-Fc in normal retinas caused obvious decrease in expression of GluR2 proteins on the surface of RGCs. Similar results was obtained in the COH rat retinas without injection of EphB2-Fc. In addition, by using whole-cell patch-clamp techniques in retinal slices, our data showed that the current-voltage (Ⅰ-Ⅴ) relationship of AMPA receptor-mediated excitatory synaptic curents (eEPSCs) recorded in RGCs displayed an inward rectification in the EphB2-Fc injected retinas, while the Ⅰ-Ⅴ relationship obtained in control retinas was linear. These results suggest that activation of EphB/ephrinB reverse signaling induced a downregulation of GluR2-containing AMPA receptor on the RGCs. (8) Pre-injection of PP2 (100 μM), a potent src tyrosine kinase inhibitor, efficiently prevented the downregulation of membrane GluR2 on the RGCs both in the COH and the EphB2-Fc injected rats. The effect was through src signaling pathway, which was confirmed by the fact that PP2 significantly prevented the increase in tyrosine phosphorylated src kinase and GluR2 by Western blot analysis. Moreover, pre-injection of PP2 prevented the RGCs apoptosis both in the EphB2-Fc injected retinas and the COH retinas.In conclusion, a robust upregulation of EphB1 and ephrinB2 proteins was found in COH rats retinas. The change in EphB1 expression was restricted in Muller cells, while ephrinB2 was both in Muller cells and RGCs. The enhanced expression of EphB1 was happened prior to that of ephrinB2, suggesting an activation of EphB/ephrinB reverse signaling in RGCs. Intravitreal injection of EphB2-Fc caused RGCs apoptosis in normal retinas through activating tyrosine kinase src. Activated src increased the phosphorylation of GluR2, then downregulated GluR2-containing AMPA receptors on the surface of RGCs, thus increasing Ca2+ influx. In contrast, pre-injection of PP2 inhibited the src kinase activity and the GluR2 phosphorylation, and stablized GluR2 expression on the surface of RGCs, thus protecting RGC from apoptosis both in the COH rats and the EphB2-Fc injected rats. All these results suggest that the activation of the EphB/ephrinB reverse signaling may contribute to RGC injury in the rat COH model by down-regulating GluR2-containing AMPA receptors.