EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

Abstract It was previously shown that EphB/ephrinB reverse signaling in retinal ganglion cells (RGCs) is activated and involved in RGC apoptosis in a rat chronic ocular hypertension (COH) model. In the present work, we first show that ephrinB/EphB forward signaling was activated in COH retinas, and RGC apoptosis in COH retinas was reduced by PP2, an inhibitor of ephrinB/EphB forward signaling. We further demonstrate that treatment of cultured Müller cells with ephrinB1-Fc, an EphB1 activator, or intravitreal injection of ephrinB1-Fc in normal rats induced an increase in phosphorylated EphB levels in these cells, indicating the activation of ephrinB/EphB forward signaling, similar to those in COH retinas. The ephrinB1-Fc treatment did not induce Müller cell gliosis, as evidenced by unchanged GFAP expression, but significantly up-regulated mRNA and protein levels of tumor necrosis factor-α (TNF-α) in Müller cells, thereby promoting RGC apoptosis. Production of TNF-α induced by the activation of ephrinB/EphB forward signaling was mediated by the NR2B subunit of NMDA receptors, which was followed by a distinct PI3K/Akt/NF-κB signaling pathway, as pharmacological interference of each step of this pathway caused a reduction of TNF-α production, thus attenuating RGC apoptosis. Functional analysis of forward and reverse signaling in such a unique system, in which ephrin and Eph exist respectively in a glial element and a neuronal element, is of theoretical importance. Moreover, our results also raise a possibility that suppression of ephrinB/EphB forward signaling may be a new strategy for ameliorating RGC apoptosis in glaucoma.