EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Muller cells in an experimental glaucoma model

Deficiency of glutamate transporter GLAST in Muller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Muller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Muller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-kappa B pathways since EphA4-Fc treatment of cultured Muller cells reduced the levels of p-Akt/Akt and NF-kappa B p65, which were reversed by transfecting Si-EFNA3. In Muller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-kappa B p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-kappa B inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Muller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Muller cells via inhibiting PI3K/Akt/NF-kappa B pathways, thus contributing to RGC damage in glaucoma.