Involvement of EphB/Ephrin-B Signaling in Axonal Survival in Mouse Experimental Glaucoma

As glaucoma is a leading cause of blindness worldwide, the underlying disease mechanisms that impact visual function are of substantial interest. From a consideration of the early patterns of visual field loss in patients, the optic nerve head (ONH) is generally regarded as an important site of pathogenesis resulting in retinal ganglion cell (RGC) axon injury. Support for this view also comes from experimental findings of significant structural remodeling1–7 and obstruction of axoplasmic transport4,8–11 at the ONH early in the course of disease. In addition, genetic evidence indicates that when RGC soma apoptosis is prevented, intraretinal axons are largely preserved until they reach the ONH, where massive degeneration ensues.12 Efforts have been undertaken to identify genes differentially expressed at the glaucomatous ONH to shed some light on the pathways involved in axon injury and survival.13–17 In this study, we focused on the potential role of the Eph/ephrin family of cell surface signaling molecules in optic axon degeneration after glaucomatous injury. The Eph receptor tyrosine kinases and their ephrin ligands function not only in developmental axonal guidance, cell migration, and morphogenesis, but also in adult synaptic plasticity, homeostasis, and cancer.18–22 In addition, altered Eph and ephrin expression has been reported in many central nervous system pathologies23–25 and genetic studies have demonstrated a functional role for Eph/ephrin signaling in modulating axon survival and regrowth after spinal cord26 and optic nerve injury.27 A key feature of the Eph/ephrin system is that signal transduction occurs bidirectionally22,28 and involves both forward and reverse signaling. In forward signaling, ephrin binding to Eph molecules triggers autophosphorylation of the Eph tyrosine kinase domain and a subsequent signaling cascade. In reverse signaling, the binding of an Eph with an ephrin molecule activates signaling pathways within the ephrin-expressing cell. Among glaucoma-related changes at the ONH, a finding that is consistently observed across multiple animal models and in glaucoma patients is the upregulation of EphB/ephrin-B gene expression and protein signaling. The expression of a number of Eph and ephrin family members is upregulated in cultured ONH astrocytes derived from human patients,13,29 and in several different animal models of glaucoma including monkey,29 the DBA/2J mouse model of pigmentary glaucoma,17,30 and the laser-induced ocular hypertension (LIOH) model in CD-1 mice.31 In mice, where this process has been examined in the greatest detail, EphB/ephrin-B upregulation is tightly correlated with axon loss,30 and occurs early in disease, preceding or coinciding with the initial morphologic signs of axon damage.31 This upregulation of EphB/ephrin-B gene expression has been found to be associated with increased active protein signaling in both axons and glia at the ONH.31 Furthermore, morphologically normal axons exhibit higher levels of ephrin-B reverse signaling, whereas this signaling pathway is downregulated in aberrant axons.31 Despite these correlational findings, whether Eph-ephrin signaling plays a functional role in disease remains unknown. In the present study, we subjected mouse mutants lacking EphB2 (EphB2−/−) or EphB3 (EphB3−/−) protein or mutants with engineered alleles of EphB2 (EphB2lacZ/lacZ) to glaucomatous optic nerve damage induced by LIOH. EphB2 and EphB3 were chosen as the genes of interest because their mRNAs were shown to be upregulated at the ONH as early as 1 to 2 days after LIOH treatment.31 As substantial data indicate axon dysfunction and degeneration precede retinal ganglion cell body loss,32–34 we focused our analysis on the integrity of axons in the optic nerve. Mice totally deficient in EphB2 or EphB3 both exhibited more severe axon degeneration compared with wild type littermates, suggesting that the EphB/ephrin-B pathway normally operates to moderate axon loss in LIOH-induced experimental glaucoma. Exogenous application of EphB2 recombinant protein attenuated axon degeneration in LIOH-treated optic nerve explants, further supporting the involvement of EphB/ephrin-B signaling in glaucomatous optic nerve pathophysiology.