Activation of multiple Eph receptors on neuronal membranes correlates with the onset of optic neuropathy

Background: Optic neuropathy is a major cause of irreversible blindness, yet the molecular determinants that contribute to neuronal demise have not been fully elucidated. Several studies have identified 'ephrin signaling' as one of the most dysregulated pathways in the early pathophysiology of optic neuropathy with varied etiologies. Developmentally, gradients in ephrin signaling coordinate retinotopic mapping via repulsive modulation of cytoskeletal dynamics in neuronal membranes. Little is known about the role ephrin signaling plays in the post-natal visual system and its correlation with the onset of optic neuropathy.

Methods: Postnatal mouse retinas were collected for mass spectrometry analysis for erythropoietin-producing human hepatocellular (Eph) receptors. Optic nerve crush (ONC) model was employed to induce optic neuropathy, and proteomic changes during the acute phase of neuropathic onset were analyzed. Confocal and super-resolution microscopy determined the cellular localization of activated Eph receptors after ONC injury. Eph receptor inhibitors assessed the neuroprotective effect of ephrin signaling modulation.

Results: Mass spectrometry revealed expression of seven Eph receptors (EphA2, A4, A5, B1, B2, B3, and B6) in postnatal mouse retinal tissue. Immunoblotting analysis indicated a significant increase in phosphorylation of these Eph receptors 48 h after ONC. Confocal microscopy demonstrated the presence of both subclasses of Eph receptors within the retina. Stochastic optical reconstruction microscopy (STORM) super-resolution imaging combined with optimal transport colocalization analysis revealed a significant co-localization of activated Eph receptors with injured neuronal cells, compared to uninjured neuronal and/or injured glial cells, 48 h post-ONC. Eph receptor inhibitors displayed notable neuroprotective effects for retinal ganglion cells (RGCs) after six days of ONC injury.

Conclusions: Our findings demonstrate the functional presence of diverse Eph receptors in the postnatal mammalian retina, capable of modulating multiple biological processes. Pan-Eph receptor activation contributes to the onset of neuropathy in optic neuropathies, with preferential activation of Eph receptors on neuronal processes in the inner retina following optic nerve injury. Notably, Eph receptor activation precedes neuronal loss. We observed a neuroprotective effect on RGCs upon inhibiting Eph receptors. Our study highlights the importance of investigating this repulsive pathway in early optic neuropathies and provides a comprehensive characterization of the receptors present in the developed retina of mice, relevant to both homeostasis and disease processes.