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

Abstract

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.

Keywords: Axonal guidance; Eph receptor; Ephrin signaling; Neurodegeneration; Neuropathy; Neuroprotection; Optic nerve; Optic nerve crush; Optic neuropathy; Retinal ganglion cell.

Fig. 1

Proteomic analysis of dissected retinal tissue of male C57BL/6J WT mice at 14 days (N = 5 biological replicates, pooled), two months (N = 5 biological replicates, pooled), and 12 months of age (N = 5 biological replicates, pooled), using mass spectrometry data independent acquisition (MS-DIA). Temporal expression of Eph receptors and ephrin ligands in the postnatal retinas

Fig. 2

Proteomic quantification of Eph receptors 24 h and 48 h post optic nerve crush (ONC). Western blot detection and quantification of phosphorylated Eph receptors/b-actin from dissected whole retinal tissue 24 h and 48 h post-ONC. The geometric means and geometric standard deviations (N = 3 biological replicates) are graphed. A P value of less than 0.05 is considered statistically significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001

Fig. 3

Proteomic quantification phosphorylated Eph receptors 24 h and 48 h post optic nerve crush (ONC). Western blot detection and quantification of Eph receptors/b-actin from dissected whole retinal tissue 24 h and 48 h post-ONC. The geometric means and geometric standard deviations (N = 3 biological replicates) are graphed. An arrow indicates the band of interest. A P value of less than 0.05 is considered statistically significant. *P ≤ 0.05, **P ≤ 0.01

Fig. 4

Phosphorylation of multiple EphA- and EphB-class receptors localized within the inner retina in early traumatic optic neuropathy. Immunofluorescent microscopy of retinas a Activated Eph receptors A2, A3, and A4, 24 h and 48 h post optic nerve crush (ONC); b Activated Eph receptors B1 and B2, 24 h and 48 h post-ONC. Scale bar at 50 μm. (N = 1 biological replicates), one biological replicate is depicted. CNTRL: uninjured control); DAPI, 4′,6-diamidino-2-phenylindole; GLUL, glutamine synthetase; TUBB3, Tubulin beta 3; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer

Fig. 5

Super-resolution imaging and co-localization analysis of multiple EphA-class receptors in neuronal and glial cells within the inner plexiform layer (IPL) of 48 h optic nerve crush (ONC) retinas (injured). a Stochastic optical reconstruction microscopy (STORM) imaging of phosphorylated Eph receptors A2, A3, and A4 (pEphAs) in injured retinas. b Optical transport colocalization (OTC) analysis comparing the localization of pEphAs to neuronal cells (TUBB3) in uninjured and injured retinas. c OTC analysis comparing the localization of pEphBs to neuronal cells (TUBB3) and glial cells (GLUL) in injured retinas. N = 3 biological replicates are graphed; One biological replicate is depicted in the representative image. Mann–Whitney U tests and a P value of less than 0.05 is considered statistically significant. ***P ≤ 0.001. Scale bar at 10 μm

Fig. 6

Super-resolution imaging and co-localization analysis of multiple EphB-class receptors in neuronal and glial cells within the inner plexiform layer (IPL) of 48 h optic nerve crush (ONC) retinas (injured). a Stochastic optical reconstruction microscopy (STORM) imaging of phosphorylated Eph receptors B1 and B2 (pEphBs) in injured retinas. b Optical transport colocalization (OTC) analysis comparing the localization of pEphBs to neuronal cells (TUBB3) in injured and uninjured retinas. c OTC analysis comparing the localization of pEphBs to neuronal cells (TUBB3) and glial cells (GLUL) in injured retinas. N = 3 biological replicates are graphed; One biological replicate is depicted in the representative image. Mann–Whitney U tests and a P value of less than 0.05 is considered statistically significant. ***P ≤ 0.001. Scale bar at 10 μm

Fig. 7

The dynamic change in retinal ganglion cell dendritic arborization detected by confocal microscopy post optic nerve crush (ONC) injury. a Quantification of retinal ganglion cell dendritic arborization by Sholl analysis two days after ONC injury (N = 3 biological replicates). b Quantification of retinal ganglion cell dendritic arborization by Sholl analysis six days after ONC injury (N = 3 biological replicates are graphed; One biological replicate is depicted in the representative image). Mann–Whitney U tests and a P value of less than 0.05 is considered statistically significant. ***P ≤ 0.001. Scale bar at 50 μm

Fig. 8

Pan Eph receptor inhibitors demonstrate neuroprotective properties, preserving retinal ganglion cell dendritic arborization as observed by confocal microscopy following six days post optic nerve crush (ONC) injury. a Quantification of retinal ganglion cell dendritic arborization by Sholl analysis six days after ONC injury. Animals treated with 50 μM of UniPR129 (N = 3 biological replicates). 25% DMSO in phosphate buffered saline (PBS) was used as a vehicle control. b Quantification of retinal ganglion cell dendritic arborization by Sholl analysis six days after ONC injury. Animals treated with 2 μM of NVP-BHG712 (N = 3 biological replicates). 25% DMSO in PBS was used as a vehicle control. c Quantification of retinal ganglion cell dendritic arborization by Sholl analysis six days after ONC injury. Animals treated with a combination of both UniPR129 (50 μM) and NVP-BHG712 (2 μM) (N = 3 biological replicates are graphed; One biological replicate is depicted in the representative image). Mann–Whitney U tests and a P value of less than 0.05 is considered statistically significant. **P ≤ 0.01, ****P ≤ 0.0001. Scale bar at 50 μm