Inhibition of CRMP2 Phosphorylation Suppresses Microglia Activation in the Retina and Optic Nerve and Promotes Optic Nerve Regeneration After Optic Nerve Injury

Abstract

As the primary connection between the eye and brain, the optic nerve plays a pivotal role in visual information transmission. Injuries to the optic nerve can occur for various reasons, including trauma, glaucoma, and neurodegenerative diseases. Retinal ganglion cells (RGCs), a type of neurons that extend axons through the optic nerve, can rapidly respond to injury and initiate cell death. Additionally, following optic nerve injury microglia, which serve as markers of neuroinflammation, transition from a resting state to an activated state. The phosphorylation of collapsin response mediator protein2 (CRMP2) in the semaphorin 3A (Sema3A) signalling pathway affects several processes, including axon guidance and neuron regeneration. In this study, we used an optic nerve crush (ONC) mouse model to investigate the effects of suppressing CRMP2 phosphorylation on microglia activation. We found that CRMP2 phosphorylation inhibitor suppressed RGCs loss and promoted neuronal regeneration following ONC. In addition, CRMP2 S522A mutant (CRMP2 KI) mice exhibited decreased microglial activation in both the retina and optic nerve following ONC. These results suggest that inhibiting the phosphorylation of CRMP2 can alleviate the loss of RGCs and microglial activation after optic nerve injury, providing insight into the development of treatments for optical neuropathies and neurodegenerative diseases.

Keywords: Collapsin mediator protein 2; Microglia; Optic nerve injury; Phosphorylation.

Fig. 1

HupA suppresses RGC death after ONC. A, C Immunostaining for Anti-RBPMS in flat mount retinas of HupA untreated/treated mice 2 or 4 weeks after ONC. B, D Quantification of RGCs 2 or 4 weeks after ONC. Data are presented as the mean ± SEM, statistical comparison was performed using students’ t-tests; Scale bar = 100 μm. (n = 4; ****p < 0.0001)

Fig. 2

CRMP2 phosphorylation inhibition by CRMP2 KI causes the inhibition of microglia proliferation in the retina and optic nerve. A Anti-iba1 and Hoechst Immunostaining on the retina of CRMP2 KI and WT mice 3 days after ONC. B Quantification of the number of activated microglia per mm² of retina. C The ratio of Iba1 positive signal in the GCL on RGC numbers in each section. D Immunostaining for anti-Iba1 and Hoechst in the optic nerves of CRMP2 KI and WT mice 3 days after ONC. E Quantification of the number of activated microglia per mm² of optic nerve. Scale bar = 50 μm. Data are presented as the mean ± SEM, statistical comparison was performed using one-way ANOVA, with a Tukey’s multiple comparisons post-hoc test (n = 6; *p < 0.05; **p < 0.001; ****p < 0.0001)

Fig. 3

CRMP2 phosphorylation inhibition by HupA causes the inhibition of microglia proliferation in the retina and optic nerve. A Immunostaining for Iba1 and Hoechst of HupA untreated/treated retina 3 days after ONC. B Quantification of the number of activated microglia per mm² of retina. C The ratio of Iba1 positive signal in the GCL on RGC numbers in each section. D Immunostaining for Iba1 and Hoechst in the optic nerve of HupA-treated and untreated WT mice 3 days after ONC. E Quantification of the number of activated microglia per mm² of optic nerve. Data are presented as the mean ± SEM, statistical comparison was performed using one-way ANOVA, with a Tukey’s multiple comparisons post-hoc test, Scale bar = 50 μm. (n = 6, *p < 0.05; ***p < 0.001; ****p < 0.0001)

Fig. 4

HupA promotes optic nerve regeneration after ONC. A Representative images of anti-GAP43 immunostaining on flat mount retina samples from HupA-treated/untreated mice 4 weeks after ONC. B Quantification of fluorescence intensity (n = 4, Scale bar = 50 μm). C, E Western blot of HupA untreated/treated optic nerves. The HupA treatment period lasted 4 weeks. D, F Quantification of Tuj1/β-actin and GAP43/β-actin. (n = 4, Scale bar = 200 μm) Data are presented as the mean ± SEM, statistical comparison was performed using one-way ANOVA, with a Tukey’s multiple comparisons post-hoc test (*p < 0.05; **p < 0.01; ***p < 0,001; ****p < 0. 0001; ns: no significant)

Fig. 5

BDA tracing of HupA-treated WT mice 4 weeks after ONC. A BDA tracing of HupA untreated/treated optic nerves. The HupA treatment period lasted 4 weeks. Red asterisks mark the injury sites. Scale bar = 500 μm. B Magnification of different distances from the crush site. Scale bar = 100 μm. C Quantification of regenerating axons at the indicated distances beyond the crush site in optic nerves in HupA untreated/treated mice (n = 4) Data are presented as the mean ± SEM, statistical comparison was performed using one-way ANOVA, with a Tukey’s multiple comparisons post-hoc test (****p < 0. 0001; ns: no significant)