Constitutively active Ras promotes protection and axon regeneration in retinal ganglion cells after optic nerve injury

Abstract

Purpose : Axons in the optic nerve do not usually regenerate when they are injured, causing permanent loss of vision. However, recent studies indicate that stimulation of PI3K/Akt and RAF/MEK/ERK signaling promotes axon regeneration and neuroprotection. In this study, we examined the effects of constitutively active Ras, K-Ras^V12, which powerfully activates these signaling, on retinal ganglion cell (RGC) protection and axon regeneration using an optic nerve crush (ONC) model.

Methods : K-Ras^V12 was generated by site-directed mutagenesis and incorporated into an adeno-associated viral serotype 2 (AAV2) vector. AAV2-K-Ras^V12 was injected intravitreally in C57BL/6J mice. One or two weeks after the injection, ONC was performed, and one or eight weeks later, cholera toxin B subunit (CTB647)-labeled regenerating axons were analysed. The number of RGCs was determined by immunolabeling using anti-RBPMS antibody in flatmounted retinas. EdU (50mg/kg) was intraperitoneally injected daily for examination of cell proliferation. Electron microscopy was performed for detailed histological analysis of axons.

Results : AAV2-K-Ras^V12 treatment demonstrated increased number of RGCs compared with the control after ONC. As Ras is an oncogene, we examined the potential of cell proliferation, but found that RGCs were not proliferated by the AAV2-K-Ras^V12 treatment. These data showed that the increased RGC number after ONC with AAV2-K-Ras treatment was due to neuroprotection. Furthermore, AAV2-K-Ras^V12 treatment induced significant amounts of RGC axon regeneration even after 1 week, and some regenerating axons reached the optic chiasm after 8 weeks. These data indicated that AAV2-K-Ras^V12 treatment induces powerful RGC axon regeneration. In addition, we found that AAV2-K-Ras^V12 -induced regenerated axons were not myelinated, suggesting that combinatory treatment with promoting myelination may be required for functional recovery.

Conclusions : AAV2-K-Ras^V12 may be useful for treatment of CNS axon injury in future, and it may be a good tool to study mechanisms for RGC axon regeneration and protection.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.