Receptor interacting protein 3-mediated autophagic cell death contributes to acute optic nerve injury

Abstract

Purpose : This study aimed to elucidate the involvement of autophagic cell death during the acute phase of experimental optic nerve injury and explore the underlying molecular mechanism.

Methods : A mouse model of optic nerve crush injury was established. Autophagic cell death was evaluated at various time intervals (6h, 12h, 1d, 3d, 7d) following injury through transmission electron microscopy Western blotting for key autophagic markers. Cellular localization of autophagy was determined via double immunostaining of autophagy machinery and cell-specific markers. The role of receptor-interacting protein kinase 3 (RIP3) in autophagy regulation was investigated using RIP3-deficient mice, and the efficacy of an RIP3-specific inhibitor, GSK-872, was assessed through intravitreal injection for treatment analysis.

Results : Autophagy was engaged during the acute phase of optic nerve crush injury, with peak activation observed at 6 hours post- injury. Excessive autophagy activation precipitated autophagic cell death, leading to retinal inflammation. Autophagy was primarily located in microglia and to a lesser extent in retinal ganglion cells. Silencing of RIP3 ameliorated autophagic cell death in microglia and mitigated retinal inflammation. Furthermore, GSK-872 treatment substantially attenuated autophagy activation and enhanced visual function in optic nerve injury mice.

Conclusions : This study reveals the excessive activation of autophagy in the early phase of optic nerve injury, leading to autophagic cell death and disease progression. These findings contribute to a deeper comprehension of autophagy regulation and may highlight potential targets for early intervention in optic nerve injury.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Autophagy is activated in the early stages of optic nerve crush injury. (A-B) Transmission electron microscopy revealing the presence of autophagosomes with a double-membrane structure. Scale bar=2μm. (C) Western blot analysis was performed to assess the protein levels of LC3 II and LC3 I at different time points. *P < 0.05, **P < 0.01.

 

Colocalization of Beclin-1 and TUNEL staining. Double-positive cells indicated autophagic cell death. GSK-872 was used for inhibition of autophagy. ***P < 0.001.