Anti-lipid peroxidation promotes significant optic nerve regeneration and neuroprotection in mouse glaucoma models

Abstract

Purpose : This study aims to test the role of lipid peroxidation in RGC survival and optic nerve (ON) regeneration under traumatic and glaucomatous conditions in mice.

Methods : Using retrograde regenerating axon tracing with Smart-Seq2 single-cell sequencing and RGC-specific RiboTag-RNA Seq, we found a common upregulated gene GPX4 in regenerating RGCs and glaucomatous RGCs. Taking advantage of AAV2-mouse mSncg promoter-driven gene expression in RGCs, we evaluated the axon regeneration in the ON after crush injury (ONC) and glaucomatous protective effect of RGC somata and axons after overexpressing GPX4 in silicone oil-induced ocular hypertension (SOHU) mouse glaucoma model. We also applied Liproxstatin-1, a well-known inhibitor of lipid peroxidation, to further demonstrate the effect of anti-lipid peroxidation in glaucoma optic nerve regeneration and neuroprotection. In addition to histological analysis with wholemount retina, CTB-tracing of regenerating axons of the ON, mouse vision improvement was examined by optokinetic tracking response (OKR), in vivo optical coherence tomography (OCT) imaging, and electrophysiological RGC function by pattern electroretinogram (PERG).

Results : GPX4, a key anti-lipid peroxidation enzyme, is significantly upregulated in regenerating RGCs and glaucomatous RGCs. We found that AAV2-mediated RGC-specific overexpression of both mitochondrial GPX4 (MitoGPX4) and Cytosol GPX4 (CytoGPX4) promotes significant ON regeneration (p<0.0001) in the ONC model, and CytoGPX4 shows better effect than MitoGPX4. Consistently, intraperitoneal injection of Liproxstatin-1 also induces axon regeneration in the ONC model (p<0.0001). Next, we further test the neuroprotection effects of anti-lipid peroxidation in the SOHU glaucoma model. Encouragingly, MitoGPX4 also promotes remarkable survival of both RGC somata and axons, evidenced by both in vivo OCT imaging and histological quantification of RGC somata and axons (p<0.05); and preserves visual function evidenced by the OKR and PERG (p<0.001).

Conclusions : Our results indicate that lipid peroxidation may play a critical role in neurodegeneration after traumatic and glaucomatous ON injuries. And that inhibition of lipid peroxidation by AAV-mediated GPX4 gene modulation or chemical inhibitor represents a promising therapeutic strategy for axon regeneration and neuroprotection in glaucoma and other CNS axonopathies.

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