Abstract
Purpose :
Age-related macular degeneration (AMD) involves increased oxidative stress burden and RPE cell death. In particular, lipid peroxidation chain reactions amplify oxidative burden. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPx4), which specifically reduces oxidized lipids. This is imperative within the retina, which contains high concentrations of polyunsaturated fatty acids. We hypothesize that GPx4 knockout within the RPE will lead to degeneration akin to AMD. We also tested whether a deuterated form of docosahexanoic acid (D-DHA) is protective in this model, as deuteration renders the lipid resistant to oxidation. Deuterium is a non-radioactive isotope with an established safety profile in medical imaging and therapeutics for neurodegenerative disease. Since DHA is the most concentrated polyunsaturated fatty acid in photoreceptor outer segments, we targeted this species for isotope reinforcement.
Methods :
C57BL/6J mice with a floxed GPx4 gene were mated with RPE65-cre/ER mice and administered tamoxifen to induce GPx4 knockout. Retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscope (cSLO) and optical coherence tomography (OCT). Retinal function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical (IHC) analyses, electron microscopy, and quantitative polymerase chain reaction assays.
Results :
Progressive photoreceptor layer thinning was noted in GPx4 KO mice. Interestingly, GPx4 KO mice displayed a negative C wave two weeks after enzyme knockout. En face IHC revealed diminished RPE cell count in GPx4 KO mice, correlating with RPE cell death seen early in AMD pathogenesis. Increased carboxyethylpyrrole staining in photoreceptor outer segments and mRNA expression of Nrf2-activated genes in the RPE and photoreceptors indicate oxidative stress in GPx4 KO mice. Electron microscopy reveals reduction in mitochondrial cristae similar to AMD. Mice given D-DHA in the diet before tamoxifen were markedly protected against degeneration.
Conclusions :
RPE-specific knockout of GPx4 recapitulates key features of AMD. This degeneration is prevented by oral delivery of D-DHA prior to enzyme knockout. These results support the concept that GPx4 KO in RPE could serve as a model for AMD, and for D-DHA as a therapeutic for oxidative stress-mediated diseases, such as AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.