Abstract
Purpose :
RPE oxidative stress has been implicated in age-related macular degeneration (AMD). Self-propagating lipid peroxidation chain reactions can be particularly pernicious. Glutathione peroxidase 4 (Gpx4) is an antioxidant enzyme that reduces oxidized lipids, and its down-regulation is part of the ferroptosis programmed cell death pathway. To determine if Gpx4 is essential for RPE viability, we studied RPE-specific Gpx4 knockout mice. Further, we tested whether an orally delivered, oxidation-resistant, deuterated form of docosahexanoic acid (D-DHA) is protective in this model. Deuterium is a stable isotope that’s been used safely in medical imaging and pharmacology. We targeted DHA because it’s the most abundant polyunsaturated acid in photoreceptor outer segments, which the RPE phagocytose daily.
Methods :
C57BL/6J mice with a floxed GPx4 gene were mated with RPE65-cre/ER mice. Mice were given Tamoxifen to induce Gpx4 knockout. Retinal phenotypes were assessed via in vivo imaging using confocal scanning laser ophthalmoscope (cSLO) and optical coherence tomography (OCT), as well as by electroretinography. After euthanasia, retinas were further studied by immunohistochemical (IHC) analyses, plastic sectioning, and quantitative polymerase chain reaction (qPCR) assays.
Results :
GPx4 knockout was specific to the RPE, as indicated by qPCR. IHC on samples collected one month after Tamoxifen injection revealed that GPx4 knockout animals had increased levels of the iron binding protein ferritin and oxidized DHA, assayed by an anti-carboxyethylpyrrole antibody. Microglia infiltrated the outer segment layer. Plastic sections showed RPE and photoreceptor degeneration, consisting of RPE thinning to 25% of normal and ONL thinning to approximately 50% of normal. Mice given D-DHA in the diet before Tamoxifen were completely protected against degeneration visible on cSLO and OCT, and partially protected when the diet was given after Tamoxifen.
Conclusions :
GPx4 is critical for health and functioning of retinal pigment epithelial cells, and, secondarily, photoreceptors. Knockout of GPx4 in the RPE produces oxidative stress, which can be rescued with a diet containing D-DHA. This supports the concept of D-DHA as a therapeutic for AMD. Further, since ferroptosis involves GPx4 downregulation, our data suggest ferroptosis or iron toxicity could contribute to AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.