Mechanism Of All-trans-retinal-mediated Ferroptosis In Photoreceptor Cells

Objective:The present study aims to explore whether atRAL can induces ferroptosis in the photoreceptor cells and its mechanism of action.Methods:First,661W photoreceptor cells treated with atRAL.Then the cell viability of 661W photoreceptor cells was detected by MTS method and cell morphology was photographed under a research-grade inverted microscope.The qRT-PCR analysis of ferroptosis related genes and iron homeostasis-related genes.And ferroptosis related protein were detected by western blot.Iron levels was examined by FeRhoNox-land ICP-MS.The Click-iT lipid peroxidation imaging kit and flow cytometry was used to analyze lipid peroxidation.Transmission electron microscopy and MitoTracker Red CMXRos was used to analyze mitochondria.GSH levels were measured by a GSH assay kit.H2DCFDA was used for the measurement of ROS production.Then we use GSH,the ferroptosis inhibitor Fer-1,and the iron-chelating agent DFO to further study.And then histological examination of mouse retina was performed by using H&E staining in neural retina of Abca4-/-Rdh8-/-mice after light exposure.Ferroptosis inhibitor Fer-1 was intraperitoneal administration to further study.lipid peroxidation in mouse retina was determined by immunofluorescence staining of lipid peroxidation marker acrolein.Results:In this study,we reveal that the overload of atRAL leads to photoreceptor degeneration through activating ferroptosis,a nonapoptotic form of cell death.Ferroptosis of photoreceptor cells induced by atRAL resulted from increased ferrous ion(Fe2+),elevated ACSL4 expression,system Xc-inhibition,and mitochondrial destruction.Fe2+ overload,tripeptide glutathione(GSH)depletion,and damaged mitochondria in photoreceptor cells exposed to atRAL provoked reactive oxygen species(ROS)production,which,together with ACSL4 activation,promoted lipid peroxidation and thereby evoked ferroptotic cell death.Moreover,exposure of photoreceptor cells to atRAL activated COX2,a well-accepted biomarker for ferroptosis onset.In addition to GSH supplement,inhibiting either Fe2+ by deferoxamine mesylate salt(DFO)or lipid peroxidation with ferrostatin-1(Fer-1)protected photoreceptor cells from ferroptosis caused by atRAL.Abca4-/-Rdh8-/-mice exhibiting defects in atRAL clearance is an animal model for dry AMD and STGD1.We observed that ferroptosis was indeed present in neural retina of Abca4-/-Rdh8-/mice after light exposure.More importantly,photoreceptor atrophy and ferroptosis in light-exposed Abca4-/-Rdh8-/-mice were effectively alleviated by intraperitoneally injected Fer-1,a selective inhibitor of ferroptosis.Conclusion:Our study suggests that ferroptosis is one of the important pathways of photoreceptor cell death in retinopathies arising from excess atRAL accumulation.And ferroptosis involves light-induced photoreceptor degeneration in Abca4-/-Rdh8-/-mice.Ferroptosis inhibitor alleviates light-induced photoreceptor degeneration and ferroptosis in Abca4-/-Rdh8-/-mice.