Proteome changes in ferroptosis‐induced retinal pigment epithelial cell death in age‐related macular degeneration

Abstract

Aims/Purpose: Ferroptosis is known to be involved in the pathogenesis of age‐related macular degeneration (AMD). We elucidated the potential roles and related pathways of ferroptosis in an in vitro AMD cell culture model and the mass spectrometry (MS)‐based proteomics analysis.Methods: To mimic ferroptosis in AMD, ARPE‐19 cells were treated with erastin (ERS; 1, 2, 5 and 10 μM) or RSL3 (1, 2, 4, and 6 μM) for 24 h. Cells without treatment were used as control. Cell viability and glutathione peroxidase 4 (Gpx4) expression levels were used to determine the optimal AMD model. The MS‐based proteomics approach was employed to profile the protein expressions in ferroptotic RPE cells compared to controls. Next, bioinformatics analysis identified the significantly affected functions and pathways of the differentially expressed proteins.Results: Cell viability and Gpx4 expression levels of ARPE‐19 cells were decreased in a dose‐dependent manner after induction with ERS (1, 2, 5 and 10 μM) and RSL3 (1, 2, 4, and 6 μM). Doses corresponding to ~60% cell viability (5 μM ERS and 2 μM RSL3) were selected for the proteomics analysis. A total of 1356 proteins were identified in all groups, with 300 and 209 significantly (p < 0.05) differentially expressed proteins in the ERS and RSL3 group, respectively. The induction of ferroptosis in this optimized model with ERS (p = 6.3 × 10−3) and RSL3 (p = 1.5 × 10−4), and disruption of iron homeostasis signalling pathway (p = 1.9 × 10−4 in RSL3) were further confirmed by proteomics analysis. Iron metabolism‐related proteins ferritin light chain and heme oxygenase 1 were significantly up‐regulated in the RSL3 group. Notably, a large cluster of proteins were highly significantly implicated in the eukaryotic initiation factor 2 signalling pathway (p = 2.0 × 10−30 in ERS and 1.0 × 10−5 in RSL3), which was correlated with ferroptosis. Furthermore, the upregulation of autophagy‐related Ras‐related protein Ral‐A and involvement of mTOR signalling pathway were found in both groups (p = 3.2 × 10−6 in ERS and p = 1.9 × 10−3 in RSL3).Conclusions: In summary, this study established an optimum model for ferroptosis and demonstrated that it is involved in RPE cell death and is autophagy‐dependent. Our results also suggested that the disruption of iron uptake and intracellular storage in RPE cells are involved in AMD progression.