GRP78 is a potential therapeutic target for ferroptosis in dry age-related macular degeneration (dAMD) by improving mitochondrial function

Abstract

Purpose : Ferroptosis is implicated in the pathogenesis of dAMD, but the exact mechanism remains unclear. The 78 kDa glucose-regulated protein (GRP78) was selected as a potential target against ferroptosis from our proteomics and bioinformatics analyses. We tested the hypothesis that stimulation of GRP78 slows down retinal ferroptosis progression in a dAMD cell model.

Methods : ARPE-19 cells were treated with different concentrations (1, 2.5, 5, 10, 20, and 30 mM) of sodium iodate (SI) for 24 h to establish dAMD model. Ferroptosis in the model was assessed with ferroptosis inhibitors (N=5) and fluorescent probe FerroOrange (N=6). The protective effect of GRP78 against ferroptosis in dAMD was confirmed by treating cells with binding immunoglobulin protein inducer X (BIX, GRP78 inducer). ARPE-19 cells were treated with or without BIX together with SI for indicated time. Next, MTS assay detected cell viability (N=6) and FerroOrange was used to detect intracellular Fe²⁺ level (N=8). Fluorescent probe DCFH-DA (N=7), mitochondrial ROS detection assay (N=4) and JC-1 staining assay (N=5) were used to evaluate mitochondrial function. One-way ANOVA was used for statistical analysis.

Results : The SI treatment at 10 mM, which corresponded to 39.8% decrease in cell viability (p<0.01) and 60.9% increase in intracellular Fe²⁺ level (p<0.01), was selected for further experiments. Ferroptosis inhibitors deferoxamine mesylate (DFO) and ferrostatin-1(Fer-1) significantly prevented SI-induced cell death from 62.0% to 76.1% (DFO, p<0.01) and 75.2% (Fer-1, p<0.01). BIX treatment rescued the SI-induced decrease in cell viability from 62.8% to 76.0% (1nM, p<0.01) and 77.2% (10nM, p<0.01), as well as reduced the SI-induced increase of intracellular Fe²⁺ levels from 162.8% to 133.4% (1nM, p<0.01) and 130.3% (10nM, p<0.01). Similarly, BIX decreased intracellular ROS level from 243.5% to 219.9% (1nM, p<0.01) and 207.7% (10nM, p<0.01), decreased mitochondrial ROS level from 280.9% to 234.9% (1nM, p<0.01), and increased mitochondrial membrane potential from 46.0% to 69.8% (1nM, p=0.21) and 96.6% (10nM, p<0.01).

Conclusions : GRP78 inducer could against SI-induced ferroptosis in a dAMD cell model by significant improvement of mitochondrial function. The results suggest that ferroptosis in dAMD may mitochondria-dependent, providing a potential therapeutic target for the disease.

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