Abstract
Purpose :
Purpose: Oxidative stress is a major contributor to age-dependent changes in RPE cells associated with retinal degradation. While photoreceptor outer segments (POS) are frequently implicated as a source of oxidative stress, lysosomal dysfunction can increase oxidative stress in other cells. This study asked whether lysosomal alkalinization is itself a trigger for oxidative stress in RPE cells and whether the presence of POS alters this.
Methods :
Oxidative stress was assessed using assays for 4-hydroxynonenal (HNE), Bodipy C11 and the CellRox indicator. The lysosoomotropic drug chloroquine was chosen to increase lysosomal pH given its clinical relevance. Levels of oxidative stress were determined in iPS-RPE and ARPE-19 cells.
Results :
Oxidative stress in RPE cells was assessed using multiple approaches. Levels of lipid peroxidation byproduct HNE were assessed as it is elevated in lipofuscin-associated proteins from human RPE and in drusen. Raising lysosomal pH with CQ was sufficient to double HNE levels, although addition of POS did not increase HNE levels either alone or in the presence of chloroquine. As oxidized lipids are frequently implicated in RPE pathogenesis, the effect of lysosomal alkalinization on oxidized lipids was assessed with Bodipy C11, a fatty acid analog whose fluorescence changes from red to green when oxidized by oxy-radicals and peroxynitrite. Addition of chloroquine increased the green Bodipy C11 signal in a punctate pattern. A positive signal was also detected with indicator CellRox following elevation of lysosomal pH. Restoration of lysosomal pH with acid nanoparticles removed the CellRox staining, suggesting lysosomal alkalization was sufficient to increase oxidative stress in RPE cells.
Conclusions :
Results from three separate assays strongly support the conclusion that elevation of lysosomal pH is sufficient to increase oxidative stress in RPE cells. These findings support those seen in vivo, but the use of isolated RPE cells confirms the stress is not due to POS. Thiese findings also suggests that restoring luminal acidity with acid nanoparticles or other tools should also decrease levels of oxidative stress in RPE cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.