Abstract
The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt’s disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.
Highlights
In some aging diseases, the accumulation of autofluorescent lipofuscin granules can signify an impaired clearance of waste material by lysosomes
Initial experiments to determine whether ticagrelor decreased lysosomal pH in retinal pigmented epithelial (RPE) cells from ABCA4−/− mice were carried out by adding ticagrelor to the drinking water
Previous work suggest lysosomal pH must be measured from freshly isolated RPE cells from ABCA4−/− mice, as the effect of lipofuscin on the lysosomal pH is altered by each cell division (Guha et al, 2014a)
Summary
The accumulation of autofluorescent lipofuscin granules can signify an impaired clearance of waste material by lysosomes. Retinal pigmented epithelial (RPE) cells are sensitive to perturbations in lysosomal enzyme activity, as they are responsible for phagocytosing the lipid-rich photoreceptor outer segment (POS) tips that are shed daily. A2E can lead to elevation of lysosomal pH, the delay between drug application and alkalinization suggests an indirect pathway (Holz et al, 1999; Liu et al, 2008; Toops et al, 2015). This alkalinization may reduce lysosomal activity and contribute to a secondary accumulation of oxidized lipid waste
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