The P2Y12 Receptor Antagonist Ticagrelor Reduces Lysosomal pH and Autofluorescence in Retinal Pigmented Epithelial Cells From the ABCA4-/- Mouse Model of Retinal Degeneration

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.

Oral Delivery of the P2Y12Receptor Antagonist Ticagrelor Prevents Loss of Photoreceptors in an ABCA4−/−Mouse Model of Retinal Degeneration

“…Expression of a second gene regulated by TFEB, LAMP2, showed a similar decrease that approached significance (not shown). This supports previous direct measurements of lysosomal pH performed previously, which demonstrate acidification of lysosomes after oral delivery of ticagrelor 14…”

“…First, the effect of ticagrelor treatment on expression of LAMP1 mRNA in the RPE of ABCA4 −/− mice was tested. Our previous findings indicate that oral treatment with ticagrelor lowers the lysosomal pH of RPE cells14 and proposed that lowering lysosomal pH of RPE cells protects the health of the outer retina 9. The expression of lysosomal genes such as LAMP1 are often inversely related to lysosomal activity, with the transcription factor E-Box (TFEB) linking decreased lysosomal degradation with increased expression of lysosomal genes in a classic negative feedback loop 20.…”

“…Several lines of evidence implicate changes in lysosomal function in the photoreceptor protection associated with ticagrelor. RPE cells express P2Y 12 receptors, and the lysosomal pH of isolated RPE cells was elevated by P2Y 12 receptor agonists and decreased by P2Y 12 receptor antagonists 14. Treatment with ticagrelor significantly lowered lysosomal pH and partially reduced autofluorescence in RPE cells of ABCA4 −/− mice when measured ex vivo 14.…”

“…The P2Y 12 receptor is coupled to G i proteins that inhibit adenylate cyclase, and thus, blocking of the P2Y 12 receptor elevates cytoplasmic cAMP 13. Inhibition of the P2Y 12 receptor was recently shown to lower lysosomal pH in RPE cells and reduce autofluorescence in cultured RPE cells fed photoreceptor outer segments while treated with the lysosomotropic agent chloroquine 14. In this initial study, the addition of the P2Y 12 receptor antagonist ticagrelor to mouse chow also lowered the pH of lysosomes in RPE cells and reduced autofluorescence 14.…”

“…Inhibition of the P2Y 12 receptor was recently shown to lower lysosomal pH in RPE cells and reduce autofluorescence in cultured RPE cells fed photoreceptor outer segments while treated with the lysosomotropic agent chloroquine 14. In this initial study, the addition of the P2Y 12 receptor antagonist ticagrelor to mouse chow also lowered the pH of lysosomes in RPE cells and reduced autofluorescence 14. Although this suggested that ticagrelor given orally could target lysosomes of RPE cells in ABCA4 −/− mice, the effects on vision were not determined.…”