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Wennan Lu, Alan M Laties, Leif Carlsson, Claire H. Mitchell; Antagonism of P2Y12 Receptor by Ticagrelor Leads to Acidification of Lysosomes in Retinal Pigmented Epithelial (RPE) Cells from ABCA4-/- Mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2034.
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Retinal Pigmented epithelial (RPE) cells play a key role in age-related macular degeneration (AMD), and the accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to the pathology. Degradative lysosomal enzymes work optimally at acidic pH levels, and elevated lysosomal pH is found in RPE cells from the ABCA4-/- mouse model. Elevation of cAMP lowers lysosomal pH in RPE cells and clears autofluorescent waste material. Cellular cAMP levels are increased pharmacologically by agonists for receptors coupled to stimulatory Gs subunits, or antagonists for receptors coupled to inhibitory Gi subunits. As the P2Y12 receptor for ADP is coupled to Gi and inhibited by the antagonist ticagrelor, we asked whether ticagrelor could restore lysosomal acidity in compromised RPE cells.
Lysosomal pH was measured in the ARPE19 cell line and in RPE cells from ABCA4-/- mice using the LysoSensor dye. Autofluorescence was determined using flow cytometry or from RPE wholemounts. Ticagrelor (148mg/kg/day) was added to standard mouse chow for the indicated 6wks. Complement factor H (CFH) and Equilibrative nucleoside transporter (ENT1) levels were determined with qPCR.
PCR confirmed expression of P2Y12 receptors in RPE cells. The P2Y12 antagonist ARC69931 lowered lysosomal pH in vitro in ARPE cells activated with P2Y12 agonist MesADP. The lipofuscin-like autofluorescence produced by exposing RPE cells to chloroquine and photoreceptor outer segments was reduced by ARC69931. The P2Y12 antagonist ticagrelor was chosen to expand the study in vivo given that its systemic use is well tolerated by patients. In vivo treatment of 7-8 month old ABCA4-/- mice with ticagrelor for 4 days significantly lowered lysosomal pH in RPE cells whereas extending treatment to 28 days reduced lysosomal pH in 18-24 month old mice. Ticagrelor treatment decreased expression of mRNA for CFH and ENT1 in 8 month old ABCA4-/- mice. Autofluorescence levels in RPE cells were decreased in some trials but not all.
Stimulating the P2Y12 receptor on RPE cells effectively restores lysosomal pH in compromised RPE cells. The ability of per oral ticagrelor to lower lysosomal pH in RPE cells of older ABCA4-/- mice may be of clinical relevance given the advanced age of patients with AMD and the widespread use of the drug.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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