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Jeong A Choi, BO-RA SEO, Jae-Young Koh, Young Hee Yoon; Protective effects of zinc and cAMP against A2E-induced toxicity in ARPE19 cells: Possible involvement of lysosomal acidification. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2480.
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Dry age-related macular degeneration (AMD) is characterized by accumulation of drusen and degeneration of photoreceptor cells and retinal pigment epithelial (RPE) cells. It has been proposed that dysfunctional lysosomes in RPE cells contributes to dry AMD pathology by hindering the degradation of shed photoreceptor membranes. We have previously shown that raising intracellular zinc levels can restore lysosomal acidity, and several studies have shown that raising cAMP levels may restore acidity and degradative functions of lysosomes. In the present study, we examined the effects of zinc and cAMP on lysosomal alkalization and dysfunction in an in vitro model of AMD.
To induce lysosomal dysfunction in ARPE19 (human RPE cell line), we used A2E (lipofuscin derivative) and chloroquine (CQ, lysosomal alkalizing drug). We quantitatively assessed A2E-induced cell death by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium. In addition, we observed the effects of zinc and dibutyryl cAMP on lysosomal acidity and degradative functions in A2E-treated ARPE19 cells. Lysosomal pH of the cells treated with cAMP or clioquinol (ClioQ, zinc ionophore) was measured by using Lysosensor and LysoTracker.
Twenty-four hours after A2E treatment, ARPE19 cells exhibited autofluorescence throughout the cell body, and showed significant amount of cell death (54.6 ± 4.6 % LDH release). A2E plus CQ-treated cell showed increased cell death. Addition of zinc or dibutyryl cAMP significantly reduced cell death by 20 – 30% in both cases (P < 0.05). A2E was seen to accumulate in lysosomes, and LysoTracker signals faded, signifying lysosomal alkalization. Moreover, both zinc and cAMP decreased A2E autofluorescence and restored lysosomal pH back to the acidic range.
Our results support the possibility that adequate levels of zinc or cAMP may help overcome A2E-induced toxicity in ARPE19 cells that contribute to the pathogenesis of AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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