title: Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E.
abstract: In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors.forename: Khandakar A S M
authors.initials: KA
authors.lastname: Saadat
authors.affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Murakami
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors.forename: Xue
authors.initials: X
authors.lastname: Tan
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nomura
authors.affiliation: Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Aichi 467-8601, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yasukawa
authors.affiliation: Okada Eye Clinic, Kanagawa 234-0054, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Okada
authors.affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yanagi
keywords: 3MA, 3-methyladenine; AMD, age-related macular degeneration; Autophagy; Lipofuscin; Mitochondria; RPE, retinal pigment epithelial cells; Reactive oxygen species; Retinal pigment epithelium; SEM, scanning electron microscopy; STGD, Stargardt disease; TEM, transmission electron microscopy; mROS, mitochondrial reactive oxygen species