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Jeehyun Yoon, Ae Jin Choi, Hyunjung J Lim, Hyewon Chung; Regulation of Exosomes by Autophagy in Retinal Pigment Epithelium: an Implication of the Formation of Drusen. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3453.
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The formation of drusen has been considered a risk factor for developing age-related macular degeneration (AMD). Although some of the molecular components of drusen are known, there is little understanding of the cell biology that leads to the formation of drusen. Previous research found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Here we investigated the relationship between the regulation of autophagy and the release of exosomes in the retinal pigment epithelium (RPE).
ARPE-19 cells were transfected with GFP-LC3 plasmid DNA and labeled with DII (1,10-dioctadecyl-3,3,3’3’-tetramethylindocarbocyanine perchlorate) dye (lipophilic tracers for exosomes staining). ARPE-19 cells were exposed to oxidative stress (H2O2, 200 µM) with co-treatment of 3-methyladenine (3-ma) or siRNA against Atg5 as inhibition of autophagy. Rapamycin or torin was used as stimulation of autophagy. Exosome pellets from conditioned media (CM) from ARPE-19 cells exposed to 200 µM H2O2 for 24 h were obtained with ExoQuickTM Exosome Precipitation Solution. Immunoblotting for multivesicular and late endosomal membrane marker proteins (Tsg101 and CD63) were performed. Atg7flox/flox;Best1-Cre mice were exposed to bright light. Immunoblotting and immunoflourescence (IF) of the RPE and retina were carried out.
GFP-LC3 presented a punctuate distribution and partially co-localized with DII labeled exosomes in ARPE-19 cells exposed to H2O2. Inhibition of autophagy with 3-ma or siRNA against Atg5 increased the expression levels of Tsg101 and CD63 in exosomes derived from ARPE-19 cells exposed to H2O2, whereas decreased expression levels of these proteins were found in ARPE-19 cells exposed to rapamycin or torin by immunoblotting and IF. Tsg101 and CD63 expression were increased in the RPE and retina from bright light-exposed Atg7flox/flox;Best1-Cre mice compared to control mice by immunoblotting and IF.
The present study indicates that increasing autophagy activity in a discretely controlled manner may decrease exocytotic activity of the RPE, thus preventing the formation of drusen in patients with AMD.
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