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Pei Xu, Zhen-Yang Zhao, Bo Yu, Bo Long, Jiyang Cai, Yan Chen; Age-dependent changes in clearance of phagocytosed photoreceptor outer segments in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3448.
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Retinal pigment epithelial (RPE) cells are the most active phagocytes that are responsible for the removal of daily shed photoreceptor outer segments (POS). Incomplete degradation of ingested POS results in gradual accumulation of lipofuscin in RPE phagolysosomes. Increasing lipofuscin with age in RPE is considered to be one of the causative factors for blindness in patients with age-related macular degeneration (AMD). Emerging evidence suggests that maturation of phagosomes depends on molecular components of the autophagic pathway; and autophagy is closely related to aging through its housekeeping function in the degradation and recycling of cytoplasmic components and damaged organelles. The goal of the study was to determine the functional relationship between autophagy and phagocytosis; and how aging influences the trafficking and lysosome-dependent turnover of POS in RPE.
Porcine POS were purified by sucrose gradient centrifugation. Cultured human RPE cells were fed with POS for 16 hours and chased for 8 hours to monitor the turnover of phagocytosed POS. The amount and subcellular localization of rhodopsin were measured by Western blot and confocal microscopy. To inhibit autophagic and endolysosomal pathways, cells were treated with chemical inhibitors, 3-methyladenine (3-MA) or wortmannin, or transfected with siRNA targeting Rab7, Atg3, Atg5, or LC3. RPE/choroid flat-mounts from young and aged mice were co-stained with rhodopsin and Rab7 or LAMP2 to determine the colocalization of POS with endosome or lysosome, respectively.
Inhibiting autophagy by 3-MA and wortmannin reduced the degradation of POS. The remaining POS formed aggregates in lysosomes. Similar effects were achieved by down-regulating Rab7, Atg3, Atg5, or LC3. Phagocytosed POS were co-localized with the late endosome marker Rab7 and were more quickly degraded by young RPE than the aged one. Rab7 level was increased but Atg5 and Atg3 levels were decreased in aged RPE.
Young RPE had higher capacity in clearance of POS than aged RPE. Autophagy activity is likely decreased with aging. Inhibiting the endolysosomal pathway led to reduced degradation of POS. The results indicate that autophagy is essential for both self-renewal and clearance of phagocytosed POS in the RPE. The interactions among autophagy, phagocytosis and endocytic vesicular trafficking are important for RPE function.
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