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Jingyu Yao, Lin Jia, Shameka Shelby, Anna Ganios, Debra Thompson, David Zacks; Circadian rhythm of basal autophagy and its regulation in the RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1813. doi: https://doi.org/.
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To characterize the circadian changes in the basal level of autophagy and its regulation in the retinal pigment epithelium (RPE).
Wild-type C57BL/6 mice were bred and reared under standard 12 h light / 12 h dark conditions. At age 2 months, the RPE/choroid was collected from 3 groups of animals. In group 1, animals were sacrificed every 3 h over a 24 h period. In group 2, at 3 h after the lights turned on, animals were moved to the dark and sacrificed at 1, 4 and 7 h later. In group 3, after the regular 12 h dark period, animals were continuously kept in darkness and RPE/choroid samples were collected every 3 h. To test the effect of photoreceptor outer segment (POS) ingestion on the autophagic activity of RPE cells, we incubated cultured RPE-J cells with bovine POS for up to 12 h. Herbimycin A (HA), an inhibitor of RPE phagocytosis which inhibits cytoplasmic Src-tyrosine kinase, was used to treat POS fed RPE-J cells to evaluate the effect on autophagy. The levels of autophagy proteins LC3, Atg5, Atg12, beclin1 and p62, and the time course of LC3-I to LC3-II conversion were evaluated by western blot and immunohistochemistry (IHC).
Basal levels of autophagy in RPE/choroid followed a circadian rhythm. In group 1, we observed two peaks of autophagy, one at 7 h after the onset of the light period and one at 7 h after the onset of the dark period. In groups 2 and 3, when the normal 12 h light / 12 h dark cycling condition was interrupted, the shape of the basal autophagy curve remained unchanged. Autophagy in cultured RPE-J cells increased significantly after they were fed with POS. IHC staining of LC3 and Atg5 detected increasing signal that was also seen on western blots. Autophagic activity increased when the POS dosage increased from 2 to 10 POS/cell. HA treatment suppressed the increase in LC3 protein level in POS fed RPE-J cells.
Basal autophagy in mouse RPE/choroid follows a bimodal circadian rhythm with one peak at 7 h after the onset of the light period and one at 7 h after the onset of the dark period. Autophagy in cultured RPE cells is increased by POS, suggesting that it is regulated by the uptake of POS rather than by light itself. Further work is needed to understand the interactive role between autophagy and phagocytosis in RPE cells, and to unravel the signaling pathways and intermediates that connect these two degradation pathways.
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