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Bo Yu, Pei Xu, Zhen-Yang Zhao, Yan Chen; Phagocytosis of photoreceptor outer segments regulates mTORC1 activity in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):267. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of metabolism and integrates environmental cues to adjust cellular metabolic activities in a timely manner. Retinal pigment epithelial (RPE) cells remove daily shed photoreceptor outer segments (POS) by phagocytosis. Degradation of ingested POS and recycling its components generate demanding metabolic loads to the RPE. In this study, we investigated whether POS can stimulate mTORC1 activity in the RPE and explored the underlying molecular mechanisms.
RPE mTORC1 activity was monitored during morning burst of OS disc shedding, the time when synchronized phagocytosis occurs. RPE cells were harvested from C57BL6/J mice started 30 minutes before light on to 3 hours afterwards. mTORC1 activity was analyzed by monitoring the phosphorylation status of downstream substrate proteins S6 and 4EBP-1. Subcellular localization of mTORC1 components was measured by immunostaining of RPE flat mounts. For mechanistic studies, cultured human RPE cells were treated with purified porcine POS. To investigate the roles of lysosomes in POS-induced mTORC1 activation, cells were either treated with lysosome inhibitor chloroquine, or transfected with small interference RNA targeting p18, V-ATPase6V0C, V-ATPase6V1A, or SLC38A9. The activity and distribution of mTORC1 were then measured by western blot or immunostaining.
RPE mTORC1 was transiently activated during morning burst of phagocytosis. Treating RPE cells with purified POS also led to activation of mTORC1 in culture. Co-localization of rhodopsin and LAMP1 with mTOR, p18, or RagA was detected by immunostaining, indicating their distribution on phagolysosomes. In contrast to the response to nutrient signals, disrupting proton gradients of the lysosome or downregulating the V-ATPase transporter protein did not affect POS-induced mTORC1 activation.
POS are physiological stimuli of mTORC1 in the RPE. The daily fluctuation in mTORC1 activity responding to POS can be critical in maintaining normal functions of the RPE and retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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