Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium.
PURPOSE
Inhibiting mechanistic target of rapamycin (mTOR) by pharmacological or genetic approaches can extend lifespan in mammals. The kinase activity of mTOR is controlled by upstream regulatory proteins and its subcellular localization. The purpose of this study was to characterize age-related alterations and functional consequences of mTOR signaling in the postmitotic RPE cells.


METHODS
Activity of mTOR complex 1 (mTORC1) was monitored by measuring phosphorylation status of its downstream effector protein S6, in either cultured human RPE cells or RPE explants prepared from mice at different ages. Subcellular distribution of mTOR was investigated by immunofluorescent staining of RPE culture or flatmount. The signaling of mTORC1 was modulated by either overexpression of a small guanosine triphosphatase, Ras homolog enriched in brain (Rheb), or disruption of the Ragulator complex with small interference RNA targeting p18. The effects of mTOR pathway on degradation of phagocytosed photoreceptor outer segments (POS) were determined by measuring the turnover rate of rhodopsin.


RESULTS
Aged RPE cells had more lysosome-associated mTOR and had increased response to amino acid stimulation. The lysosome distribution was essential for mTORC1 function, as disruption of the Ragulator complex abolished mTORC1 activation by amino acids. Increased mTORC1 activity caused decreased rate of degradation of internalized POS in the RPE.


CONCLUSIONS
Aging changes the subcellular localization and function of mTOR in the RPE. Increased mTORC1 inhibits POS degradation and may further exacerbate lysosome dysfunction of aged RPE.
['Yu|Bo|B|', 'Xu|Pei|P|', 'Zhao|Zhenyang|Z|', 'Cai|Jiyang|J|', 'Sternberg|Paul|P|', 'Chen|Yan|Y|']
D000818:Animals D015153:Blotting, Western D002338:Carotenoids D002478:Cells, Cultured D016922:Cellular Senescence D006801:Humans D008247:Lysosomes D000076222:Mechanistic Target of Rapamycin Complex 1 D051379:Mice D046912:Multiprotein Complexes D055214:Retinal Photoreceptor Cell Outer Segment D055213:Retinal Pigment Epithelium D015398:Signal Transduction D058570:TOR Serine-Threonine Kinases
aging retinal pigment epithelium mTOR signaling lysosome eye