title: Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, United States.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, United States.
authors.forename: Pei
authors.initials: P
authors.lastname: Xu
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, United States.
authors.forename: Zhenyang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, United States.
authors.forename: Jiyang
authors.initials: J
authors.lastname: Cai
authors.affiliation: Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Sternberg
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002338:Carotenoids / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D046912:Multiprotein Complexes / Q000502:physiology*; D055214:Retinal Photoreceptor Cell Outer Segment / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000502:physiology*
chemical_list: D046912:Multiprotein Complexes; D002338:Carotenoids; C113102:rhodopin; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases
keywords: aging; eye; lysosome; mTOR; retinal pigment epithelium; signaling