Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium

Bo Yu, Pei Xu, Zhenyang Zhao, Jiyang Cai, Paul Sternberg, Yan Chen

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8638-8650
Number of pages13
JournalInvestigative Ophthalmology and Visual Science
Volume55
Issue number12
DOIs
StatePublished - 2014

Fingerprint

Retinal Pigment Epithelium
Sirolimus
Lysosomes
Aminoacylation
S 6
Rhodopsin
Guanosine
RNA Interference
Phagocytosis
Mammals
Proteins
Phosphotransferases
Phosphorylation
Pharmacology
Staining and Labeling
Amino Acids
Brain

Keywords

  • Aging
  • Eye
  • Lysosome
  • mTOR
  • Retinal pigment epithelium
  • Signaling

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium. / Yu, Bo; Xu, Pei; Zhao, Zhenyang; Cai, Jiyang; Sternberg, Paul; Chen, Yan.

In: Investigative Ophthalmology and Visual Science, Vol. 55, No. 12, 2014, p. 8638-8650.

Research output: Contribution to journalArticle

Yu, Bo ; Xu, Pei ; Zhao, Zhenyang ; Cai, Jiyang ; Sternberg, Paul ; Chen, Yan. / Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium. In: Investigative Ophthalmology and Visual Science. 2014 ; Vol. 55, No. 12. pp. 8638-8650.
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N2 - 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.

AB - 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.

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