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 language | English (US) |
|---|---|
| Pages (from-to) | 8638-8650 |
| Number of pages | 13 |
| Journal | Investigative Ophthalmology and Visual Science |
| Volume | 55 |
| Issue number | 12 |
| DOIs | |
| State | Published - 2014 |
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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 journal › Article
}
TY - JOUR
T1 - Subcellular distribution and activity of mechanistic target of rapamycin in aged retinal pigment epithelium
AU - Yu, Bo
AU - Xu, Pei
AU - Zhao, Zhenyang
AU - Cai, Jiyang
AU - Sternberg, Paul
AU - Chen, Yan
PY - 2014
Y1 - 2014
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.
KW - Aging
KW - Eye
KW - Lysosome
KW - mTOR
KW - Retinal pigment epithelium
KW - Signaling
UR - http://www.scopus.com/inward/record.url?scp=84939781204&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84939781204&partnerID=8YFLogxK
U2 - 10.1167/iovs.14-14758
DO - 10.1167/iovs.14-14758
M3 - Article
VL - 55
SP - 8638
EP - 8650
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 12
ER -