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Mandy Losiewicz, Lynda Elgahzi-Cras, Patrice E Fort, Steven Abcouwer, Thomas W Gardner; The Rat Retina Exhibits High Level of mTOR Complexes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):246. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mechanistic target of rapamycin (mTOR) kinase exists in the mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). mTORC1 includes the regulatory-associated protein of mTOR (RAPTOR), PRAS40, DEPTOR and LST8. mTORC2 contains the rapamycin-insensitive companion of mTOR (RICTOR), Sin1, PROTOR and LST8. These complexes represent key control points for protein synthesis and growth responses. However, little is known about their expression in retinal cells or roles in retinal physiology. We previously showed that diabetes reduced retinal mTORC2 activity and protein synthesis. The aim of this study was to further define the expression of these complexes and their downstream effectors in normal rat retina.
The retinal contents of mTORC1-associated and mTORC2-associated proteins and the phosphorylation of downstream targets of these complexes were assessed by immunoblotting and co-immunoprecipitation of retinal lysates, and by immunofluorescence of retinal sections from control Sprague-Dawley rats.
Immunoblotting studies reveal that the retina contains approximately twice the amounts of mTOR, PROTOR, Sin1, Sin1 phospho-Thr86, and the mTORC1 regulating proteins, TSC1 and TSC2, versus brain and liver. Retinal levels of PRAS40, and PRAS40 phospho-Ser183 were intermediate between brain and liver. Retinal Raptor co-precipitated with TSC2, PRAS40 and PRAS40 phospho-Ser183, while TSC2, DEPTOR, Sin1 phospho-Thr86, PRAS40 and PRAS40 phospho-Thr246 all co-precipitated with Rictor. Immunofluorescence studies show that mTOR was detected throughout the ganglion cell layer (GCL). Raptor was detected between the outer plexiform layer (OPL) and GCL. Rictor co-localized with neurofilament L in the nerve fiber layer. S6 ribosomal protein phospho-Ser240/244, a downstream target of mTORC1, was detected throughout the GCL and OPL. S6 phospho-Ser235/236 was detected in a small number of cells in the GCL and inner nuclear layer (INL). The mTORC2 target, Akt phospho-Ser473, was weakly detected in the OPL, and Akt phospho-Thr308 was observed in the OPL. A second mTORC2 target, protein kinase C alpha (PKCα) phospho-Ser657, was detected at the junction of the GCL and IPL and in the OPL.
The mTORC complexes are relatively highly expressed in normal retina compared with brain and liver. Collectively, these findings are compatible with an important role for mTOR complexes in normal retinal physiology.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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