mTORC1 and mTORC2 expression in inner retinal neurons and glial cells

Abstract

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

Keywords: Protein synthesis; Raptor; Retina; Retinal ganglion cells; Rictor; mTORC.

Figure 1.. mTOR complex components show different expression patterns in retina, brain, and liver.

Western blot analysis of mTOR complex components in normal rat tissues. Equal amounts of lysate (40 μg) were loaded for analysis; n = 5 for all tissues. All data are normalized to actin levels and are shown as average ± SEM. Statistical analysis was performed using the Kruskal-Wallis Test (for nonparametric data). (A) mTOR, (B) raptor, (C) rictor, (D) TSC1, (E) TSC2, (F) DEPTOR, (G) SIN1, (H) P-Sin1 T86, (I) Protor-2, (J) PRAS40, (K) S183 P-PRAS40, and (L) T246 P-PRAS40.

Figure 1.. mTOR complex components show different expression patterns in retina, brain, and liver.

Western blot analysis of mTOR complex components in normal rat tissues. Equal amounts of lysate (40 μg) were loaded for analysis; n = 5 for all tissues. All data are normalized to actin levels and are shown as average ± SEM. Statistical analysis was performed using the Kruskal-Wallis Test (for nonparametric data). (A) mTOR, (B) raptor, (C) rictor, (D) TSC1, (E) TSC2, (F) DEPTOR, (G) SIN1, (H) P-Sin1 T86, (I) Protor-2, (J) PRAS40, (K) S183 P-PRAS40, and (L) T246 P-PRAS40.

Figure 2.. Retina, brain and liver exhibit varying mTORC composition

Raptor (A-F) and rictor (G-L) immunoprecipitations (IP) using equal amounts (500 μg) of protein in retina, brain, and liver of control Sprague-Dawley rats. n = 4 for all tissues. All co-immunoprecipitation data are normalized to appropriate (raptor or rictor) IP protein levels and are shown as average ± SEM. Statistical analysis was performed using the Kruskal-Wallis Test (for nonparametric data). Raptor IPs blotted for (A) raptor, (B) mTOR, (C) TSC2, (D) PRAS40, (E) P-PRAS40 S183, (F) P-PRAS40 T246, and rictor IP’s blotted for (G) rictor, (H) mTOR, (I) TSC2, (J) Sin1, (K) P-SIN1 T86, and (L) DEPTOR.

Figure 3.. Expression patterns of mTOR, raptor, rictor and ribosomal protein S6 proteins in glial cells, RGC and the NFL in normal central human retina.

Representative immunofluorescence in human central retina for mTOR (A-C), raptor (D-F), rictor (G-I) and S6 (J) in green, with neurofilament heavy (NF-H), glial fibrillary acidic protein (GFAP), and vimentin in red. Arrows and arrowheads highlight costained cells. Nuclei were counterstained with Hoechst. Scale bars, 50μm.

Figure 4.. Expression of mTOR, raptor and rictor proteins in glial cells, RGC and the NFL of normal rat retina.

Representative immunostaining for mTOR (A-E), raptor (F-J), rictor (K-O) in green and neurofilament light (NF-L), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), vimentin and retinal binding protein multiple splice (Rbpms) in red. Arrows highlight costained cells. Nuclei were counterstained with Hoechst. Scale bars, 50μm.

Figure 5.. Expression patterns of phosphorylated ribosomal protein S6 in glial cells and RGC of normal rat retina.

Representative images of S6 ribosomal protein (A-C) and Ser240/244phospho-S6 (D-F) in green, and retinal binding protein multiple splice (Rbpms) (panels A, D), glutamine synthase (GS) (panels B, E), and neurofilament light (NF-L) (panels C, F) in red. Nuclei were counterstained with Hoechst. Scale bars, 50μm.

Figure 6.. Expression patterns of mTOR, raptor and rictor proteins in glial cells, RGC and the NFL of normal mouse retina.

Representative expression patterns of mTOR (A-D), raptor (E-H),rictor (I-L) in green with neurofilament light (NF-L) (panels A, E, I), glutamine synthetase (GS) (panels B, F, J), glial fibrillary acidic protein (GFAP) (panels C, G, K), and retinal binding protein multiple splice (Rbpms) (panels D, H, L). Nuclei were counterstained with Hoechst. Scale bars, 50 μm.

Figure 7.. Expression patterns of S6 ribosomal protein and phosphorylated ribosomal protein S6 in glial cells and RGC of normal mouse retina.

Representative S6 expression (A-B) and phosphorylated Ser240/244 (C-D) in green with retinal binding protein multiple splice (Rbpms) (panels A, C) and glutamine synthetase (GS) (panels B, D) in red. Nuclei were counterstained with Hoechst. Scale bars, 50 μm.

Figure 8.. Summary of mTORC pathway expression in control human, rat and mouse retinas by layer.

Schematic representation of the overall localization of expression of the primary components of mTORC1 (mTOR, raptor and S6) and mTORC2 (mTOR and rictor)(Left panel), and higher magnification representation of the same complex in the inner retina with specific localization of mTORC1 mostly in ganglion cells and mTORC2 in the glial cells (right panel). Bold text = high immunoreactivity; regular text: relatively low immunoreactivity. Nerve Fiber Layer (NFL), Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner Nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Outer Segments/Internal Segments (OS/IS), Retinal Pigment Epithelium (RPE).