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Mandy Losiewicz, Lynda Elghazi, Dejuan Kong, Diane Fingar, Raju V S Rajala, Patrice E Fort, Steven F Abcouwer, Thomas W Gardner; Retinal Ganglion Cell Protein Synthesis is Regulated by Glycolysis, mTORC1 Signaling, and Diabetes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6429.
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© ARVO (1962-2015); The Authors (2016-present)
Normal retina exhibits a high basal rate of protein synthesis that decreases in response to glycolysis inhibition and during diabetes. Mechanistic target of rapamycin complex 1 (mTORC1), which contains the core kinase mTOR and partner protein raptor, functions as a central effector of the PI3K signaling pathway and protein synthesis. The goal of this study was to assess the cellular localization of this complex and its role in cellular specific protein synthesis.
mTOR, raptor and S6 ribosomal proteins were localized by immunofluorescence (IF) in retinal sections of male C57BL/6J mice. Retinal protein synthesis was localized and quantified by the SUnSET method, using intraperitoneal injection (i.p.) of puromycin (200 mg/kg) and antibody-mediated detection of puromycinylated nascent proteins. Glycolysis was inhibited by 2-deoxyglucose (2DG) treatment (500 mg/kg, i.p.) and diabetes was induced with streptozotocin (65 mg/kg, i.p. daily for 5 days). 18S and 28S ribosomal RNA was localized by in situ hybridization. For conditional knockout (cKO) of mTORC1 in the ganglion cell layer (GCL), mTORfl/fl and Rptorfl/fl mice were intravitreally injected with AAV2-Cre or negative control AAV2-CreΔC vectors.
mTOR, raptor, and S6 proteins were located primarily in retinal ganglion cells (RGC) and the nerve fiber layer, where the highest levels of 18S and 28S rRNAs were also detected. Retinal protein synthesis was highest in RGC and photoreceptor outer segments. Systemic inhibition of glycolysis with 2DG reduced RGC protein synthesis by 33%. Western blot analysis of whole retina showed a 65% decrease in protein synthesis in 12-week diabetic mice. cKO of mTOR and Rptor reduced RGC protein synthesis by 52% and 77%, respectively.
Collectively, these novel findings reveal a previously unappreciated high level of metabolic activity in RGC and suggest important roles for glycolysis and mTORC1 signaling in the control of RGC protein synthesis. Understanding the regulation of retinal homeostatic mechanisms may be key to maintaining vision in patients with diabetes and other retinal diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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