Characterization of Retinoid-Binding Protein 3 (RBP3): A Protective Factor Against Diabetic Retinopathy Identified in People with Extreme Diabetes Duration

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PROJECT SUMMARY/ABSTRACT Although treatment exists for late-stage diabetic retinopathy (DR) and macular edema, interventions to inhibit DR onset and worsening, other than glycemic control, have generally not been successful. To identify novel DR therapeutic targets, we studied Joslin 50-Year Medalists (N=1019), all of whom have type 1 diabetes (T1D) for 50-87 years. The presence of DR protective factors is supported by a bimodal distribution of DR in this cohort; 41% of Medalists have no-mild DR and 47% have quiescent proliferative DR (QPDR) despite no significant difference in glycemic control. Longitudinal data for up to 60 years shows that Medalists protected from proliferative DR (PDR) did not experience DR worsening after their first 17 years of diabetes. Mass spectrometry of post-mortem retina and vitreous found a novel protective factor, interphotoreceptor retinol- binding protein 3 (RBP3), to be elevated in Medalists with no-mild DR despite poor glycemic control. Our paper in Science Transl. Medicine (2019) confirmed that RBP3 is elevated in the retina and vitreous of Medalists with no-mild DR versus Medalists and non-Medalists with QPDR, and that RBP3 in the retina and vitreous of diabetic individuals is lower than in non-diabetic controls. RBP3 overexpression in in vivo studies by lentivirus subretinal injection, embryonically by transgene targeting photoreceptors or intravitreous injection of recombinant RBP3, inhibited retinal VEGF and IL-6 expression and normalized vascular permeability, electroretinogram changes and acellular capillaries in diabetic rodents. Mechanistic studies showed that in Muller and endothelial cells, RBP3 binds to cell surface proteins including GLUT-1 to decrease glucose uptake and glycolytic flux, neutralizing adverse actions of hyperglycemia. We developed a sensitive and specific ELISA assay that showed RBP3 levels in the vitreous and serum (at 1/1000 of vitreous levels) were correlated with each other and with DR severity, and inversely correlated with vitreous VEGF. RBP3 expression in photoreceptor cells was reduced by high glucose, possibly due to protein kinase C (PKC)  activation and inhibition of serum reactive factor (SRF) transcription factor via the Akt pathway. Preliminary studies of RBP3 subdomains show structure-function activities for inhibiting glucose uptake by binding to GLUT-1 transporters and reducing VEGF and IL-6 expression in Muller cells. The specific aims proposed are: Sp. Aim 1: To characterize and compare RBP3 levels in the retina, vitreous and serum as a potential biomarker for DR in T1D and T2D patients at the Joslin Diabetes Center, with validation in the Finland FinnDiane T1D cohort and DRCR Protocol T (T2D) cohort. Sp. Aim 2: To determine the mechanism for hyperglycemia-induced downregulation of RBP3 expression in photoreceptors in vivo and in a photoreceptor cell line by activation of PKC and deactivation of the Akt/mTOR/S6K pathway and SRF transcription factor. Sp. Aim 3: To define structure-function relationships between the RBP3 full length protein and its subdomains with regard to interaction with GLUT-1 and glucose uptake in Müller and retinal endothelial cells.