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Ruchira Singh, David Kuai, Jackie Meyer, Molly Smith, Kyle Wallace, Amelia Verhoeven, David M Gamm; Targeting protein degradation pathways in macular degeneration using a human iPS cell model of Best disease. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2677.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) dysfunction in degrading photoreceptor outer segments (POS) is linked to increased accumulation of autofluorescent material in several maculopathies, including Best disease (BD). Similarly, we previously demonstrated that human induced pluripotent stem cell (hiPSC)-derived RPE from two patients with BD (BD-hiPSC-RPE) possessed delayed degradation of POS and increased autofluorescence accumulation after chronic POS feeding. In the present study, we used this model system to determine 1) if alterations in specific protein degradation pathways are associated with delayed digestion of POS and 2) whether we could pharmacologically target autofluorescent material accumulation
hiPSC-RPE monolayers were derived from two BD patients and unaffected siblings (Ctrl). Western blotting, immunocytochemistry, ELISA and fluorescence-based assays were utilized to evaluate expression, localization and/or activity of key enzymes involved in lysosomal, ubiquitin-proteasomal and autophagy-mediated protein degradation before and after chronic POS feeding. Exosomes secreted by RPE in the apical and basal media were isolated by differential ultracentrifugation and probed for protein content. Time course experiments were used to evaluate the effect of drugs that selectively targeted protein degradation pathways. This analysis included rate of POS degradation and amount of autofluorescent material accumulation in RPE cells after chronic POS feeding.
Differences in the expression and/or activity of specific proteins/enzymes involved in intracellular protein degradation was observed 1) between BD and Ctrl hiPSC-RPE and 2) before and after POS feeding. Differences were also seen in the amount of exosome released from BD vs. Ctrl hiPSC-RPE both prior to and after chronic POS feeding. Application of selected drugs increased the rate of POS degradation in BD hiPSC-RPE. Furthermore, continuous drug-treatment decreased the amount of autofluorescent material after chronic POS feeding in this BD hiPSC-RPE model.
Our results show that we can treat a key mechanistic defect in BD, delayed POS degradation, by targeting protein degradation pathways. Furthermore, by modulating the rate of POS digestion in this BD hiPSC-RPE model, we can reduce the accumulation of autofluorescent material in RPE, a pathological manifestation of various macular dystrophies.
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