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M. B. Grant, J. Cai, L. Wu, P. Thampi, H. Vittal Rao, M. E. Boulton; Dysregulation of -Secretase Increases Oxidative Stress-Induced Transepithelial Permeability and Apoptosis in the RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4175.
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The goal of this study was to investigate whether dysregulation of γ-secretase in the RPE enhances susceptibility to oxidative stress and increases transepithelial permeability leading to a reduction in the functional capacity of the RPE.
ARPE19 and primary human RPE cells were grown to confluence on culture flasks or Transwell inserts. Expression of the γ-secretase complex proteins [presenilin-1, nicastrin, APH-1, PEN-2] were determined by Western blot in whole cell lysates and purified mitochondrial preparations. Measurements of reactive oxygen species (ROS), permeability, apoptosis and junctional integrity was assessed in cells exposed to H2O2 in the presence of pigment epithelial-derived factor (PEDF) (which we have shown enhances γ-secretase activity [Cai et al, J Biol Chem, 2006]) or the γ-secretase inhibitor L685458. ROS was determined by ELISA. Changes in permeability were measured by transepithelial electrical resistance (TER) and the flux of FITC-dextran across the monolayer. Apoptosis was verified by FACS analysis of Bcl-2 and BAX labelled cells. Presenilin-1 was immunoprecipitated and then Western blot for N-cadherin, β-catenin and occludin was performed.
Presenilin-1 and Nicastrin were localized to both the mitochondria and junctional complexes in human RPE cells. In mitochondria, γ-secretase was increased by PEDF and reduced by both oxidative stress and γ-secretase inhibition. Elevation of mitochondrial γ-secretase activity was able to suppress the generation of endogenous superoxide anions. FACS analysis demonstrated that PEDF caused a greater than 50% reduction in H2O2-induced apoptotic RPE cell death and that this protection was absent in the presence of a γ-secretase inhibitor. γ-secretase protection against RPE apoptosis correlated with increased expression of the anti-apoptotic protein Bcl-2. Oxidative stress significantly increased RPE transepithelial permeability and caused disassociation of junctional complexes. This could be blocked by enhancing γ-secretase activity. Immunoprecipitation studies demonstrated a direct association between presenilin-1, N-cadherin, β-catenin and occludin suggesting that γ-secretase can act to stabilize junctional complexes.
γ-secretase can regulate oxidative stress-induced transepithelial permeability and apoptosis in RPE cells and may represent a therapeutic target in AMD.
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