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Paloma B. Liton, Nallathambi Jeyabalan, Kristine Porter, David L. Epstein; Oxidative Stress Induces Lysosomal Basification And Decreased Autophagic Flux In Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2748.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects of chronic oxidative stress in the autophagy lysosomal pathway in trabecular meshwork (TM) cells.
Confluent cultures of TM cells were grown for 1, 2, and 3 weeks under physiological (5% O2) and chronic oxidative stress (40% O2) conditions. Autophagy dynamics were monitored by confocal microscopy, using a recombinant adenovirus containing the tandem fluorescence tagged LC3 (tfLC3); immunoblotting, by monitoring the LC3-I to LC3-II conversion; electron microscopy; and flow cytometry, using the CytoID Autophagy detection kit. Protein expression levels were quantified by WB analysis using specific antibodies against an array of Atgs, LAMP1, p62, LC3, Beclin, Rab7, CTSB, and CTSD. mRNA expression levels of beclin, Atg4, Atg5, Atg7, LC3, LAMP1, and LAMP2 were monitored by qPCR. Cathepsin activities were assayed using fluorogenic substrates. Lysosomal pH was monitored using Lysosensor Yellow-Blue dextran. Leupeptin and bafilomycin were used to pharmacologically inhibit lysosomal degradation.
WB analysis showed increased levels of the autophagosome marker LC3-II, as well as LAMP1 and single-chain CTSB in the oxidatively stressed cultures. The levels of LC3-II further increased in the presence of leupeptin and bafilomycin at week 1, but experienced no changes at week 2 and significantly diminished at week 3 in the cells grown at 40% O2. Cyto ID demonstrated the occurrence of autophagy at weeks 2 and 3 in the stressed cultures. Confocal microscopy showed increased number of autophagosomes and autolysosomes with oxidative stress. qPCR analysis showed the down-regulation of Atg7 and upregulation of LAMP1 and LAMP2 in the stressed cultures. Cells grown at 40% O2 displayed increased lysosomal pH (p=0.0125) accompanied by decreased cathepsin activities
Altogether, our results indicate that while TM cells respond to oxidative challenge by activation of the autophagic lysosomal degradative pathway, chronic exposure to oxidative stress leads to lysosomal basification and defective proteolytic activation of lysosomal enzymes with subsequent decrease of the autophagic flux. Given the critical role of lysosomal function in cellular and tissue homeostasis, including the degradation of phagocytosed material, we propose that diminished autophagic flux induced by oxidative stress represents one of the factors leading to progressive failure of cellular TM function with age, and this may be involved in the pathogenesis of primary open angle glaucoma. We further propose that pharmacological modulation of autophagy might represent a novel therapeutic strategy to minimize the impact of oxidative stress and restore TM function in aging and in disease.
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