Purchase this article with an account.
Kristine M. Porter, Yizhi Lin, David L. Epstein, Paloma B. Liton; Autophagy Dynamics In Oxidatively Stressed Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4635.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Increased oxidative damage has been hypothesized to be involved in the pathogenesis of primary open angle glaucoma. Here we investigate the role of the autophagic lysosomal degradative pathway as a defense mechanism against oxidative stress in trabecular meshwork (TM) cells.
Porcine TM cells were subjected to chronic oxidative stress using the hyperoxic model. Autophagic activity was monitored by analyzing autophagosome formation and maturation by electron microscopy, immunoblotting (LC3-I to LC3-II conversion), and confocal microscopy using a recombinant adenovirus containing the tandem fluorescence tagged LC3 (tfLC3) reporter gene. The following parameters were quantified by flow cytometry using the fluorogenic probes indicated within parentheses: intralysosomal oxidized material, lysosomal content (lysotracker red), and SA-β-galactosidase (FDG). Cathepsin activities were assayed using fluorogenic substrates. Rapamycin, bafilomycin A1, chloroquine, and 3-MA were used to pharmacologically modulate different steps within the autophagic pathway.
PTM cells treated with rapamycin (1 µM) showed a significant increase in LC3-II levels (> 2-fold), as well as tfLC3 punctuate staining by confocal microscopy starting 30 min post-treatment, followed by a decrease after 120 min. PTM cells exposed to chronic oxidative stress showed higher presence of autophagolysosomes and LC3-II levels (7.2±1.3 fold, p=0.001). Inhibition of lysosomal degradation using chloroquine (0.3 µM) or bafilomycin (0.1 µM) indicated that such increased levels of LC3-II were due to autophagy induction rather than decrease autophagy flux. These results were further confirmed using the autophagy inhibitor 3-MA. Incubation of TM cells with 3-MA (100 nM) resulted in decreased levels of LC3-II, as well as lipofuscin content (25.83±2.13, p=0.002), lysosomal mass (53.47±6.64, p=0.006) and SA-β-Gal staining (51.61±5.11, p=0.003) in the stressed cultures.
Our results validate the use of LC3 as a marker to monitor autophagy dynamics in TM cells and demonstrate that TM cells respond to an oxidative challenge by inducing the autophagic lysosomal pathway. Pharmacological modulation of autophagy might represent a novel therapeutic approach to promote the degradation of oxidized material and organelles, and thus restore TM function in aging and in disease.
This PDF is available to Subscribers Only