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J Mario Wolosin, Andy Want, Stephanie Gillespie, Zheng Wang, Robert Ritch, Audrey M Bernstein; Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3005.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that exfoliation syndrome (XFS), a systemic disease that causes the most common recognizable form of glaucoma, involves autophagy dysfunction.
Fibroblasts derived from Tenon’s tissue (TFs) obtained at the time of filtration surgery in exfoliation glaucoma patients were compared to age-matched TF derived from primary open-angle glaucoma (POAG) trabeculectomy or strabismus surgery. Nomarski and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize lysosomes, endosomes, Golgi, and microtubules. Light scattering, Cyto-ID and JC1 flow cytometry were used to measure relative cell size, autophagic flux and mitochondrial membrane potential (MMPT), respectively. Image analysis of cells transduced with a LC3B GFP-Cherry tandem plasmid was used to identify active autolysomes. Enhanced autophagy was induced by serum withdrawal.
XFS TFs displayed a different cell size and morphology in culture and a forward scatter that was 1.35-fold larger (p=0.05) than POAG TF, corresponding to an 80% larger cell volume. In extended 3D cultures, XFS-TFs accumulated 8-10 times more Fibulin-5 than the POAG cells, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes, lysosomes and LOXL1 to the perinuclear area and in development of a compact microtubule organizing center. Autophagic flux as measured by Cyto-ID dye revealed that the autophagic flux rate in XFS-TFs was 53% (p=0.01) lower than in POAG-TFs. Images and red to green ratios in cells transduced with the LC3B GFP-Cherry tandem plasmid revealed decreased autolysosome content and/or acidity in XFS cells. Finally, the proportion of cells with diminished MMPT was 3-8 larger in the XFS than in the POAG cells in serum or serum-free conditions (p = 0.02), consistent with impaired mitophagy.
TFs from XFS patients have diminished capacity for degradation processes essential for the maintenance of cell health. This newly identified handicap of XFS cells may lead to a reduced ability to process misfolded nascent protein and/or endocytosed peri-cellular denatured proteinateous material thereby accelerating the development of extra-cellular XFS material.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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