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      Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients

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          Abstract

          Purpose

          To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma.

          Methods

          Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-ID TM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal.

          Results

          In culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8–10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3–8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02).

          Conclusions

          Our results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.

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          Most cited references41

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          How to interpret LC3 immunoblotting.

          Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.
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            Protein turnover via autophagy: implications for metabolism.

            Autophagy is a process of cellular "self-eating" in which portions of cytoplasm are sequestered within double-membrane cytosolic vesicles termed autophagosomes. The autophagosome cargo is delivered to the lysosome, broken down, and the resulting amino acids recycled after release back into the cytosol. Autophagy occurs in all eukaryotes and can be up-regulated in response to various nutrient limitations. Under these conditions, autophagy may become essential for viability. In addition, autophagy plays a role in certain diseases, acting to prevent some types of neurodegeneration and cancer, and in the elimination of invading pathogens. We review the current information on the mechanism of autophagy, with a focus on its role in protein metabolism and intracellular homeostasis.
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              Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.

              Elastic fibers are components of the extracellular matrix and confer resilience. Once laid down, they are thought to remain stable, except in the uterine tract where cycles of active remodeling occur. Loss of elastic fibers underlies connective tissue aging and important diseases including emphysema. Failure to maintain elastic fibers is explained by a theory of antielastase-elastase imbalance, but little is known about the role of renewal. Here we show that mice lacking the protein lysyl oxidase-like 1 (LOXL1) do not deposit normal elastic fibers in the uterine tract post partum and develop pelvic organ prolapse, enlarged airspaces of the lung, loose skin and vascular abnormalities with concomitant tropoelastin accumulation. Distinct from the prototypic lysyl oxidase (LOX), LOXL1 localizes specifically to sites of elastogenesis and interacts with fibulin-5. Thus elastin polymer deposition is a crucial aspect of elastic fiber maintenance and is dependent on LOXL1, which serves both as a cross-linking enzyme and an element of the scaffold to ensure spatially defined deposition of elastin.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 July 2016
                2016
                : 11
                : 7
                : e0157404
                Affiliations
                [1 ]Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, United States of America
                [2 ]Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, United States of America
                [3 ]Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, United States of America
                [4 ]Einhorn Clinical Research Center, New York Eye and Ear Infirmary of Mount Sinai, New York, NY, 10003, United States of America
                University of Iowa, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AMB JMW RR. Performed the experiments: AW SG ZW. Analyzed the data: AMB JMW AW CI RG. Contributed reagents/materials/analysis tools: AMB JMW CI RG. Wrote the paper: AMB JMW RR.

                Article
                PONE-D-15-55129
                10.1371/journal.pone.0157404
                4938507
                27391778
                f1d42d8e-d7f5-40c5-89e6-4acea26baeaa
                © 2016 Want et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 December 2015
                : 27 May 2016
                Page count
                Figures: 7, Tables: 1, Pages: 21
                Funding
                Funded by: The Research to Prevent Blindness
                Award Recipient :
                Funded by: The MYS Family U.S. Charitable Foundation, Inc
                Award Recipient :
                Funded by: The Bright Focus Foundation
                Award Recipient :
                Funded by: The Glaucoma Foundation
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000053, National Eye Institute;
                Award ID: EY024942
                Award Recipient :
                Funded by: The Research to Prevent Blindness
                Award Recipient :
                Funded by: The MYS Family U.S. Charitable Foundation, Inc.
                Award Recipient :
                Funded by: The Bright Focus Foundation
                Award Recipient :
                Funded by: The Glaucoma Foundation
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000053, National Eye Institute;
                Award ID: EY018748
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: T32 GM062754
                Award Recipient :
                This work was supported by The Research to Prevent Blindness (AMB, JMW, and CI), The MYS Family U.S. Charitable Foundation, Inc., The Bright Focus Foundation, and The Glaucoma Foundation (AMB and JMW), NIH-NEI R01 EY024942 (AMB), NIH-NEI R01 EY018748 (JMW), NIH training grant T32 GM062754 (SRG), NIH-NEI EY022639 (CI). Microscopy and image analysis was performed at the Microscopy CORE. The authors would like to thank Leica and Mount Sinai’s Microscopy CORE for providing access to the Leica TCS SP8 STED3X. The authors gratefully acknowledge the contribution of the surgeons who supplied surgical specimens: Dr. Celso Tello, (Manhattan Eye and Ear and Throat Hospital); Dr. Sung Chul Park (Manhattan Eye and Ear and Throat Hospital); Dr. Jeffrey Liebmann (Columbia University Medical Center); Dr. Brian Campollataro (New York Eye and Ear Infirmary of Mount Sinai). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Lysosomes
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Death
                Autophagic Cell Death
                Biology and Life Sciences
                Biochemistry
                Bioenergetics
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Biochemistry
                Proteins
                Extracellular Matrix Proteins
                Medicine and Health Sciences
                Ophthalmology
                Eye Diseases
                Glaucoma
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Endosomes
                Physical Sciences
                Physics
                Electromagnetic Radiation
                Light
                Light Scattering
                Physical Sciences
                Physics
                Scattering
                Light Scattering
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Custom metadata
                All relevant data are within the paper.

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