Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients.
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 in...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4938507?pdf=render |
| _version_ | 1831738535487471616 |
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| author | Andrew Want Stephanie R Gillespie Zheng Wang Ronald Gordon Carlo Iomini Robert Ritch J Mario Wolosin Audrey M Bernstein |
| author_facet | Andrew Want Stephanie R Gillespie Zheng Wang Ronald Gordon Carlo Iomini Robert Ritch J Mario Wolosin Audrey M Bernstein |
| author_sort | Andrew Want |
| collection | DOAJ |
| description | 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-IDTM 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. |
| first_indexed | 2024-12-21T13:24:07Z |
| format | Article |
| id | doaj.art-ea9e71ff556b4328b17537f479e9c9bd |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T13:24:07Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-ea9e71ff556b4328b17537f479e9c9bd2022-12-21T19:02:31ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01117e015740410.1371/journal.pone.0157404Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients.Andrew WantStephanie R GillespieZheng WangRonald GordonCarlo IominiRobert RitchJ Mario WolosinAudrey M BernsteinPURPOSE: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-IDTM 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.http://europepmc.org/articles/PMC4938507?pdf=render |
| spellingShingle | Andrew Want Stephanie R Gillespie Zheng Wang Ronald Gordon Carlo Iomini Robert Ritch J Mario Wolosin Audrey M Bernstein Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. PLoS ONE |
| title | Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. |
| title_full | Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. |
| title_fullStr | Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. |
| title_full_unstemmed | Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. |
| title_short | Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients. |
| title_sort | autophagy and mitochondrial dysfunction in tenon fibroblasts from exfoliation glaucoma patients |
| url | http://europepmc.org/articles/PMC4938507?pdf=render |
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