High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis
Purpose: Our previous study indicated that mitochondrial DNA (mtDNA) damage and mutations are crucial to the progressive loss of retinal ganglion cells (RGCs) in a glaucomatous rat model. In this study, we examined whether high pressure could directly cause mtDNA alterations and whether the latter c...
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doaj-666de11f8d2549c0849686eade9db2aa2020-11-24T21:03:50ZengFrontiers Media S.A.Frontiers in Cellular Neuroscience1662-51022016-11-011010.3389/fncel.2016.00254223142High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosisSheng-Hai Zhang0Sheng-Hai Zhang1Feng-Juan Gao2Zhong-Mou Sun3Ping Xu4Jun-Yi Chen5Xing-Huai Sun6Xing-Huai Sun7Xing-Huai Sun8Ji-Hong Wu9Ji-Hong Wu10Ji-Hong Wu11Eye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan UniversityShanghai Key Laboratory of Visual Impairment and RestorationEye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan UniversityWesleyan University, MiddletownEye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan UniversityEye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan UniversityEye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan UniversityShanghai Key Laboratory of Visual Impairment and RestorationKey Laboratory of Myopia, Ministry of Health, Fudan UniversityEye & ENT Hospital, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan UniversityShanghai Key Laboratory of Visual Impairment and RestorationKey Laboratory of Myopia, Ministry of Health, Fudan UniversityPurpose: Our previous study indicated that mitochondrial DNA (mtDNA) damage and mutations are crucial to the progressive loss of retinal ganglion cells (RGCs) in a glaucomatous rat model. In this study, we examined whether high pressure could directly cause mtDNA alterations and whether the latter could lead to mitochondrial dysfunction and RGC death.Methods: Primary cultured rat RGCs were exposed to 30 mm Hg of hydrostatic pressure (HP) for 12, 24, 48, 72, 96, and 120 hours. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and POLG expressions were also analyzed. The RGCs were then infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting POLG (POLG-shRNA), and mtDNA alterations as well as mitochondrial function, including complex I/III activities and ATP production were subsequently studied at appropriate times. Finally, RGC apoptosis and the mitochondrial-apoptosis pathway-related protein cleaved caspase-3 were detected using a Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and western blotting, respectively. Results: mtDNA damage was observed as early as 48 hours after the exposure of RGCs to HP. At 120 h after HP, mtDNA damage and mutations significantly increased, reaching >40% and 4.8±0.3-fold, respectively, compared with the control values. Twelve hours after HP, the expressions of OGG1, MYH and POLG mRNA in the RGCs were obviously increased 5.02±0.6-fold (p<0.01), 4.3±0.2-fold (p<0.05), and 0.8±0.09-fold p<0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 hours after HP (p<0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (p<0.01), while complex I/III activities gradually decreased (p<0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (p<0.05). Increases in the apoptosis of RGCs and cleaved caspase-3 protein expression were observed after mtDNA damage and mutations.Conclusions: High pressures could directly cause mtDNA alterations, leading to mitochondrial dysfunction and RGC death.http://journal.frontiersin.org/Journal/10.3389/fncel.2016.00254/fullHydrostatic PressureMutationRetinal Ganglion CellsMitochondrial dysfunctionmitochondrial DNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Sheng-Hai Zhang Sheng-Hai Zhang Feng-Juan Gao Zhong-Mou Sun Ping Xu Jun-Yi Chen Xing-Huai Sun Xing-Huai Sun Xing-Huai Sun Ji-Hong Wu Ji-Hong Wu Ji-Hong Wu |
spellingShingle |
Sheng-Hai Zhang Sheng-Hai Zhang Feng-Juan Gao Zhong-Mou Sun Ping Xu Jun-Yi Chen Xing-Huai Sun Xing-Huai Sun Xing-Huai Sun Ji-Hong Wu Ji-Hong Wu Ji-Hong Wu High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis Frontiers in Cellular Neuroscience Hydrostatic Pressure Mutation Retinal Ganglion Cells Mitochondrial dysfunction mitochondrial DNA |
author_facet |
Sheng-Hai Zhang Sheng-Hai Zhang Feng-Juan Gao Zhong-Mou Sun Ping Xu Jun-Yi Chen Xing-Huai Sun Xing-Huai Sun Xing-Huai Sun Ji-Hong Wu Ji-Hong Wu Ji-Hong Wu |
author_sort |
Sheng-Hai Zhang |
title |
High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis |
title_short |
High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis |
title_full |
High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis |
title_fullStr |
High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis |
title_full_unstemmed |
High pressure-induced mtDNA alterations in retinal ganglion cells and subsequent apoptosis |
title_sort |
high pressure-induced mtdna alterations in retinal ganglion cells and subsequent apoptosis |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cellular Neuroscience |
issn |
1662-5102 |
publishDate |
2016-11-01 |
description |
Purpose: Our previous study indicated that mitochondrial DNA (mtDNA) damage and mutations are crucial to the progressive loss of retinal ganglion cells (RGCs) in a glaucomatous rat model. In this study, we examined whether high pressure could directly cause mtDNA alterations and whether the latter could lead to mitochondrial dysfunction and RGC death.Methods: Primary cultured rat RGCs were exposed to 30 mm Hg of hydrostatic pressure (HP) for 12, 24, 48, 72, 96, and 120 hours. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and POLG expressions were also analyzed. The RGCs were then infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting POLG (POLG-shRNA), and mtDNA alterations as well as mitochondrial function, including complex I/III activities and ATP production were subsequently studied at appropriate times. Finally, RGC apoptosis and the mitochondrial-apoptosis pathway-related protein cleaved caspase-3 were detected using a Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and western blotting, respectively. Results: mtDNA damage was observed as early as 48 hours after the exposure of RGCs to HP. At 120 h after HP, mtDNA damage and mutations significantly increased, reaching >40% and 4.8±0.3-fold, respectively, compared with the control values. Twelve hours after HP, the expressions of OGG1, MYH and POLG mRNA in the RGCs were obviously increased 5.02±0.6-fold (p<0.01), 4.3±0.2-fold (p<0.05), and 0.8±0.09-fold p<0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 hours after HP (p<0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (p<0.01), while complex I/III activities gradually decreased (p<0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (p<0.05). Increases in the apoptosis of RGCs and cleaved caspase-3 protein expression were observed after mtDNA damage and mutations.Conclusions: High pressures could directly cause mtDNA alterations, leading to mitochondrial dysfunction and RGC death. |
topic |
Hydrostatic Pressure Mutation Retinal Ganglion Cells Mitochondrial dysfunction mitochondrial DNA |
url |
http://journal.frontiersin.org/Journal/10.3389/fncel.2016.00254/full |
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