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High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis

View Article: PubMed Central - PubMed

ABSTRACT

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 h. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and polymerase gamma (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 h 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 h 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.

No MeSH data available.


Related in: MedlinePlus

RGC apoptosis increased after transfection of lenti-shPOLG. (A) Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay for detecting apoptotic cell death. Representative microscopic images showing TUNEL-positive cells in the RGCs treated with lenti-shPOLG or with lenti-scrambled shRNA. Red, TUNEL; blue, DAPI. Scale bars, 50 μm. (B) Quantitative analysis of RGC apoptosis determined by TUNEL assay. The data are expressed as survival cell counts. (C) The active form of caspase-3 increased as determined by western blot using an antibody to cleaved caspase-3. Values are presented as the means ± SEMs. **P < 0.01, ***P < 0.001, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.
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Figure 7: RGC apoptosis increased after transfection of lenti-shPOLG. (A) Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay for detecting apoptotic cell death. Representative microscopic images showing TUNEL-positive cells in the RGCs treated with lenti-shPOLG or with lenti-scrambled shRNA. Red, TUNEL; blue, DAPI. Scale bars, 50 μm. (B) Quantitative analysis of RGC apoptosis determined by TUNEL assay. The data are expressed as survival cell counts. (C) The active form of caspase-3 increased as determined by western blot using an antibody to cleaved caspase-3. Values are presented as the means ± SEMs. **P < 0.01, ***P < 0.001, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.

Mentions: Next, we assessed the effects of mtDNA damage and mutation on RGC apoptosis. Thirty-six percentage (p < 0.01) and 57% (p < 0.001) of RGCs were positive for TUNEL staining 5 and 10 days after transfected with POLG-shRNA, respectively. Indicating the occurrence of cell apoptosis, while no obvious apoptosis was observed in the control group (Figures 7A,B). Meanwhile, western blotting analysis showed that the amount of cleaved caspase-3 was increased 2.6-fold and 6.5-fold 5 and 10 days after transfected with POLG-shRNA, respectively, compared to the controls (p < 0.001, Figure 7C).


High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis
RGC apoptosis increased after transfection of lenti-shPOLG. (A) Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay for detecting apoptotic cell death. Representative microscopic images showing TUNEL-positive cells in the RGCs treated with lenti-shPOLG or with lenti-scrambled shRNA. Red, TUNEL; blue, DAPI. Scale bars, 50 μm. (B) Quantitative analysis of RGC apoptosis determined by TUNEL assay. The data are expressed as survival cell counts. (C) The active form of caspase-3 increased as determined by western blot using an antibody to cleaved caspase-3. Values are presented as the means ± SEMs. **P < 0.01, ***P < 0.001, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.
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Figure 7: RGC apoptosis increased after transfection of lenti-shPOLG. (A) Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay for detecting apoptotic cell death. Representative microscopic images showing TUNEL-positive cells in the RGCs treated with lenti-shPOLG or with lenti-scrambled shRNA. Red, TUNEL; blue, DAPI. Scale bars, 50 μm. (B) Quantitative analysis of RGC apoptosis determined by TUNEL assay. The data are expressed as survival cell counts. (C) The active form of caspase-3 increased as determined by western blot using an antibody to cleaved caspase-3. Values are presented as the means ± SEMs. **P < 0.01, ***P < 0.001, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.
Mentions: Next, we assessed the effects of mtDNA damage and mutation on RGC apoptosis. Thirty-six percentage (p < 0.01) and 57% (p < 0.001) of RGCs were positive for TUNEL staining 5 and 10 days after transfected with POLG-shRNA, respectively. Indicating the occurrence of cell apoptosis, while no obvious apoptosis was observed in the control group (Figures 7A,B). Meanwhile, western blotting analysis showed that the amount of cleaved caspase-3 was increased 2.6-fold and 6.5-fold 5 and 10 days after transfected with POLG-shRNA, respectively, compared to the controls (p < 0.001, Figure 7C).

View Article: PubMed Central - PubMed

ABSTRACT

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 h. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and polymerase gamma (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 h after the exposure of RGCs to HP. At 120 h after HP, mtDNA damage and mutations significantly increased, reaching &gt;40% and 4.8 &plusmn; 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 &plusmn; 0.6-fold (p &lt; 0.01), 4.3 &plusmn; 0.2-fold (p &lt; 0.05), and 0.8 &plusmn; 0.09-fold (p &lt; 0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 h after HP (p &lt; 0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (p &lt; 0.01), while complex I/III activities gradually decreased (p &lt; 0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (p &lt; 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.

No MeSH data available.


Related in: MedlinePlus