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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

Transfection of lenti-shPOLG into cultured RGCs led to mitochondrial dysfunction. (A) Representative images of JC-1 staining of astrocytes at 10 days (left panel). Quantitative analysis of mitochondrial membrane depolarization by flow cytometry after JC-1 staining (right panel). Values are expressed as percentages, with the red/green fluorescence ratio values of lenti-SC shRNA-transfected RGCs set at 100%. Scale bars, 50 μm. (B) Mitochondrial ATP production rates in RGCs were analyzed at different time points after transfection with lenti-shPOLG or SC shRNA, using a luciferase-based assay. All of the values in these figures are presented as the means ± SEMs from three or four independent experiments. *P < 0.05, 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 6: Transfection of lenti-shPOLG into cultured RGCs led to mitochondrial dysfunction. (A) Representative images of JC-1 staining of astrocytes at 10 days (left panel). Quantitative analysis of mitochondrial membrane depolarization by flow cytometry after JC-1 staining (right panel). Values are expressed as percentages, with the red/green fluorescence ratio values of lenti-SC shRNA-transfected RGCs set at 100%. Scale bars, 50 μm. (B) Mitochondrial ATP production rates in RGCs were analyzed at different time points after transfection with lenti-shPOLG or SC shRNA, using a luciferase-based assay. All of the values in these figures are presented as the means ± SEMs from three or four independent experiments. *P < 0.05, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.

Mentions: To further determine whether increased mtDNA alterations could lead to mitochondrial dysfunction, we used a lentivirus to transfect POLG-small hairpin RNA (shRNA) into RGCs to block repair of mtDNA alterations, as previously reported (Tewari et al., 2012); subsequently, verification was undertaken by real-time PCR and western blotting. The results showed that the mRNA and protein expression of POLG was significantly decreased 10 days after transfection (p < 0.01, Figure 4A). In the lenti-shPOLG-transfected RGCs, mtDNA damage increased, as shown by the decrease in the relative amplification of long (13.4 kb) fragments of mtDNA, compared with short (210 bp) fragments of mtDNA (Figure 4B). The mutation frequency of mtDNA at both 1427 and 8335 sites was also significantly increased (p < 0.01) at 4 days after transfection (Figures 4C,D). Then, we examined the changes in mitochondrial function. The activities of complex I and complex III were decreased by 32.6 ± 2.3% (p < 0.05) and 46.8 ± 2.8% (p < 0.01), respectively, at 10 days after transfection with shPOLG (Figures 5A,B). To further characterize the mitochondrial dysfunction, we used the JC-1 fluorescent probe to measure Δψm, as previously published (Almeida and Bolaños, 2001). The ratio of the fluorescence of the aggregate and monomer forms of JC-1 reflected a decrease in Δψm in shPOLG-treated astrocytes (Figure 6A). We performed the kinetic measurement of ATP production. As expected, the MAPR in the cells transfected with shPOLG decreased in a time-dependent manner, with levels amounting to 57.5 ± 3.7% of those in the SC-transfected cells at 9 days (p < 0.05, Figure 6B).


High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis
Transfection of lenti-shPOLG into cultured RGCs led to mitochondrial dysfunction. (A) Representative images of JC-1 staining of astrocytes at 10 days (left panel). Quantitative analysis of mitochondrial membrane depolarization by flow cytometry after JC-1 staining (right panel). Values are expressed as percentages, with the red/green fluorescence ratio values of lenti-SC shRNA-transfected RGCs set at 100%. Scale bars, 50 μm. (B) Mitochondrial ATP production rates in RGCs were analyzed at different time points after transfection with lenti-shPOLG or SC shRNA, using a luciferase-based assay. All of the values in these figures are presented as the means ± SEMs from three or four independent experiments. *P < 0.05, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.
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Related In: Results  -  Collection

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Figure 6: Transfection of lenti-shPOLG into cultured RGCs led to mitochondrial dysfunction. (A) Representative images of JC-1 staining of astrocytes at 10 days (left panel). Quantitative analysis of mitochondrial membrane depolarization by flow cytometry after JC-1 staining (right panel). Values are expressed as percentages, with the red/green fluorescence ratio values of lenti-SC shRNA-transfected RGCs set at 100%. Scale bars, 50 μm. (B) Mitochondrial ATP production rates in RGCs were analyzed at different time points after transfection with lenti-shPOLG or SC shRNA, using a luciferase-based assay. All of the values in these figures are presented as the means ± SEMs from three or four independent experiments. *P < 0.05, compared with control RGCs transfected with lenti-scrambled shRNA. Sh, lenti-shPOLG-transfected RGCs; SC, lenti-scrambled shRNA-transfected RGCs.
Mentions: To further determine whether increased mtDNA alterations could lead to mitochondrial dysfunction, we used a lentivirus to transfect POLG-small hairpin RNA (shRNA) into RGCs to block repair of mtDNA alterations, as previously reported (Tewari et al., 2012); subsequently, verification was undertaken by real-time PCR and western blotting. The results showed that the mRNA and protein expression of POLG was significantly decreased 10 days after transfection (p < 0.01, Figure 4A). In the lenti-shPOLG-transfected RGCs, mtDNA damage increased, as shown by the decrease in the relative amplification of long (13.4 kb) fragments of mtDNA, compared with short (210 bp) fragments of mtDNA (Figure 4B). The mutation frequency of mtDNA at both 1427 and 8335 sites was also significantly increased (p < 0.01) at 4 days after transfection (Figures 4C,D). Then, we examined the changes in mitochondrial function. The activities of complex I and complex III were decreased by 32.6 ± 2.3% (p < 0.05) and 46.8 ± 2.8% (p < 0.01), respectively, at 10 days after transfection with shPOLG (Figures 5A,B). To further characterize the mitochondrial dysfunction, we used the JC-1 fluorescent probe to measure Δψm, as previously published (Almeida and Bolaños, 2001). The ratio of the fluorescence of the aggregate and monomer forms of JC-1 reflected a decrease in Δψm in shPOLG-treated astrocytes (Figure 6A). We performed the kinetic measurement of ATP production. As expected, the MAPR in the cells transfected with shPOLG decreased in a time-dependent manner, with levels amounting to 57.5 ± 3.7% of those in the SC-transfected cells at 9 days (p < 0.05, Figure 6B).

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