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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

Real-time PCR analysis of the mRNA levels of the mtDNA repair/replication enzymes OGG1 (A), MYH (B) and polymerase gamma (POLG; C) in the cultured RGCs under an increased HP. The data are expressed as normalized ratios (Ctrl = 1). Each sample was run in triplicate. (n = 5–7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. ctrl, control with normal pressure.
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Figure 2: Real-time PCR analysis of the mRNA levels of the mtDNA repair/replication enzymes OGG1 (A), MYH (B) and polymerase gamma (POLG; C) in the cultured RGCs under an increased HP. The data are expressed as normalized ratios (Ctrl = 1). Each sample was run in triplicate. (n = 5–7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. ctrl, control with normal pressure.

Mentions: OGG1, MYH and POLG are important mtDNA repair/replication enzymes. At 12 h of HP exposure, we found that the expression of OGG1, MYH and POLG mRNA in the RGCs were 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), respectively. Subsequently, OGG1 and MYH mRNA levels declined to the levels observed in RGCs incubated under normal pressure (Figures 2A,B), whereas POLG decreased by approximately 1.9 ± 0.4-fold (Figure 2C), compared with control RGCs. In contrast, the mitochondrial accumulation of OGG1, MYH and POLG proteins was significantly decreased at 72 h of HP exposure and remained lower after 120 h of high-pressure insult (Figure 3).


High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis
Real-time PCR analysis of the mRNA levels of the mtDNA repair/replication enzymes OGG1 (A), MYH (B) and polymerase gamma (POLG; C) in the cultured RGCs under an increased HP. The data are expressed as normalized ratios (Ctrl = 1). Each sample was run in triplicate. (n = 5–7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. ctrl, control with normal pressure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121242&req=5

Figure 2: Real-time PCR analysis of the mRNA levels of the mtDNA repair/replication enzymes OGG1 (A), MYH (B) and polymerase gamma (POLG; C) in the cultured RGCs under an increased HP. The data are expressed as normalized ratios (Ctrl = 1). Each sample was run in triplicate. (n = 5–7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. ctrl, control with normal pressure.
Mentions: OGG1, MYH and POLG are important mtDNA repair/replication enzymes. At 12 h of HP exposure, we found that the expression of OGG1, MYH and POLG mRNA in the RGCs were 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), respectively. Subsequently, OGG1 and MYH mRNA levels declined to the levels observed in RGCs incubated under normal pressure (Figures 2A,B), whereas POLG decreased by approximately 1.9 ± 0.4-fold (Figure 2C), compared with control RGCs. In contrast, the mitochondrial accumulation of OGG1, MYH and POLG proteins was significantly decreased at 72 h of HP exposure and remained lower after 120 h of high-pressure insult (Figure 3).

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