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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

Western blot analysis showed that protein expressions of OGG1, MYH and POLG in the mitochondria of cultured RGCs decreased after exposure to increased HP. Cox IV was used as a control to ensure equal protein loading. Band density is expressed in normalized ratios (Ctrl = 1; n = 7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. HP, increased hydrostatic pressure; ctrl, control with normal pressure.
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Figure 3: Western blot analysis showed that protein expressions of OGG1, MYH and POLG in the mitochondria of cultured RGCs decreased after exposure to increased HP. Cox IV was used as a control to ensure equal protein loading. Band density is expressed in normalized ratios (Ctrl = 1; n = 7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. HP, increased hydrostatic pressure; 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
Western blot analysis showed that protein expressions of OGG1, MYH and POLG in the mitochondria of cultured RGCs decreased after exposure to increased HP. Cox IV was used as a control to ensure equal protein loading. Band density is expressed in normalized ratios (Ctrl = 1; n = 7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. HP, increased hydrostatic pressure; ctrl, control with normal pressure.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121242&req=5

Figure 3: Western blot analysis showed that protein expressions of OGG1, MYH and POLG in the mitochondria of cultured RGCs decreased after exposure to increased HP. Cox IV was used as a control to ensure equal protein loading. Band density is expressed in normalized ratios (Ctrl = 1; n = 7/time point/group). *P < 0.05, **P < 0.01. Values are the means ± SEMs. HP, increased hydrostatic pressure; 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