All animals (obtained from SLAC Laboratory Animal Co., Ltd. Shanghai, China) were handled and housed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures followed the Declaration of Helsinki, accorded with the guidelines of Fudan University on the ethical use of animals, and all endeavors were done to minimize animal suffering. A total of 420 rats were used.

The isolation protocol for purified RGCs followed our previously described two-step immunopanning-magnetic separation method (Wu et al., 2015; Gao et al., 2016). Briefly, 2-day-old Sprague-Dawley rats were sacrificed to obtain the retinas. The retinas were removed and dissociated in 4.5 units/mL of papain solution (#3126, Worthington, Lakewood, NJ, USA). The cell suspensions were then transferred to a petri dish coated with anti-macrophage antibody (#CLAD51240; Cedarlane Laboratories, Hornby, ON, Canada) and CD90.1 MicroBeads (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) successively, and RGCs were then collected by an MS column (Miltenyi Biotec GmbH) in the magnetic fields of a MiniMACS (Miltenyi Biotec GmbH). The isolated RGCs were then seeded onto 6-well plates treated with poly-D-lysine (#P6407; Sigma-Aldrich, St. Louis, MO, USA) and mouse-laminin (#3400-010-01; Trevigen Inc., Gaithersburg, MD, USA). The cultures were maintained at 37°C in a humidified environment containing 10% CO₂. To mimic chronically elevated IOP, the cells were exposed to 30 mm Hg of HP using a closed, pressurized chamber equipped with a manometer for 12, 24, 48, 72 and 120 h, as we previously reported (Wu et al., 2013). Subsequently, the cells were collected for further analysis.

To induce mtDNA mutations and damage in vitro, the cultured RGCs were transfected with lentivirus-small harpin polymerase gamma (shPOLG; Santa Cruz Biotechnology, CA, USA). Lentivirus-scrambled RNA was used as a control (Lentivirus-SC RNA, Santa Cruz Biotechnology). As previously reported (Tewari et al., 2012), the cells were incubated with the lentivirus-shPOLG or SC RNA for 4 days, washed with phosphate-buffered saline (PBS, Gibco, Grand Island, NY, USA), and incubated for 6 additional days. The transfection efficiency was evaluated by quantifying POLG gene expression. mtDNA was examined using Long-extension (LX)-PCR and mtDNA mutation assays. Mitochondrial function was assessed by examining complex activity, ATP production and mitochondrial membrane potential (Δψm).

The cultured RGCs were harvested and mitochondria were isolated using Mitochondria Isolation Kit (Pierce Biotechnology, Rockford, IL, USA) as previously reported (Wu et al., 2015). The isolated mitochondrial pellet was resuspended in 100 μL of mannitol (210 mM), sucrose (70 mM), HEPES (5 mM), EGTA (1 mM) and fatty-acid-free bovine serum albumin (BSA, 0.5% (w/v) Sigma-Aldrich, St. Louis, MO, USA) solution.

RGC protein extractions and Western blot analysis were performed as previously reported (Wu et al., 2015). Briefly, cultured RGCs were lysed and total proteins were extracted on ice by use of cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and then the proteins were separated by SDS-polyacrylamide gel electrophoresis and the separated proteins were electrotransferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk for 20 min, the membranes were incubated overnight at 4°C with primary antibodies against DNA POLG (Abcam, Cambridge, MA, USA) and cleaved caspase-3 antibody (Abcam, Cambridge, MA, USA). The loading controls included β-actin (Abcam, Cambridge, MA, USA) for the total proteins and Cox IV (Molecular Probes, Rockford, IL, USA) for mitochondria. Secondary antibodies included HRP-conjugated goat anti-rabbit antibody (Millipore, MA, USA) and HRP-conjugated goat anti-mouse antibody (Millipore, MA, USA). The resulting western blots were exposed to film (Hyperfilm ECL, Thermo Fisher Scientific, Rockford, IL, USA) and were analyzed using the Kodak Imaging System (Kodak 440CF). The intensity of the band was quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).

The total DNA and mtDNA were extracted from the RGCs as described in our previously reported (Wu et al., 2015). Shortly, the total DNA was isolated using the DNeasy blood and tissue kit (QIAGEN, Duess eldorf, Germany). mtDNA were obtained from the isolated mitochondria by use of mitochondrial lysis buffer containing proteinase K (0.2 mg/ml), SDS (0.5%), Tris-HCl (10 mM) and 0.05 M EDTA. The amount of DNA was determined using the Quant-iT dsDNA assay kit (Invitrogen, Carlsbad, CA, USA).

mtDNA mutation frequency was detected by random mutation capture assay as described in our published article (Wu et al., 2015). In summary, two primer pairs were designed for PCR reactions. One is flanking the Taq1 sites at mtDNA positions 1427 and 8335, the other primer pair does not flank a TaqI restriction sites as controls. PCR amplification was performed in 25 μL reactions, containing 2 μL of 10 μM forward and reverse primers, 0.2 μL of uracil DNA glycosylase (New England Biolabs, Beverly, MA, USA), 12.5 μL 2× Brilliant SYBR Green I Master Mix (Stratagene, La Jolla, CA, USA) and 3.3 μL H₂O. Then the PCR product was digested with TaqI restriction enzyme and the mtDNA mutation frequency was determined by analysis of the band size of gel electrophoresis. The mutation frequency was showed as the mutation number per million bases.

LX-PCR assay was performed to determine DNA damage using GeneAmp XL PCR kit (Applied Biosystems, CA, USA) as previously described (Wu et al., 2015). DNA damage was quantified by comparing the ratio between the long and short fragments of PCR amplicons (mtDNA = 210 bp/13.4 kb).

The total RNA was extracted by use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was generated using a SuperScript First-Strand Synthesis kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The mRNA expressions of Polg, Ogg1 and Myh were assessed by SYBR Green-based real-time PCR using an Applied Biosystems, 7500fast (Life Technologies, Grand Island, NY, USA). The β-actin was acted as a normalized control. Amplifications were performed in duplicate well in two independent experiments, and the transcripts were quantified by the ΔΔC (t) method.

Mitochondrial membrane potential (Δψm) was evaluated using MitoProbe JC-1 dye (Invitrogen), according to the manufacturer’s specifications. In brief, RGCs cultured in a six-well plate with different treatments were incubated with JC-1 for 30 min and were visualized using an inverted fluorescence microscope (Leica; green: 488 nm excitation/530 nm emission; red: 550 nm excitation/590 nm emission). Quantitative analysis was performed using ImageJ software, and Δψm was indicated by the ratio of the mean red fluorescence to the mean green fluorescence. According to this assay, mitochondrial depolarization was indicated by an increase in the green-to-red fluorescence intensity ratio.

The activities of mitochondrial complex I and III were detected using previously described methods (Birch-Machin and Turnbull, 2001; Rosca et al., 2005; Yao et al., 2011; Wu et al., 2015) with a Synergy H1 plate reader (BioTek Winooski, VT, USA) using the kinetics mode of Gen5 software (BioTek). Briefly, complex I activity was determined by following the decrease in absorbance to the NADH oxidation at 340 nm. Complex III activity was measured by monitoring the cytochrome c reduction at 550 nm. Enzymatic activities are expressed as nmol/mg protein/min.

Rate of ATP production was assayed using bioluminescence assay kit (ENLITEN ATP assay system; Promega, Madison, WI, USA), followed by the manufacturer’s protocol in our lab (Wu et al., 2015). The ATP content was measured using a microplate reader (BioTek Synergy HT) and was standardized to the total proteins of mitochondria. The results were calculated from three independent ATP standards run in duplicate.

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed according to the manufacturer’s instructions (in situ Cell Detection Kit; Roche, Mannheim, Germany). The methods for TUNEL were performed as previously described (Galvao et al., 2015). Cultured rat RGCs were fixed in 4% (w/v) paraformaldehyde (PFA) at 4°C and were blocked with 3% BSA and 0.1% Triton X-100 for 1 h at RT. The nuclei were counter-stained with DAPI. The preparations were mounted using Glycergel mounting medium (Dako, Denmark) and were visualized using a confocal microscope (Leica).

All data are expressed as the means ± SEMs and were analyzed using SigmaStat software. All data were tested by the Shapiro-Wilk test and only variables that were normally distributed; the data were then analyzed of variance, followed by two-tailed unpaired Student’s test. Values of p < 0.05 were considered the threshold for statistical significance.

To determine whether high pressure itself directly induced mtDNA damage and mutations previously observed in RGCs in glaucomatous rat models, we conducted an in vitro experiment to investigate the effects of HP on the mitochondrial genome using cultured RGCs. The exposure of RGCs to high pressure (HP) induced mtDNA damage (indicated by the ratio of the 210 bp and 13.4 kb mtDNA band) as early as 48 h (p < 0.05) after onset. The damage continued to increase and was significantly increased (>40%) at 120 h (Figure 1A). Next, we examined the frequency of mtDNA mutations in the RGCs by random mutation capture assay. An increase in mtDNA mutations (1427 and 8335) was observed when the duration of HP was extended beyond 48 h, reaching levels 4.8 ± 0.3-fold (p < 0.01), compared with the control values at 120 h (Figure 1B). Furthermore, we found no significant increase in ROS throughout the duration of the experiment (data not shown).

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).

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).

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).