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

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.

Overexpression of parkin protects retinal ganglion cells in experimental glaucoma

“…Intraocular pressure (IOP) elevation is considered to be the major risk factor for glaucoma and so far the only treatable factor 2 . However, lowering IOP is not always sufficient to prevent progression of glaucomatous optic neuropathy 3 . Recent studies have shown that impairment of mitochondrial dynamics may play a key role in glaucomatous RGC loss 4 , 5 .…”

A lab-on-a-chip model of glaucoma

“…These changes include increased apoptosis, changes in cell morphology and gene expression. Neuronal cells including PC12 (Agar et al, 2000; Tök et al, 2014) and RGC-5 cell lines (Agar et al, 2006; Ju et al, 2009a; Ju et al, 2007; Lee et al, 2010; Liu et al, 2012), primary cultures of RGC (Sappington et al, 2006; Zhang et al, 2016), optic nerve head astrocytes (Liu and Neufeld, 2001; Liu and Neufeld, 2003; Mandal et al, 2010; Miao et al, 2010; Ricard et al, 2000; Salvador‐Silva et al, 2004; Salvador‐Silva et al, 2001; Tezel et al, 2001; Yang et al, 2004), Müller cells (Yu et al, 2012a; Yu et al, 2011; Yu et al, 2012b), or microglial cells (Madeira et al, 2015; Sappington and Calkins, 2006; Sappington and Calkins, 2008), and more complex preparations such as organotypic co-cultures of the retinal cells (Obazawa et al, 2004; Osborne et al, 2015; Tezel and Wax, 2000) or eye cup preparations (Ishikawa et al, 2010; Ishikawa et al, 2011; Reigada et al, 2008; Zhang et al, 2007) have been exposed to elevated pressure.…”

Quercetin Declines Apoptosis, Ameliorates Mitochondrial Function and Improves Retinal Ganglion Cell Survival and Function in In Vivo Model of Glaucoma in Rat and Retinal Ganglion Cell Culture In Vitro

“…The mitochondrial membrane potential (∆ψm) was evaluated using MitoProbe JC-1 dye (Invitrogen, Carlsbad, CA, USA) as previously described (Zhang et al, 2016 ). In brief, RGCs with different treatments were incubated with JC-1 at 37°C for 30 min while protected from light and assessed via FACS (Becton Dickinson, San Jose, CA, USA) and a confocal fluorescence microscope (Leica; green: 488 nm excitation/530 nm emission; red: 550 nm excitation/590 nm emission).…”