AUTHOR=Zhang Sheng-Hai , Gao Feng-Juan , Sun Zhong-Mou , Xu Ping , Chen Jun-Yi , Sun Xing-Huai , Wu Ji-Hong 

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

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=10

YEAR=2016

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2016.00254

DOI=10.3389/fncel.2016.00254

ISSN=1662-5102

ABSTRACT=<p><bold>Purpose</bold>: 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.</p><p><bold>Methods</bold>: 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.</p><p><bold>Results</bold>: 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 ± 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 (<italic>p</italic> &lt; 0.01), 4.3 ± 0.2-fold (<italic>p</italic> &lt; 0.05), and 0.8 ± 0.09-fold (<italic>p</italic> &lt; 0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 h after HP (<italic>p</italic> &lt; 0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (<italic>p</italic> &lt; 0.01), while complex I/III activities gradually decreased (<italic>p</italic> &lt; 0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (<italic>p</italic> &lt; 0.05). Increases in the apoptosis of RGCs and cleaved caspase-3 protein expression were observed after mtDNA damage and mutations.</p><p><bold>Conclusions</bold>: High pressures could directly cause mtDNA alterations, leading to mitochondrial dysfunction and RGC death.</p>