High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis.
High Pressure-Induced mtDNA Alterations in Retinal Ganglion Cells and Subsequent Apoptosis.
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高压诱导视网膜神经节细胞 mtDNA 改变和随后的细胞凋亡
DOI：
10.3389/fncel.2016.00254
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发表时间：
2016
影响因子：
5.3
通讯作者：
Wu JH
中科院分区：
文献类型：
--
作者：
Zhang SH;Gao FJ;Sun ZM;Xu P;Chen JY;Sun XH;Wu JH
目的：我们先前的研究表明，线粒体DNA（mtDNA）的损伤和突变是至关重要的进行性损失的视网膜神经节细胞（RGC）在一个昏迷的大鼠模型。在这项研究中，我们研究了高压是否会直接导致线粒体DNA的改变，以及后者是否会导致线粒体功能障碍和RGC死亡。方法：将原代培养的大鼠视网膜神经节细胞置于30 mm Hg的静水压力（HP）中，分别作用12、24、48、72、96和120 h。线粒体DNA的改变和线粒体DNA修复/复制酶OGG 1，MYH和聚合酶γ（POLG）的表达也进行了分析。然后用靶向POLG的慢病毒小发夹RNA（shRNA）表达载体（POLG-shRNA）感染RGCs，随后在适当的时间研究mtDNA的改变以及线粒体功能，包括复合物I/III的活性和ATP的产生。最后，RGC凋亡和神经细胞凋亡途径相关蛋白裂解caspase-3分别使用末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）法和蛋白质印迹法检测。结果：HP作用于RGCs后48 h即出现线粒体DNA损伤。在HP后120 h，mtDNA损伤和突变显著增加，与对照值相比分别达到>40%和4.8 ± 0.3倍。HP后12 h，RGCs中OGG 1、MYH和POLG mRNA的表达分别增加5.02 ± 0.6倍（p < 0.01）、4.3 ± 0.2倍（p < 0.05）和0.8 ± 0.09倍（p < 0.05）。Western blot分析显示，HP后72和120 h，三种酶的蛋白水平均下降（p < 0.05）。POLG-shRNA干扰后，线粒体DNA损伤和突变明显增加（p < 0.01），复合体I/III活性逐渐降低（p < 0.05）。POLG-shRNA转染后第5天和第6天，细胞膜电位和ATP产生分别出现相应的降低（p < 0.05）。线粒体DNA损伤和突变后，RGCs凋亡和切割的caspase-3蛋白表达增加。结论：高压可直接引起线粒体DNA的改变，导致线粒体功能障碍和RGC死亡。
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.
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影响因子：
5.6
作者：
Ciccone S;Maiani E;Bellusci G;Diederich M;Gonfloni S
通讯作者：
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作者：
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