Changes in Mitochondrial Morphology and Bioenergetics in Human Lymphoblastoid Cells With Four Novel OPA1 Mutations
Changes in Mitochondrial Morphology and Bioenergetics in Human Lymphoblastoid Cells With Four Novel OPA1 Mutations
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DOI：
10.1167/iovs.14-16288
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发表时间：
2015-04-01
影响因子：
4.4
通讯作者：
Lin, An-Lo
中科院分区：
文献类型：
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作者：
Kao, Shu-Huei;Yen, May-Yung;Lin, An-Lo
目的.在常染色体显性视神经萎缩（ADOA）患者中已报告了视神经萎缩1基因（OPA 1）突变。OPA 1在线粒体动力学和细胞凋亡中起重要作用。OPA 1突变与生物能量学变化之间的联系尚未完全解决。本研究的目的是探讨OPA 1基因突变对线粒体肾小管网络和生物能量学的影响。我们建立了来自四个携带不同OPA 1突变的ADOA家族、未受影响的亲属（内部对照细胞系）和无关的正常对照（正常对照细胞系）的淋巴母细胞样细胞系。通过逆转录-PCR和实时定量PCR分析OPA 1剪接变体和mRNA。通过Western印迹法检测蛋白质亚型。通过共聚焦显微镜观察线粒体网络。使用Seahorse XF 24通量分析仪评估线粒体生物能量学。线粒体膜电位和氧化损伤进行了分析，通过流式细胞仪。OPA 1突变细胞系显示OPA 1 mRNA和蛋白表达、线粒体膜电位和ATP合成显著降低。在OPA 1中间结构域和GT3效应结构域突变的细胞中观察到OPA 1长同种型的显著缺陷。共聚焦显微镜显示OPA 1突变细胞的线粒体碎片增加。OPA 1突变细胞也显示出减少的耗氧量，并经历糖酵解以产生ATP。此外，OPA 1基因突变导致了氧化损伤的积累。我们的实验表明，OPA 1突变诱导线粒体断裂，解偶联线粒体呼吸，并引起功能失调的生物能量。然而，各种OPA 1突变之间没有显著差异。
PURPOSE. Mutations in the optic atrophy 1 gene (OPA1) have been reported in patients with autosomal dominant optic atrophy (ADOA). OPA1 plays important roles in mitochondrial dynamics and cell apoptosis. The link between OPA1 mutations and changes in bioenergetics is still not fully resolved. The aim of this study was to investigate the effects of OPA1 mutations on the mitochondrial tubular network and bioenergetics.METHODS. We established lymphoblastoid cell lines from four ADOA families harboring different OPA1 mutations, unaffected relatives (internal control cell lines), and unrelated normal controls (normal control cell lines). OPA1 splice variants and mRNA were analyzed by reverse transcription-PCR and quantitative real-time PCR. Protein isoforms were examined by Western blotting. The mitochondrial network was visualized by confocal microscopy. Mitochondrial bioenergetics were assessed using a Seahorse XF24 flux analyzer. Mitochondrial membrane potential and oxidative damage were analyzed by flow cytometry.RESULTS. OPA1 mutant cell lines showed significant decreases in OPA1 mRNA and protein expression, mitochondrial membrane potential, and ATP synthesis. A marked deficiency of the long isoform of OPA1 was observed in cells with OPA1 mutations in the middle domain and GTPase effector domain. Confocal microscopy revealed increased mitochondrial fragmentation in OPA1 mutant cells. OPA1 mutant cells also displayed reduced oxygen consumption and underwent glycolysis to produce ATP. Moreover, OPA1 mutations caused the accumulation of oxidative damage.CONCLUSIONS. Our experiments demonstrated that OPA1 mutations induced mitochondrial fragmentation, uncoupled mitochondrial respiration, and elicited dysfunctional bioenergetics. However, there were no significant differences among the various OPA1 mutations.