Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model
Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model
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DOI：
10.1371/journal.pone.0052637
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发表时间：
2012-12-19
期刊：
影响因子：
3.7
通讯作者：
Neutzner, Albert
中科院分区：
文献类型：
--
作者：
Fang, Lei;Hemion, Charles;Neutzner, Albert
目的：使用暴露于青光眼相关应激条件的神经元前体细胞体外模型，研究线粒体泛素连接酶 MARCH5 对线粒体形态和诱导细胞凋亡的影响。方法：用 MARCH5、MARCH5(H43W)、Dpr1(K38A) 或载体对照表达构建体转染的 RGC5 细胞暴露于 30 mmHg 的高压、线粒体电子传递引起的氧化应激链（ETC）抑制，或缺氧-复氧条件。线粒体标记细胞色素 c 染色和光激活 GFP (PAGFP) 扩散测定后，分析了 RGC5 细胞的线粒体形态。通过分析细胞色素 c 从线粒体释放到胞浆中并进行流式细胞术，可以确定这些细胞中细胞凋亡的诱导情况。结果：将 RGC5 细胞暴露于氧化应激条件以及升高的压力下，导致对照细胞以及表达 MARCH5 的细胞中线粒体网络的断裂。在表达失活MARCH5(H43W)或失活Drp(K38A)的细胞中，线粒体断裂被显着阻断，并且线粒体形态与正常条件下的对照细胞相当。通过细胞色素 c 释放和 DNA 染色评估，RGC5 细胞暴露于高压或氧化应激条件下会诱导细胞凋亡，而显性失活 MARCH5(H43W) 或 Drp1(K38A) 的表达确实显着延迟细胞死亡。 结论：通过干扰线粒体裂变机制防止线粒体断裂，可保护神经元细胞在暴露于与青光眼发病机制生理相关的应激源后免于程序性细胞死亡。
Purpose: To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.Methods: RGC5 cells transfected with expression constructs for MARCH5, MARCH5(H43W), Dpr1(K38A) or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.Results: Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5(H43W) or inactive Drp(K38A), mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5(H43W) or Drp1(K38A) did significantly delay cell death.Conclusion: Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.