MRC Toxicology Unit, University of Leicester, Leicester, LE1 9HN, UK.
J Biochem Mol Toxicol. 2010 Sep-Oct;24(5):335-41. doi: 10.1002/jbt.20343.
Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP-1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l-Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP-1 activity using overexpression of the dominant negative DRP-1(K38A) slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild-type DRP-1. In contrast, responses to l-Glu appeared DRP-1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca(2+) uptake or the ability of l-Glu to stimulate excitotoxic Ca(2+) overload. Finally, apoptosis-induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death.
线粒体碎片化被认为是细胞凋亡起始过程中的一个重要事件。在本研究中，我们使用单细胞成像技术，研究了线粒体裂变蛋白 DRP-1 在经历坏死或凋亡性细胞死亡的原代海马神经元中线粒体形态和线粒体碎片化中的作用。用 500 nM 星形孢菌素（凋亡）或 30 μM 谷氨酸盐（兴奋性坏死）处理神经元会导致线粒体的碎片化和浓缩，尽管发生的时间框架明显不同，但外观上似乎大致相似。在暴露于凋亡刺激的神经元中，通过过表达显性负性 DRP-1（K38A）抑制 DRP-1 活性，与过表达野生型 DRP-1 相比，减缓了线粒体碎片化的速度并降低了总细胞死亡。相比之下，对 l-Glu 的反应似乎与 DRP-1 无关。同样，线粒体网络的裂变/融合状态的改变不会改变线粒体 Ca2+摄取或 l-Glu 刺激兴奋性 Ca2+超载的能力。最后，观察到凋亡诱导的线粒体碎片化与 Bax 向线粒体膜的募集同时发生。相比之下，在谷氨酸兴奋性毒性期间，Bax 仍留在细胞质隔室中。我们得出结论，不同的途径导致坏死和凋亡性神经元细胞死亡期间出现碎片化的线粒体。
