Possible involvement of mitochondrial uncoupling protein-2 in cytotoxicity mediated by acquired N-methyl-D-aspartate receptor channels
Possible involvement of mitochondrial uncoupling protein-2 in cytotoxicity mediated by acquired N-methyl-D-aspartate receptor channels
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DOI：
10.1016/j.neuint.2012.03.019
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发表时间：
2012-09-01
影响因子：
4.2
通讯作者：
Yoneda, Yukio
中科院分区：
文献类型：
--
作者：
Fukumori, Ryo;Takarada, Takeshi;Yoneda, Yukio
我们先前已经表明，在原代培养的大鼠海马神经元中，N-甲基-D-天冬氨酸（NMDA）受体（NMDAR）激活后观察到的神经毒性的机制可能涉及线粒体膜电位破坏。在这项研究中，我们试图证明线粒体解偶联蛋白-2（UCP 2）作为NMDA神经毒性的决定因素的关键作用，通过使用获得的NMDAR通道人工编排在HEK 293细胞。在UCP 2过表达的细胞中，免疫反应性UCP 2仅在用线粒体标记物MitoTracker染色的细胞内位置检测到。在获得NMDAR通道的细胞中，暴露于NMDA或钙离子载体A23187同样导致Fluo-3成像测定的细胞溶质Ca 2+水平显著增加，而与UCP 2的过表达无关。相比之下，NMDA，而不是A23187，是显着更有效地增加线粒体Ca 2+水平测定Rhod-2荧光成像在细胞转染NMDAR亚基和UCP 2表达载体比那些没有UCP 2过表达。UCP 2的过表达显著增加了具有获得性NMDAR通道的培养物中碘化丙啶染色的细胞数量，但未能显著影响暴露于A23187的细胞中的碘化丙啶染色的细胞数量。免疫细胞化学和免疫沉淀分析同样揭示了获得性NMDAR通道和UCP 2过表达的HEK 293细胞中GluN 1亚基和UCP 2之间可能的相互作用。这些结果表明，UCP 2可以发挥的作用，作为一个决定因素的神经毒性介导的NMDAR通过一个机制与未鉴定的相互作用的重要GluN 1亚基对线粒体Ca 2+水平的调节神经元。(C)2012爱思唯尔有限公司保留所有权利。
We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-D-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca2+ levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca2+ levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca2+ levels in neurons. (C) 2012 Elsevier Ltd. All rights reserved.