Sino-UK Joint Laboratory of Brain Function and Injury, Xinxiang Medical University, Xinxiang, China.
Faculty of Biological Sciences, School of Biomedical Sciences, University of Leeds, Leeds, UK.
J Cell Physiol. 2019 Apr;234(4):3647-3660. doi: 10.1002/jcp.27134. Epub 2018 Sep 19.
Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H O induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn ] , and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H O increased concentration-dependently the [Zn ] and caused lysosomal dysfunction and Zn loss and, furthermore, mitochondrial Zn accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3',5,5'-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn uptake in isolated mitochondria, which was prevented by TPEN. H O -induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H O -induced ROS generation, lysosomal dysfunction and Zn release, and mitochondrial Zn accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn release, and mitochondrial Zn accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.
瞬时受体电位 melastatin 2 (TRPM2) 通道被活性氧 (ROS) 激活，在迟发性神经元细胞死亡中起关键作用，通过改变细胞内 Zn 稳态导致缺血后脑损伤，但对其机制仍知之甚少。在这里，我们表明 H 2 O 2 诱导神经母细胞瘤 SH-SY5Y 细胞死亡具有显著的延迟，这依赖于 TRPM2 通道和增加的 [Zn 2+ ] ，因此我们使用该细胞模型来研究 ROS 诱导的 TRPM2 介导的迟发性细胞死亡的机制。H 2 O 2 浓度依赖性地增加 [Zn 2+ ] ，并导致溶酶体功能障碍和 Zn 丢失，此外，线粒体 Zn 积累、碎片化和 ROS 生成。这些作用被以下方法抑制：用 PJ34 和 3,3',5,5'-四叔丁基二苯醌抑制多聚（腺嘌呤二核苷酸核糖）聚合酶-1 依赖性 TRPM2 通道激活；用 2-氨基乙氧基二苯硼酸盐（2-APB）和 N-（对戊基肉桂酰基）邻氨基苯甲酸抑制 TRPM2 通道；用 N,N,N,N-四（2-吡啶基甲基）乙二胺（TPEN）螯合 Zn 2+ 。巴弗洛霉素诱导的溶酶体功能障碍也导致线粒体 Zn 积累、碎片化和 ROS 生成，这些作用被 PJ34 或 2-APB 抑制，表明这些线粒体事件依赖于 TRPM2，是溶酶体功能障碍的后果。在分离的线粒体中检测到线粒体 TRPM2 表达，并暴露于 ADPR 诱导的 Zn 摄取，该摄取被 TPEN 抑制。APOC 和二苯碘二酮，即烟酰胺腺嘌呤二核苷酸磷酸氢（NADPH）氧化酶（NOX）抑制剂、GKT137831（一种 NOX1/4 特异性抑制剂）或 Gö6983（一种蛋白激酶 C（PKC）抑制剂）抑制 H 2 O 2 诱导的迟发性细胞死亡。此外，抑制 PKC/NOX 可防止 H 2 O 2 诱导的 ROS 生成、溶酶体功能障碍和 Zn 释放以及线粒体 Zn 积累、碎片化和 ROS 生成。总之，这些结果支持 TRPM2 通道在连接 PKC/NOX 介导的 ROS 生成、溶酶体 Zn 释放和线粒体 Zn 积累以及 ROS 生成中起关键作用，形成 ROS 诱导的迟发性细胞死亡的恶性正反馈信号机制。
