Departamento de Bioquímica y Biología Molecular y Genética, E.U. Enfermería y Terapia Ocupacional, Universidad de Extremadura, 10071 Cáceres, Spain.
Free Radic Biol Med. 2010 May 15;48(10):1370-81. doi: 10.1016/j.freeradbiomed.2010.02.024. Epub 2010 Mar 2.
Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases.
尽管氧化应激是帕金森病发病机制的基础，但在氧化剂暴露导致细胞死亡后，参与信号转导的信号分子仍未明确，这使得难以确定具有治疗相关性的分子靶点。我们在人类多巴胺能神经母细胞瘤 SH-SY5Y 细胞中研究了这个问题，这些细胞暴露于帕金森病毒素百草枯（PQ）中。这种毒素引发了活性氧物种和细胞死亡的剂量依赖性增加，这与 ASK1 的激活以及应激激酶 p38 和 JNK 的激活相关。使用 ASK1 的干扰 RNA 和两种用于 JNK 和 p38 的成熟药物抑制剂，证明了这些激酶在引导 PQ 神经毒性方面的相关性。维生素 E 的预孵育大大减弱了 PQ 的毒性作用，阻断了 ASK1 途径，并防止了氧化应激和细胞死亡。在寻找可能抵消 PQ 诱导的 ASK1 激活的生理途径时，我们分析了转录因子 Nrf2 的作用，Nrf2 是氧化还原平衡的主要调节剂，及其靶标硫氧还蛋白（Trx），Trx 结合并抑制 ASK1。Nrf2 缺陷型小鼠胚胎成纤维细胞（MEFs）中无法检测到 Trx 水平，而 Keap1 缺陷型 MEFs 以及用萝卜硫素（SFN）处理的 SH-SY5Y 细胞中 Trx 水平持续升高。与这些数据一致，Nrf2 缺陷型 MEFs 对 PQ 诱导的细胞死亡更为敏感，而 Keap1 缺陷型 MEFs 和用 SFN 孵育的 SH-SY5Y 细胞对 PQ 诱导的细胞死亡更为抵抗。这项研究确定了 ASK1/JNK 和 ASK1/p38 作为两种在氧化应激条件下参与细胞死亡激活的关键途径，并确定了 Nrf2/Trx 轴作为阻断这些途径和保护免受氧化剂暴露（如帕金森病和其他神经退行性疾病中发现的）的新靶点。
