Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250, USA.
Toxicol Appl Pharmacol. 2011 Nov 1;256(3):314-23. doi: 10.1016/j.taap.2011.07.021. Epub 2011 Aug 6.
The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ(D327A) and kinase dead PKCδ(K376R) or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ(D327A) protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.
神经毒素 6-羟多巴胺（6-OHDA）被用于研究帕金森病（PD）中多巴胺能神经元选择性退化的细胞和分子机制。氧化应激和半胱天冬酶的激活导致多巴胺能神经元的 6-OHDA 诱导的凋亡性细胞死亡。在本研究中，我们试图系统地描述细胞培养和 PD 动物模型中涉及 6-OHDA 诱导的多巴胺能退化的关键下游信号分子。用 6-OHDA（100μM）处理中脑多巴胺能神经元 N27 细胞 24h 后，线粒体活性显著降低，胞质细胞色素 c 增加，随后 caspase-9 和 caspase-3 相继激活。用自由基清除剂 MnTBAP（10μM）共同处理可显著减弱 6-OHDA 诱导的半胱天冬酶活性。有趣的是，用 caspase-3 特异性抑制剂 Z-DEVD-FMK（50μM）处理可完全抑制 6-OHDA 诱导的蛋白激酶 C 德尔塔（PKCδ）的蛋白水解切割和激活。此外，用 caspase-3 切割位点抗性突变体 PKCδ（D327A）和激酶失活 PKCδ（K376R）表达或 PKCδ 的 siRNA 介导的敲低可防止 6-OHDA 诱导的神经元细胞死亡，表明 caspase-3 依赖性 PKCδ 促进氧化应激诱导的多巴胺能退化。siRNA 抑制 PKCδ 表达也可有效保护 N27 细胞免受 6-OHDA 诱导的凋亡性细胞死亡。6-OHDA 注射的 C57 黑小鼠黑质中也观察到 PKCδ 的切割，但在对照动物中没有观察到。PKCδ （D327A）蛋白的病毒介导传递可防止小鼠黑质中 6-OHDA 诱导的 PKCδ 激活。总的来说，这些结果强烈表明 PKCδ 的蛋白水解激活是多巴胺能退化的关键下游事件，这些结果对于开发治疗 PD 的新治疗策略可能具有重要的转化价值。
