Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa 50011.
Toxicol Sci. 2019 Jun 1;169(2):333-352. doi: 10.1093/toxsci/kfz049.
Chronic exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). Previously, we showed that dieldrin induces dopaminergic neurotoxicity by activating a cascade of apoptotic signaling pathways in experimental models of PD. Here, we systematically investigated endosulfan's effect on the interplay between apoptosis and autophagy in dopaminergic neuronal cell models of PD. Exposing N27 dopaminergic neuronal cells to endosulfan rapidly induced autophagy, indicated by an increased number of autophagosomes and LC3-II accumulation. Prolonged endosulfan exposure (>9 h) triggered apoptotic signaling, including caspase-2 and -3 activation and protein kinase C delta (PKCδ) proteolytic activation, ultimately leading to cell death, thus demonstrating that autophagy precedes apoptosis during endosulfan neurotoxicity. Furthermore, inhibiting autophagy with wortmannin, a phosphoinositide 3-kinase inhibitor, potentiated endosulfan-induced apoptosis, suggesting that autophagy is an early protective response against endosulfan. Additionally, Beclin-1, a major regulator of autophagy, was cleaved during the initiation of apoptotic cell death, and the cleavage was predominantly mediated by caspase-2. Also, caspase-2 and caspase-3 inhibitors effectively blocked endosulfan-induced apoptotic cell death. CRISPR/Cas9-based stable knockdown of PKCδ significantly attenuated endosulfan-induced caspase-3 activation, indicating that the kinase serves as a regulatory switch for apoptosis. Additional studies in primary mesencephalic neuronal cultures confirmed endosulfan's effect on autophagy and neuronal degeneration. Collectively, our results demonstrate that a functional interplay between autophagy and apoptosis dictate pesticide-induced neurodegenerative processes in dopaminergic neuronal cells. Our study provides insight into cell death mechanisms in environmentally linked neurodegenerative diseases.
慢性暴露于杀虫剂与帕金森病（PD）的病因发病机制有关。此前，我们已经表明，狄氏剂通过在 PD 的实验模型中激活一系列凋亡信号通路诱导多巴胺能神经毒性。在这里，我们系统地研究了硫丹对 PD 多巴胺能神经元细胞模型中凋亡与自噬相互作用的影响。将 N27 多巴胺能神经元细胞暴露于硫丹中会迅速诱导自噬，表现为自噬体数量增加和 LC3-II 积累。长时间暴露于硫丹（>9 小时）会引发凋亡信号，包括半胱天冬酶-2 和 -3 的激活以及蛋白激酶 C 德尔塔（PKCδ）的蛋白水解激活，最终导致细胞死亡，这表明自噬在硫丹神经毒性中先于凋亡发生。此外，使用磷酯酰肌醇 3-激酶抑制剂wortmannin 抑制自噬会增强硫丹诱导的细胞凋亡，表明自噬是对硫丹的早期保护反应。此外，自噬的主要调节因子 Beclin-1 在凋亡细胞死亡开始时被切割，并且切割主要由半胱天冬酶-2 介导。此外，半胱天冬酶-2 和 -3 抑制剂可有效阻止硫丹诱导的凋亡细胞死亡。基于 CRISPR/Cas9 的 PKCδ 稳定敲低显著减弱了硫丹诱导的 caspase-3 激活，表明该激酶作为凋亡的调节开关。在原代中脑神经元培养物中的进一步研究证实了硫丹对自噬和神经元变性的影响。总之，我们的结果表明，自噬和凋亡之间的功能相互作用决定了多巴胺能神经元细胞中农药诱导的神经退行性过程。我们的研究为环境相关神经退行性疾病中的细胞死亡机制提供了新的见解。
