Organophosphate pesticide chlorpyrifos impairs STAT1 signaling to induce dopaminergic neurotoxicity: Implications for mitochondria mediated oxidative stress signaling events.
Organophosphate pesticide chlorpyrifos impairs STAT1 signaling to induce dopaminergic neurotoxicity: Implications for mitochondria mediated oxidative stress signaling events.
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DOI：
10.1016/j.nbd.2018.05.019
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发表时间：
2018-09
影响因子：
6.1
通讯作者：
Kanthasamy A
中科院分区：
文献类型：
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作者：
Singh N;Lawana V;Luo J;Phong P;Abdalla A;Palanisamy B;Rokad D;Sarkar S;Jin H;Anantharam V;Kanthasamy AG;Kanthasamy A
农业环境中使用的有机磷 (OP) 农药毒死蜱 (CPF) 会导致发育和神经损伤。最近使用体外细胞培养模型的研究表明，CPF 暴露与线粒体介导的氧化应激反应和多巴胺能细胞死亡呈正相关。然而，线粒体活性氧（ROS）导致多巴胺能细胞死亡的机制仍不清楚。因此，我们假设转录因子 STAT1 通过线粒体介导的氧化应激机制导致凋亡性多巴胺能细胞死亡。在这里，我们发现，通过 MTS 测定和 DNA 片段化测量，将多巴胺能神经元细胞（例如 N27 细胞（永生化小鼠中脑多巴胺能细胞））暴露于 CPF 会导致凋亡细胞死亡呈剂量依赖性增加。在 CPF 处理的人多巴胺能神经元细胞（LUHMES 细胞）中也观察到类似的效果，线粒体功能障碍也随之增加。此外，CPF(10μM)诱导的STAT1活化的时间依赖性增加与线粒体跨膜电位的崩溃、ROS生成的增加、蛋白激酶Cδ(PKCδ)的蛋白水解裂解、线粒体基础耗氧率(OCR)的抑制相一致，伴随着ATP连接的OCR和储备能力的减少、Bax/Bcl-2比率的增加和自噬的增强。此外，通过染色质免疫沉淀 (ChIP)，我们证明 STAT1 与 NOX1 和 Bax 启动子区域中的推定调节序列结合，响应 N27 细胞中的 CPF。有趣的是，非磷酸化 STAT1 突变体（STAT1Y701F 和 STAT1S727A）的过表达（而非 STAT1 WT 构建体）减弱了 PKCδ 的裂解，并最终减弱了 CPF 处理细胞中的细胞死亡。此外，小干扰RNA敲低证明STAT1是N27细胞中CPF处理后自噬和线粒体介导的促凋亡细胞信号转导事件的关键调节因子。最后，出生后大鼠 (PND 27-61) 口服 CPF (5mg/kg) 会引起运动缺陷和黑质纹状体多巴胺能神经变性，同时诱导 STAT1 依赖性促凋亡细胞信号转导事件。相反，mitoapocynin（一种线粒体靶向抗氧化剂）和 CPF 联合治疗可以挽救运动缺陷，并通过消除 STAT1 依赖性促凋亡细胞信号转导事件来恢复多巴胺能神经元的存活。综上所述，我们的研究确定了 STAT1 调节线粒体介导的氧化应激反应、PKCδ 激活和自噬的新机制。在这种情况下，STAT1 中酪氨酸 701 和丝氨酸 727 的磷酸化被发现对于 PKCδ 裂解至关重要。通过减弱线粒体衍生的 ROS，mitoapocynin 可能具有逆转 CPF 诱导的多巴胺能神经毒性和相关神经行为缺陷以及神经退行性疾病的治疗应用。
The organophosphate (OP) pesticide chlorpyrifos (CPF), used in agricultural settings, induces developmental and neurological impairments. Recent studies using in vitro cell culture models have reported CPF exposure to have a positive association with mitochondria-mediated oxidative stress response and dopaminergic cell death; however, the mechanism by which mitochondrial reactive oxygen species (ROS) contribute to dopaminergic cell death remains unclear. Therefore, we hypothesized that STAT1, a transcription factor, causes apoptotic dopaminergic cell death via mitochondria-mediated oxidative stress mechanisms. Here we show that exposure of dopaminergic neuronal cells such as N27 cells (immortalized murine mesencephalic dopaminergic cells) to CPF resulted in a dose-dependent increase in apoptotic cell death as measured by MTS assay and DNA fragmentation. Similar effects were observed in CPF-treated human dopaminergic neuronal cells (LUHMES cells), with an associated increase in mitochondrial dysfunction. Moreover, CPF (ΙΟμΜ) induced time-dependent increase in STAT1 activation coincided with the collapse of mitochondrial transmembrane potential, increase in ROS generation, proteolytic cleavage of protein kinase C delta (PKCδ), inhibition of the mitochondrial basal oxygen consumption rate (OCR), with a concomitant reduction in ATP-linked OCR and reserve capacity, increase in Bax/Bcl-2 ratio and enhancement of autophagy. Additionally, by chromatin immunoprecipitation (ChIP), we demonstrated that STAT1 bound to a putative regulatory sequence in the NOX1 and Bax promoter regions in response to CPF in N27 cells. Interestingly, overexpression of non-phosphorylatable STAT1 mutants (STAT1Y701F and STAT1S727A) but not STAT1 WT construct attenuated the cleavage of PKCδ and ultimately cell death in CPF-treated cells. Furthermore, small interfering RNA knockdown demonstrated STAT1 to be a critical regulator of autophagy and mitochondria- mediated proapoptotic cell signaling events after CPF treatment in N27 cells. Finally, oral administration of CPF (5mg/kg) in postnatal rats (PNDs 27–61) induced motor deficits, and nigrostriatal dopaminergic neurodegeneration with a concomitant induction of STAT1-dependent proapoptotic cell signaling events. Conversely, co-treatment with mitoapocynin (a mitochondrially-targeted antioxidant) and CPF rescued motor deficits, and restored dopaminergic neuronal survival via abrogation of STAT1-dependent proapoptotic cell signaling events. Taken together, our study identifies a novel mechanism by which STAT1 regulates mitochondria-mediated oxidative stress response, PKCδ activation and autophagy. In this context, the phosphorylation of Tyrosine 701 and Serine 727 in STAT1 was found to be essential for PKCδ cleavage. By attenuating mitochondrial-derived ROS, mitoapocynin may have therapeutic applications for reversing CPF-induced dopaminergic neurotoxicity and associated neurobehavioral deficits as well as neurodegenerative diseases.
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影响因子：
9.5
作者：
Boengler K;Hilfiker-Kleiner D;Heusch G;Schulz R
通讯作者：
Schulz R
DOI：
10.4161/jkst.25666
发表时间：
2013-10-01
期刊：
JAK-STAT
影响因子：
--
作者：
Bourke LT;Knight RA;Latchman DS;Stephanou A;McCormick J
通讯作者：
McCormick J
影响因子：
2.9
作者：
Crumpton, TL;Seidler, FJ;Slotkin, TA
通讯作者：
Slotkin, TA
影响因子：
6.6
作者：
Choi, Dong-Hee;Cristovao, Ana Clara;Kim, Yoon-Seong
通讯作者：
Kim, Yoon-Seong
中枢神经系统血清素能和多巴胺能突触活性在产前或新生儿毒性菌暴露后成年。
影响因子：
10.4
作者：
通讯作者：
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