Background

Over-exposure to manganese (Mn) causes irreversible movement disorders with signs and symptoms similar, but not identical, to idiopathic Parkinson's disease (IPD). Recent data suggest that Mn toxicity occurs in dopaminergic (DA) neurons, although the mechanism remains elusive. This study was designed to investigate whether Mn interfered the apoptotic signaling transduction cascade in DA neurons.

Methods

Mouse midbrain dopaminergic MN9D cells were exposed to Mn in a concentration range of 0, 400, 800, or 1200 μM as designated as control, low, medium, and high exposure groups, respectively. The flow cytometry with Annexin V/PI double staining and immunohistochemistry were used to assess the apoptosis.

Results

Data indicated that Mn exposure caused morphological alterations typical of apoptosis, increased apoptotic cells by 2–8 fold, and produced reactive oxidative species (ROS) by 1.5–2.2 fold as compared to controls (p < 0.05). Studies by qPCR and Western blot revealed that Mn exposure significantly increased the protein expression of extracellular signal-regulated kinase-5 (ERK5) and mitogen-activated ERK kinase-5 (MEK5) (p < 0.05). The presence of BIX02189, a specific inhibitor of MER/ERK, caused a much greater cytotoxicity, i.e., higher cell death, more ROS production, and worsened apoptosis, than did the treatment with Mn alone. Following Mn exposure, the expression of a downstream effector Bcl- 2 was reduced by 48 % while those of Bax and Caspase-3 were increased by 266.7 % and 90.1 %, respectively, as compared to controls (p < 0.05).

Conclusion

Taken together, these data provide the initial evidence that the signaling transduction cascade mediated by MEK5/ERK5 is responsible to Mn-induced cytotoxicity; Mn exposure, by suppressing anti-apoptotic function while facilitating pro-apoptotic activities, alters neuronal cell’s survival and functionally inhibits DA production by MN9D cells.

中文翻译：

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MEK5/ERK5 信号通路参与锰诱导的多巴胺能 MN9D 细胞损伤。

背景

过度接触锰 (Mn) 会导致不可逆的运动障碍，其体征和症状与特发性帕金森病 (IPD) 相似但不相同。最近的数据表明，锰毒性发生在多巴胺能（DA）神经元中，尽管其机制仍然难以捉摸。本研究旨在探讨 Mn 是否干扰 DA 神经元中的凋亡信号转导级联。

方法

将小鼠中脑多巴胺能 MN9D 细胞暴露于浓度范围为 0、400、800 或 1200 μM 的 Mn，分别指定为对照组、低暴露组、中暴露组和高暴露组。采用Annexin V/PI双染流式细胞仪和免疫组化方法评估细胞凋亡。

结果

数据表明，与对照组相比，锰暴露引起典型的细胞凋亡形态改变，凋亡细胞增加 2-8 倍，产生的活性氧化物质 (ROS) 增加 1.5-2.2 倍（p < 0.05）。qPCR 和蛋白质印迹研究表明，锰暴露显着增加了细胞外信号调节激酶 5 (ERK5) 和丝裂原激活的 ERK 激酶 5 (MEK5) 的蛋白表达 (p < 0.05)。BIX02189（一种 MER/ERK 的特异性抑制剂）的存在，与单独使用 Mn 的治疗相比，会导致更大的细胞毒性，即更高的细胞死亡、更多的 ROS 产生和更严重的细胞凋亡。与对照组相比，接触 Mn 后，下游效应子 Bcl-2 的表达减少了 48%，而 Bax 和 Caspase-3 的表达分别增加了 266.7% 和 90.1%（p < 0.05）。

结论

总而言之，这些数据提供了初步证据，表明 MEK5/ERK5 介导的信号转导级联是 Mn 诱导的细胞毒性的原因。Mn 暴露通过抑制抗凋亡功能同时促进促凋亡活性，改变神经元细胞的存活并在功能上抑制 MN9D 细胞产生 DA。