抑制素蛋白Prohibitin 1在锰致脉络丛上皮细胞周期阻滞效应中的分子调控机制及对多巴胺神经元的影响

The pathological injury of brain parenchyma caused by chronic manganism is irreversible, moreover, the early diagnosis, prevention and treatment of chronic manganism are still difficult to achieve. The most prominent site of synthesis of growth differentiation factor-15 (GDF-15, a trophic factor for nigral dopaminergic neurons) within the brain is the choroid plexus (CP), which locates in the third, fourth and lateral ventricles, produces cerebrospinal fluid (CSF), and secretes GDF-15 into the CSF, from where the molecule can penetrate through the ependymal layer into the parenchyma. Choroid plexus surely plays an essential protection role in the pathogenic mechanism of chronic manganism. We reported that manganese chloride could induce cell cycle arrest in choroidial epithelial cells in vitro, but whether this effect has anything to do with the up-regulation of protein prohibitin 1 (PHB1), which was identified and verified in our previous proteomics study of CP manganese (Mn)-associated differential expressed proteins both in vivo and in vitro, has not been described. The underlying mechanisms involved are poorly characterized. Thus we assume that, the arrest effect of Mn on cell cycle in choroidial epithelial cells may possibly depend on regulation of PHB1 both at transcriptional and post-translational level, which might be associated with signal transduction pathways including the p53 and Rb pathway in nuclear and Ras-Raf-1, PI3K pathway on cell mebrane. Therefore, in this study we are going to explore the molecular regulatory mechanism of PHB1 in manganese induced cell cycle arrest in choroidial epithelial cells in vitro. Furthmore, the CP-Specific PHB1 conditional knockout mice will be applied to elucidate the relation between the arrest effect of cell cycle in epithelial cell induced by Mn, and the injury restoration of dopaminergic neurons in vivo. Finally, in a human study, the concentration of PHB1 and GDF-15 in CSF of welder workers will be detected, which aims to probe the potential biomarker and indicator for early diagnosis and monitoring progression of chronic manganism. Above all, from a new viewpoint of choroid plexus, this project will undoubtedly pave the way for developing the theory of manganese-related neurotoxicity mechanism and will provide a novel insight into the early prevention and control of manganism.

慢性锰中毒的脑实质损伤不可逆，早期诊治尚难实现。脑室中的脉络丛分泌脑脊液，并合成多巴胺神经元特异性营养因子GDF-15，发挥关键作用。我们已报导锰引起脉络丛上皮细胞周期阻滞，但不清楚是否与我们筛检发现的抑制素蛋白（PHB1）上调有关。我们设想，PHB1在转录和翻译后水平调控锰致脉络丛上皮细胞周期阻滞，可能涉及核内p53、Rb和膜上Ras-Raf-1、PI3K信号通路，不利于GDF-15的合成分泌，影响多巴胺神经元的损伤修复。本项目拟在细胞水平应用基因点突变等技术，揭示PHB1在锰致脉络丛上皮细胞周期阻滞中的调控机制；在整体水平应用条件性基因敲除小鼠探明PHB1调控的锰致细胞周期阻滞效应与多巴胺神经元损伤修复的关系；在锰暴露人群水平，测定脑脊液PHB1和GDF-15的变化，探索锰中毒早期潜在标志物及干预靶点。本研究将从脉络丛这个新视点，为锰毒性机制理论贡献新认知，为锰中毒早期防治提供新思路。

锰（Manganese, Mn）是人体内的必需微量元素，但过量锰暴露能引起机体的毒性效应。锰可由日常膳食摄入，故锰缺乏的情况很少见；而由职业因素过量接触锰及其化合物引起的职业性慢性锰中毒 (Manganism) 并不少见，而且，汽车尾气等生活环境锰暴露造成的危害更是不容忽视。慢性锰中毒确诊时多是晚期阶段，即便停止接触锰，中枢神经系统中以黑质纹状体多巴胺神经元为主体的脑实质病理性损伤仍会进行性加重，难以恢复和治愈。其发病机制至今尚未完全阐明，故临床上早期诊治尚难实现。脑室中的脉络丛（choroid plexus，CP）组织合成、分泌脑脊液，包括多巴胺神经元特异性营养因子GDF-15等多种成分，而且其上皮细胞是血－脑脊液屏障的组织基础，对脑实质组织具有营养和保护作用。从其解剖位置和功能来看，CP是锰最终引起脑实质多巴胺神经元毒性损伤的中间环节，在锰中毒的发病过程中起关键作用。本课题组发现氯化锰（Manganese chloride，MnCl2）导致脉络丛上皮细胞周期阻滞，引起脉络丛上皮细胞中与细胞周期调控相关的抑制素蛋白1(prohibitin1, PHB1)的基因与蛋白表达水平上调。体外研究也证实了PHB1上调是MnCl2所引起的永生化脉络丛上皮细胞系Z310细胞周期阻滞的主要调控因素。我们进一步探明，锰作用下PHB1上调表达的亚细胞分布和定位特征，MnCl2处理后，上调表达的PHB1蛋白主要位于胞浆，同时发现胞核内PHB1降低，提示MnCl2能够引起Z310细胞中PHB1蛋白升高并从胞核向胞浆移动，这种亚细胞水平的PHB1空间重定位（translocation）改变是锰毒性依赖性的；胞浆和胞核中的PHB1均与p53、Rb有共定位（Co-localization）关系，并且PHB1与二者在染锰前后均可能存在相互作用；胞浆中的PHB1主要存在于线粒体上。另外，MnCl2作用下，能够改变人血清、大鼠血清和脑脊液、Z310细胞及其培养上清液中PHB1和GDF-15的表达水平，提示PHB1和GDF-15的体内外变化具有一定的毒理学意义。本研究从脉络丛这个新视点，为锰毒性机制理论贡献新认知，为锰中毒早期防治提供新思路。