线粒体自噬在甲基叔丁基醚致神经系统损伤中的作用与机制

Methyl tert-butyl ether (MTBE) is one of occupational and environmental toxicants. Neurotoxicity of MTBE is focused by researchers. We find that MTBE can cause some neurological lesions, such as hypomnesia and spatial disorientation in MTBE workers. However, the pathogenic mechanism is still unclear. There are evidences indicate that MTBE can permeate into brain tissue directly through the blood-brain barrier. Then MTBE induce oxidative stress, mitochondrial dysfunction and mitophagy. SIRT3 and GCN5L1, two vital mitochondrial regulation enzymes of acetylation, may play important roles in regulating mitophagy. This project intends to combine vitro experiments and population research. After SIRT3 and GCN5L1 silenced by RNA interference, respectively, oxidative stress, mitophagy and its associated proteins modified by acetylation, and the expression of mitophagy proteins (mainly including receptors of mitophagy and the proteins of PINK1/Parkin pathway) are observed in cell experiment to analyze the level of mitophagy induced by MTBE and investigate the roles of SIRT3 and GCN5L1 in this process. In occupational population of MTBE, the neurological injuries, oxidative stress and expressions of mitophagy proteins are detected to discuss the level of mitophagy of MTBE workers. This research will provide a theoretical basis for elucidating the neurotoxicity mechanisms of MTBE and prevention and cure of its occupational hazard.

汽油添加剂甲基叔丁基醚（MTBE）是一种广泛使用的职业与环境毒物，其神经毒性作用逐渐受到学者关注。本课题组在MTBE职业人群中调查发现，MTBE可引起记忆力减退、空间定向障碍等神经损伤症状，但机制不清。MTBE可通过血脑屏障，引起线粒体功能障碍和线粒体自噬发生。线粒体蛋白乙酰化是调控线粒体自噬的重要机制之一，定位于线粒体的调控线粒体自噬的蛋白SIRT3和GCN5L起着的关键作用。但MTBE诱导线粒体自噬发生的机制还有待进一步研究。本项目拟利用RNAi分别沉默SH-SY5Y细胞中SIRT3和GCN5L1后，观察线粒体自噬及其相关蛋白表达及乙酰化水平；在MTBE作业工人中观察神经系统损害、分析血液中线粒体自噬水平及其相关蛋白的表达情况，探讨SIRT3和GCN5L1作为MTBE接触的生物标志物的意义。本研究将为阐明MTBE的神经毒作用机制及其职业危害的防治提供理论依据。

汽油添加剂甲基叔丁基醚（methyl tert-butyl ether, MTBE）是神经退行性疾病的环境致病因素之一，主要机制与MTBE诱导线粒体损伤有关，而线粒体自噬是清除受损线粒体的主要途径。因此，本研究从线粒体自噬为切入点，用不同浓度的MTBE（0、0.25、0.5、1mM）染毒SH-SY5Y细胞24h，检测线粒体损伤、线粒体自噬及其调控机制，探讨线粒体自噬与神经损伤之间的关系。结果显示，MTBE染毒后，细胞线粒体ROS水平升高、线粒体脊数量减少、线粒体膜模糊、线粒体融合蛋白表达下降，表明MTBE可造成线粒体损伤。线粒体自噬是清除受损线粒体的主要途径，本项目进一步检测了线粒体自噬情况，结果显示：电镜下发现典型的双层膜自噬体结构；随MTBE染毒剂量升高，线粒体自噬标志性蛋白LC3-II/LC3-I、Beclin1、Parkin、Pink1和p62表达升高，但线粒体与溶酶体共定位现象减少，表明MTBE可诱导线粒体自噬发生，但线粒体自噬降解功能存在障碍。本项目进一步检测了自噬溶酶体途径的核心调控因子TFEB，发现随MTBE染毒剂量的增加，TFEB在胞浆中表达上调，在胞核表达下降，提示，MTBE导致线粒体自噬功能紊乱可能与TFEB核转位被抑制有关。此外，本项目还检测了线粒体自噬蛋白乙酰化修饰的重要调控基因SIRT3和GCN5L1，但二者均未检出，该部分结果尚需进一步优化条件。线粒体自噬通畅可及时清除受损的线粒体，而线粒体自噬功能受阻，则会影响其清除功能，加剧线粒体和细胞的损伤。结果显示随着MTBE染毒剂量的增加，Aβ表达水平显著升高，提示，Aβ蛋白沉积可能与MTBE诱导线粒体自噬功能紊乱有关。上述研究结果表明，MTBE可通过诱导线粒体自噬功能紊乱导致Aβ蛋白沉积，在该过程中TFEB可能发挥重要的调控作用。本研究为阐明MTBE的神经毒作用机制及其职业危害的防治提供了理论依据。