TFEB介导溶酶体功能障碍在PBDE-47发育神经毒性中作用及机制研究

Due to its high body burdens in infants and young children, the 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) has received worldwide concern for developmental neurotoxicity. However, the underlying mechanisms still remain unclear. Our previous studies have shown that apoptosis and abnormal autophagy played a key role in PBDE-47-induced developmental neurotoxicity. Lysosome could induce cell death via lysosomal apoptosis and autophagy-lysosome pathways. Recently we found that PBDE-47 affected the function of lysosome and down-regulated the expression of transcription factor EB (TFEB), which is a key factor responsible for lysosomal biogenesis and function. Therefore, we hypothesize that PBDE-47 induces lysosome-dependent cell death through lysosomal dysfunction by reducing the biosynthesis and/or the activity of TFEB, which contributes to PBDE-47-induced developmental neurotoxicity. In this project, both in vivo and in vitro models will be established by drug intervention, gene silencing and plasmid transfection. The role of TFEB and lysosome dysfunction in the developmental neurotoxicity of PBDE-47 will be studied. Meanwhile, their relationship will be investigated. The results will further elucidate the targets and the molecular mechanisms of PBDE-47-induced developmental neurotoxicity, and provide scientific basis to improve the prevention and the control of PBDE-47-induced developmental neurotoxicity.

2,2',4,4'-四溴联苯醚（PBDE-47）在婴幼儿体内高负荷而产生的发育神经毒性已引起高度关注，但其发育神经毒性作用机制尚未阐明。我们前期研究发现，PBDE-47引起的细胞凋亡及自噬异常与其神经毒性密切相关。溶酶体可通过溶酶体-凋亡通路及自噬-溶酶体通路引起细胞死亡，且我们最近也发现，PBDE-47影响溶酶体功能，降低调节溶酶体合成和功能的转录因子EB（TFEB）表达，据此推测PBDE-47可能通过影响TFEB的合成和/或活性导致溶酶体功能障碍，引起溶酶体依赖的细胞死亡，从而参与PBDE-47发育神经毒性的发生发展。本项目拟采用药物干预、基因沉默及质粒转染等方法建立动物和细胞模型，研究TFEB和溶酶体障碍在PBDE-47发育神经毒性中的作用，并揭示三者之间的内在联系，进一步阐明PBDE-47发育神经毒性的作用靶点和分子机制，为其发育神经毒性的预防和控制提供科学依据。

PBDE-47的发育神经毒性已受到广泛关注，明确PBDE-47的毒性效应机制对暴露PBDE-47的早期风险评价具有重要意义。本项目通过建立PBDE-47染毒的动物模型和神经细胞模型，在自噬异常与PBDE-47的神经毒性的关系上开展了一系列PBDE-47神经毒性及其干预的机制研究。主要研究结果包括：（1）围产期低剂量PBDE-47暴露可致子代大鼠神经行为缺损，其机制与自噬障碍和过度凋亡有关。（2）PBDE-47发育神经毒性与其引起自噬体与溶酶体融合过程受阻，导致自噬流紊乱有关。TFEB可与STX17相互作用并促进自噬体与溶酶体融合，从而改善PBDE-47所致的自噬流异常，恢复自噬通量，提高PC12细胞存活率。（3）围产期暴露于低剂量PBDE-47可通过触发成年雌性大鼠甲状腺内质网应激,溶酶体功能失调致甲状腺球蛋白的转换异常，最终导致成年雌性大鼠甲状腺功能异常。（4）妊娠期暴露于低剂量PBDE-47可致雌性母鼠血清代谢组异常，并导致成年后代的性别特异性体重增加。（5）妊娠期和哺乳期暴露于低剂量PBDE-47后可致成年大鼠精子数量减少和活力受损，其机制与甲状腺激素异常升高有关。（6）PBDE-47可通过影响线粒体融合分裂动力学稳态致线粒体形态异常，引起细胞过度凋亡，进而导致神经元丢失和一系列的神经行为缺损。上述研究结果不仅阐明了自噬异常与PBDE-47神经毒性之间的关系，也从分子水平阐明了TFEB与溶酶体功能障碍以及自噬体与溶酶体融合障碍在PBDE-47神经毒性发生机制中的作用，为寻找PBDE-47神经毒性的敏感效应生物学标志、阻断PBDE-47神经毒性发生发展提供了新的理论依据。同时，在本课题的资助下，结合本课题获得的研究结果和国内外研究进展，对线粒体动力学改变在PBDE-47神经毒性中的作用也开展了初步研究，发现PBDE-47可致PC12细胞线粒体融合分裂异常和线粒体生物发生障碍，提示PBDE-47神经毒性可能与线粒体动力学异常有关，为进一步深入开展PBDE-47神经毒性作用机制研究提供了新的线索。