Zap1-like蛋白在果蝇脑内调控锌稳态的机制及对Aβ沉积的影响

Numerous evidences suggest that zinc dishomeostasis is closely related to β-amyloid (Aβ) deposition in the AD brain. Using AD Drosophila model, we first found that inhibition of the expression of zinc importer dZip1 in the brain of Drosophila could significantly reduce the accumulation of Aβ protein, ameliorate the disease of AD Drosophila, indicating that the control of zinc homeostasis may be an effective way to slow down the progression of AD. However, our knowing of brain zinc metabolic pathways is still quite poor. Seen from the molecular mechanisms of maintaining yeast zinc homeostasis we know that Zap1 controls the expression of Zips and ZnTs, which are key proteins for yeast cytoplasmic zinc import and export. We proposed that the regulation of Zap1 expression from the source may play a more significant role in zinc homeostasis. The project intends to start from Zap1-like transcription factors, by using qRT-PCR, RNAi interference, immunohistochemistry, EMSA, confocal laser scanning, X-ray fluorescence analysis and other techniques to reveal the molecular mechanism of dZAPL(Drosophila Zap1-like protein) in the regulation of zinc homeostasis in the Drosophila nervous system, and the dZips and/or dZnTs whose expression are under the control of dZAP1L. Furthermore, the project will disclose the regulation effects of the expression of dZAP1L and hZAP1L (Human Zap1-like protein) on the zinc level and distribution in the Drosophila brain tissues, and the interventional mechanism and effect of their expression on the disease of AD flies, which would be verified in the APP Mouse AD model, in order to provide new targets and theoretical basis for the treatment of AD.

众多实验证据表明脑锌代谢紊乱与AD大脑β-淀粉样蛋白(Aβ)沉积密切相关。应用AD果蝇模型，我们首次发现抑制果蝇脑锌吸收蛋白dZip1的表达能明显减少Aβ蛋白的累积，改善AD果蝇的病症，表明控制锌稳态可能是延缓AD的一有效途径。但目前有关大脑锌代谢途径的知识还相当匮乏。从维持酵母锌稳态的分子机制中可知Zap1管控着胞质锌转运关键蛋白Zips和ZnTs的表达，从源头上调控Zap1的表达可能对锌稳态的维持起到更为显著的作用。本项目拟用qRT-PCR、RNAi干扰、免疫组化、凝胶阻滞、激光共聚焦、X射线荧光分析等技术揭示果蝇Zap1类蛋白dZAP1L在中枢神经系统调控锌稳态的工作机制，阐明dZAP1L和人hZAP1L表达对果蝇脑组织锌离子水平和分布的影响和对AD果蝇发病影响和干预机制，并用小鼠AD模型进行验证，以期为AD治疗提供新的靶点和理论依据。

众多研究表明脑锌代谢紊乱与AD大脑β-淀粉样蛋白(Aβ)沉积密切相关，我们前期利用果蝇AD模型发现通过遗传调控锌转运蛋白Zip1的表达可显著减少Aβ累积，改善AD果蝇的病症。在此基础上我们设想从调控锌稳态的转录因子入手有可能作用更为显著，但目前负责锌匮乏调控的Zap1转录因子只在酵母中有较为详细的研究，在果蝇及其他高等生物和人还未发现其同源基因。本研究利用生物信息学方法在果蝇基因组中发现了一类酵母Zap1蛋白基因，命名为dZap1L（dZap1-Like 蛋白），并深入研究了其功能机制及与AD发病的关系，发现了一些重要的新功能和作用机制。1.dZap1L是一类酵母Zap1转录因子基因，在果蝇脑神经系统不仅对锌稳态，而且对多种微量金属特别是铁稳态具有显著的调控作用；2.dZap1L在脑神经系统可能通过调控或影响dZip1，dZip3，dZnT1，dZip99c和dZnT35c的表达影响脑锌水平，通过调控或影响MCO1，MCO3，Malvolio以及 Transferrin1、Transferrin2、Transferrin3的表达影响脑铁水平；3.通过调控dZap1L的表达可以显著影响AD果蝇模型的寿命、行为以及认知能力，而这些可能是主要通过影响果蝇脑锌、铁离子水平，进而影响Aß沉积及神经元死亡来实现的，通过精细调控dZap1L表达水平有望对改善AD病症取得非常积极的效果。本研究为全面阐释果蝇脑神经系统金属稳态机制填补了一项空白，同时为预防和治疗AD提出了新的思路和治疗靶位。