MT3调制Aβ聚集及脑内胰岛素信号通路异常的分子机制研究

Alzheimer’s disease (AD) is fundamentally a metabolic disease with substantial and progressive responsiveness to insulin stimulation. Amyloid-beta peptide (Aβ) is the main constituent of the senile plaques in the brains of AD patients, produced as soluble protein and can be converted into aggregates (such as oligomers and fibrils). Growing evidence supports the concept that the etiology of AD is the brain insulin resistance induced by the binding events between Aβ aggregates and insulin signaling proteins or membranes. Metallothionein-3 (MT3), which is especially enriched in the central nervous system, is capable of modulating the aggregation of Aβ and Aβ-induced toxicity. However, the Aβ aggregation mechanism remains debatable and the modulation effect from MT3 is unknown. In this proposal, the central theme is the study of MT3-involved Aβ aggregation and its effect on aberrant brain insulin signaling. Specifically, the binding sites and the binding constants of Aβ to MT3 will be confirmed. The aggregation of Aβ in the presence of MT3 will be investigated respectively in solution and membrane/solution interface, to gain insight into the interplay between MT3-involved aggregation and the role of aggregates in membrane damage. Meanwhile, the interations of Aβ and insulin signaling proteins (IR, IDE, IRS, PI3K) will be investigated. A major emphasis will be placed on the understanding of the structure-function/protein interactions of Aβ aggregates. Finally, cytotoxicity of the Aβ aggregates will be studied on hippopotamus neurons of SD rats. We envision that a comprehensive description about the fundamental aspect of MT3-involved Aβ/protein and Aβ/membrane interactions, as well as the related neurotoxicity of the various aggregates, will evolve from our project. Such information will consolidate the understanding of the aggregation properties of Aβ and their involvements in aberrant brain insulin signaling in AD. It will also provide insight into animal model studies and clinical assays.

胞外β淀粉样多肽（Aβ）聚集体通过多个途径导致脑内胰岛素信号通路异常，这是引发阿尔茨海默病（AD）的主要病因之一。作为结合蛋白，金属硫蛋白3（MT3）能调制Aβ的聚集，进而影响其在胰岛素信号通路中的毒性作用。但是，目前Aβ的聚集机制尚不完善，MT3的调制作用研究更是空白。针对上述问题，本项目拟结合实验与理论计算，确定Aβ与MT3作用的结合位点和结合常数，揭示溶液中和磷脂膜上MT3调制Aβ聚集的分子机制；通过评估膜通透性和电学性质，建立Aβ聚集体诱导膜损伤的分子机制。同时，研究Aβ与胰岛素信号通路相关蛋白（IR、IDE、IRS、PI3K等）的作用，确认MT3对络合物结构和生物功能的影响；通过SD大鼠海马神经元毒性研究，建立“Aβ聚集体—神经毒性”之间的构效关系。最终完善MT3调制Aβ聚集和脑内胰岛素信号通路异常的分子模型，阐明MT3作为结合蛋白的神经保护机制，为AD的病理研究提供理论指导。

为了探讨金属硫蛋白3（MT3）在阿尔茨海默病病理中所起的作用，本项目深入研究了β淀粉样多肽（Aβ）Aβ与金属硫蛋白3（MT3）的相互作用及其对细胞的毒性，系统研究了溶液组成、pH、金属离子等因素对Aβ/MT3络合物形成及其细胞毒性的影响。确定了Aβ/MT3相互作用的结合位点分别为Aβ的Phe4和Tyr10、MT3的TCPCP序列，揭示了溶液中MT3通过空间位阻调制Aβ聚集的分子机制；通过研究金属离子的影响，发现Zn-MT3通过与Aβ复合实现对其原位保护而毒化的Cu-MT3与Aβ分离从而实现动态调控；研究了相关儿茶酚胺类神经递质（如DA、NE）在阿尔茨海默病病理中的作用机制，揭示了DA、NE等神经递质过量释放造成的细胞伤害以及ATP、MT3通过金属络合、抗氧化实现的神经保护机制。同时，建立了一系列用于临床Aβ分型检测的新方法，包括：通过构筑多孔支撑的双层磷质膜体系，实现了对淀粉样蛋白跨膜聚集行为的实时电学监测，获得了电场变化对淀粉样蛋白聚集行为的影响；构筑基于核酸适配体的电化学检测芯片，实现了对脑脊液中低分子量Aβ寡聚体的灵敏检测；通过共免疫捕获和纳米粒子放大，实现了对脑脊液中磷酸化Aβ比例的有效检测等。建立了一个以电化学和光谱方法为基础、结合分离技术与DFT计算工作站的研究神经退行性疾病致病机制的技术平台。系列研究成果为阿尔茨海默病的病理研究提供了理论指导。