TIMP-2对阿尔茨海默病血脑屏障损伤的保护作用及机制研究

The blood-brain barrier (BBB) plays an important role in maintaining the normal physiological function of the nervous system. Recent imaging and biomarker studies suggest an early BBB dysfunction in AD detectable before cognitive decline. The matrix metalloproteinases 2 (MMP-2) is considered as a key molecules involved in BBB breakdown. Active MMP-2 is stringently regulated by endogenous tissue inhibitor of metalloproteinases 2 (TIMP-2). A balanced interaction between MMP-2/TIMP-2 is essential for the development and extracellular matrix homeostasis. Thus, inhibition of MMP-2 is considered as a potential therapeutic target in AD. Recent study demonstrated that the systemic pools of TIMP2 were necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevented long-term potentiation, indicating TIMP2 might be involved in the pathogenesis of AD. .In the preliminary study, we found that TIMP-2 expression was significantly reduced in the plasma of APP/PS1 mice compared to that in the wild-type mice. Thus, we hypothesized that TIMP-2 might involve in the pathogenesis of AD by regulating BBB function. In the present project, firstly we will observe the effect of TIMP-2 on functional BBB formation in the cortex development. Then, the role of TIMP2 on BBB dysfunction of AD will be investigated by using a variety of animal and cell models (gene overexpression and knockout). Lastly, the mechanisms of TIMP2 on BBB dysfunction of AD will be detected. The study will provide the strong scientific evidences for targeting TIMP-2 to prevent or delay AD. This project will contribute to further clarify the pathogenesis of Alzheimer’s disease, and TIMP-2 might act as a drug target for AD-induced BBB dysfunction.

血脑屏障（BBB）对维持神经系统正常生理功能具有重要作用。在AD患者中，BBB损伤的发生早于AD的病理改变和临床症状，可能为AD发生的始动环节。因此，改善BBB损伤已成为AD治疗的重要策略。MMP-2降解紧密连接和基底膜蛋白损伤BBB，在AD中发挥重要作用。TIMP-2作为MMP-2的内源性抑制剂，能够调节AD小鼠脑内基因表达水平，并逆转学习记忆障碍。由此提出科学假说：TIMP-2可能是AD发生发展的关键因素。TIMP-2缺失导致MMP-2/TIMP-2失衡，进而激活MMP-2造成BBB完整性破坏、通透性增加、转运系统功能异常，最终引起AD脑内突触可塑性降低及认知功能减退。本项目以TIMP-2为靶点，利用多种动物和细胞模型（基因过表达或敲除）系统考察TIMP-2对AD血脑屏障损伤的保护作用，并揭示其作用机制。本项目的开展将为阐明AD发病机制提供理论基础，更为AD药物研发提供新的思路和靶点。

血脑屏障（BBB）对维持神经系统正常生理功能具有重要作用。在AD患者中，BBB损伤的发生早于AD的病理改变和临床症状，可能为AD发生的始动环节。因此，改善BBB损伤已成为AD治疗的重要策略。TIMP-2作为MMP-2的内源性抑制剂，前期研究结果显示，TIMP-2可改善老龄小鼠认知障碍。本项目以TIMP-2为靶点，利用多种动物和细胞模型系统考察TIMP-2对AD血脑屏障损伤的保护作用，并揭示其作用机制。体外结果显示，TIMP-2敲除导致细胞旁通透性增加、跨膜电阻减低，损害血管内皮细胞屏障功能；导致连接复合体组分ZO-1、Occludin和Claudin-5表达下调；影响MMP-2活性。采用TIMP-2敲除鼠进一步发现，TIMP-2敲除老龄鼠血脑屏障通透性增加，脑微血管紧密连接复合体表达缺失、且随月龄增加而加重。机制研究采用IP-MS结合CO-IP以及血管内皮功能检测实验，证实 Integrin α3β1是TIMP-2在血管内皮细胞发挥关键作用的膜受体。RNA-Seq结果表明，TIMP-2敲除影响黏附因子ICAM-2、转运蛋白Cav1以及多种AD相关基因的转录。采用整体和离体AD模型探索TIMP-2对AD导致的血脑屏障损伤的改善作用，给予TIMP-2重组蛋白可逆转Aβ及缺氧损伤模型导致的血管内皮屏障功能损伤，以及连接复合体表达下调；AD转基因小鼠敲除TIMP-2进一步加重认知障碍以及血管内皮细胞连接复合体表达缺失，而靶向血管内皮细胞表达TIMP-2可以上调连接复合体表达。本项目系统研究了TIMP-2在生理和AD病理情况下对血脑屏障功能的调控作用及机制，为阐明AD发病机制提供理论基础，更为AD药物研发提供新的思路和靶点。