脑靶向抑制DMT1功能阻抑阿尔茨海默病转基因小鼠脑内β淀粉样蛋白沉积和tau蛋白异常磷酸化的分子机制

One of the reasons accounting for metal dyshomeostasis in AD brains is the abnormal distribution and expression of the divalent metal transporter, DMT1, which is also the key factor that causes abeta production and accumulation and neuronal injury. Therefore, DMTI has become the therapeutic target for AD. In our previous work, we have found that inhibiting the metal-transport function of DMTI can reduce the expression of abeta and abnormal phosphorylation of tau as well as prevent the neuronal and synaptic injury caused by abeta oligomers. All these studies would become the foundation of AD therapy theoretically and technically. The study subject of this project is PS1/APP/tau transgenic mice. We would administrate the Ferristatin and Ebselen gel through nasal mucosa or through caudal injection carrying brain-target chitosan nanoparticles. And we also tend to knock out gene slc11a2 in PS1/APP mice using neuron-specific promoters. Then detect a series of indicators, including abeta senile, NTFs and neuronal apoptosis. With the in vitro experiments, not only can the research reveal the involvement of DMTI in AD pathogenesis, it also can contribute a lot to the identification of DMT1 as a therapeutic target of AD and to the evidence supporting how to choose a practical and effective way for AD treatment.

DMT1分布和表达异常是阿尔茨海默病(AD)脑内金属离子稳态失衡的原因之一，也是引起Aβ产生和沉积并造成神经元损伤的关键因素，因此DMT1已经成为AD治疗的重要靶点。在前期工作中，我们发现干扰DMT1功能不但能减少Aβ生成和tau蛋白异常磷酸化，还能拮抗可溶性Aβ寡聚体对神经元和突触的损伤。本项目以PS1/APP/tau三转基因小鼠为研究对象，DMT1小分子抑制物Ferristatin和Ebselen凝胶制剂经鼻黏膜吸收和脑靶向壳寡糖纳米粒经尾静脉注射给药，并构建中枢神经系统slc11a2基因Knock-down的PS1/APP双转基因小鼠。对小鼠脑内Aβ老年斑形成、NFTs缠结和神经元凋亡等相关指标进行检测，结合体外实验，不仅进一步揭示DMT1参与AD发病的机制，还可以为明确DMT1能否作为AD治疗靶点以及选择有效给药途径和治疗策略提供理论依据和技术支持。

以β淀粉样蛋白（Aβ）为核心构成的老年斑以及tau蛋白异常磷酸化形成的神经原纤维缠结被认为是阿尔茨海默病（Alzheimer‘s Disease，AD）的主要病理特征，越来越多的证据表明，二价金属离子及其转运蛋白DMT1在AD发生发展中起到至关重要的作用。我们通过构建并繁育中枢神经系统特异性敲除DMT1基因slc11a2的小鼠模型，侧脑室注射毒性Aβ寡聚体，另外选择AD转基因小鼠给予DMT1抑制剂Ebselen腹腔注射，建立起两种DMT1功能特异性抑制的AD动物模型后，应用Morris水迷宫和Nesting检测模型小鼠的学习记忆能力和社会行为能力；应用免疫组织化学、免疫荧光染色、Westernblot等方法检测模型小鼠脑内Aβ老年斑、APP水解相关酶类及酶切产物、tau蛋白不同位点的过度磷酸化及tau磷酸化相关蛋白激酶的表达情况。同时选择AD小鼠饲喂MnCl2构建锰中毒AD小鼠模型，应用Nesting实验检测模型小鼠的社会行为能力损害情况，应用免疫组织化学、免疫荧光染色、Westernblot等方法检测模型小鼠脑内Aβ老年斑、APP水解相关酶类及酶切产物的变化。通过条件性共培养小胶质细胞和过表达人APPsw突变基因的N2a细胞，考察锰离子代谢失衡通过刺激小胶质活化和炎症因子的释放，促进APP淀粉样代谢途径相关水解酶的活性增强及代谢产物产生。重要实验结果：1）特异性抑制DMT1的二价金属离子转运功能缓解AD发生发展过程中的Aβ形成和tau蛋白过度磷酸化。2）二价金属离子锰的稳态失衡也可以加重AD的发生发展进程。3）DMT1可以作为防治AD的潜在靶点。这些研究结果为进一步揭示金属离子代谢紊乱及其转运体DMT1参与AD发生的机理，以及确立将DMT1调节金属离子稳态作为AD治疗的新策略提供了充分的理论依据。