APP新剪切产物Aη-α调控阿尔茨海默症病理进程的神经毒性机制研究

The exact course of Alzheimer’s disease (AD) is still unclear. However, the failure of β-amyloid (Aβ)-target anti-AD drugs in the clinical studies indicated that Aβ hypothesis may not be able to fully explain the onset of AD. Aη-α is a novel proteolytic fragment of Aβ precursor protein (APP). We have previously found that Aη-α could assemble into oligomer with high neurotoxicity. Moreover, Aη-α could increase the aggregation of Aβ. Hippocampal injection of Aη-α induced advanced cognitive impairments in AD transgenic mice. These results suggested that Aη-α might play a role in the pathological progress of AD. However, it is still unknown how Aη-α assembles into oligomer, how Aη-α produces neurotoxicity, and whether Aη-α regulates the pathological progress of AD. In this project, the mechanisms underlying the assembly of Aη-α oligomer will be elucidated by using molecular dynamics study and point mutation test. Moreover, Aη-α oligomer-induced neurotoxicity via synaptic impairments will be studied in primary neuronal cultures, hippocampal slices, and normal mice, respectively. Furthermore, the regulation of AD pathological progress by Aη-α will be explored in APP/PS1 transgenic mice with hippocampal over-expression of Aη-α. In addition, Aη-α oligomer inhibitors will be screened. And the disease-modifying effects of Aη-α oligomer inhibitors will be investigated. Our results will not only elucidate the neurotoxic mechanisms of Aη-α underlying the regulation of AD pathological progress, but also provide a novel molecular target for further AD drug development.

阿尔茨海默症（AD）病因尚不明确，以β-淀粉样蛋白（Aβ）为靶标的药物试验失败提示主流Aβ假说可能无法全面概括AD发病机理。Aη-α是新发现的Aβ前体蛋白（APP）剪切产物。我们前期发现Aη-α易组装成高神经毒性的寡聚体，还能促进Aβ聚集，海马注射Aη-α可诱导AD小鼠提前出现认知障碍，提示Aη-α可能参与AD病理进程。但Aη-α如何组装、怎样产生神经毒性以及能否调控AD病理进程这些关键问题尚不清楚。本项目拟首先应用分子模拟结合定点突变实验阐明Aη-α寡聚体组装的机制；进一步在细胞、脑片、动物三个层面证明Aη-α寡聚体诱导突触损伤产生神经毒性，通过APP/PS1转基因小鼠海马过表达Aη-α从正面探究其对AD病理进程的调控作用；筛选Aη-α寡聚体抑制剂从反面验证阻止Aη-α毒性、逆转AD病理进程的可行性。研究结果有望明确Aη-α调控AD病理进程的神经毒性机制，为AD药物研发提供新的分子靶标。

项目组圆满完成各项任务及指标。研究期间在ACS Chemical Neuroscience等国际期刊共发表基金资助SCI论文16篇，其中第一标注15篇；授权发明专利2项；培养研究生3名；参加学术会议4次。项目组1）制备Aη-α蛋白，在Aβ寡聚化的缓冲体系中，培养经HFIP预处理的Aη-α单体，发现其可形成寡聚体，且形貌为球形颗粒；2）应用计算机分子模拟手段解析Aη-α组装时的分子间相互作用，明确Aη-α特异的组装机制；3）通过慢病毒感染方式，在APP/PS1转基因小鼠等海马过表达Aη-α，确定Aη-α表达与海马PSD-95、Synapsin-I、BDNF等表达负相关，与神经炎症、认知障碍以及突触损伤正相关，显示Aη-α 可能调控 AD病理进程；4）发现多个可抑制寡聚体组装的小分子化合物，验证通过降低寡聚体神经毒性阻止AD病理进程的可行性：发现来自染料的CSB6B可调节寡聚体组装，阻止AD相关神经炎症和神经损伤，在AD动物模型发挥抗认知障碍活性；发现天然化合物钩吻碱甲对抗寡聚体蛋白诱导的AD模型小鼠认知障碍，由于钩吻碱甲已被报道具有镇痛等抗神经疾病作用，且发现的抗AD作用浓度远低于其毒性浓度，我们猜测其可能用于AD治疗；发现临床用于治疗痉挛性疼痛的药物间苯三酚可抑制淀粉样蛋白聚集体形成，并降解预先形成的聚集体，提示其可能发展为治疗神经退行性疾病的药物；合成具有抑制寡聚体活性的PLGA-PEG-岩藻黄素纳米颗粒，纳米颗粒可通过血脑屏障，在AD动物中较低浓度就能阻止寡聚体诱导的神经毒性和神经炎症，具有进一步开发价值；通过分子设计、化学合成等方法获得9-methylfascaplysin、tacrine-hupyridone和tacrine-dicolylamine二聚体等小分子化合物，这些化合物体外可有效抑制淀粉样蛋白组装，阻止小鼠认知障碍和运动障碍，尤其以他克林为主要基团合成的二聚体化合物阻止淀粉样蛋白聚集活性强，提示他克林基团可能有效作用AD毒性蛋白，为其进一步开发抗AD化合物提供依据。以上结果有望明确Aη-α调控AD病理进程的神经毒性机制，发现新的抗AD药物。