Molecular and cell biological studies on the production, aggregation and clearance of Alzheimer amyloid β peptides.

本研究的目的是阐明被认为是阿尔茨海默病（AD）致病蛋白的β淀粉样蛋白肽的产生、积累和清除过程，从而为阿尔茨海默病（AD）的疾病修饰治疗创造一个新的概念。通过对产生a β肽的膜蛋白酶γ-分泌酶的研究，我们试图阐明a β产生的机制，并阻断AD的最上游原因。具体来说，我们开发了含有烯酮片段的新型γ分泌酶抑制剂，并分析了这些化合物的作用机制。使用Cys扫描方法，我们发现早老素1的6/7跨膜结构域（后者是γ-分泌酶的催化亚基）形成亲水的催化孔结构，我们进一步使用单颗粒分析分析了γ-分泌酶和相关蛋白酶SPP的结构。我们发现CLAC，一种新的淀粉样斑块成分蛋白，在体外抑制a β纤维的形成，并参与转基因小鼠大脑中淀粉样斑块的压实。我们还利用永生化的脑内皮细胞细胞系表明，LRP1参与了所有肽穿过血脑屏障的清除。因此，我们利用蛋白质组学分析、RNAi和小分子筛选等尖端技术，阐明了Aβ沉积的关键步骤，即γ-分泌酶、淀粉样蛋白结合蛋白和Aβ清除，为开发新的AD治疗策略提供了线索。

The aim of this research project is to elucidate the process of production, accumulation and clearance of amyloid β peptides, that are regarded as pathogenic proteins in Alzheimer's disease (AD), thereby creating a novel concept for the disease-modifying therapy for AD. Through the studies of γ-secretase, a membrane protease that produces Aβ peptide, we tried to clarify the mechanism of Aβ production and to block the most upstream cause of AD. Specifically we developed novel γ-secretase inhibitors harboring an enone-moiety and analyzed the mechanism of action of these compounds. Using Cys scan approach, we showed that the 6/7 transmembrane domains of presenilin 1, the latter being the catalytic subunit of γ-secretase, form a hydrophilic catalytic pore structure, We further analyzed the structure of γ-secretase and a related protease, SPP, using single particle analysis. We showed that CLAC, a novel amyloid plaque component protein, inhibits Aβ fibril formation in vitro, and is involved in the compaction of amyloid plaques in the brains of transgenic mice. We also showed that LRP1 is involved in the clearance of All peptides across the blood brain barrier, using an immortalized cell line of brain endothelial cells. We thus elucidated the key steps of Aβ deposition, i.e., γ-secretase, amyloid binding protein and Aβ clearance, using cutting edge technologies including proteomic analysis, RNAi and small molecule screen, providing clues to the development of novel therapeutic strategies for AD.