Clarification of Molecular Mechanism For-AmyloidβGeneration in Alzheimer's Disease

淀粉样蛋白β-肽（a β）被认为在阿尔茨海默病（AD）的发病机制中起核心作用。我们项目的目的是验证Aβ假说的有效性，并开发新的治疗AD的策略。Aβ是通过β- γ分泌酶对Aβ前体蛋白（APP）的顺序蛋白水解而产生的。我们关注γ-分泌酶的生物学，因为Aβ的致病性依赖于γ-分泌酶的裂解位点。(a) a β生成的调控机制分析：先前我们发现γ-分泌酶是一种含有早老素（PS）的大分子复合物，一种新的蛋白质nicastrin （Nct）是该复合物的重要组成部分。在这个项目中，我们报道了PS:Nct复合物对于Notch切割和APP切割是必不可少的（Nat Cell Biol 2001），额外的组分PEN-2对于γ-分泌酶复合物的激活至关重要（J Neurochem, press）， alcadin是γ-分泌酶的一种新型底物，参与了fe65介导的转录调节（J Biol Chem, press）。为了研究临床PS突变增强致病性a β (a β42/43)产生的机制，我们对影响γ分泌酶活性的突变体PS 1进行了随机诱变筛选。我们成功地鉴定了只产生Aβ42/43种的独特突变体（准备发表）。这些突变体有助于分析Aβ42/43代的分子基础，并开发抑制Aβ42/43代的新策略。(b) Aβ假说的有效性检验：我们已经尝试培育出具有AD症状和病理特征的转基因猴。这些转基因猴子不仅可用于验证Aβ假说的有效性，还可用于评估新开发的AD治疗方法的效力和副作用。

Amyloidβ-peptides (Aβ) have been proposed to play a central role in the pathogenesis of Alzheimer disease (AD). The aim of our project is to test the validity of Aβ hypothesis and develop the novel therapeutic strategy for AD. Aβ are generated through the sequential proteolysis of Aβ precursor protein (APP)by β-and γ-secretase. We focus on the biology of γ-secretase, because the pathogenicity of Aβ is dependent on the cleavage site of γ-secretase. (a) Analyses of regulatory mechanism for Aβ generation: Previously we revealed that γ-secretase is a macromolecular complex containing presenilin (PS), and that a novel protein nicastrin (Nct) is an essential constituent of this complex. During this project, we have reported that the PS:Nct complex is indispensable for Notch cleavage as well as APP cleavage (Nat Cell Biol 2001), that the additional constituent PEN-2 is critical for activation of γ-secretase complex (J Neurochem, in press), and that Alcadein is a novel substrate for γ-secretase and involved in Fe65-mediated transcriptional regulation (J Biol Chem, in press). To examine the mechanism for enhancement of pathogenic Aβ (Aβ42/43) generation by clinical PS mutations, we performed a random mutagenesis screen to search for mutant PS 1 which affect the γ-secretase activity. We successfully identified the unique mutants that exclusively generated Aβ42/43 species (in preparation for publication). These mutants are useful to analyze the molecular basis for Aβ42/43 generation and to develop the new strategy for inhibiting Aβ42/43 generation. (b) Test for the validity of Aβ hypothesis: We have been tried to develop transgenic monkeys exhibiting symptom and pathology characteristic to AD. The transgenic monkeys should be useful not only for testing the validity of Aβ hypothesis but also for evaluating the potency and adverse effect of newly developed AD therapies.