Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard University, Boston, MA, USA.
Mol Brain. 2008 Nov 4;1:15. doi: 10.1186/1756-6606-1-15.
Deposition of amyloid-β protein (Aβ) is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from γ-secretase cleavage of amyloid precursor protein (APP). In addition to APP, γ-secretase also cleaves other type I integral membrane proteins, including the Notch receptor, a key molecule involved in embryonic development.
To explore selective γ-secretase inhibitors, a combination of five methods was used to systematically determine these inhibitors' profiles on the γ-secretase cleavage of APP and Notch. When two potent γ-secretase inhibitors, compound E (cpd E) and DAPT, were used in a conventional in vitro γ-secretase activity assay, cpd E completely blocked Aβ generation from the cleavage of substrate APP C100, but only had a minor effect on Notch cleavage and NICD generation. Next, cpd E and DAPT were applied to HEK293 cells expressing a truncated Notch substrate NotchDeltaE. Both cpd E and DAPT were more potent in blocking Aβ generation than NICD generation. Third, a reporter construct was created that carried the NICD targeting promoter with three Su(H) binding sequences followed by the luciferase gene. We found that the inhibition of NICD generation by cpd E and DAPT was consistent with the reduced expression of luciferase gene driven by this Notch targeting promoter. Fourth, levels of "Notch-Aβ-like" (Nβ*) peptide derived from two previously reported chimeric APP with its transmembrane domain or the juxtamembrane portion replaced by the Notch sequence were quantified. Measurement of Nβ* peptides by ELISA confirmed that EC₅₀'s of cpd E were much higher for Nβ* than Aβ. Finally, the expression levels of Notch target gene her6 in cpd E or DAPT-treated zebrafish were correlated with the degree of tail curvature due to defective somitogenesis, a well characterized Notch phenotype in zebrafish.
Our ELISA-based quantification of Aβ and Nβ* in combination with the test in zebrafish provides a novel approach for efficient cell-based screening and in vivo validation of APP selective γ-secretase inhibitors.
淀粉样蛋白-β（Aβ）的沉积是阿尔茨海默病（AD）的主要病理标志。Aβ是由γ-分泌酶切割淀粉样前体蛋白（APP）产生的。除了 APP，γ-分泌酶还切割其他类型的 I 型整合膜蛋白，包括 Notch 受体，这是胚胎发育中关键的分子。
为了探索选择性 γ-分泌酶抑制剂，采用了五种方法的组合来系统地确定这些抑制剂对 APP 和 Notch 的 γ-分泌酶切割的谱。当两种有效的 γ-分泌酶抑制剂，化合物 E（cpd E）和 DAPT，用于传统的体外 γ-分泌酶活性测定时，cpd E 完全阻断了底物 APP C100 的切割产生的 Aβ，但对 Notch 切割和 NICD 生成只有轻微影响。接下来，cpd E 和 DAPT 应用于表达截短 Notch 底物 NotchDeltaE 的 HEK293 细胞。cpd E 和 DAPT 都比 NICD 生成更有效地阻断 Aβ 的生成。第三，创建了一个携带 NICD 靶向启动子的报告构建体，该启动子带有三个 Su(H)结合序列，其后是荧光素酶基因。我们发现，cpd E 和 DAPT 对 NICD 生成的抑制作用与由这个 Notch 靶向启动子驱动的荧光素酶基因的表达降低一致。第四，定量了两个先前报道的嵌合 APP，其跨膜结构域或近膜部分被 Notch 序列取代后产生的“Notch-Aβ 样”（Nβ*）肽的水平。通过 ELISA 测量 Nβ肽证实，cpd E 的 EC₅₀ 对 Nβ的作用比 Aβ高得多。最后，在 cpd E 或 DAPT 处理的斑马鱼中 Notch 靶基因 her6 的表达水平与由于体节形成缺陷导致的尾巴弯曲程度相关，这是斑马鱼中 Notch 表型的一个很好的特征。
我们基于 ELISA 的 Aβ 和 Nβ*的定量与在斑马鱼中的测试相结合，为 APP 选择性 γ-分泌酶抑制剂的有效细胞筛选和体内验证提供了一种新方法。
