Total inactivation of γ-secretase activity in presenilin-deficient embryonic stem cells
Total inactivation of γ-secretase activity in presenilin-deficient embryonic stem cells
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DOI：
10.1038/35017105
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发表时间：
2000-07-01
影响因子：
21.3
通讯作者：
De Strooper, B
中科院分区：
文献类型：
--
作者：
Herreman, A;Serneels, L;De Strooper, B
* 神经元细胞生物学实验室，CME，KU Leuven 和法兰德斯大学间生物技术研究所 (VIB4)，Herestraat 49, 3000 Leuven，Belgium† Thromb–X，NV，Herestraat 49, 3000 Leuven，Belgium‡ 电子邮件：Bart。毁灭者@医学。库勒文。交流。一段时间以来，β-淀粉样蛋白前体蛋白 (β-APP)、Notch-1 等蛋白质如何在膜平面上裂解，这一问题一直困扰着细胞生物学家和阿尔茨海默病研究人员。 γ-分泌酶对 β-APP 的加工被认为是一个重要的治疗靶点，因为它构成了淀粉样蛋白 β 肽 (Aβ) 释放的最后一步，Aβ 是阿尔茨海默病患者大脑中淀粉样斑块的主要成分。最近，γ-分泌酶活性与两种蛋白质相关：早老素-1 (PS1) 和早老素-2 (PS2；参考文献 1、2)。早老素是嵌入细胞内膜的强疏水性蛋白质，其基因的错义突变会导致一种罕见的、侵袭性的阿尔茨海默病3。有人提出，早老素跨膜结构域中的两个天冬氨酸残基构成了γ分泌酶2的催化位点。反对这一假设的一个论点是，在 PS1 或 PS2 缺陷小鼠的细胞中观察到重要的残余 γ-分泌酶活性1, 4。因此，问题是完全缺乏 PS1 和 PS2 的细胞是否维持这种 γ-分泌酶活性。不幸的是，PS 缺失小鼠在胚胎发生早期死亡（大约在发育 9.5 天后；E9.5），从而不可能产生足够的细胞来执行所需的任务。 生化实验4.因此，我们从 PS1+/–PS2–/–小鼠 4 交配获得的 PS 无效囊胚中产生了胚胎干细胞的多能系。我们使用聚合酶链式反应 (PCR) 4 和 Southern blotting 对细胞系进行基因分型（图 1a）。双缺陷（PS-null）细胞系的32%产率略高于预期的25%；我们通过蛋白质印迹证实了 PS1（图 1b）和 PS2（数据未显示）的缺失。我们观察到内源性 β-和 α-分泌酶裂解的 β-APP 羧基末端残基的积累，这是 γ-分泌酶的直接底物（图 1b 和参考文献 1）。然后，我们使用重组塞姆利基森林病毒 (SFV) 在胚胎干细胞中表达含有 K595N/M596L（瑞典语）突变的 β-APP695。这种突变显着增加了 Aβ5、6 的产生，有利于我们的检测中的检测。与单敲除细胞获得的结果相反，在 PS1–/–PS2–/– 细胞的条件培养基中未检测到 Aβ（图 1c）。通过连续稀释，我们确定即使 Aβ 的产量<野生型细胞中的 0.5%，我们也能检测到它（图 1c）。我们使用针对 42 个氨基酸形式的 Aβ1 的酶联免疫吸附测定 (ELISA) 进一步证实不存在 γ 分泌酶活性。 mNotchΔ E 是 PS1 依赖性 γ 分泌酶 7, 8 的另一种底物。该构建体的处理模仿全长 Notch-1，并导致 Notch 胞内结构域 (NICD) 的释放。 NICD 易位到细胞核并与 CBF-1 家族的转录因子一起激活多个基因，包括 HES-1（参考文献 9）。因此，我们在 HES-1 启动子（由 R. Kopan 和 A. Israel 友情提供）的控制下，用编码 mNotchΔ E 和荧光素酶的质粒共转染胚胎干细胞，并测量荧光素酶活性的诱导10。作为对照，我们还用编码 NICD 的质粒转染胚胎干细胞。在 PS1+/+ PS2–/– 细胞中观察到 mNotchΔ E 诱导 HES-1 活性，但在 PS1–/– 细胞中未观察到
* Neuronal Cell Biology Laboratory, CME, KU Leuven and Flanders Interuniversitary Institute for Biotechnology (VIB4), Herestraat 49, 3000 Leuven, Belgium† Thromb–X, NV, Herestraat 49, 3000 Leuven, Belgium‡ e–mail: Bart. Destrooper@ med. kuleuven. ac. be he question of how proteins such as the β-amyloid precursor protein (β-APP), Notch-1 and others can be cleaved in the plane of the membrane has challenged cell biologists and researchers into Alzheimer’s disease for some time. Processing of β-APP by γ-secretase is considered an important therapeutic target, as it constitutes the final step in the release of the amyloid βpeptide (Aβ), the principal constituent of the amyloid plaques in the brains of Alhzeimer’s patients. Recently, γ-secretase activity was linked to two proteins, presenilin-1 (PS1) and presenilin-2 (PS2; refs 1, 2). Presenilins are strongly hydrophobic proteins embedded in intracellular membranes, and missense mutations in their genes cause a rare, aggressive form of Alzheimer’s disease3. It has been proposed that two aspartate residues in the transmembrane domains of presenilins constitute the catalytic site of γsecretase2. One argument against this hypothesis is the important residual γ-secretase activity observed in cells derived from PS1-or PS2-deficient mice1, 4. The question is therefore whether cells that are completely devoid of PS1 and PS2 maintain this γ-secretase activity or not.Unfortunately, PS-null mice die early in embryogenesis (after roughly 9.5 days of development; E9. 5), making it impossible to generate sufficient cells to carry out the required biochemical experiments4. We therefore generated pluripotent lines of embryonic stem cells from PS-null blastocysts obtained by mating of PS1+/–PS2–/–mice4. We genotyped the cell lines using the polymerase chain reaction (PCR) 4 and Southern blotting (Fig. 1a). The 32% yield of double-deficient (PS-null) cell lines was slightly higher than the expected 25%; we confirmed the absence of PS1 (Fig. 1b) and PS2 (data not shown) by western blotting. We observed the accumulation of endogenous β-and α-secretase-cleaved β-APP carboxy-terminal stubs, the direct substrates for γ-secretase (Fig. 1b and ref. 1). We then expressed β-APP695 harbouring the K595N/M596L (Swedish) mutation in the embryonic stem cells, using recombinant Semliki Forest virus (SFV). This mutation markedly increases production of Aβ5, 6, facilitating its detection in our assays. In contrast to results obtained with single-knockout cells, no Aβ was detected in the conditioned media of PS1–/–PS2–/–cells (Fig. 1c). Using serial dilutions, we established that we would have detected Aβ even if its production was< 0.5% of that in wildtype cells (Fig. 1c). We further confirmed the absence of γ-secretase activity using an enzyme-linked immunosorbent assay (ELISA) specific for the 42-amino-acid form of Aβ1. mNotch∆ E is another substrate for PS1-dependent γsecretase7, 8. Processing of this construct mimics that of full-length Notch-1, and results in release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates, together with transcription factors of the CBF-1 family, several genes, including HES-1 (ref. 9). We therefore co-transfected the embryonic stem cells with plasmids encoding mNotch∆ E and luciferase under the control of the HES-1 promoter (kindly provided by R. Kopan and A. Israel), and measured induction of luciferase activity10. As a control, we also transfected embryonic stem cells with plasmids encoding the NICD. Induction of HES-1 activity with mNotch∆ E was observed in PS1+/+ PS2–/–cells, but not in PS1–/–