背景:载脂蛋白E4(APOE4)是阿尔茨海默病最普遍的遗传危险因素。一些研究表明,APOE4与其受体的结合与它们在细胞内区室中的内在化和积累有关。重要的是,这种现象也发生在其他人身上,非ApoE受体。基于这些观察,我们假设APOE4病理效应是由不同受体(APOER2,LRP1,IR,VEGFR)。
目的:研究APOE基因型对受体蛋白水平和区室化的影响。
方法:从APOE3或APOE4靶向替代小鼠制备原代小鼠神经元,或者APOE-KO小鼠.在这些神经元中评估了特异性受体蛋白水平,利用免疫荧光染色。此外,通过细胞表面生物素化测定和ELISA评估这些受体的表面膜蛋白水平。通过双重染色和共聚焦显微镜评估受体与细胞内区室的共定位,其次是共定位分析。最后,用CRISPR/Cas9系统敲低LRP1或APOER2以检查它们在介导APOE4对受体的作用中的作用。
结果:我们的结果显示APOE4受体水平较低,特别是在膜表面。此外,APOE4以两种模式影响这些受体的分隔:第一种是在LRP1中观察到的,并且与许多细胞内区室中受体水平降低有关。第二个是与其他受体一起获得的,并且与它们在早期内体中的积累和在晚期内体中的减少有关。
结论:这些结果提供了一个统一的机制,其中APOE4驱动各种受体的下调,在不同的APOE4相关病理过程中发挥重要作用。
BACKGROUND: Apolipoprotein E4 (APOE4) is the most prevalent genetic risk factor of Alzheimer\'s disease. Several studies suggest that APOE4 binding to its receptors is associated with their internalization and accumulation in intracellular compartments. Importantly, this phenomenon also occurs with other, non-ApoE receptors. Based on these observations, we hypothesized that APOE4 pathological effects are mediated by impairment in the life cycle of distinct receptors (APOER2, LRP1, IR, VEGFR).
OBJECTIVE: To examine the effects of APOE genotype on receptors protein levels and compartmentalization.
METHODS: Primary mouse neurons were prepared from APOE3 or APOE4 targeted replacement mice, or APOE-KO mice. Specific receptors protein levels were evaluated in these neurons, utilizing immunofluorescent staining. Additionally, surface membrane protein levels of those receptors were assessed by cell surface biotinylation assay and ELISA. Receptors\' colocalization with intracellular compartments was assessed by double staining and confocal microscopy, followed by colocalization analysis. Finally, LRP1 or APOER2 were knocked-down with CRISPR/Cas9 system to examine their role in mediating APOE4 effects on the receptors.
RESULTS: Our results revealed lower receptors\' levels in APOE4, specifically on the membrane surface. Additionally, APOE4 affects the compartmentation of these receptors in two patterns: the first was observed with LRP1 and was associated with decreased receptor levels in numerous intracellular compartments. The second was obtained with the other receptors and was associated with their accumulation in early endosomes and their decrease in the late endosomes.
CONCLUSIONS: These results provide a unifying mechanism, in which APOE4 drives the down regulation of various receptors, which plays important roles in distinct APOE4 related pathological processes.