Intraneuronal accumulation of amyloid-β (Aβ) is an early pathological signum of Alzheimer’s disease, and compartments of the endolysosomal system have been implicated in both seeding and cell–cell propagation of Aβ aggregation. We have studied how clathrin-independent mechanisms contribute to Aβ endocytosis, exploring pathways that are sensitive to changes in membrane tension and the regulation of Rho GTPases. Using live cell confocal microscopy and flow cytometry, we show the uptake of monomeric Aβ(1-42) into endocytic vesicles and vacuole-like dilations, following relaxation of osmotic pressure-induced cell membrane tension. This indicates Aβ(1-42) uptake via clathrin independent carriers (CLICs), although overexpression of the bar-domain protein GRAF1, a key regulator of CLICs, had no apparent effect. We furthermore report reduced Aβ(1-42) uptake following overexpression of constitutively active forms of the Rho GTPases Cdc42 and RhoA, whereas modulation of Rac1, which is linked to macropinosome formation, had no effect. Our results confirm that uptake of Aβ(1-42) is clathrin- and dynamin-independent and point to the involvement of a new and distinct clathrin-independent endocytic mechanism which is similar to uptake via CLICs or macropinocytosis but that also appear to involve yet uncharacterized molecular players.

中文翻译：

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膜张力敏感的内吞作用和Rho GTPases在摄取阿尔茨海默氏病肽Aβ（1-42）中的作用。

淀粉样蛋白-β（Aβ）的神经内神经蓄积是阿尔茨海默氏病的早期病理征兆，并且溶酶体系统的各个区室与Aβ聚集的播种和细胞间传播都有牵连。我们研究了不依赖网格蛋白的机制如何促进Aβ内吞，探索了对膜张力变化和Rho GTPases调节敏感的途径。使用活细胞共聚焦显微镜和流式细胞仪，我们显示了在渗透压引起的细胞膜张力松弛后，单体Aβ（1-42）被摄入内吞囊泡和液泡样扩张。这表明通过网格蛋白独立载体（CLIC）吸收Aβ（1-42），尽管过表达棒域蛋白GRAF1（CLIC的关键调节剂）没有明显作用。我们还报告说，Rho GTPases Cdc42和RhoA的组成型活性形式过表达后，Aβ（1-42）摄取减少，而与大胶体形成有关的Rac1调节则没有作用。我们的结果证实，Aβ（1-42）的摄取与网格蛋白和动力蛋白无关，并指出了一种新的独特的与网格蛋白无关的内吞机制的参与，该机制类似于通过CLIC或巨胞饮的摄取，但似乎还涉及没有特征的分子参与者。