Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA.
Neurodegener Dis. 2018;18(1):26-37. doi: 10.1159/000486199. Epub 2018 Feb 7.
Sorting nexins (SNXs) have diverse functions in protein sorting and membrane trafficking. Recently, single-nucleotide polymorphisms in SNX3 were found to be associated with Alzheimer disease. However, it remains unknown whether SNX3 participates in amyloid (A)β peptide production.
To examine the role of SNX3 in Aβ production and APP processing.
The effect of increased expression of SNX3 was studied in HEK293T cells. Aβ peptides were measured by immunoassay. Protein-protein association was analyzed by a bimolecular fluorescence complementation (BiFC) assay. APP uptake was measured with an α-bungarotoxin-binding assay, and flow cytometry was used to measure cell surface APP levels.
We found that overexpression of SNX3 in HEK293T cells decreases the levels of secreted Aβ and soluble N-terminal APP fragments (sAPPβ). The reduction correlated with a decreased association of APP with BACE1, as revealed by BiFC. This effect may, in part, be explained by a reduced internalization of APP; SNX3 overexpression reduced APP internalization as determined by an α-bungarotoxin-binding assay, and caused increased APP levels on the cell surface, as shown by flow cytometry. In addition, SNX3 overexpression increased the cellular levels of full-length APP.
These results provide evidence that SNX3 regulates Aβ production by influencing the internalization of APP.
分选连接蛋白（SNXs）在蛋白质分选和膜运输中具有多种功能。最近，发现 SNX3 的单核苷酸多态性与阿尔茨海默病有关。然而，SNX3 是否参与淀粉样β肽（Aβ）的产生仍不清楚。
研究 SNX3 在 Aβ 产生和 APP 加工中的作用。
在 HEK293T 细胞中研究了 SNX3 表达增加的影响。通过免疫测定法测量 Aβ 肽。通过双分子荧光互补（BiFC）测定分析蛋白质-蛋白质相互作用。用α-银环蛇毒素结合测定法测量 APP 摄取，并用流式细胞术测量细胞表面 APP 水平。
我们发现，HEK293T 细胞中 SNX3 的过表达降低了分泌的 Aβ 和可溶性 N 端 APP 片段（sAPPβ）的水平。这种减少与 APP 与 BACE1 的结合减少有关，BiFC 揭示了这一点。这种作用部分可以通过 APP 的内化减少来解释；SNX3 的过表达通过 α-银环蛇毒素结合测定法减少了 APP 的内化，并且通过流式细胞术显示细胞表面的 APP 水平增加。此外，SNX3 的过表达增加了全长 APP 的细胞内水平。
这些结果提供了证据表明 SNX3 通过影响 APP 的内化来调节 Aβ 的产生。
