Department of Biochemistry, University of Zurich , Winterthurerstrasse 190, CH-8057 Zurich , Switzerland.
Biol Open. 2012 Aug 15;1(8):761-74. doi: 10.1242/bio.20121578. Epub 2012 Jun 27.
Endocytosis of amyloid-β precursor protein (APP) is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.
内吞淀粉样前体蛋白（APP）被认为是β-分泌酶 BACE1 产生淀粉样蛋白 Aβ肽的主要底物来源。蛋白水解裂解的不可逆性意味着 APP 从其合成部位到细胞表面存在有效的补充途径。我们最近发现，APP 与钙连蛋白-1 密切相关地离开反式高尔基体网络，钙连蛋白-1 是一种跨膜货物对接蛋白，用于 Kinesin-1 介导的囊泡运输。在这里，我们使用细胞内运输细胞器的选择性免疫分离、免疫细胞化学、实时成像和 RNAi 来描述钙连蛋白-1在神经元 APP 运输中的功能。我们发现 APP 与钙连蛋白-1一起沿着轴突被共运输到生长锥中央区域的早期内体，这些载体排除了α-分泌酶 ADAM10。有趣的是，钙连蛋白-1/APP 细胞器包含 BACE1，表明 APP 在其顺行路径上过早切割。然而，我们发现钙连蛋白-1/APP 细胞器中的 APP 是稳定的。我们进一步分析了培养的海马神经元中 APP 的囊泡运输，其中钙连蛋白-1通过 RNAi 减少。我们发现 APP 与 ADAM10 在轴突和生长锥中的共定位明显增加，同时 APP 的蛋白水解加工和 Aβ分泌增加。这表明，钙连蛋白-1依赖性 APP 运输能力的降低导致 APP 错误分拣到额外的轴突载体中，因此，未受保护的 APP 与它的细胞外结构域蛋白酶过早相遇。综上所述，我们的结果将钙连蛋白-1/APP 细胞器描述为 APP 受保护的顺行轴突运输载体。
