Background We recently demonstrated an endolysosomal accumulation of the β-secretase-derived APP C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models. Moreover, we showed that the treatment with the γ-secretase inhibitor (D6) led to further increased endolysosomal APP-CTF levels, but also revealed extracellular APP-CTF-associated immunostaining. We here hypothesized that this latter staining could reflect extracellular vesicle (EV)-associated APP-CTFs and aimed to characterize these γ-secretase inhibitor-induced APP-CTFs. Methods EVs were purified from cell media or mouse brains from vehicle- or D6-treated C99 or APPswedish expressing cells/mice and analyzed for APP-CTFs by immunoblot. Combined pharmacological, immunological and genetic approaches (presenilin invalidation and C99 dimerization mutants (GXXXG)) were used to characterize vesicle-containing APP-CTFs. Subcellular APP-CTF localization was determined by immunocytochemistry. Results Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains. Surprisingly, EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight (HMW) APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts. Accordingly, the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869. By combined pharmacological, immunological and genetic approaches, we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83. Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network, whereas oligomers were confined to endosomes and lysosomes, thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs. The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due to γ-secretase blockade, since it similarly occurred in presenilin-deficient fibroblasts. Further, our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis, γ-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN. Conclusions This is the first study to demonstrate the presence of oligomeric APP-CTFs in AD mouse models, the levels of which are selectively enriched in endolysosomal compartments including exosomes and amplified by γ-secretase inhibition. Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset, progression and spreading.

中文翻译：

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靶向 γ-分泌酶触发了来自阿尔茨海默细胞和小鼠模型的脑细胞外囊泡中寡聚 APP-CTF 的选择性富集。

背景 我们最近在阿尔茨海默病 (AD) 小鼠模型的大脑中证明了 β-分泌酶衍生的 APP C 末端片段 (CTF) C99 的内溶酶体积累。此外，我们发现用 γ-分泌酶抑制剂 (D6) 治疗导致内溶酶体 APP-CTF 水平进一步增加，但也揭示了细胞外 APP-CTF 相关的免疫染色。我们在这里假设后一种染色可以反映细胞外囊泡 (EV) 相关的 APP-CTF，并旨在表征这些 γ-分泌酶抑制剂诱导的 APP-CTF。方法 从载体或 D6 处理的 C99 或 APPswedish 表达细胞/小鼠的细胞培养基或小鼠脑中纯化 EV，并通过免疫印迹分析 APP-CTF。结合药理，免疫学和遗传学方法（早老素失效和 C99 二聚化突变体 (GXXXG)）用于表征含有囊泡的 APP-CTF。通过免疫细胞化学确定亚细胞APP-CTF定位。结果 与来自对照细胞/大脑的 EV 相比，来自 AD 细胞或小鼠模型的纯化 EV 富含 APP-CTF。令人惊讶的是，来自 D6 处理细胞的 EV 不仅显示出增加的 C99 和 C99 衍生的 C83 水平，而且还显示出在全细胞提取物中几乎无法检测到的更高分子量 (HMW) APP-CTF 免疫反应性。因此，HMW APP-CTFs 的细胞内水平被外泌体抑制剂 GW4869 放大。通过结合药理学、免疫学和遗传学方法，我们确定这些 HMW APP-CTF 对应于由 C99 和/或 C83 组成的寡聚 APP-CTF。免疫细胞化学分析表明，单体主要定位于跨高尔基网络，而寡聚体仅限于内体和溶酶体，从而为电动汽车中 HMW APP-CTF 的选择性恢复提供了解剖学支持。D6 诱导的 APP-CTF 寡聚化和亚细胞错误定位确实是由于 γ-分泌酶阻断，因为它同样发生在早老素缺陷的成纤维细胞中。此外，我们的数据表明，除了通过阻止 C99 蛋白水解来促进 APP-CTF 寡聚化之外，抑制 γ-分泌酶还导致 SorLA 介导的 HMW APP-CTF 从内体区室逆行转运到 TGN 的缺陷。结论 这是第一项证明 AD 小鼠模型中存在寡聚 APP-CTF 的研究，其水平选择性地富集在内溶酶体区室中，包括外泌体，并通过抑制γ-分泌酶而放大。未来的研究应该评估这些外泌体相关的 APP-CTF 在 AD 发病、进展和传播中的假定贡献。