Fas-associated factor 1 (FAF1) has been implicated in Parkinson’s disease (PD) and activates the cell death machinery in the cytosol. However, the presence of extracellular FAF1 has not been studied. Serum-free conditioned medium (CM) from FAF1-transfected SH-SY5Y cells was concentrated and analyzed by western blotting. Exosomes were isolated from CM by ultracentrifugation and analyzed by western blotting, electron microscopy and nanoparticle tracking analysis. Soluble FAF1 from CM was immunodepleted using anti-FAF1 antibody. Transmission of secreted FAF1 was examined by transwell assay under a confocal microscope. CM-induced cell death was determined by measuring propidium iodide (PI) uptake using a flow cytometer. FAF1 was secreted from SH-SY5Y cells via exocytosis and brefeldin A (BFA)-resistant secretory pathways. Furthermore, FAF1 was secreted as a vesicle-free form and a genuine exosome cargo in the lumen of exosomes. In addition, FAF1 increased the number of exosomes, suggesting a regulatory role in exosome biogenesis. Extracellular FAF1 was transmitted via endocytosis to neighboring cells, where it induced cell death through apoptotic and necrotic pathways. This study presents a novel route by which FAF1 induces neuronal death through cell-to-cell transmission.

中文翻译：

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FAF1 的新功能，通过细胞间传递诱导多巴胺能神经元死亡。

Fas 相关因子 1 (FAF1) 与帕金森病 (PD) 有关联，并激活细胞质中的细胞死亡机制。然而，尚未研究细胞外 FAF1 的存在。来自 FAF1 转染的 SH-SY5Y 细胞的无血清条件培养基 (CM) 被浓缩并通过蛋白质印迹分析。通过超速离心从 CM 中分离外泌体，并通过蛋白质印迹、电子显微镜和纳米颗粒跟踪分析进行分析。来自 CM 的可溶性 FAF1 使用抗 FAF1 抗体进行免疫消耗。在共聚焦显微镜下通过 transwell 测定检查分泌的 FAF1 的传输。CM 诱导的细胞死亡是通过使用流式细胞仪测量碘化丙啶 (PI) 摄取来确定的。FAF1 通过胞吐作用和布雷菲尔德菌素 A (BFA) 抗性分泌途径从 SH-SY5Y 细胞分泌。此外，FAF1 以无囊泡形式分泌，是外泌体腔中真正的外泌体货物。此外，FAF1 增加了外泌体的数量，表明在外泌体生物发生中具有调节作用。细胞外 FAF1 通过内吞作用传递到邻近细胞，在那里它通过凋亡和坏死途径诱导细胞死亡。这项研究提出了 FAF1 通过细胞间传递诱导神经元死亡的新途径。