Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein.
Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein.
复制标题
DOI：
10.3389/fnins.2017.00172
复制
发表时间：
2017
影响因子：
4.3
通讯作者：
McLean PJ
中科院分区：
文献类型：
--
作者：
Delenclos M;Trendafilova T;Mahesh D;Baine AM;Moussaud S;Yan IK;Patel T;McLean PJ
导致细胞毒性的α-突触核蛋白（αsyn）的错误折叠和聚集是帕金森病（PD）和相关突触核蛋白病的标志。最近的大量证据表明，αsyn可以通过涉及细胞外囊泡（EV）（如外泌体）的非常规胞吐作用从神经元细胞中释放。α-syn在细胞间的转移被认为是PD疾病传播的重要机制。迄今为止，外泌体运输机制，包括释放和细胞-细胞传递，尚未完全描述。为了深入了解所涉及的机制，从分泌αsyn寡聚体的稳定细胞的条件培养基中纯化外泌体。一种新的双分子蛋白互补分析用于检测含有αsyn寡聚体的外泌体。用含有α-syn寡聚体或“游离”非外泌体相关α-syn寡聚体的外泌体处理p53细胞，并监测内化。我们证明了细胞来源的外泌体相关的αsyn寡聚体可以被受体细胞有效地内化。有趣的是，从条件培养基中分离的无外泌体的α-syn寡聚体没有内化，而是保持与细胞外表面结合。为了研究外泌体摄取所需的内吞途径，使用了小窝蛋白依赖性、网格蛋白依赖性和巨胞饮途径的不同药理学抑制剂。令人惊讶的是，这些途径似乎都没有在外泌体相关的αsyn寡聚体的内化中发挥重要作用。最后，硫酸肝素蛋白聚糖（HSPGs）的作用外泌体相关的αsyn内化进行了研究，使用遗传学方法。尽管先前的研究表明HSPG可以调节纤维状αsyn的内化，但在我们的手中，遗传操作并没有减弱外泌体相关αsyn寡聚体的内化，这表明外泌体相关αsyn通过另一种内吞途径内化，该途径尚未阐明。
Misfolding and aggregation of alpha-synuclein (αsyn) resulting in cytotoxicity is a hallmark of Parkinson's disease (PD) and related synucleinopathies. The recent body of evidence indicates that αsyn can be released from neuronal cells by nonconventional exocytosis involving extracellular vesicles (EVs) such as exosomes. The transfer of αsyn between cells has been proposed to be an important mechanism of disease propagation in PD. To date, exosome trafficking mechanisms, including release and cell-cell transmission, have not been fully described. To gain insight into the mechanisms involved, exosomes were purified from conditioned media of stable cells secreting αsyn oligomers. A novel bimolecular protein complementation assay was used to detect exosomes containing αsyn oligomers. Recipient cells were treated with exosomes containing αsyn oligomers or “free” non-exosome-associated αsyn oligomers and internalization was monitored. We demonstrate that cell-derived exosome-associated αsyn oligomers can be efficiently internalized by recipient cells. Interestingly exosome-free αsyn oligomers isolated from conditioned medium were not internalized but remained bound to the extracellular surface. To investigate the endocytic pathway(s) required for the exosome uptake different pharmacological inhibitors of caveolin-dependent, clathrin-dependent, and macropinocytosis pathways were utilized. Surprisingly, none of these pathways appear to play a significant role in the internalization of exosome-associated αsyn oligomers. Finally, the role of heparin sulfate proteoglycans (HSPGs) in exosome-associated αsyn internalization was investigated using genetic approach. Despite previous studies showing HSPGs can modulate internalization of fibrillar αsyn, genetic manipulations did not attenuate internalization of exosome-associated αsyn oligomers in our hands, suggesting that exosome-associated αsyn is internalized via an alternative endocytic pathway(s) that has yet to be elucidated.