Department of Neurology, Richard T Johnson Division of Neuroimmunology and Neurological Infections, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Department of Biological Chemistry, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Glia. 2020 Jan;68(1):128-144. doi: 10.1002/glia.23708. Epub 2019 Aug 30.
Extracellular vesicles have now emerged as key players in cell-to-cell communication. This is particularly important in the central nervous system, where glia-neuron cross-talk helps maintain normal neuronal function. Astrocyte-derived extracellular vesicles (ADEVs) secreted constitutively promote neurite outgrowth and neuronal survival. However, extracellular stimuli can alter the cargo and downstream functions of ADEVs. For example, ADEVs secreted in response to inflammation contain cargo microRNAs and proteins that reduce neurite outgrowth, neuronal firing, and promote neuronal apoptosis. We performed a comprehensive quantitative proteomic analysis to enumerate the proteomic cargo of ADEVs secreted in response to multiple stimuli. Rat primary astrocytes were stimulated with a trophic stimulus (adenosine triphosphate, ATP), an inflammatory stimulus (IL-1β) or an anti-inflammatory stimulus (IL10) and extracellular vesicles secreted within a 2 hr time frame were collected using sequential ultracentrifugation method. ADEVs secreted constitutively without exposure to any stimulus were used a control. A tandem mass tag-based proteomic platform was used to identify and quantify proteins in the ADEVs. Ingenuity pathway analysis was performed to predict the downstream signaling events regulated by ADEVs. We found that in response to ATP or IL10, ADEVs contain a set of proteins that are involved in increasing neurite outgrowth, dendritic branching, regulation of synaptic transmission, and promoting neuronal survival. In contrast, ADEVs secreted in response to IL-1β contain proteins that regulate peripheral immune response and immune cell trafficking to the central nervous system.
细胞外囊泡现在已成为细胞间通讯的关键参与者。这在中枢神经系统中尤为重要，其中神经胶质细胞-神经元的相互作用有助于维持正常的神经元功能。星形胶质细胞衍生的细胞外囊泡（ADEVs）持续分泌，促进神经突生长和神经元存活。然而，细胞外刺激可以改变 ADEVs 的货物和下游功能。例如，响应炎症而分泌的 ADEVs 包含减少神经突生长、神经元放电和促进神经元凋亡的货物 microRNAs 和蛋白质。我们进行了全面的定量蛋白质组学分析，以列举响应多种刺激分泌的 ADEVs 的蛋白质组货物。用营养刺激物（三磷酸腺苷，ATP）、炎症刺激物（IL-1β）或抗炎刺激物（IL10）刺激原代大鼠星形胶质细胞，并使用连续超速离心法收集在 2 小时时间范围内分泌的细胞外囊泡。未暴露于任何刺激而持续分泌的 ADEVs 用作对照。使用串联质量标签（tandem mass tag，TMT）基于蛋白质组学平台来鉴定和定量 ADEVs 中的蛋白质。进行了 IPA 分析以预测 ADEVs 调节的下游信号事件。我们发现，响应于 ATP 或 IL10，ADEVs 包含一组参与增加神经突生长、树突分支、调节突触传递和促进神经元存活的蛋白质。相比之下，响应于 IL-1β 分泌的 ADEVs 包含调节外周免疫反应和免疫细胞向中枢神经系统迁移的蛋白质。
