Project description:Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.

Project description:Ectopic adenosine triphosphate synthase (eATP synthase) was found on the cell surface to generate extracellular ATP in various cancer cells. Recently using proteomics approach, several reports identified ATP synthase in extracellular vesicles (EVs) which are important in cell-cell communications. However, the functions of ATP synthase in EVs are still unclear. Here we increased the expression of eATP synthase on the plasma membrane of lung cancer A549 cells via 0.1% FBS starvation and collected EVs including microvesicles and exosomes from the culture medium using serial centrifugation. Both of them were assuredly isolated and confirmed by transmission electron microscopy, nano tracking analysis, and specific EV markers. We also used western blot to demonstrate the presence of ATP synthase in EVs. Besides, dot blot experiment revealed that the F1 portion faced towards extracellular space using anti-ATP beta subunit antibody. Under starvation, the expression of eATP synthase was enhanced on cell surface and exosomes as well as increased EV secretion. To further investigate the functions, we first elucidated whether eATP synthase on EV membrane produced ATP by ATP luciferase analysis. Our data showed that there was no significant ATP production. Moreover, we performed proteomic analysis using dimethyl labeling and LC-MS/MS to compare the difference between EVs released by A549 control and starvation. We identified a total of 1504 and 869 proteins in microvesicles and exosomes, respectively. Using 1.5- and 0.67-fold change as the thresholds, there were 171 and 46 differential expressed proteins in microvesicles and exosomes, respectively. Seven ATP synthase subunits in microvesicles and five in exosomes were identified. The proteomic data were further analyzed using gene set enrichment analysis (GSEA) within the C2 (curated gene sets) subclasses and setting 0.25 as FDR cutoff to find out biological functions related to eATP synthase. Among these up-regulated terms, metabolism, cell cycle and apoptosis were enriched, which indicating that starvation influences cell behavior. Additionally, the immune response is upregulated strikingly in exosomes within C2 gene set. One of these immune response pathways, MHC-1 antigen presentation, was previously proved to interact with eATP synthase which could be recognized by several immune cells.

Dataset Information

ATP MODIFIES THE PROTEOME OF EXTRACELLULAR VESICLES RELEASED BY MICROGLIA AND INFLUENCES THEIR ACTION ON ASTROCYTES

Publications

ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes.

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