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Proteome analysis of epithelial-mesenchymal transition extracellular vesicles: exosomes, microparticles and shed midbody remnants

ABSTRACT: Cell-derived extracellular vesicles (EVs) are evolutionary-conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs) and shed midbody remnants (lEVs/sMB-Rs). Here we conducted large-scale purification of exosomes, MPs and sMBRs isolated from MDCK and 21D1 cells, and performed comprehensive proteomics analysis to differentiate these EV subtypes. Findings from this study suggests that sMBRs may hold crucial clues to the regulation of EMT and the involvement of different subtypes of EVs based on their dynamic proteome landscape in this process.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Canis Lupus Familiaris (ncbitaxon:9615)

SUBMITTER: David Greening

PROVIDER: MSV000093044 | MassIVE | Wed Oct 04 20:44:00 BST 2023

REPOSITORIES: MassIVE

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Project description:Background : In mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described. Methods : Here, we isolated EVs secreted by the microglial BV-2 cells after treatment with GAGs purified from urines of Sanfilippo patients (MPS-GAGs EVs) or from age-matched healthy subjects (WT-GAGs EVs) to explore the EVs’ proteins and small RNA profiles using LC–MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following WT-GAGs- or MPS-GAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy. Results : Functional enrichment analysis for both proteomics and RNA sequencing data from MPS-GAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with MPS-GAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines. Conclusions : This study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies.

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Proteome analysis of epithelial-mesenchymal transition extracellular vesicles: exosomes, microparticles and shed midbody remnants

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