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.

Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.

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.

			Action	DRS
	211210_JeAU_StTR_nE_LFQ_n1-3_100k_CTL2_2763.d.7z	Other
	211210_JeAU_StTR_nE_LFQ_n1-3_100k_CTL2_2781.d.7z	Other
	211210_JeAU_StTR_nE_LFQ_n1-3_100k_CTL2x3_2796.d.7z	Other
	211210_JeAU_StTR_nE_LFQ_n1-3_100k_CTL3_2782.d.7z	Other
	211210_JeAU_StTR_nE_LFQ_n1-3_100k_CTL4_2783.d.7z	Other

Dataset Information

Extracellular vesicles derived from MPSIII microglia cell model impair neurite growth

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Publications

Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization.

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