Dataset Information

A novel method for isolation of spontaneously-released brain small extracellular vesicles

ABSTRACT: Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing an advantageous tool for Precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity as well diverse individual clinical profile often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles. Based on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we hereby present an efficient purification method that captures a more physiologically relevant, small EV-enriched population spontaneously released by mouse and human brain tissue. The spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Martina Samiotaki

LAB HEAD: Ioannis Sotiropoulos

PROVIDER: PXD031947 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implications for stress-driven brain pathology.

Gomes Patrícia A PA Bodo Cristian C Nogueras-Ortiz Carlos C Samiotaki Martina M Chen Minghao M Soares-Cunha Carina C Silva Joana M JM Coimbra Bárbara B Stamatakis George G Santos Liliana L Panayotou George G Tzouanou Foteini F Waites Clarissa L CL Gatsogiannis Christos C Sousa Nuno N Kapogiannis Dimitrios D Costa-Silva Bruno B Sotiropoulos Ioannis I

Cell communication and signaling : CCS 20230213 1

<h4>Background</h4>Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatmen ...[more]

PMID: 36782237

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Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To understand the role of ARF6 in human ECs, first we compared GFP-AAV treated human iECs differentiated from ARF6-KO vs WT iPSCs, next we compared ARF6-GFP-AAV vs GFP-AAV treated iECs differentiated from WT iPSCs. During the ARF6-KO vs WT iEC comparison we found 983 vs 741 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins revealed mRNA processing among the most significantly affected biological processes. During the ARF6-GFP-AAV vs GFP-AAV treated WT iEC comparison we found 1106 vs 1218 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed endocytic recycling, retrograde transport (endosome to Golgi), and ER-Golgi vesicle-mediated transport among the most significantly affected biological processes. Specifically, ARF6 and its binding-protein GGA2, DNM1L and several subunits of the Conserved oligomeric Golgi complex (COG) were upregulated. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe, a major regulator of brain lipid metabolism. Proteomic analysis of BECs from Apoe deficient mice revealed a signature of accelerated aging. To explore the role of APOE in human endothelial cells, in this experiment we compared human iECs differentiated from APOE-KO and WT iPSCs and found 326 significantly altered proteins. Enrichment analyses of significantly downregulated proteins revealed vesicle-mediated transport and vesicle fusion to be among the most significantly affected biological processes. Specifically, among the 34 significantly altered vesicle-mediated transport proteins 26 were downregulated. Accordingly, we found reduced levels of endocytosis of FM1-43FX in newly formed vesicles in APOE-KO iECs.

Dataset Information

A novel method for isolation of spontaneously-released brain small extracellular vesicles

Publications

A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implications for stress-driven brain pathology.

Similar Datasets

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