Abstract

ABSTRACT Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Highlights

  • Extracellular vesicles (EVs) are nano-sized, lipid bilayer-delimited particles that are released by various cells

  • We evaluated the effects of tissue perfusion and post-mortem interval before final brain-derived EVs (bdEVs) extraction on various parameters of the recovered bdEVs

  • It should noted that this 10K fraction contains EVs of various sizes, but that purity might be low and that some large EVs were likely removed by the prior filtration

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Summary

Introduction

Extracellular vesicles (EVs) are nano-sized, lipid bilayer-delimited particles that are released by various cells. They can package and deliver molecules such as RNAs and proteins and are involved in multiple physiological and pathological pathways by serving as messengers in cell-tocell communication. EVs are released by all neural cells[1,2,3], including neurons, oligodendrocytes, 21 astrocytes, and microglia. They can carry disease-associated agents such as amyloid-beta (Aβ)[4,5,6,7] and tau[6,7,8] proteins, which may promote neurodegenerative and inflammatory diseases. EVs may exert protective functions in the CNS by distributing anti-inflammatory factors[9,10,11]

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