Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space. Here we describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin. We also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, we compared this novel iodixanol-based high-resolution density step gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered was similar, the iodixanol-based gradient better separated distinct EV species as compared with the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.
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All data needed to evaluate the conclusions of the paper are present in the paper. No datasets or custom code were generated in this study. All single datapoints are reported in the respective graphs, when possible (Figs. 4,5) and as Excel Source Data files. Raw, uncropped blots for Figs. 2,3 are provided as pdf source data files. Additional data related to this paper may be requested from the authors. Source data are provided with this paper.
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This work was supported by the National Institute on Aging (grant numbers AG017617, AG056732 and AG057517) and the National Institute on Drug Abuse (grant number DA044489). The authors thank M. Pawlik and S. DeRosa for the animal husbandry and G. Ferrari for coordinating and managing our laboratory.
The authors declare no competing interests.
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Nature Protocols thanks Éric Boilard, Ashok Shetty and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
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Extended Data Fig. 1 Purified brain EVs fixed with PFA and stained with uranyl acetate show a distinctive cup-shape morphology.
Representative photomicrograph of sucrose fraction c brain EVs isolated from a 12-month-old female mouse and visualized after negative stain. Note the cup shape of fixed EVs and the absence of contaminating material. Scale bar, 200 nm. All animal procedures were performed following the National Institutes of Health guidelines with approval from the Institutional Animal Care and Use Committee at the Nathan S. Kline Institute for Psychiatric Research.
Extended Data Fig. 2 Representative cryo-EM photomicrograph of contaminating crystals during acquisition of mitovesicles (iodixanol fraction 8 EVs).
Crystals are visualized as electron-dense bodies that are either amorphous (orange arrowheads) or polygonal, for instance, hexagonal or cubic (red arrowheads). The white arrow indicates a typical mitovesicle, which is characterized by the presence of a double membrane. The amorphous and polygonal dark structures in this case are the same contaminant, caused by moisture from the air that has frozen during the freezing process. Scale bar, 200 nm. All animal procedures were performed following the National Institutes of Health guidelines with approval from the Institutional Animal Care and Use Committee at the Nathan S. Kline Institute for Psychiatric Research.
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D’Acunzo, P., Kim, Y., Ungania, J.M. et al. Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues. Nat Protoc 17, 2517–2549 (2022). https://doi.org/10.1038/s41596-022-00719-1
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