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Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues

Abstract

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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Fig. 1: Overview of the procedure used to isolate brain EV subpopulations.
Fig. 2: A 0.22 µm filtration step is crucial for EV quality and yield.
Fig. 3: High-resolution iodixanol-based column fractionates subtypes of EVs that are not separated by sucrose-based column.
Fig. 4: NTA analyses of sucrose and iodixanol-based EV fractions.
Fig. 5: Mitovesicles kept at 37 °C produce ATP through the electron transport chain.

Data Availability

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.

References

  1. van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).

    PubMed  Google Scholar 

  2. Del Conde, I., Shrimpton, C. N., Thiagarajan, P. & Lopez, J. A. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 106, 1604–1611 (2005).

    PubMed  Google Scholar 

  3. D’Acunzo, P. et al. Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome. Sci. Adv. https://doi.org/10.1126/sciadv.abe5085 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    CAS  PubMed  Google Scholar 

  5. Perez-Gonzalez, R. et al. Neuroprotection mediated by cystatin C-loaded extracellular vesicles. Sci. Rep. 9, 11104 (2019).

    PubMed  PubMed Central  Google Scholar 

  6. Kaur, G. et al. Cystatin C prevents neuronal loss and behavioral deficits via the endosomal pathway in a mouse model of down syndrome. Neurobiol. Dis. 120, 165–173 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Liu, S., Hossinger, A., Gobbels, S. & Vorberg, I. M. Prions on the run: how extracellular vesicles serve as delivery vehicles for self-templating protein aggregates. Prion 11, 98–112 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Guo, B. B., Bellingham, S. A. & Hill, A. F. The neutral sphingomyelinase pathway regulates packaging of the prion protein into exosomes. J. Biol. Chem. 290, 3455–3467 (2015).

    CAS  PubMed  Google Scholar 

  9. Guo, B. B., Bellingham, S. A. & Hill, A. F. Stimulating the release of exosomes increases the intercellular transfer of prions. J. Biol. Chem. 291, 5128–5137 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhang, X. et al. Potential transfer of polyglutamine and CAG-repeat RNA in extracellular vesicles in Huntington’s disease: background and evaluation in cell culture. Cell Mol. Neurobiol. 36, 459–470 (2016).

    PubMed  PubMed Central  Google Scholar 

  11. Guix, F. X. et al. Detection of aggregation-competent tau in neuron-derived extracellular vesicles. Int. J. Mol. Sci. https://doi.org/10.3390/ijms19030663 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Perez-Gonzalez, R., Gauthier, S. A., Kumar, A. & Levy, E. The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space. J. Biol. Chem. 287, 43108–43115 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Perez-Gonzalez, R. et al. A pleiotropic role for exosomes loaded with the amyloid beta precursor protein carboxyl-terminal fragments in the brain of Down syndrome patients. Neurobiol. Aging 84, 26–32 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Perez-Gonzalez, R. et al. Extracellular vesicles: where the amyloid precursor protein carboxyl-terminal fragments accumulate and amyloid-β oligomerizes. FASEB J. 34, 12922–12931 (2020).

    CAS  PubMed  Google Scholar 

  15. Mathews, P. M. & Levy, E. Exosome production is key to neuronal endosomal pathway integrity in neurodegenerative diseases. Front. Neurosci. 13, 1347 (2019).

    PubMed  PubMed Central  Google Scholar 

  16. Gauthier, S. A. et al. Enhanced exosome secretion in Down syndrome brain—a protective mechanism to alleviate neuronal endosomal abnormalities. Acta Neuropathol. Commun. 5, 65 (2017).

    PubMed  PubMed Central  Google Scholar 

  17. D’Acunzo, P. et al. Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction. Dev. Neurobiol. 79, 656–663 (2019).

    PubMed  PubMed Central  Google Scholar 

  18. Peng, K. Y. et al. Apolipoprotein E4 genotype compromises brain exosome production. Brain 142, 163–175 (2019).

    PubMed  Google Scholar 

  19. Barreto, B. R. et al. Cocaine modulates the neuronal endosomal system and extracellular vesicles in a sex-dependent manner. Neurochem. Res. https://doi.org/10.1007/s11064-022-03612-1 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Cataldo, A. M. et al. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer’s disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am. J. Pathol. 157, 277–286 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang, Y. et al. Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1. Nat. Commun. 12, 1731 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Nuriel, T. et al. The endosomal–lysosomal pathway is dysregulated by APOE4 expression in vivo. Front. Neurosci. 11, 702 (2017).

    PubMed  PubMed Central  Google Scholar 

  23. Norman, M. et al. L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma. Nat. Methods 18, 631–634 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Melki, I. et al. Platelets release mitochondrial antigens in systemic lupus erythematosus. Sci. Transl. Med. https://doi.org/10.1126/scitranslmed.aav5928 (2021).

    Article  PubMed  Google Scholar 

  25. Thul, P. J. et al. A subcellular map of the human proteome. Science https://doi.org/10.1126/science.aal3321 (2017).

    Article  PubMed  Google Scholar 

  26. Uhlen, M. et al. Proteomics. Tissue-based map of the human proteome. Science 347, 1260419 (2015).

    PubMed  Google Scholar 

  27. Uhlen, M. et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248–1250 (2010).

    CAS  PubMed  Google Scholar 

  28. Kim, K. M. et al. Mitochondrial RNA in Alzheimer’s disease circulating extracellular vesicles. Front. Cell Dev. Biol. 8, 581882 (2020).

    PubMed  PubMed Central  Google Scholar 

  29. Liangsupree, T., Multia, E. & Riekkola, M. L. Modern isolation and separation techniques for extracellular vesicles. J. Chromatogr. A 1636, 461773 (2021).

    CAS  PubMed  Google Scholar 

  30. Perez-Gonzalez, R. et al. A method for isolation of extracellular vesicles and characterization of exosomes from brain extracellular space. Methods Mol. Biol. 1545, 139–151 (2017).

    CAS  PubMed  Google Scholar 

  31. Vella, L. J. et al. A rigorous method to enrich for exosomes from brain tissue. J. Extracell. Vesicles 6, 1348885 (2017).

    PubMed  PubMed Central  Google Scholar 

  32. Ruan, Z. et al. Alzheimer’s disease brain-derived extracellular vesicles spread tau pathology in interneurons. Brain https://doi.org/10.1093/brain/awaa376 (2020).

    Article  PubMed Central  Google Scholar 

  33. Crescitelli, R., Lasser, C. & Lotvall, J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat. Protoc. 16, 1548–1580 (2021).

    CAS  PubMed  Google Scholar 

  34. Brinkman, J. E. & Sharma, S. Physiology, Body Fluids. In StatPearls (2020).

  35. Qian, X. et al. Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 28, 69–80 (2000).

    CAS  PubMed  Google Scholar 

  36. Brewer, G. J. & Torricelli, J. R. Isolation and culture of adult neurons and neurospheres. Nat. Protoc. 2, 1490–1498 (2007).

    CAS  PubMed  Google Scholar 

  37. Lam, D. M. Biosynthesis of acetylcholine in turtle photoreceptors. Proc. Natl Acad. Sci. USA 69, 1987–1991 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Miyazawa, T. Enzymatic resolution of amino acids via ester hydrolysis. Amino Acids 16, 191–213 (1999).

    CAS  PubMed  Google Scholar 

  39. Varughese, K. I. et al. Crystal structure of a papain-E-64 complex. Biochemistry 28, 1330–1332 (1989).

    CAS  PubMed  Google Scholar 

  40. Hussain, R. Z. et al. Defining standard enzymatic dissociation methods for individual brains and spinal cords in EAE. Neurol. Neuroimmunol. Neuroinflamm. 5, e437 (2018).

    PubMed  PubMed Central  Google Scholar 

  41. Volovitz, I. et al. A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells. BMC Neurosci. 17, 30 (2016).

    PubMed  PubMed Central  Google Scholar 

  42. Bonneh-Barkay, D. & Wiley, C. A. Brain extracellular matrix in neurodegeneration. Brain Pathol. 19, 573–585 (2009).

    CAS  PubMed  Google Scholar 

  43. Crescitelli, R. et al. Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. J. Extracell. Vesicles 9, 1722433 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Pérez-González, R., Gauthier, S. A., Kumar, A. & Levy, E. The exosome-secretory pathway transports amyloid precursor protein carboxyl terminal fragments from the cell into the brain extracellular space. J. Biol. Chem. 287, 43108–43115 (2012).

    PubMed  PubMed Central  Google Scholar 

  45. Corballis, M. C. Left brain, right brain: facts and fantasies. PLoS Biol. 12, e1001767 (2014).

    PubMed  PubMed Central  Google Scholar 

  46. Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445 e418 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kugeratski, F. G. et al. Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker. Nat. Cell Biol. 23, 631–641 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Rao, L., Perez, D. & White, E. Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135, 1441–1455 (1996).

    CAS  PubMed  Google Scholar 

  49. Thery, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Chuo, S. T., Chien, J. C. & Lai, C. P. Imaging extracellular vesicles: current and emerging methods. J. Biomed. Sci. 25, 91 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization: P.D. and E.L.; methodology: P.D., Y.K. and R.P.-G.; formal analysis: P.D.; investigation: P.D., C.G. and J.M.U.; writing original and revised drafts: P.D. and E.L.; visualization: P.D. and E.L.; supervision: E.L.; project administration: E.L. and P.D.; funding acquisition: E.L.

Corresponding author

Correspondence to Efrat Levy.

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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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Related links

Key references using this protocol

D’Acunzo, P. et al. Sci. Adv. 7, eabe5085 (2021): https://doi.org/10.1126/sciadv.abe5085

Zhang, Y. et al. Nat. Commun. 12, 1731 (2021): https://doi.org/10.1038/s41467-021-22003-8

Perez-Gonzalez, R. et al. J. Biol. Chem. 287, 43108–43115 (2012): https://doi.org/10.1074/jbc.M112.404467

Extended data

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.

Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

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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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