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Tau Protein pp 273–284Cite as

Flow Cytometry Analysis and Quantitative Characterization of Tau in Synaptosomes from Alzheimer’s Disease Brains

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1523))

Abstract

Synaptosomes, resealed nerve terminals that form when tissue is homogenized in isotonic medium, are a model system that has been a key source of knowledge about neurotransmission. Synaptosomes contain mitochondria, cytoskeletal proteins, and release neurotransmitters; many have postsynaptic elements. Cryopreservation at the time of autopsy makes it possible to prepare synaptosomes from human samples. Flow cytometry is a powerful analytic technique that precisely measures fluorescence on a cell-by-cell basis, and also indicates particle size and complexity with a routine parameter that measures light scattering. We describe here a procedure for flow cytometry analysis of tau in synaptosomes, a procedure that enables (1) “purification” of synaptosomes from the P-2 fraction (crude synaptosomes) by gating on particle size, and (2) quantitative measure of tau immunofluorescence in individual terminals. Application of flow cytometry to study of synaptosomes has yielded important information, not possible with routine biochemistry, about synaptic pathology in Alzheimer’s disease.

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References

  1. Gouras GK, Tampellini D, Takahashi RH, Capetillo-Zarate E (2010) Intraneuronal beta-amyloid accumulation and synapse pathology in Alzheimer’s disease. Acta Neuropathol 119:523–541. doi:10.1007/s00401-010-0679-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dunkley PR, Jarvie PE, Robinson PJ (2008) A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3:1718–1728. doi:10.1038/nprot.2008.171

    Article  CAS  PubMed  Google Scholar 

  3. Wolf ME, Kapatos G (1989) Flow cytometric analysis of rat striatal nerve terminals. J Neurosci 9:94–105

    CAS  PubMed  Google Scholar 

  4. Woo J, Baumann A, Arguello V (2014) Recent advancements of flow cytometry: new applications in hematology and oncology. Expert Rev Mol Diagn 14:67–81. doi:10.1586/14737159.2014.862153

    Article  CAS  PubMed  Google Scholar 

  5. Wolf ME, Kapatos G (1989) Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum. J Neurosci 9:106–114

    CAS  PubMed  Google Scholar 

  6. Wolf ME, Kapatos G (1989) Stimulation of D2 dopamine receptors decreases intracellular calcium levels in rat anterior pituitary cells but not striatal synaptosomes: a flow cytometric study using indo-1. Synapse 4:353–370. doi:10.1002/syn.890040411

    Article  CAS  PubMed  Google Scholar 

  7. Gylys KH, Fein JA, Cole GM (2000) Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry. J Neurosci Res 61:186–192

    Article  CAS  PubMed  Google Scholar 

  8. Fein JA, Sokolow S, Miller CA, Vinters HV, Yang F, Cole GM, Gylys KH (2008) Co-localization of amyloid beta and tau pathology in Alzheimer’s disease synaptosomes. Am J Pathol 172:1683–1692. doi:10.2353/ajpath.2008.070829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gylys KH, Fein JA, Yang F, Cole GM (2004) Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex. Cytometry A 60:90–96. doi:10.1002/cyto.a.20031

    Article  PubMed  Google Scholar 

  10. Gylys KH, Fein JA, Yang F, Wiley DJ, Miller CA, Cole GM (2004) Synaptic changes in Alzheimer’s disease: increased amyloid-beta and gliosis in surviving terminals is accompanied by decreased PSD-95 fluorescence. Am J Pathol 165:1809–1817

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Battista N, Bari M, Finazzi-Agrò A, Maccarrone M (2002) Anandamide uptake by synaptosomes from human, mouse and rat brain: inhibition by glutamine and glutamate. Lipids Health Dis 1:1

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kanekiyo T, Cirrito JR, Liu C-C, Shinohara M, Li J, Schuler DR, Shinohara M, Holtzman DM, Bu G (2013) Neuronal clearance of amyloid-β by endocytic receptor LRP1. J Neurosci 33:19276–19283. doi:10.1523/JNEUROSCI.3487-13.2013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rausch JL, Johnson ME, Li J, Hutcheson J, Carr BM, Corley KM, Gowans AB, Smith J (2005) Serotonin transport kinetics correlated between human platelets and brain synaptosomes. Psychopharmacology (Berl) 180:391–398. doi:10.1007/s00213-005-2178-6

    Article  CAS  Google Scholar 

  14. Dodd PR, Hardy JA, Baig EB, Kidd AM, Bird ED, Watson WE, Johnston GA (1986) Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain. Neurochem Pathol 4:177–198

    Article  CAS  PubMed  Google Scholar 

  15. Jones PB, Adams KW, Rozkalne A, Spires-Jones TL, Hshieh TT, Hashimoto T, von Armin CAF, Mielke M, Bacskai BJ, Hyman BT (2011) Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain. PLoS One 6, e14586. doi:10.1371/journal.pone.0014586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sokolow S, Luu SH, Headley AJ, Hanson AY, Kim T, Miller CA, Vinters HV, Gylys KH (2011) High levels of synaptosomal Na(+)-Ca(2+) exchangers (NCX1, NCX2, NCX3) co-localized with amyloid-beta in human cerebral cortex affected by Alzheimer’s disease. Cell Calcium 49:208–216. doi:10.1016/j.ceca.2010.12.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sokolow S, Henkins KM, Bilousova T, Gonzalez B, Vinters HV, Miller CA, Cornwell L, Poon WW, Gylys KH (2015) Pre-synaptic C-terminal truncated tau is released from cortical synapses in Alzheimer’s disease. J Neurochem 133:368–379. doi:10.1111/jnc.12991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Begley JG, Butterfield DA, Keller JN, Koppal T, Drake J, Mattson MP (1998) Cryopreservation of rat cortical synaptosomes and analysis of glucose and glutamate transporter activities, and mitochondrial function. Brain Res Brain Res Protoc 3:76–82

    Article  CAS  PubMed  Google Scholar 

  19. Pankiewicz JE, Guridi M, Kim J, Asuni AA, Sanchez S, Sullivan PM, Holtzman DM, Sadowski MJ (2014) Blocking the apoE/Aβ interaction ameliorates Aβ-related pathology in APOE ε2 and ε4 targeted replacement Alzheimer model mice. Acta Neuropathol Commun 2:75. doi:10.1186/s40478-014-0075-0

    PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. UCLA School of Nursing and Mary S. Easton Center for Alzheimer’s Research at UCLA, Box 956919, Factor Building, Los Angeles, CA, 90095-6919, USA

    Karen Hoppens Gylys Ph.D. & Tina Bilousova

Authors
  1. Karen Hoppens Gylys Ph.D.
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  2. Tina Bilousova
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Corresponding author

Correspondence to Karen Hoppens Gylys Ph.D. .

Editor information

Editors and Affiliations

  1. UMR CNRS 8576, Villeneuve d’Ascq, France

    Caroline Smet-Nocca

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Gylys, K.H., Bilousova, T. (2017). Flow Cytometry Analysis and Quantitative Characterization of Tau in Synaptosomes from Alzheimer’s Disease Brains. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 1523. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6598-4_16

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  • DOI: https://doi.org/10.1007/978-1-4939-6598-4_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6596-0

  • Online ISBN: 978-1-4939-6598-4

  • eBook Packages: Springer Protocols

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