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
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
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
Wolf ME, Kapatos G (1989) Flow cytometric analysis of rat striatal nerve terminals. J Neurosci 9:94–105
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
Wolf ME, Kapatos G (1989) Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum. J Neurosci 9:106–114
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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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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