Flow Cytometry Analysis and Quantitative Characterization of Tau in Synaptosomes from Alzheimer's Disease Brains - PubMed (original) (raw)

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

Karen Hoppens Gylys et al. Methods Mol Biol. 2017.

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.

Keywords: Cytoskeleton; FACs; Flow cytometry; HT7 antibody; Neurodegeneration; Tau fragment.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Synaptosome preparation. (a) Homogenization of fresh brain tissue in isotonic sucrose solution induces formation of synaptosomes—avulsion and resealing of synaptic terminals, which may contain some postsynaptic elements and sometimes astrocyte end-feet. (b) Schematic illustration of synaptosome-enriched (P2) fraction preparation from brain homogenate

Fig. 2

Fig. 2

Flow cytometry analysis of synaptosomal preparations. (a) An example of size standard acquisition for FSC-based size gate establishment. (b) Background labeling in presence of isotype control antibodies (negative control): only 1.8 % from the total 10,000 events collected within applied size gates show positive signal with average intensity 46.3 relative fluorescence units (RFU). (c) Presynaptic marker (SNAP25)-positive events within the size gate (positive control): 97.6 % positive events with average intensity 345.8 RFU. (d) An example of p-tau (Ser369/Ser404 phosphorylation-dependent epitopes accessed by PHF-1 antibody) signal in a synaptosomal fraction from normal human brain cortex: 9.4 % of p-tau positive events with average intensity 17.2 RFU. (e) An example of p-tau (PHF-1) signal in a synaptosomal preparation from brain of a subject with a late stage of Alzheimer’s disease: 42.5 % of p-tau positive events with average intensity 76.9 RFU

References

    1. Gouras GK, Tampellini D, Takahashi RH, Capetillo-Zarate E. Intraneuronal beta-amyloid accumulation and synapse pathology in Alzheimer’s disease. Acta Neuropathol. 2010;119:523–541. doi: 10.1007/s00401-010-0679-9. -DOI -PMC -PubMed
    1. Dunkley PR, Jarvie PE, Robinson PJ. A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc. 2008;3:1718–1728. doi: 10.1038/nprot.2008.171. -DOI -PubMed
    1. Wolf ME, Kapatos G. Flow cytometric analysis of rat striatal nerve terminals. J Neurosci. 1989;9:94–105. -PMC -PubMed
    1. Woo J, Baumann A, Arguello V. Recent advancements of flow cytometry: new applications in hematology and oncology. Expert Rev Mol Diagn. 2014;14:67–81. doi: 10.1586/14737159.2014.862153. -DOI -PubMed
    1. Wolf ME, Kapatos G. Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum. J Neurosci. 1989;9:106–114. -PMC -PubMed

MeSH terms

Substances

LinkOut - more resources