Elsevier

Journal of Neuroscience Methods

Volume 312, 15 January 2019, Pages 73-83
Journal of Neuroscience Methods

Mass synaptometry: High-dimensional multi parametric assay for single synapses

https://doi.org/10.1016/j.jneumeth.2018.11.008Get rights and content

Highlights

  • Mass synaptometry method for single synapse multiplexed molecular profiling.

  • Synapse by Time Of Flight (SynTOF) panel of 34 antibodies for wide application.

  • Optimized synaptosome preparation and CyTOF acquisition parameters.

  • Proof of principle validation in 4 Lewy body disease and 2 Alzheimer’s disease cases.

Abstract

Background

Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline.

New method

We report “Mass Synaptometry” for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells.

Result

Here we overcome challenges for single synapse analysis by optimizing synaptosome preparations, generating a ‘SynTOF panel,’ recalibrating acquisition settings, and applying computational analyses. Through the analysis of 390,000 individual synaptosomes, we also provide proof-of principle validation by characterizing changes in synaptic diversity in Lewy Body Disease (LBD), Alzheimer’s disease and normal brain.

Comparison with existing method(s)

Current imaging methods to study synapses in humans are capable of analyzing a limited number of synapses, and conventional flow cytometric techniques are typically restricted to fewer than 6 parameters. Our method allows for the simultaneous detection of 34 parameters from tens of thousands of individual synapses.

Conclusion

We applied Mass Synaptometry to analyze 34 parameters simultaneously on more than 390,000 synaptosomes from 13 human brain samples. This new approach revealed regional and disease-specific changes in synaptic phenotypes, including validation of this method with the expected changes in the molecular composition of striatal dopaminergic synapses in Lewy body disease and Alzheimer’s disease. Mass synaptometry enables highly parallel molecular profiling of individual synaptic terminals.

Introduction

Alzheimer’s disease (AD) and Lewy body disease (LBD), which includes Dementia with Lewy Bodies (DLB) and Parkinson’s disease (PD), are proposed to initiate with regional synaptic injury and degeneration (Bellucci et al., 2012; Braak and Braak, 1991; McKeith et al., 2017; Overk and Masliah, 2014; Scott et al., 2010; Wishart et al., 2006). Synaptic alterations, especially presynaptic changes, are cardinal feature of neurodegenerative diseases, including LBD, and AD, and strongly correlate with cognitive decline (DeKosky and Scheff, 1990; Dickson et al., 1995; Masliah et al., 1991; Scheff and Price, 2003; Sze et al., 1997; Terry, 1996; Terry et al., 1991). Although there is substantial support for this hypothesis from animal models (Dietrich et al., 2018; Duyckaerts et al., 2008; Galli et al., 2014; Jucker, 2010; Zhu et al., 2017), data on human synaptic changes derive mostly from electron microscopy, Golgi stains, or tissue homogenates. Thus a major gap exists regarding comprehensive molecular characterization of individual synapses from human brain.

One approach to this challenge is multiplex analysis of synaptosomes: pinched-off, re-sealed, mostly presynaptic terminals used widely in neurophysiology and neuropharmacology for decades (Hebb and Whittaker, 1958). Like extracellular vesicles, such as exosomes and microvesicles, synaptosomes are smaller than a cell and bound by the cell of origin’s lipid bilayer. Several labs, including ours, have adapted conventional flow cytometric analysis of synaptosomes from human cerebral cortex (Sokolow et al., 2012), hippocampus (Bilousova et al., 2016; Gylys and Bilousova, 2017), and striatum (Postupna et al., 2017, 2014) and demonstrated, despite using different samples, probes, and instruments, that cytometry of human synaptosomes is a robust and reproducible technique. Importantly, comparison to non-human primate brain collected with no agonal state and no post mortem interval demonstrated that protein in human synaptosomes from rapid brain autopsy is intact (Postupna et al., 2014). However, despite this advance of analyzing thousands of individual synaptic particles in a single run, to date multiplexing has involved a limited number of probes (Bilousova et al., 2016; Gylys and Bilousova, 2017; Postupna et al., 2017, 2014; Sokolow et al., 2012). As with cytometry in immunology and cancer biology, the repertoire of probes needed to assess comprehensively the type of synapse, pathologic proteins (including protein products of risk genes discovered in GWAS), and markers of stress and injury far exceeds what is achievable with conventional flow cytometry.

In contrast to fluorescence-based cytometry, mass cytometry—also known as cytometry by time-of-flight mass spectrometry (CyTOF)—uses antibody probes conjugated to metal ions coupled with time-of-flight mass spectrometry (Amir el et al., 2013; Anchang et al., 2016; Bandura et al., 2009; Bendall et al., 2012), overcoming many of the multiplexing limitations of conventional flow cytometry. Indeed, CyTOF has been used by us and others to great effect in oncology and immunology research to reveal tissue and cellular diversity (Bendall et al., 2011; Chevrier et al., 2017; Gonzalez et al., 2018; Hamers et al., 2017; Korin et al., 2017; Lavin et al., 2017; Mrdjen et al., 2018; Simmons et al., 2015; Spitzer et al., 2015; Spitzer and Nolan, 2016; Wong et al., 2015). Here we describe a novel technique, termed mass synaptometry, which adapts mass cytometry and synaptosome preparation to achieve high throughput molecular characterization of individual synapses. Although we have applied our novel approach to the investigation of individual human synapses, our work also serves as a template for others focused on mass cytometry of subcellular individual events such as platelets or extracellular vesicles.

Section snippets

Collection and preservation of brain tissue

Human samples were obtained from rapid (post mortem interval < 8 h.) brain autopsy by the University of Washington Alzheimer’s Disease Research Center or the Pacific Udall Center. Brain regions (caudate, cerebral cortex and cerebellum) approximately 0.5 cm3 in size were collected, minced with two razor blades, transferred to cryo tubes containing 1 ml of 0.32 M sucrose with 10% DMSO, and mixed by shaking. Following gradual cooling to -80 °C the samples were stored in liquid nitrogen, or at

Overview of mass synaptometry

As illustrated in Fig. 1, Fig. 2, we pursued a multi-step approach to enable analysis of single synapses. Briefly, we optimized synaptosome preparation for CyTOF; compiled a “synapse by time-of-flight (SynTOF)” panel of 34 conjugated antibodies that probed cell type, synapse type, protein products of several AD or LBD risk genes, and markers of injury/response to injury (Tables 1 and S1); established acquisition parameters (Figure S1); and applied bioinformatics tools to visualize and analyze

Discussion

Tissue heterogeneity has presented a significant challenge to the study of biomarkers, proteostasis, and the dissection of the regulatory programs in development and diseases. With the advent of single cell technologies, significant progress has been made in immunology and oncology in resolving tissue heterogeneity and the continuum of cellular diversity. CyTOF based technologies, in particular, have allowed the generation of single cell multi-parametric high-dimensional data. However, such

Competing financial interests

The authors declare no competing financial interests.

Author contributions

CG and TM designed the study. CG and RF performed CyTOF experiments. CG and NP prepared synaptosomes. CG and TM generated mass synaptometry antibody panel. CG optimized and validated method and protocol. CK and NP performed the rapid autopsies and provided pathologic evaluations. MA and SB provided expertise in CyTOF operation and data analysis. CG, RF, MA, SB, DT, and EF interpreted and analyzed the data. CG, KM, and TM wrote the manuscript. All co-authors critically revised the manuscript. TM

Acknowledgements

This work was supported by grants from the NIH: P50 NS062684, RF1 AG053959</GS1>, <GN1>R01 AG056287, P50 AG005136</GN3>, <GN1>R01AG057915, DP2 EB024246, and P50 AG047366, and by the Nancy and Buster Alvord Endowment. We thank M. Holden and M. Leipold of the Stanford Human Immune Monitoring Core for their assistance and guidance, and A. Beller from the University of Washington Department of Pathology for administrative support.

References (75)

  • N. Postupna et al.

    Human striatal dopaminergic and regional serotonergic synaptic degeneration with lewy body disease and inheritance of APOE epsilon4

    Am. J. Pathol.

    (2017)
  • N.O. Postupna et al.

    Flow cytometry analysis of synaptosomes from post-mortem human brain reveals changes specific to Lewy body and Alzheimer’s disease

    Lab. Invest.

    (2014)
  • S.W. Scheff et al.

    Synaptic pathology in Alzheimer’s disease: a review of ultrastructural studies

    Neurobiol. Aging

    (2003)
  • M.H. Spitzer et al.

    Mass cytometry: single cells, many features

    Cell

    (2016)
  • M.T. Wong et al.

    Mapping the diversity of follicular helper t cells in human blood and tonsils using high-dimensional mass cytometry analysis

    Cell Rep.

    (2015)
  • J.D. Altman et al.

    Phenotypic analysis of antigen-specific T lymphocytes

    Science

    (1996)
  • A.D. Amir el et al.

    viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia

    Nat. Biotechnol.

    (2013)
  • B. Anchang et al.

    Visualization and cellular hierarchy inference of single-cell data using SPADE

    Nat. Protoc.

    (2016)
  • D.R. Bandura et al.

    Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry

    Anal. Chem.

    (2009)
  • S.C. Bendall et al.

    Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum

    Science

    (2011)
  • E. Bereczki et al.

    Synaptic markers of cognitive decline in neurodegenerative diseases: a proteomic approach

    Brain

    (2018)
  • W.A. Bonner et al.

    Fluorescence activated cell sorting

    Rev. Sci. Instrum.

    (1972)
  • H. Braak et al.

    Neuropathological stageing of Alzheimer-related changes

    Acta Neuropathol.

    (1991)
  • W.M. Caudle et al.

    Proteomic identification of proteins in the human brain: towards a more comprehensive understanding of neurodegenerative disease

    Proteomics Clin. Appl.

    (2008)
  • S. Chevrier et al.

    An immune atlas of clear cell renal cell carcinoma

    Cell

    (2017)
  • A. Datta et al.

    An iTRAQ-based proteomic analysis reveals dysregulation of neocortical synaptopodin in Lewy body dementias

    Mol. Brain

    (2017)
  • S.T. DeKosky et al.

    Synapse loss in frontal cortex biopsies in Alzheimer’s disease: correlation with cognitive severity

    Ann. Neurol.

    (1990)
  • K. Dietrich et al.

    Synaptic alterations in mouse models for alzheimer Disease-A special focus on N-Truncated abeta 4-42

    Molecules

    (2018)
  • C. Duyckaerts et al.

    Alzheimer disease models and human neuropathology: similarities and differences

    Acta Neuropathol.

    (2008)
  • S. Galli et al.

    Deficient Wnt signalling triggers striatal synaptic degeneration and impaired motor behaviour in adult mice

    Nat. Commun.

    (2014)
  • K.H. Gylys et al.

    Flow cytometry analysis and quantitative characterization of tau in synaptosomes from alzheimer’s disease brains

    Methods Mol. Biol.

    (2017)
  • K.H. Gylys et al.

    Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry

    J. Neurosci. Res.

    (2000)
  • A.A.J. Hamers et al.

    Diversity of human monocyte subsets revealed by CyTOF mass cytometry

    J. Immunol.

    (2017)
  • L.A. Hansen et al.

    Frontal cortical synaptophysin in Lewy body diseases: relation to Alzheimer’s disease and dementia

    J. Neurol. Neurosurg. Psychiatry

    (1998)
  • J.R. Heath et al.

    Single-cell analysis tools for drug discovery and development

    Nat. Rev. Drug Discov.

    (2016)
  • C.O. Hebb et al.

    Intracellular distributions of acetylcholine and choline acetylase

    J. Physiol.

    (1958)
  • E.M. Hol et al.

    Neuronal expression of GFAP in patients with Alzheimer pathology and identification of novel GFAP splice forms

    Mol. Psychiatry

    (2003)
  • Cited by (22)

    View all citing articles on Scopus
    View full text