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