A method to study human synaptic protein-protein interactions by using flow cytometry coupled to proximity ligation assay (Syn-FlowPLA)

Michela Marcatti; Danielle Jamison; Anna Fracassi; Wen-Ru Zhang; Agenor Limon; Giulio Taglialatela

doi:10.1016/j.jneumeth.2023.109920

A method to study human synaptic protein-protein interactions by using flow cytometry coupled to proximity ligation assay (Syn-FlowPLA)

J Neurosci Methods. 2023 Aug 1:396:109920. doi: 10.1016/j.jneumeth.2023.109920. Epub 2023 Jul 17.

Authors

Michela Marcatti¹, Danielle Jamison², Anna Fracassi¹, Wen-Ru Zhang², Agenor Limon¹, Giulio Taglialatela³

Affiliations

¹ Mitchell Center for Neurodegenerative Disease, Department of Neurology, University of Texas Medical Branch at Galveston, USA.
² Mitchell Center for Neurodegenerative Disease, Department of Neurology, University of Texas Medical Branch at Galveston, USA; Department of Pharmacology and Toxicology, University of Texas Medical Branch at Galveston, USA.
³ Mitchell Center for Neurodegenerative Disease, Department of Neurology, University of Texas Medical Branch at Galveston, USA. Electronic address: gtaglial@utmb.edu.

PMID: 37459899
DOI: 10.1016/j.jneumeth.2023.109920

Abstract

Background: Synapses are highly specialized sites characterized by intricate networks of protein-protein interactions (PPIs) important to maintain healthy synapses. Therefore, mapping these networks could address unsolved questions about human cognition, synaptic plasticity, learning, and memory in physiological and pathological conditions. The limitation of analyzing synaptic interactions in living humans has led to the development of methods to isolate synaptic terminals (synaptosomes) from cryopreserved human brains.

New method: Here, we established a method to detect synaptic PPIs by applying flow cytometric proximity ligation assay (FlowPLA) to synaptosomes isolated from frozen human frontal cortex (FC) and hippocampus (HP) (Syn-FlowPLA).

Results: Applying this method in synaptosomes, we were able to detect the known post-synaptic interactions between distinct subtypes of N-methyl-D-aspartate glutamate receptors (NMDARs) and their anchoring postsynaptic density 95 protein (PSD95). Moreover, we detected the known pre-synaptic interactions between the SNARE complex proteins synaptosomal-associated protein of 25 kDa (SNAP25), synaptobrevin (VAMP2), and syntaxin 1a (STX1A). As a negative control, we analyzed the interaction between mitochondrial superoxide dismutase 2 (SOD2) and PSD95, which are not expected to be physically associated.

Comparison with existing methods: PPIs have been studied in vitro primarily by co-immunoprecipitation, affinity chromatography, protein-fragment complementation assays (PCAs), and flow cytometry. All these are valid approaches; however, they require more steps or combination with other techniques. PLA technology identifies PPIs with high specificity and sensitivity.

Conclusions: The Syn-FlowPLA described here allows rapid analyses of PPIs, specifically within the synaptic compartment isolated from frozen autopsy specimens, achieving greater target sensitivity. Syn-FlowPLA, as presented here, is therefore a useful method to study human synaptic PPI in physiological and pathological conditions.

Keywords: Flow cytometry; Human synapses; Method; PLA; Protein-protein-interaction; Synaptosomes.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Flow Cytometry
Humans
Neuronal Plasticity
Presynaptic Terminals
Synapses* / metabolism
Synaptosomes* / metabolism

Abstract

Publication types

MeSH terms

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