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

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


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.

Original languageEnglish (US)
Article number109920
JournalJournal of Neuroscience Methods
StatePublished - Aug 1 2023


  • Flow cytometry
  • Human synapses
  • Method
  • PLA
  • Protein-protein-interaction
  • Synaptosomes

ASJC Scopus subject areas

  • General Neuroscience


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