Abstract
Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction.
Highlights
Synapses are the cellular units for information transfer in the central nervous system.The mammalian brain is comprised of functionally diverse synapse types connecting different neuronal populations into networks that enable complex behavior and responses to external and internal cues
Peroxidases that have been used for proximity labeling are horseradish peroxidase (HRP) and APEX/APEX2, which are metalloenzymes that catalyze the oxidation of organic substrates by hydrogen peroxide (H2 O2 )
APEX2, a more active variant of the engineered peroxidase APEX [51], is suitable for intracellular and extracellular applications, while HRP is only active in the oxidizing environments of the secretory pathway and the cell surface due to its structurally essential disulfide-bonds
Summary
The mammalian brain is comprised of functionally diverse synapse types connecting different neuronal populations into networks that enable complex behavior and responses to external and internal cues. Understanding this diversity will be fundamental to defining brain connectivity [1,2,3]. Synapse function is instructed by the diverse proteomic composition of these specialized neuronal. These molecular components guide synaptogenesis, maturation, and differentiation in development and in adulthood [4,5]. Function and composition are specific to neuron identity, local connectivity, and regional specificity and contribute to a diversified and specialized synapse proteome within the brain [3]. Different states of the diseased brain, e.g., neuro-degeneration or addiction, may further alter synapse composition and function [8,9,10,11]
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