Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

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.

Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes

“…Previous work has shown that Kcc2 is located at or in close proximity to excitatory synapses (Gulyás et al, 2001;Chamma et al, 2012;Mahadevan et al, 2017). In agreement with this we detected well characterized excitatory synapse proteins such as Cntn1 (Cijsouw et al, 2018), and Grm2/3 (Ting et al, 2016;Spence et al, 2019). Interestingly, we also identified several proteins associated with inhibitory synapses including Cacna1e (Nakamura et al, 2016), and Cyfip1 (Supplementary Table S1; Davenport et al, 2019).…”

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

“…This defect is reminiscent of the reduction in AZ length and number at perisomatic inhibitory synapses in CA1 pyramidal neurons in Tnr KO mice 62 . Together, these observations suggest that IgSF8 regulates synapse number and length, possibly via an interaction with TenR, which localizes to the excitatory synaptic cleft of cultured neurons 108 .…”

Novel Hyperthermophilic Crenarchaeon Thermofilum adornatum sp. nov. Uses GH1, GH3, and Two Novel Glycosidases for Cellulose Hydrolysis

“…Intensity-based absolute quantitation (iBAQ) quantitative values of detected proteins were obtained and normalized as summed iBAQ values within one sample, which represent the molar abundance or relative iBAQ (riBAQ) of an identified protein within a sample. For each detected protein, the log2 riBAQ value was calculated (Cijsouw et al, 2018). To determine the similarity of the samples' expression profiles, a hierarchical clustering of the samples based on the correlation coefficients of the riBAQ values was performed.…”