Validation of a gene construct for proximity labeling of excitatory synaptic cleft proteins

The synaptic proteome undergoes continuous remodeling in response to activity-dependent cues. In addition, pathological states, including neurodegenerative diseases, impinge on synapse structure. In this regard, proteomics offer the opportunity to characterize the molecular players of synaptic plasticity using an unbiased data-driven approach. However, traditional methods based on subcellular fractionation (e.g., preparation of synaptosomes) or on co-immunoprecipitation using a specific synaptic protein as the bait are limited by a low signal-to-noise ratio, as well as by the tendency to retrieve multiple false positives. These problems can be overcome by employing proximity labeling, which takes advantage of the TurboID engineered biotin ligase to covalently label proteins in specific subcellular compartments. Here, we designed a construct for tethering TurboID at excitatory synapses. In addition, a Cre recombinase-dependent system was employed to restrict the time window for expressing this construct. This thesis aimed at validating (i) the correct pattern of expression of TurboID at excitatory synapses, both in vitro and in vivo, and (ii) its biotinylating activity. These experiments lay the ground for subsequent in vivo use of TurboID to characterize the changes in the synaptic cleft proteome in mouse models of neurodegeneration.