Identification of learning-specific changes in the synaptic proteome via genetically encoded probes

Memory formation is supported by functional, biochemical and structural changes within neural circuits. Potentiation of excitatory synaptic transmission has a key role, relying on extensive rearrangements in the synaptic protein content. However, determining in vivo state-dependent modifications in response to specific conditions remains challenging, due to the difficulty of selectively tagging synapses subjected to activity-dependent plasticity and to isolate their protein content. To address this issue, we employed the SynActive (SA) approach to express genetically encoded reporters at potentiated synapses, a feature conferred by regulatory sequences from 5’ and 3’ UTRs of the immediate-early gene Arc mRNA, combined with synapse-targeting protein sequences fused to the encoded reporter. In our pilot study, I cloned a SA-version of a FLAG-tagged postsynaptic hub protein PSD-95 under the control of the activity-dependent ESARE promoter to obtain expression at potentiated synapses and used a constitutively expressed PSD-95-FLAG driven by human synapsin (hSyn) promoter as a term of comparison. After validation in hippocampal cultures, adeno-associated viral vectors (AAVs) carrying both constructs were delivered to the CA1 region of the hippocampus of mice, later challenged with contextual fear conditioning (CxFC) paradigm to trigger learning-dependent synaptic potentiation. Hippocampi were dissected and the PSD-95 interactome was immunoprecipitated and analyzed via mass spectrometry (MS). The outcome was the generation of the first dataset of the PSD-95 interactome of potentiated synapses. Moreover, I extended the characterization of the potentiated synapses proteome to synaptic cleft proteins of potentiated synapses. miniTurbo biotin ligase was fused to postsynaptic protein Neuroligin-1 to achieve extracellular expression and proximity labeling of nearby proteins and expressed under SA control, with a doxycycline-regulated transcriptional control. After in vitro validation, I used an AAV to deliver the probe to CA1 region of the hippocampus of mice. After biotin supplementation and exposure to CxFC, hippocampi were dissected and processed for immunoprecipitation of biotinylated proteins. MS will reveal the proteomic map of both pre- and post-synaptic sides of potentiated synapses. This approach enhances understanding of memory mechanisms and supports therapeutic target discovery in animal models of neurodevelopmental and neurodegenerative disorders.