Mapping the Proteome of the Synaptic Cleft Through Reporter Proteins

Synapses are specialized cellular junctions that connect neurons into the brain circuits that underlie cognitive processes and behavior. Synaptic junctions are heterogeneous in molecular composition and function, reflecting diverse health and disease states. Aberrations in neuronal connectivity occur in brain disorders, and synaptic changes in addiction-relevant brain regions are correlated with drug seeking and relapse. Progress over the past years has shown that trans-synaptic adhesion molecules mediate synapse formation and differentiation, including in brain regions relevant for addiction, and we recently reported that a mouse model lacking a synaptogenic adhesion protein exhibits altered addiction-relevant behaviors. This agrees with human genetic data that support that mutations in synaptic adhesion proteins can be linked to addictive behaviors.

Aiming to define the mechanisms that control synapse maturation and remodeling, we hypothesize that synaptic cell adhesion molecules modulate the molecular composition of synapse-organizing complexes in the synaptic cleft when synapses undergo experience-dependent changes. Further, we hypothesize that disease states and exposure to psychostimulants impact the molecular makeup of the cleft to alter signaling and synaptic function. The objective of this pilot project is to apply a proteomic approach to define the cleft composition of synapse populations. To test our hypothesis, we aim to use cleft reporter proteins that allow for the labeling of synaptic surface proteins. We will combine this molecular labeling method with affinity purification and unbiased analysis by quantitative proteomics. Labeled synaptic membrane protein targets will be purified and subjected to quantitative proteomics for identification at the Yale/NIDA Neuroproteomics Center.

This approach has the potential to probe the synaptic cleft as a compartment rather than individual protein-protein interactions, and to untangle the heterogeneity of synapses in different brain regions. Once established, the molecular remodeling of the synaptic cleft during plasticity can be investigated. Further, this approach can be applied to diverse neuronal populations. This includes neurons in addiction-relevant brain regions which can determine how drugs of abuse may impact the synaptic cleft and alter neuronal communication. We expect this to reveal novel candidate proteins that can underlie acute and chronic synaptic changes after psychostimulant exposure.