Mapping Synaptic Proteomes during Learning and Memory Consolidation Using Proximity Labeling

Achieving proficiency in a specific task requires persistent practice and training. One of the most intriguing
questions in neuroscience asks how such continuous engagement enables the brain to remember and retain new
skills or memories effectively. Previous studies revealed that repeated stimulation of the same neural pathways
strengthens their connections, a phenomenon known as long-term potentiation (LTP). During LTP, synapses
undergo structural and functional reorganization, which enhances neurotransmission and consolidates memories
and learned behaviors. While key molecular components involved in LTP have been identified, our understanding
of the complete protein landscape and the complex interactions governing synaptic remodeling remains elusive.
To map the protein compositions of pre- and postsynaptic terminals of excitatory neurons undergoing LTP
requires a multifaceted molecular approach that is unbiased, proteome-wide, spatially precise, cell type-specific,
and LTP-specific. However, no existing method possesses all these attributes. To address the unmet need, we
propose to develop a proximity labeling tool that can label endogenous proteins within synaptic compartments in
an LTP-dependent manner. Our strategy uses CaMKIIa (calcium/calmodulin-dependent protein kinase II alpha),
an established key driver of LTP, to control the activity of the proximity labeling enzyme TurboID. Once activated
at potentiated synapses, TurboID will tag proximal endogenous proteins during a user-defined time window of 10-
30 minutes, enabling us to identify and map synaptic proteins at different LTP phases through subsequent
proteomic analysis. We will begin our studies in dissociated neuronal culture, then progress to acute brain slices
and finally awake behaving animals. Ultimately, this study should provide both a novel molecular tool for
spatiotemporally-specific profiling of LTP-associated proteomes, and molecular insight into the mechanisms of
adaptive behavior and learning.