MOLECULAR RECOGNITION AT THE NEURONAL SYNAPSE

The human brain contains 100 billion neurons, each forming ~7,000 connections with other neurons. Despite this complexity, neural circuits are assembled with remarkable specificity, such that each neuron establishes defined connections required for proper circuit function. The molecular mechanisms that enable this specificity remain incompletely understood. One important contributor to this process is the structure and function of cell adhesion molecules (CAMs), a diverse class of proteins localized at both pre- and postsynaptic membranes. CAMs form homo- or heterophilic trans-synaptic complexes that maintain physical proximity between synaptic partners and play critical roles in synapse formation, consolidation, and differentiation. In this work, we investigate two key mechanisms that expand CAM molecular diversity beyond the genetic code: alternative splicing and N-glycosylation. We examine how these post-transcriptional and post-translational modifications regulate synaptic recognition by focusing on the synaptic CAM Teneurin-3. First, we developed and validated an affinity purification–mass spectrometry workflow optimized for membrane-bound CAMs, enabling robust interactome profiling from minimal amounts of mouse brain tissue. Using Teneurin-3 as bait, we characterized its interactome and identified several potential novel interaction partners. Next, we show that alternative splicing of Teneurin-3 modulates its binding specificity toward known interactants. Finally, we investigate the role of N-glycosylation in Teneurin-3 expression and stability using a comprehensive, site-specific glycomutant library, revealing that proper folding of Teneurin-3 depends on N-glycosylation of different extracellular domains. Together, these findings highlight alternative splicing and glycosylation as key molecular determinants of CAM function and provide new insight into the mechanisms underlying synaptic specificity.