Bioengineering Selective Synaptic Destruction: An Approach with a Chimeric Protein with Binding and Repulsion Properties

Current tools for studying and manipulating synaptic connectivity often lack the specificity needed to target individual microcircuits without affecting neighboring connections. To address this, we developed CadPlexin, a bioengineered chimeric protein that enables selective synaptic elimination by combining the extracellular domain of Drosophila DE-Cadherin with the transmembrane and intracellular domains M. musculus Plexin-B2. CadPlexin is designed to mimic semaphorin-triggered Plexin activation through cadherin-mediated binding, initiating repulsive signaling and cytoskeletal remodeling. We validated CadPlexin in Cos7 cells using co-plating and antibody clustering assays. Co-plating CadPlexin cells demonstrated characteristic cell-cell isolation and confocal imaging and phalloidin quantification confirmed actin filament formation characteristic of Plexin signaling. Additional experiments showed CadPlexin can bind DE-Cadherin heterophilically, though this does not induce repulsion, underscoring its signaling specificity. CadPlexin was then introduced into primary cultured hippocampal neurons, where full-length expression, proper localization, and robust neurite outgrowth were observed—marking a key milestone in demonstrating its viability in a physiologically relevant system. The construct successfully integrated into post-mitotic neurons, co-localizing with both DE-Cadherin and Plexin-B2 domains. While no overt repulsive phenotype was detected under current conditions, these results validate CadPlexin’s structural stability and expression in neurons and lay the foundation for further functional testing. A modest reduction in CadPlexin-positive cell density suggests a possible role in modulating neuronal viability or synaptic engagement, highlighting new directions for future investigation. To enhance translational potential, a lentiviral version of CadPlexin is under development for in vivo use. One application involves targeting glutamatergic projections from the Pedunculopontine Nucleus (PPN) to dopaminergic neurons in the Substantia Nigra pars compacta (SNc), enabling precise synapse-specific disconnection in addiction-related circuits. This thesis establishes CadPlexin as a modular, genetically encoded tool for selective synaptic destruction. Its precision and adaptability make it a promising asset for manipulating neural circuits in both research and disease models.