EphA3 Domains Differentially Regulate Axonal Transport, Neuronal Outgrowth, and Differentiation

The precise establishment of neural circuits in the CNS depends on environmental stimuli exerted by guidance molecules, integrating topographic cues for accurate axon connectivity. Eph receptors and ephrin ligands play essential roles in axon attraction and repulsion; however, how specific Eph motifs contribute to axonal growth and neuronal development remains unknown. Here, we evaluated the intracellular dynamics of the EphA3 receptor using full-length wild-type (WT) and truncated ligand-binding domain- (Δ-LBD) constructs. Data from N2a cells and primary hippocampal neurons expressing Δ-LBD showed increased morphological outgrowth parameters compared to WT suggesting that LBD is involved in subcellular protein localization and differentiation. While both constructs displayed a mobile vesicular phenotype, Δ-LBD showed a significant increase in dynamic transport properties in primary and human iPSC-derived neurons. Surprisingly, we found that EphA3-WT and not Δ-LBD is transferred from transfected distal axons to crossing neighboring axons, suggesting a novel local axonal mechanism of cellular membrane transfer. We propose that this axo-axonal transfer is mediated by Eph receptor interaction with its ligands. Future work will focus on elucidating the underlying pathways using specific inhibitors. To date, no report describes this transfer phenomenon between distal axons, marking a unique event in molecular signaling.