Longest neurite-specific activation of Rap1B in hippocampal neurons contributes to polarity formation through RalA and Nore1A in addition to PI3-kinase

In a developing nervous system, axon-dendrite formation is instructed by extrinsic cues, and the mechanism whereby a developing neuron interprets these cues using intracellular signaling is particularly important. Studies using dissociated hippocampal neurons have identified many signaling pathways underlying neuronal polarization. Among the components of these pathways, Rap1B is essential for axon specification in hippocampal cultures. However, spatiotemporal regulation of Rap1B activity in polarizing neurons and how it affects neuronal polarization remain unclear. Herein, we investigated spatiotemporal activity-change of Rap1B and its target molecules in hippocampal neurons. FRET imaging showed that specific activation of Rap1B was observed at the tip of a future axon. To dissect downstream signaling, we used three effector mutants of Rap1B. Expression of Rap1B-G12V/E37G and G12V/Y40C mutants resulted in supernumerary axons. The targets of Rap1B-G12V/E37G were RalA and Nore1A, whereas Rap1B-G12V/Y40C activated PI3-kinase. RalA was activated in the tip of stage 3 axons, and RalA-S28N expression reduced the fraction of neurons with supernumerary axons induced by Rap1B-G12V/E37G. Furthermore, Nore1A depletion reduced the number of cells without axons. These results indicate that specific activation of Rap1B contributes to neuronal polarization via interaction with RalA and Nore1A in addition to PI3-kinase.