Calcium plays a key role during growth cone collapse. Recently, it has been proposed that low- and high-amplitude rises in [Ca(2+)](i) result in the activation of the neurite outgrowth inhibiting proteins calcineurin and calpain, respectively. However, it remains unknown if and how these mechanisms are modulated by specific guidance cues. Here we report that the inhibitory cue Semaphorin 5B induces growth cone collapse by promoting the influx of extracellular Ca(2+). The resulting rise in [Ca(2+)](i) is characterized by a low-amplitude increase followed by a marked secondary rise, suggesting the potential involvement of both calcineurin and calpain. In support of this, inhibition of either effector attenuated Sema5B-induced collapse, a result that was augmented by the simultaneous inhibition of both targets. Furthermore, we provide evidence that Sema5B induces calpain-mediated cleavage of calcineurin. We thus show for the first time that ligand-induced growth cone collapse can activate both calcineurin- and calpain-mediated pathways concurrently.

To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.