CNP-induced cGMP signaling reduces growth cone stiffness and Ca2+ levels in embryonic DRG neurons.

A cyclic guanosine monophosphate (cGMP) signaling pathway composed of the extracellular ligand C-type natriuretic peptide (CNP), the transmembrane natriuretic peptide receptor 2 (Npr2), and the cGMP-dependent protein kinase I (cGKI) regulates axon bifurcation of embryonic dorsal root ganglion (DRG) neurons in mice. Despite the importance of this process for the development of neuronal connectivity, the underlying mechanisms are only partially understood. Axon bifurcation requires an orchestrated rearrangement of the cytoskeleton in growth cones, the highly motile structures at axon tips. In this study, we explored the effects of cGMP signaling on growth cones in fixed and living DRG explant cultures obtained from mouse embryos. The cytoskeletal organization and stiffness of growth cones was examined by fluorescence microscopy and scanning ion conductance microscopy (SICM). Activation of cGMP signaling by CNP or the membrane-permeable cGMP analog 8-Bromo-cGMP reduced growth cone and axon shaft stiffness. Experiments with DRG neurons from Npr2 knockout (KO) mice confirmed that the anti-stiffness effect of CNP was Npr2-dependent. Pharmacological disruption of the cytoskeleton revealed that growth cone stiffness was determined by F-actin content. Activation of cGMP signaling reduced F-actin content in growth cones. Next, we studied the mechanism of cGMP-mediated cytoskeletal remodeling in growth cones. Genetic deletion of vasodilator-stimulated phosphoprotein (Vasp), a phosphorylation target of cGKI that regulates actin polymerization, did not impair cGMP-induced reduction of growth cone and axon shaft stiffness in vitro and axon bifurcation in vivo. Since growth cone dynamics is also regulated by the intracellular Ca2+ concentration, we performed simultaneous imaging of cGMP and Ca2+ in living growth cones. CNP-induced cGMP elevations suppressed ATP-induced Ca2+ transients in wild-type growth cones, but not in cGKI-deficient growth cones. In summary, this study indicates that the CNP-Npr2-cGMP-cGKI axis in DRG neurons controls Ca2+ signaling, remodeling of the actin cytoskeleton, and growth cone mechanics. Thereby, it might contribute to regulating axonal branching.