Abstract
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.
Highlights
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites
We found that local application of a neutralizing antibody against NT-3 to the axon terminal inhibited axon outgrowth (Supplementary Fig. 1), indicating that the local amplification of NT-3 is required for axon formation in the stochastic model
Long-range inhibitory signals may be produced from growing axons instage 3 cultured hippocampal neurons, we assumed that visualizing these signals in a static state poses practical difficulties
Summary
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. We demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Hippocampal neurons first extend several filopodia all around the cell body (stage 1) These neurons generate multiple, morphologically similar immature neurites (i.e., minor neurites). Because axonal fate is stochastically determined in the absence of additional extracellular factors, this process is called ‘‘the stochastic model’’ of neuronal polarization Extracellular factors such as neurotrophins and insulin-like growth factor-1 play a critical role in neuronal polarization[10,11,12,13]. Attenuation of neurotrophins and/or Trks impairs neuronal polarization[11, 12], indicating that neurotrophin/Trk signaling is essential for neuronal polarization
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