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 d More than 400 CBP-interacting proteins, including Npas4, are identified d MAPK phosphorylates Npas4 and increases the interaction between Npas4 and CBP d Phosphorylation of Npas4 enhances the transcriptional activity of Npas4 d Phosphorylation of Npas4 in D1R-MSNs regulates rewardrelated learning and memory

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Dopamine (DA) activates MAPK via PKA/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R)in the nucleus accumbens (NAc), thereby regulating reward-related behavior.However, howMAPKregulates reward-relatedlearning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we performed proteomic analysis using affinity beads coated with CREBbinding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identified more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We found that MAPK phosphorylated Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impaired cocaine-induced place preference, which was rescued by Npas4-WT but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory. (Funahashi et al., Cell Reports, 2019) YIA

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