Yasuhiro Funahashi

Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27.

The neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.

Pioneering Axons Regulate Neuronal Polarization in the Developing Cerebral Cortex

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

Extracellular and Intracellular Signaling for Neuronal Polarity.

Neurons are one of the highly polarized cells in the body. One of the fundamental issues in neuroscience is how neurons establish their polarity; therefore, this issue fascinates many scientists. Cultured neurons are useful tools for analyzing the mechanisms of neuronal polarization, and indeed, most of the molecules important in their polarization were identified using culture systems. However, we now know that the process of neuronal polarization in vivo differs in some respects from that in cultured neurons. One of the major differences is their surrounding microenvironment; neurons in vivo can be influenced by extrinsic factors from the microenvironment. Therefore, a major question remains: How are neurons polarized in vivo? Here, we begin by reviewing the process of neuronal polarization in culture conditions and in vivo. We also survey the molecular mechanisms underlying neuronal polarization. Finally, we introduce the theoretical basis of neuronal polarization and the possible involvement of neuronal polarity in disease and traumatic brain injury.

CRMP‐2 directly binds to cytoplasmic dynein and interferes with its activity

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long‐distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi‐directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein‐2 (CRMP‐2) played key roles in axon elongation and neuronal polarization. CRMP‐2 was also found to associate with the anterograde motor protein Kinesin‐1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP‐2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP‐2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N‐terminus of CRMP‐2, which is the distinct side of CRMP‐2’s kinesin light chain‐binding region. Furthermore, over‐expression of the dynein‐binding fragments of CRMP‐2 prevented dynein‐driven microtubule transport in COS‐7 cells. Given that CRMP‐2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP‐2 might have an important role in axon formation, and neuronal development.

Local Application of Neurotrophins Specifies Axons Through Inositol 1,4,5-Trisphosphate, Calcium, and Ca2+/Calmodulin–Dependent Protein Kinases

Neurotrophins stimulate calcium signaling to promote axon specification. Trking a Calcium Signal to Axon Specification Neurons are polar cells with structurally and functionally distinct processes that are specialized to receive information (the dendrites) or to pass it along to other cells (the axon). Nakamuta et al. investigated the extracellular and intracellular signals involved in axon specification—the process whereby one immature neurite is “chosen” to become an axon, whereas the others become dendrites—a process that occurs in vitro in the absence of directional cues. Blocking neurotrophin signaling through Trk receptors inhibited the loss of neurite symmetry in cultured hippocampal neurons, suggesting that the autocrine or paracrine release of neurotrophins was crucial for axonal specification. Local application of neurotrophin to a given neurite induced axon specification in that neurite, enabling the authors to determine that calcium signaling was crucial to axonal specification in response to the neurotrophin NT-3 in vitro. Moreover, they used an electroporation system to confirm a role for neurotrophin and calcium signaling in axonal specification of cortical neurons in vivo. Neurons are highly polarized cells that have structurally distinct processes—the axons and dendrites—that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca2+) in an inositol 1,4,5-trisphosphate (IP3)–dependent fashion as well as local activation of the Ca2+ effector Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3–induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP3-Ca2+-CaMKK signaling in axon specification.

The role of selective transport in neuronal polarization.

Neurons are functionally and morphologically polarized and possess two distinct types of neurites: axons and dendrites. Key molecules for axon formation are transported along microtubules and accumulated at the distal end of the nascent axons. In this review, we summarize recent advances in the understanding of the mechanisms involved in selective transport in neurons. In addition, we focus on motor proteins, cargo, cargo adaptors, and the loading and unloading of cargo.

Radial Glial Cell-Neuron Interaction Directs Axon Formation at the Opposite Side of the Neuron from the Contact Site.

How extracellular cues direct axon–dendrite polarization in mouse developing neurons is not fully understood. Here, we report that the radial glial cell (RGC)–cortical neuron interaction directs axon formation at the opposite side of the neuron from the contact site. N-cadherin accumulates at the contact site between the RGC and cortical neuron. Inhibition of the N-cadherin-mediated adhesion decreases this oriented axon formation in vitro, and disrupts the axon–dendrite polarization in vivo. Furthermore, the RGC–neuron interaction induces the polarized distribution of active RhoA at the contacting neurite and active Rac1 at the opposite neurite. Inhibition of Rho–Rho-kinase signaling in a neuron impairs the oriented axon formation in vitro, and prevents axon–dendrite polarization in vivo. Collectively, these results suggest that the N-cadherin-mediated radial glia–neuron interaction determines the contacting neurite as the leading process for radial glia-guided neuronal migration and directs axon formation to the opposite side acting through the Rho family GTPases. SIGNIFICANCE STATEMENT Neurons are highly polarized cell lines typically with a single axon and multiple dendrites, which underlies the ability of integrating and transmitting the information in the brain. How is the axon–dendrite polarity of neurons established in the developing neocortex? Here we show that the N-cadherin-mediated radial glial cell–neuron interaction directs axon–dendrite polarization, the radial glial cell–neuron interaction induces polarized distribution of active RhoA and active Rac1 in neurons, and Rho–Rho-kinase signaling is required for axon–dendrite polarization. Our work advances the overall understanding of how extracellular cues direct axon–dendrite polarization in mouse developing neurons.

Neuronal polarization in vivo: Growing in a complex environment.

Neurons are one of the most polarized cell types in the body. During the past three decades, many researchers have attempted to understand the mechanisms of neuronal polarization using cultured neurons. Although these studies have succeeded in discovering the various signal molecules that regulate neuronal polarization, one major question remains unanswered: how do neurons polarize in vivo?

Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo.

Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.

ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

Axon formation is one of the most important events in neuronal polarization and is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is poorly understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, that ERK2 phosphorylates Par3 at Ser-1116, and that the phosphorylated Par3 accumulates at the axonal tips in a manner dependent upon ERK2 activity. The phosphorylation of Par3 by ERK2 inhibited the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. The knockdown of Par3 by RNA interference impaired neuronal polarization, which was rescued with RNAi-resistant Par3, but not with the phosphomimic Par3 mutant, in cultured rat hippocampal neurons and mouse cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.