JIP1 mediates anterograde transport of Rab10 cargos during neuronal polarization.

Axon development and elongation require strictly controlled new membrane addition. Previously, we have shown the involvement of Rab10 in directional membrane insertion of plasmalemmal precursor vesicles (PPVs) during neuronal polarization and axonal growth. However, the mechanism responsible for PPV transportation remains unclear. Here we show that c-Jun N-terminal kinase-interacting protein 1 (JIP1) interacts with GTP-locked active form of Rab10 and directly connects Rab10 to kinesin-1 light chain (KLC). The kinesin-1/JIP1/Rab10 complex is required for anterograde transport of PPVs during axonal growth. Downregulation of JIP1 or KLC or disrupting the formation of this complex reduces anterograde transport of PPVs in developing axons and causes neuronal polarity defect. Furthermore, this complex plays an important role in neocortical neuronal polarization of rats in vivo. Thus, this study has demonstrated a mechanism underlying directional membrane trafficking involved in axon development.

Laminin/ß1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization.

Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through ß1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization.

Nuclear factor kappaB controls acetylcholine receptor clustering at the neuromuscular junction.

At the vertebrate neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is stimulated by motor neuron-derived glycoprotein Agrin and requires a number of intracellular signal or structural proteins, including AChR-associated scaffold protein Rapsyn. Here, we report a role of nuclear factor kappaB (NF-kappaB), a well known transcription factor involved in a variety of immune responses, in regulating AChR clustering at the NMJ. We found that downregulating the expression of RelA/p65 subunit of NF-kappaB or inhibiting NF-kappaB activity by overexpression of mutated form of IkappaB (inhibitor kappaB), which is resistant to proteolytic degradation and thus constitutively keeps NF-kappaB inactive in the cytoplasma, impeded the formation of AChR clusters in cultured C2C12 muscle cells stimulated by Agrin. In contrast, overexpression of RelA/p65 promoted AChR clustering. Furthermore, we investigated the mechanism by which NF-kappaB regulates AChR clustering. Interestingly, we found that downregulating the expression of RelA/p65 caused a marked reduction in the protein and mRNA level of Rapsyn and upregulation of RelA/p65 enhanced Rapsyn promoter activity. Mutation of NF-kappaB binding site on Rapsyn promoter prevented responsiveness to RelA/p65 regulation. Moreover, forced expression of Rapsyn in RelA/p65 downregulated muscle cells partially rescued AChR clusters, suggesting that NF-kappaB regulates AChR clustering, at least partially through the transcriptional regulation of Rapsyn. In line with this notion, genetic ablation of RelA/p65 selectively in the skeletal muscle caused a reduction of AChR density at the NMJ and a decrease in the level of Rapsyn. Thus, NF-kappaB signaling controls AChR clustering through transcriptional regulation of synaptic protein Rapsyn.

Wnt/beta-catenin signaling suppresses Rapsyn expression and inhibits acetylcholine receptor clustering at the neuromuscular junction.

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.