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Trka Mediates Retrograde Semaphorin 3A Signaling Through Plexin A4 To © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1802-1814 doi:10.1242/jcs.184580 RESEARCH ARTICLE TrkA mediates retrograde semaphorin 3A signaling through plexin A4 to regulate dendritic branching Naoya Yamashita1,2,*, Masayuki Yamane1, Fumikazu Suto3 and Yoshio Goshima1,* ABSTRACT subcellular compartments of neurons – axons, dendrites and cell – Semaphorin 3A (Sema3A), a secretory semaphorin, exerts various body need to communicate with each other. biological actions through a complex between neuropilin-1 and plexin- Semaphorin 3A (Sema3A), one of the first repulsive axon As (PlexAs). Sema3A induces retrograde signaling, which is involved guidance molecules to be recognized (Raper, 2000; Tran et al., in regulating dendritic localization of GluA2 (also known as GRIA2), 2007), plays an important role in proper neuronal network formation an AMPA receptor subunit. Here, we investigated a possible by regulating not only growth cone motility but also dendritic – interaction between retrograde signaling pathways for Sema3A and development and maturation through neuropilin-1 (NRP1) Plexin- nerve growth factor (NGF). Sema3A induces colocalization of PlexA4 A (PlexA) receptor complexes (Campbell et al., 2001; Morita et al., (also known as PLXNA4) signals with those of tropomyosin-related 2006; Polleux et al., 2000; Sasaki et al., 2002; Shelly et al., 2011; kinase A (TrkA, also known as NTRK1) in growth cones, and these Suto et al., 2003; Yamashita et al., 2014). Sema3A accelerates colocalized signals were then observed along the axons. The time- endocytosis during growth cone collapse (Fournier et al., 2000; lapse imaging of PlexA4 and several TrkA mutants showed that the Hida et al., 2012), and also induces bidirectional axonal transport kinase and dynein-binding activity of TrkA were required for Sema3A- (Goshima et al., 1999, 1997; Li et al., 2004; Yamane et al., 2012). induced retrograde transport of the PlexA4–TrkA complex along the We have previously shown that Sema3A facilitates dendritic as well axons. The inhibition of the phosphoinositide 3-kinase (PI3K)–Akt as axonal transport in cultured hippocampal neurons. Sema3A signal, a downstream signaling pathway of TrkA, in the distal axon initiates signals at axonal growth cones, and these signals are suppressed Sema3A-induced dendritic localization of GluA2. The propagated to the somatodendritic compartments by retrograde knockdown of TrkA suppressed Sema3A-induced dendritic axonal transport of Sema3A and PlexA4 (also known as PLXNA4), localization of GluA2 and that suppressed Sema3A-regulated one of the PlexA isoforms. PlexA4 induces dendritic localization of dendritic branching both in vitro and in vivo. These findings suggest the AMPA receptor GluA2 (also known as GRIA2) through that by interacting with PlexA4, TrkA plays a crucial role in redirecting cis-interaction with GluA2 at the immunoglobulin-like plexin- local Sema3A signaling to retrograde axonal transport, thereby transcription-factor domain (PlexA-IPT) in the somatodendritic regulating dendritic GluA2 localization and patterning. compartment (Yamashita et al., 2014, 2016). This finding indicates that, at least among PlexAs, PlexA4 plays an important KEY WORDS: Hippocampus, Semaphorin 3A, NGF, TrkA, PlexA4, role in mediating retrograde Sema3A signaling through a form of Axonal transport signaling endosome. Thus, it is possible that the long-range signaling between the growth cone and the cell body of NGF and INTRODUCTION Sema3A might share some common components during neuronal Various neurotrophic factors, such as nerve growth factor (NGF), development. the prototypical growth factor, are required for the survival and In the present study, we provide evidence that, by acting at the maintenance of neurons. After NGF is released from the target cells, distal axons, Sema3A induces a dynein-dependent retrograde it binds to and activates its high-affinity receptor, tropomyosin- axonal transport of a TrkA–PlexA4 complex. Specifically, our related kinase (TrkA, also known as NTRK1), and is internalized findings suggest that Sema3A regulates dendritic patterning through into the responsive neuron. The signaling endosome containing retrograde transport of the TrkA–PlexA4 complex along the axons, TrkA activated by NGF is subsequently trafficked back to the cell which in turn regulates the dendritic localization of GluA2. This body where the downstream signaling is activated. This movement represents a new mode of crosstalk between the signaling pathways of NGF from the axon tip to the soma is thought to be involved in the involving axon guidance molecules and neurotrophins. long-range signaling between the growth cone and the cell body (Harrington and Ginty, 2013; Zweifel et al., 2005). In addition, RESULTS coordinated regulation of local and global neuronal signaling Sema3A induces PlexA4 and TrkA colocalization in cultured elicited by the extracellular environment is thought to be essential DRG neurons for the proper formation of neural networks. In this process, the To investigate the relationship between Sema3A and neurotrophin signaling, we first determined whether PlexA4 interacts with Trk 1Department of Molecular Pharmacology and Neurobiology, Yokohama City receptors. In HEK293T cells, we found that PlexA4 interacted University School of Medicine, Yokohama 236-0004, Japan. 2Department of 3 with TrkA and TrkC (also known as NTRK3) but not with TrkB Biology, Johns Hopkins University, Baltimore, MD 21218, USA. National Center of – Neurology and Psychiatry, National Institute of Neuroscience, Department of (also known as NTRK2) (Fig. 1A), and PlexA4 TrkA Ultrastructural Research, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. immunoprecipitation efficiency was higher than that of PlexA4– TrkC. The relative ratio of immunoprecipitated TrkC to PlexA4 *Authors for correspondence ([email protected]; [email protected]) (ratio of TrkA to PlexA4 as a percentage) was 68.7±25.1% (mean± s.e.m.; n=3), suggesting that PlexA4 has a higher affinity for TrkA Received 9 December 2015; Accepted 26 February 2016 than for TrkC. Journal of Cell Science 1802 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1802-1814 doi:10.1242/jcs.184580 Fig. 1. Sema3A induces interaction between PlexA4 and TrkA. (A) Flag-tagged PlexA4 expressed in HEK293T cells interacted with EGFP-tagged TrkA and TrkC, but not with TrkB. Immunoprecipitation (IP) was performed with anti-Flag-tag antibody. A, B and C represent TrkA, TrkB and TrkC, respectively. (B) Schematic representation of TrkA and constructed TrkA mutants. TrkA contains signal peptides (SP) and C1, LRR, C2, Ig1, Ig2, transmembrane (TM) domains and cytosolic domains. Amino acids 464–483 of the cytosolic domain make up the dynein-binding domain (DB). Full (F), full-length; ΔC1-C2 (C2), lacking the region from the C1 domain to the C2 domain; ΔC1-Ig1 (Ig1), lacking the region from the C1 domain to the Ig1 domain; K537A, catalytic inactive; Δ464–483, lacking amino acids 464–483. (C) Flag-tagged PlexA4 interacted with full-length TrkA and the C2 mutant but not with the Ig1 mutant of TrkA–EGFP. (D) After Sema3A treatment, the fluorescence clusters of PlexA4 and TrkA moved along the axons towards the cell bodies in a time-dependent manner (arrowheads). Scale bar: 10 µm. (E) Time dependence of the mean relative ratio of immunostaining intensity in the axon to that in the growth cone (n=12 neurons from three independent cultures). (F) Sema3A treatment increased the number of double-positive clusters (arrowheads) of anti-PlexA4 and anti-TrkA in the growth cones (main panels), and subsequently in the axons (rectangular panels below main image), in a time-dependent manner. The colocalization of PlexA4 and TrkAis presented using colocalization highlighter (white color). Scale bars: 5 µm (main panels), 2 µm (rectangular panels). (G) Time-dependence of mean colocalization coefficients in growth cones (GC) and axons (n=12 neurons from three independent cultures). Data are presented as mean±s.e.m. *P<0.05; **P<0.01 (one-way ANOVA compared to 0 min); #P<0.05; ##P<0.01 (one-way ANOVA compared to 3 min). Journal of Cell Science 1803 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1802-1814 doi:10.1242/jcs.184580 We used several TrkA mutants to determine the region of the et al., 2001; Zweifel et al., 2005). However, NGF neither altered the protein that interacted with PlexA4. Both full-length TrkA and TrkA localization of PlexA4 (Fig. 3A,B) nor induced the PlexA4 and mutant lacking the region from C1 to C2 (TrkA ΔC1-C2, Fig. 1B) TrkA colocalization in the growth cones and the axons (Fig. 3C,D). region interacted with PlexA4 (Fig. 1C). In contrast, a TrkA mutant These data clearly show that the interaction between PlexA4 and lacking the region from C1 to Ig1 region (TrkA ΔC1-Ig1, Fig. 1B) TrkA, and retrograde axonal transport of the PlexA4–TrkA complex did not interact with PlexA4, thereby indicating that the TrkA Ig1 occur in response to Sema3A, but not to NGF, thereby suggesting domain is required for the interaction between TrkA and PlexA4 distinct signaling pathways for Sema3A and NGF. (Fig. 1C). To further investigate the interaction between PlexA4 and TrkA, The interaction between PlexA4 and TrkA mediates we next performed immunostaining of PlexA4 and TrkA in cultured Sema3A-induced dendritic localization of GluA2 mouse dorsal root ganglion (DRG) neurons. Sema3A potently We have previously shown that the Sema3A retrograde signal induces axon repulsion or growth cone collapse of the NGF- regulates the dendritic localization of GluA2 in hippocampal sensitive neurons in the DRG (Messersmith et al., 1995). Anti- neurons by means of a PlexA4-dependent mechanism (Yamashita PlexA4 immunostaining signals were mainly observed in the et al., 2014). To examine the roles of TrkA in Sema3A signaling, we growth cones of neurons when not treated with Sema3A, which is analyzed dendritic localization of GluA2 in cultured hippocampal consistent with the results observed in cultured hippocampal neurons where TrkA was knocked down by means of small neurons (Yamashita et al., 2014). The specificity of the anti- interfering RNA (siRNA).
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