Chick Dorsal Root Ganglion Neurons Research Articles

Phosphorylation of Collapsin Response Mediator Protein 1 (CRMP1) at Tyrosine 504 residue regulates Semaphorin 3A-induced cortical dendritic growth.

Collapsin response mediator proteins (CRMPs) have been identified as mediating proteins of repulsive axon guidance cue Semaphorin-3A (Sema3A). Phosphorylation of CRMPs plays a crucial role in the Sema3A signaling cascade. It has been shown that Fyn phosphorylates CRMP1 at Tyrosine 504 residue (Tyr504); however, the physiological role of this phosphorylation has not been examined. We found that CRMP1 was the most strongly phosphorylated by Fyn among the five members of CRMPs. We confirmed Tyr504 phosphorylation of CRMP1 by Fyn. Immunocytochemistry of mouse dorsal root ganglion (DRG) neurons showed that phosphotyrosine signal in the growth cones was transiently increased in the growth cones upon Sema3A stimulation. Tyr504-phosphorylated CRMP1 also tended to increase after Sema3A simulation. Ectopic expression of a single amino acid mutant of CRMP1 replacing Tyr504 with phenylalanine (CRMP1-Tyr504Phe) suppressed Sema3A-induced growth cone collapse response in chick DRG neurons. CRMP1-Tyr504Phe expression in mouse hippocampal neurons also suppressed Sema3A but not Sema3F-induced growth cone collapse response. Immunohistochemistry showed that Tyr504-phosphorylated CRMP1 was present in the cell bodies and in the dendritic processes of mouse cortical neurons. CRMP1-Tyr504Phe suppressed Sema3A-induced dendritic growth of primary cultured mouse cortical neurons as well as the dendritic development of cortical pyramidal neurons in vivo. Fyn± ; Crmp1± double heterozygous mutant mice exhibited poor development of cortical layer V basal dendrites, which was the similar phenotype observed in Sema3a-/- , Fyn-/- , and Crmp1-/- mice. These findings demonstrate that Tyr504 phosphorylation of CRMP1 by Fyn is an essential step of Sema3A-regulated dendritic development of cortical pyramidal neurons. (247 words).

Reduction of extracellular sodium evokes nociceptive behaviors in the chicken via activation of TRPV1

Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, is mainly expressed in nociceptive primary sensory neurons. Sensitivity of TRPV1 to several stimuli is known to vary among species, specifically, the avian orthologue is nearly insensitive to capsaicin. Extracellular sodium ions ([Na+]o) regulate TRPV1 activity in mammals, but their regulatory role on chicken TRPV1 (cTRPV1) is unknown. Here, we focused on the actions of capsaicin and low [Na+]o on cTRPV1 activity. In chicken dorsal root ganglion (cDRG) neurons, capsaicin elicited [Ca2+]i increases, but its effective concentration was much higher than those in mammals. Low [Na+]o evoked [Ca2+]i increases in cDRG neurons in a decreasing [Na+]o-dependent manner and the complete removal of [Na+]o (0Na) produced maximal effects. The population of 0Na-sensitive neurons was mostly overlapped with those of proton- and capsaicin-sensitive ones. Low [Na+]o synergistically potentiated the capsaicin- and proton-induced TRPV1 activation in cDRG neurons. In HEK293 cells expressing cTRPV1 (cTRPV1-HEK), capsaicin elicited [Ca2+]i increases with an EC50 of 11.8 µM, and low [Na+]o also did. Well-defined mammalian TRPV1 antagonists hardly suppressed cTRPV1 activation by low [Na+]o. 0Na evoked outwardly rectified currents in cTRPV1-HEK. Mutagenesis analyses revealed a possible interaction of [Na+]o with the proton-binding sites of cTRPV1. The administration of capsaicin and 0Na to chick eyes elicited pain-related behaviors. These results suggest that low [Na+]o is capable of activating cTRPV1 in vitro, resulting in pain in vivo. Our data demonstrate that characterization of the cTRPV1 function is important to understand activation mechanisms of TRPV1.

Disease-associated mutations in human TUBB3 disturb netrin repulsive signaling.

Missense mutations in the human TUBB3 gene cause a variety of neurological disorders associated with defects in axon guidance and neuronal migration, but the underlying molecular mechanisms are not well understood. Recent studies have shown that direct coupling of dynamic TUBB3 in microtubules with netrin receptors is required for netrin-1-mediated axon guidance, and the interaction of netrin-1 repulsive receptor UNC5C with TUBB3 is involved in netrin-1 mediated axonal repulsion. Here, we report that TUBB3 mutations perturb netrin-1/UNC5C repulsive signaling in the developing nervous system. Among twelve mutants reported in previous studies, five of them show significantly reduced interaction with UNC5C in comparison to the wild-type TUBB3. TUBB3 mutants R262C and R62Q exhibit decreased subcellular colocalization with UNC5C in the peripheral area of the growth cone of primary mouse neurons. Netrin-1 reduces the colocalization of UNC5C with wild-type TUBB3, but not TUBB3 mutants R262C or R62Q, in the growth cone. Results from the in vitro cosedimentation assay indicate that netrin-1 inhibits cosedimentation of UNC5C with polymerized microtubules in primary mouse neurons expressing the wild-type TUBB3, but not R262C or R62Q. Expression of either R262C or R62Q not only blocks netrin-1-induced growth cone collapse and axonal repulsion of primary EGL cells in vitro, but also results in axon projections defects of chicken dorsal root ganglion neurons in ovo. Our study reveals that human TUBB3 mutations specifically perturb netrin-1/UNC5C-mediated repulsion.

Expression of pseudorabies virus-encoded long noncoding RNAs in epithelial cells and neurons.

Long noncoding RNAs (lncRNAs) play important roles in regulating eukaryotic genome replication and gene expression in diverse biological systems. Here, we identified lncRNAs transcribed from pseudorabies virus (PRV)-infected PK-15 cells. Based on high-throughput sequencing data, we obtained 87,263,926 and 93,947,628 clean reads from mock-infected and PRV-infected PK-15 cells, respectively. Through a normalized analytic protocol, we identified three novel viral lncRNAs. According to an analysis of differential expression between the mock-infected and PRV-infected cells, 4151 host lncRNAs were significantly upregulated and 2327 host lncRNAs were significantly downregulated in the latter group. Viral lncRNAs and several host lncRNAs were verified by northern blotting and real-time PCR. The findings showed that the viral lncRNA LDI might regulate the expression of IE180, a potent transcriptional activator of viral genes. Furthermore, we characterized the expression of viral lncRNAs in a culture of infected primary chicken dorsal root ganglia (DRG). Collectively, the obtained data suggest that PRV generates lncRNAs in both epithelial cells and chick DRG neurons.

The soluble form of LOTUS inhibits Nogo receptor type 1-mediated signaling induced by B lymphocyte stimulator and chondroitin sulfate proteoglycans

There are global efforts in developing therapeutic strategies for central nervous system (CNS) injuries using multimodal approaches. Nogo receptor type 1 (NgR1) has been known as a primary molecule limiting neuronal regeneration in the adult CNS. We identified lateral olfactory tract usher substance (LOTUS) as an endogenous NgR1 antagonist. Membrane-bound LOTUS interacts with NgR1 and inhibits its function by blocking its ligand binding. Five molecules including Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs) have been identified as NgR1 ligands. These ligands bind to NgR1 and activate NgR1 signaling, leading to axon growth inhibition such as growth cone collapse and neurite outgrowth inhibition. We have recently reported that the soluble form of LOTUS (s-LOTUS) also suppressed NgR1-mediated signaling induced by myelin axonal inhibitors (MAIs) including Nogo, MAG and OMgp by binding with both NgR1 and its co-receptor p75 neurotrophin receptor (p75NTR). Though s-LOTUS has been reported to suppress MAIs, whether s-LOTUS also suppresses NgR1 signaling induced by BLyS and CSPGs remains to be elucidated.Here, we show that s-LOTUS inhibits NgR1-mediated signaling induced by BLyS and CSPGs. Although treatment with s-LOTUS did not suppress BLyS-NgR1 interaction, s-LOTUS inhibited growth cone collapse and neurite outgrowth inhibition induced by BLyS and CSPGs in chick dorsal root ganglion (DRG) neurons. Furthermore, s-LOTUS compensated for the suppressive function of endogenous LOTUS in NgR1-mediated signaling in olfactory bulb neurons of lotus-knockout mice. These findings suggest that s-LOTUS is a potent therapeutic agent for neuronal regeneration in the CNS injuries.

Visualization of Clathrin-Mediated Endocytosis During Semaphorin-Guided Axonal Growth.

Semaphorin3A (Sema3A) guides axonal growth during neuronal network development. Accumulating evidence indicates that Sema3A-induced growth cone collapse and repulsion involve endocytic membrane trafficking in the growth cone. It is now possible to visualize endocytic processes in living cells using total internal reflection fluorescence microscopy (TIRFM), a powerful tool for imaging dynamic subcellular events at the plasma membrane. In this chapter, we describe a method for TIRFM observation and analysis of clathrin-mediated endocytosis in growth cones of chicken dorsal root ganglion neurons that receive an extracellular concentration gradient of Sema3A in a culture medium.

Cyclic Nucleotide Control of Microtubule Dynamics for Axon Guidance.

Graded distribution of intracellular second messengers, such as Ca(2+) and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca(2+) have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca(2+) signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca(2+)-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. Developing neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.

Optogenetic activation of axon guidance receptors controls direction of neurite outgrowth.

Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue.

Quantitative analysis of intraneuronal transport in human iPS neurons

Induced pluripotent stem (iPS) cells are promising tools to investigate disease mechanism and develop new drugs. Intraneuronal transport, which is fundamental for neuronal survival and function, is vulnerable to various pharmacological and chemical agents and is disrupted in some neurodegenerative disorders. We applied a quantification method for axonal transport by counting CM-DiI–labeled particles traveling along the neurite, which allowed us to monitor and quantitate, for the first time, intraneuronal transport in human neurons differentiated from iPS cells (iCell neurons). We evaluated the acute effects of several anti-neoplastic agents that have been previously shown to affect intraneuronal transport. Vincristine, paclitaxel and oxaliplatin decreased the number of moving particle along neurites. Cisplatin, however, produced no effect on intraneuronal transport, which is in contrast to our previous report indicating that it inhibits transport in chick dorsal root ganglion neurons. Our system may be a useful method for assessing intraneuronal transport and neurotoxicity in human iPS neurons.

Effectiveness of magnetically aligned collagen for neural regeneration in vitro and in vivo.

We investigated the effectiveness of using magnetically aligned collagen (after exposure to a maximum 8-T magnetic field) for nerve regeneration in both an in vitro and in vivo model. Neurite outgrowth from embryonic chick dorsal root ganglion (DRG) neurons was significantly greater on magnetically aligned collagen gel than on control gel, and was dependent on magnetic field strength. Silicone tubes (15 mm length) filled with collagen gel formed bridges between severed rat sciatic nerves. We prepared tubes for four groups: collagen gel only (COL), magnetically aligned collagen gel (M-COL), collagen gel mixed with Schwann cells (S-COL), and magnetically aligned collagen gel mixed with Schwann cells (M-S-COL). The ratio of infiltrating regenerated nerves was higher in the M-COL group compared to the COL group at 8 weeks post-operation. There were no significant differences between the two groups with and without Schwann cells. Compound action potentials showed higher amplitude and shorter latency in the M-COL than COL group at 12 weeks post-operation. The number and diameter of regenerated axons increased significantly in the M-COL compared with the COL group at 12 weeks post-operation. Here we demonstrated that magnetically orientated collagen promoted nerve regeneration using both an in vitro and in vivo model.

The Endocannabinoid, 2-Arachidonoyl Glycerol, Induces Growth Cone Collapse and Neurite Retraction in Growing Peripheral Sensory Neurons

Cannabis has a detrimental impact on the developing nervous system. Therefore, regular con- sumption of cannabis by pregnant and lactating woman poses a potential risk to neuronal growth in fetuses and infants. Indeed, endogenous cannabis-like molecules called endocannabinoids re- gulate many physiological processes, including neurogenesis, axon guidance, and synaptic plastic- ity through CB1 receptors. To investigate the physiological role of CB1 receptors on peripheral sensory nerve growth, the endocannabinoid 2-arachidonoyl glycerol was added to cultured chick dorsal root ganglion neurons. This compound inhibited neurite elongation and induced growth cone collapse in a dose- and time-dependent manner. These data suggest that caution should be exercised regarding maternal cannabis use during pregnancy. Because ectopic sprouting and ab- normal neuronal network connections are considered to be a cause of neuropathic pain, our cur- rent data imply an additional role of endocannabinoids as inhibitors of the formation of pain- maintenance networks.

The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches.

The axonal transport of organelles is critical for the development, maintenance, and survival of neurons, and its dysfunction has been implicated in several neurodegenerative diseases. Retrograde axon transport is mediated by the motor protein dynein. In this study, using embryonic chicken dorsal root ganglion neurons, we investigate the effects of Ciliobrevin D, a pharmacological dynein inhibitor, on the transport of axonal organelles, axon extension, nerve growth factor (NGF)-induced branching and growth cone expansion, and axon thinning in response to actin filament depolymerization. Live imaging of mitochondria, lysosomes, and Golgi-derived vesicles in axons revealed that both the retrograde and anterograde transport of these organelles was inhibited by treatment with Ciliobrevin D. Treatment with Ciliobrevin D reversibly inhibits axon extension and transport, with effects detectable within the first 20 min of treatment. NGF induces growth cone expansion, axonal filopodia formation and branching. Ciliobrevin D prevented NGF-induced formation of axonal filopodia and branching but not growth cone expansion. Finally, we report that the retrograde reorganization of the axonal cytoplasm which occurs on actin filament depolymerization is inhibited by treatment with Ciliobrevin D, indicating a role for microtubule based transport in this process, as well as Ciliobrevin D accelerating Wallerian degeneration. This study identifies Ciliobrevin D as an inhibitor of the bidirectional transport of multiple axonal organelles, indicating this drug may be a valuable tool for both the study of dynein function and a first pass analysis of the role of axonal transport.

Steering neuronal growth cones by shifting the imbalance between exocytosis and endocytosis.

Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.

Activation of PI3K and R‐ras signaling promotes the extension of sensory axons on inhibitory chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are extracellular inhibitors of axon extension and plasticity, and cause growth cones to exhibit dystrophic behaviors. Phosphoinositide 3-kinase (PI3K) is a lipid kinase activated by axon growth promoting signals. In this study, we used embryonic chicken dorsal root ganglion neurons to determine if CSPGs impair signaling through PI3K. We report that CSPGs inhibit PI3K signaling in axons and growth cones, as evidenced by decreased levels of phosphorylated downstream kinases (Akt and S6). Direct activation of PI3K signaling, using a cell permeable phosphopeptide (PI3Kpep), countered the effects of CSPGs on growth cones and axon extension. Both overnight and acute treatment with PI3Kpep promoted axon extension on CSPG-coated substrates. The R-Ras GTPase is an upstream positive regulator of PI3K signaling. Expression of constitutively active R-Ras promoted axon extension and growth cone elaboration on CSPGs and permissive substrata. In contrast, an N-terminus-deleted constitutively active R-Ras, deficient in PI3K activation, promoted axon extension but not growth cone elaboration on CSPGs and permissive substrata. These data indicate that activation of R-Ras-PI3K signaling may be a viable approach for manipulating axon extension on CSPGs.

Protein Synthesis Dependence of Growth Cone Collapse Induced by Different Nogo-A-Domains

BackgroundThe protein Nogo-A regulates axon growth in the developing and mature nervous system, and this is carried out by two distinct domains in the protein, Nogo-A-Δ20 and Nogo-66. The differences in the signalling pathways engaged in axon growth cones by these domains are not well characterized, and have been investigated in this study.Methodology/Principal FindingsWe analyzed growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 using explanted chick dorsal root ganglion neurons growing on laminin/poly-lysine substratum. Collapse induced by purified Nogo-A-Δ20 peptide is dependent on protein synthesis whereas that induced by Nogo-66 peptide is not. Nogo-A-Δ20-induced collapse is accompanied by a protein synthesis-dependent rise in RhoA expression in the growth cone, but is unaffected by proteasomal catalytic site inhibition. Conversely Nogo-66-induced collapse is inhibited ∼50% by proteasomal catalytic site inhibition.Conclusion/SignificanceGrowth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 is mediated by signalling pathways with distinguishable characteristics concerning their dependence on protein synthesis and proteasomal function.

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry-induced increase of phosphorylation of eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2-mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore-assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin-dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)-evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP-induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth.

GSK3β/Axin-1/β-Catenin Complex Is Involved in Semaphorin3A Signaling

Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3β (GSK3β). To delineate how GSK3β mediates Sema3A signaling, we here determined the substrates of GSK3β involved. Introduction of either GSK3β mutants, GSK3β-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3β. The phosphorylation of Axin-1 by GSK3β accelerates the association of Axin-1 with β-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of β-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3β, Axin-1, and β-catenin in the growth cones. The increase of β-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or β-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3β/Axin-1/β-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.

Differing Semaphorin 3A Concentrations Trigger Distinct Signaling Mechanisms in Growth Cone Collapse

Semaphorin-3A (Sema3A) is a major guidance cue in the developing nervous system. Previous studies have revealed a dependence of responses to Sema3A on local protein synthesis (PS) in axonal growth cones, but a recent study has called this dependence into question. To understand the basis of this discrepancy we used the growth cone collapse assay on chick dorsal root ganglion neurons. We show that the dependence of growth cone collapse on protein synthesis varies according to Sema3A concentration, from near-total at low concentration (<100 ng/ml) to minimal at high concentration (>625 ng/ml). Further, we show that neuropilin-1 (NP-1) mediates both PS-dependent and PS-independent collapse. Our findings are consistent with the operation of at least two distinct Sema3A signaling pathways: one that is PS-dependent, involving mammalian target of rapamycin, and one that is PS-independent, involving GSK-3β activation and operative at all concentrations of Sema3A examined. The results provide a plausible explanation for the discrepancy in PS-dependence reported in the literature, and indicate that different signaling pathways activated within growth cones can be modulated by changing the concentration of the same guidance cue.

The carboxyl-terminal region of Crtac1B/LOTUS acts as a functional domain in endogenous antagonism to Nogo receptor-1

Myelin-derived axon growth inhibitors, such as Nogo, bind to Nogo receptor-1 (NgR1) and thereby limit the action of axonal regeneration after injury in the adult central nervous system. Recently, we have found that cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS) binds to NgR1 and functions as an endogenous NgR1 antagonist. To examine the functional domain of LOTUS in the antagonism to NgR1, analysis using the deletion mutants of LOTUS was performed and revealed that the carboxyl-terminal region (UA/EC domain) of LOTUS bound to NgR1. The UA/EC fragment of LOTUS overexpressed together with NgR1 in COS7 cells abolished the binding of Nogo66 to NgR1. Overexpression of the UA/EC fragment in cultured chick dorsal root ganglion neurons suppressed Nogo66-induced growth cone collapse. These findings suggest that the UA/EC region is a functional domain of LOTUS serving for an antagonistic action to NgR1.

Syntaxin 1B Suppresses Macropinocytosis and Semaphorin 3A-Induced Growth Cone Collapse

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.