Plexin-A3 mediates semaphorin signaling and regulates the development of hippocampal axonal projections.

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Here, we show that plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, we provide evidence that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo.     

The vesicular SNARE Synaptobrevin is required for Semaphorin 3A axonal repulsion

Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.     

Gating of Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to attraction during brain development

The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. Here, we show that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. Our data therefore reveal a "gating" function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.     

The transmembrane protein Off-track associates with Plexins and functions downstream of Semaphorin signaling during axon guidance

The Plexin family of transmembrane proteins appears to function as repulsive receptors for most if not all Semaphorins. Here, we use genetic and biochemical analysis in Drosophila to show that the transmembrane protein Off-track (OTK) associates with Plexin A, the receptor for Sema 1a, and that OTK is a component of the repulsive signaling response to Semaphorin ligands. In vitro, OTK associates with Plexins. In vivo, mutations in the otk gene lead to phenotypes resembling those of loss-of-function mutations of either Sema1a or PlexA. The otk gene displays strong genetic interactions with Sema1a and PlexA, suggesting that OTK and Plexin A function downstream of Sema 1a.     

FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A.     

The role and mechanism-of-action of Sema3E and Plexin-D1 in vascular and neural development

Class 3 secreted semaphorins (Sema3A–3G) participate in many aspects of axon guidance through holoreceptor complexes that include Neuropilin-1 (Npn-1) or Neuropilin-2 and one of the four class A plexin proteins. However, unlike other Sema3 family proteins, Sema3E directly binds to Plexin-D1 without neuropilins. Its biological function was first explored in intersomitic vessel formation and since its initial discovery, Sema3E–Plexin-D1 signaling has been found to participate in the many biological systems in addition to vascular development, via seemingly different mode of actions. For example, temporal and spatial control of ligand vs. receptor results in two different mechanisms governing vascular patterning. Interactions with other transmembrane proteins such as neuropilin and VEGFR2 result in different axonal behaviors. Ligand receptor localization on pre- vs. post-synaptic neurons is used to control different types of synapse formation. Perhaps different downstream effectors will also result in different functional outcomes. Given the limited number of ligands and receptors in the genome and their multifunctional nature, we expect that more modes of action will be discovered in the future. In this review, we highlight current advances on the mechanisms of how Sema3E–Plexin-D1 interaction shapes the networks of multiple biological systems, in particular the vascular and nervous systems.     

The activity of the plexin-A1 receptor is regulated by Rac

Plexins constitute a large family of transmembrane proteins that act as receptors for the semaphorin family of ligands. They are best known for their role in growth cone guidance, although they also are widely expressed outside the nervous system. Plexins are thought to control axon guidance by modifying the growth cone cytoskeleton, and Rho GTPases have been strongly implicated in this response. However, the exact contribution of Rho proteins is unclear. Sema3A/Plexin-A1-induced growth cone collapse, for example, requires Rac activity, which is a surprising result given that this GTPase is usually associated with membrane protrusions. We show here that Sema3A-induced collapse of COS-7 cells expressing Plexin-A1 also requires Rac but not Rho activity and that the cytoplasmic tail of Plexin-A1 interacts directly with activated Rac. However, collapse induced by a constitutively activated version of Plexin-A1 does not require Rac. We propose a novel function for Rac, namely that it acts upstream of Plexin-A1 during semaphoring-induced collapse, to regulate the activity of the receptor.     

Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells

The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC “routes”). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC “target”). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.     

Semaphorin 4D/Plexin-B1 Stimulates PTEN Activity through R-Ras GTPase-activating Protein Activity,Inducing Growth Cone Collapse in Hippocampal Neurons

Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 α activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.     

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.     

Neuropilin-2 acts as a modulator of Sema3A-dependent glioma cell migration

Semaphorin 3A (Sema3A) is a secreted guidance molecule initially described in the nervous system. This protein is able to control axon growth but also effects on endothelial cells migration. Here, we report that Sema3A acts as a chemorepellent factor for the rat C6 glioma cells and three different human glioma cell lines. Interestingly, Sema3A triggered a chemoattractive response in a fourth human glioma cell line. The nature of the receptor complex ensuring the appropriate signaling was dissected in C6 cells by using function blocking antibodies and gain- or loss-of function experiments using recombinant receptors. Our results demonstrate that neuropilin-1, neuropilin-2 and PlexinA1 are necessary to trigger cell repulsion. The selective blockade of neuropilin-1 or Plexin-A1 switched the chemorepulsive effect of Sema3A into a chemoattractive one. Strikingly, blocking Neuropilin-2 suppressed Sema3A-induced cell migration while overexpression of neuropilin-2 was able to convert the chemorepulsive effect of Sema3A into a chemoattractive one. Our results not only provide additional evidence for a biological function of Sema3A in glioma migration but also reveal part of the receptor complex involved. Hence, our study describes a receptor-based plasticity in cancer cells leading to opposite migration behavior in response to the same extracellular signal.Key words: semaphorin, neuropilin, glioma, cell migration, signalling, cancer     

Neuropilin-2 acts as a modulator of Sema3A-dependent glioma cell migration

Semaphorin 3A (Sema3A) is a secreted guidance molecule initially described in the nervous system. This protein is able to control axon growth but also effects on endothelial cells migration. Here, we report that Sema3A acts as a chemorepellent factor for the rat C6 glioma cells and 3 different human glioma cell lines. Interestingly, Sema3A triggered a chemoattractive response in a fourth human glioma cell line. The nature of the receptor complex ensuring the appropriate signaling was dissected in C6 cells by using function blocking antibodies and gain- or loss-of function experiments using recombinant receptors. Our results demonstrate that neuropilin-1, neuropilin-2 and PlexinA1 are necessary to trigger cell repulsion. The selective blockade of neuropilin-1 or Plexin-A1 switched the chemorepulsive effect of Sema3A into a chemoattractive one. Strikingly, blocking Neuropilin-2 suppressed Sema3A-induced cell migration while over-expression of neuropilin-2 was able to convert the chemorepulsive effect of Sema3A into a chemoattractive one. Our results not only provide additional evidence for a biological function of Sema3A in glioma migration but also reveal part of the receptor complex involved. Hence, our study describes a receptor-based plasticity in cancer cells leading to opposite migration behavior in response to the same extracellular signal.     

Requirement of the transmembrane semaphorin Sema4C for myogenic differentiation

Semaphorins constitute a large family of signaling proteins that contribute to axonal guidance. Here we demonstrate that the transmembrane semaphorin Sema4C is up-regulated both in the early stage of differentiation of C2C12 mouse skeletal myoblasts into myotubes and during injury-induced muscle regeneration in vivo. Depletion of Sema4C in C2C12 cells resulted in marked attenuation of myotube formation. A fusion protein containing the extracellular Sema domain and a peptide corresponding to the intracellular COOH-terminal region of Sema4C each inhibited the differentiation of C2C12 cells. These findings indicate that Sema4C-mediated interaction among myoblasts plays an important role in terminal myogenic differentiation.     

Plexin/neuropilin complexes mediate repulsion by the axonal guidance signal semaphorin 3A

In the developing nervous system axons navigate with great precision over large distances to reach their target areas. Chemorepulsive signals such as the semaphorins play an essential role in this process. The effects of one of these repulsive cues, semaphorin 3A (Sema3A), are mediated by the membrane protein neuropilin-1 (Npn-1). Recent work has shown that neuropilin-1 is essential but not sufficient to form functional Sema3A receptors and indicates that additional components are required to transduce signals from the cell surface to the cytoskeleton. Here we show that members of the plexin family interact with the neuropilins and act as co-receptors for Sema3A. Neuropilin/plexin interaction restricts the binding specificity of neuropilin-1 and allows the receptor complex to discriminate between two different semaphorins. Deletion of the highly conserved cytoplasmic domain of Plexin-A1 or -A2 creates a dominant negative Sema3A receptor that renders sensory axons resistant to the repulsive effects of Sema3A when expressed in sensory ganglia. These data suggest that functional semaphorin receptors contain plexins as signal-transducing and neuropilins as ligand-binding subunits.     

Expression patterns of plexins and neuropilins are consistent with cooperative and separate functions during neural development

Background  

Plexins are a family of transmembrane proteins that were shown to act as receptors for Semaphorins either alone or in a complex together with Neuropilins. Based on structural criteria Plexins were subdivided into 4 classes, A through D. PlexinAs are mainly thought to act as mediators of repulsive signals in cell migration and axon guidance. Their functional role in vertebrates has been studied almost exclusively in the context of Semaphorin signaling, i.e. as co-receptors for class 3 Semaphorins. Much less is known about Plexins of the other three classes. Despite the fact that Plexins are involved in the formation of neuronal circuits, the temporal changes of their expression patterns during development of the nervous system have not been analyzed in detail.     

Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). Here we characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppressed R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressed the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocked the growth cone collapse. The R-Ras GAP activity was necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and was also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induced dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. We conclude that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.     

Plexin-A1 and plexin-B1 specifically interact at their cytoplasmic domains

Semaphorin 3A (Sema3A) is a member of semaphorins and functions as an axonal repulsive guidance molecule. Neuropilin-1 and plexin-As form receptor complexes for Sema3A and plexin-As are thought to initiate the intracellular signaling cascade. However, the molecule by which plexin-As transduce their signal is not well understood. We searched molecules that interact with intracellular domains of plexin-A1 by yeast two-hybrid screening and identified a 349 amino acid fragment of plexin-B1 as a plexin-A1 interacting protein. We, then, cloned mouse plexin-B1 and confirmed their interaction in a mammalian expression system. Plexin-B1 physically associated with plexin-A1, but not with plexin-A2 or A3. Northern blot analysis showed the expression of both plexin-A1 and B1 in adult brain. We propose that plexin-A1 and B1 interact in the adult brain and transduce Sema3A signaling in cooperation.     

Plexin-A4 is expressed in oligodendrocyte precursor cells and acts as a mediator of semaphorin signals

Class 3 semaphorin acts as a guidance clue for both cell migration and nerve fiber projection. The signal of class 3 semaphorin travels via a receptor complex consisting of neuropilins and Plexin-A subfamily. Although it has been reported that class 3 semaphorin acts as a repellent for oligodendrocyte precursor cells (OPCs), which migrate actively during brain development, the expression of Plexin-A subfamily has not been reported in OPCs yet. Therefore, it is currently unclear how semaphorin signals can travel in OPCs. In the present study, the expression of Plexin-A4 (PlexA4) was first demonstrated in a newly established OPC line and OPCs in developing brain. In the OPC line, repulsion for process extension was caused by both Sema3A and Sema6A, and the effect of the semaphorins was diminished in cells expressing PlexA4 lacking the cytoplasmic domain. These results strongly suggest that PlexA4 expressed in OPCs acts as a mediator of semaphorin signals.     

Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3a ^loxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.     

Identification and characterization of zebrafish semaphorin 6D

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.