Plexin Research Articles

Semaphorin 6B acts as a receptor in post-crossing commissural axon guidance

Semaphorins are a large family of axon guidance molecules that are known primarily as ligands for plexins and neuropilins. Although class-6 semaphorins are transmembrane proteins, they have been implicated as ligands in different aspects of neural development, including neural crest cell migration, axon guidance and cerebellar development. However, the specific spatial and temporal expression of semaphorin 6B (Sema6B) in chick commissural neurons suggested a receptor role in axon guidance at the spinal cord midline. Indeed, in the absence of Sema6B, post-crossing commissural axons lacked an instructive signal directing them rostrally along the contralateral floorplate border, resulting in stalling at the exit site or even caudal turns. Truncated Sema6B lacking the intracellular domain was unable to rescue the loss-of-function phenotype, confirming a receptor function of Sema6B. In support of this, we demonstrate that Sema6B binds to floorplate-derived plexin A2 (PlxnA2) for navigation at the midline, whereas a cis-interaction between PlxnA2 and Sema6B on pre-crossing commissural axons may regulate the responsiveness of axons to floorplate-derived cues.

Semaphorin 6B acts as a receptor in post-crossing commissural axon guidance.

Semaphorins are a large family of axon guidance molecules that are known primarily as ligands for plexins and neuropilins. Although class-6 semaphorins are transmembrane proteins, they have been implicated as ligands in different aspects of neural development, including neural crest cell migration, axon guidance and cerebellar development. However, the specific spatial and temporal expression of semaphorin 6B (Sema6B) in chick commissural neurons suggested a receptor role in axon guidance at the spinal cord midline. Indeed, in the absence of Sema6B, post-crossing commissural axons lacked an instructive signal directing them rostrally along the contralateral floorplate border, resulting in stalling at the exit site or even caudal turns. Truncated Sema6B lacking the intracellular domain was unable to rescue the loss-of-function phenotype, confirming a receptor function of Sema6B. In support of this, we demonstrate that Sema6B binds to floorplate-derived plexin A2 (PlxnA2) for navigation at the midline, whereas a cis-interaction between PlxnA2 and Sema6B on pre-crossing commissural axons may regulate the responsiveness of axons to floorplate-derived cues.

Transmembrane recognition of the semaphorin co-receptors neuropilin 1 and plexin A1: coarse-grained simulations.

The cancer associated class 3 semaphorins require direct binding to neuropilins and association to plexins to trigger cell signaling. Here, we address the role of the transmembrane domains of neuropilin 1 and plexin A1 for the dimerization of the two receptors by characterizing the assembly in lipid bilayers using coarse-grained molecular dynamics simulations. From experimental evidence using a two-hybrid system showing the biochemical association of the two receptors transmembrane domains, we performed molecular simulations in DOPC and POPC demonstrating spontaneously assembly to form homodimers and heterodimers with a very high propensity for right-handed packing of the helices. Inversely, left-handed packing was observed with a very low propensity. This mode of packing was observed uniquely when the plexin A1 transmembrane domain was involved in association. Potential of mean force calculations were used to predict a hierarchy of self-association for the monomers: the two neuropilin 1 transmembrane domains strongly associated, neuropilin 1 and plexin A1 transmembrane domains associated less and the two plexin A1 transmembrane domains weakly but significantly associated. We demonstrated that homodimerization and heterodimerization are driven by GxxxG motifs, and that the sequence context modulates the packing mode of the plexin A1 transmembrane domains. This work presents major advances towards our understanding of membrane signaling platforms assembly through membrane domains and provides exquisite information for the design of antagonist drugs defining a novel class of therapeutic agents.

Role of Semaphorins in Angiogenesis and Innervation in Human Intervertebral Disc Degeneration

Introduction Although the intervertebral disc (IVD) is considered the largest aneural structure within the human body, during degeneration nociceptive nerves are seen to accompany blood vessels into the IVD yet it remains unclear how this process occurs. Semaphorins are axonal guidance molecules known to repulse nerves by altering the actin cytoskeleton of growth cones. Semaphorin 3A (sema3A) has recently been localized in healthy IVDs, suggesting an inhibitory role toward neural and vascular ingrowth. This study aimed to first identify the presence of the class 3 semaphorins and sema4D shown to be involved in promoting angiogenesis and their receptors within nucleus pulposus(NP) cells and an endothelial cell line and determine whether their expression could be altered by cytokine treatment. Materials and Methods Real-time PCR (RT-PCR) was performed to investigate gene expression of class 3 semaphorins; Sema3A-3F, sema4D, their receptors plexin A1-A4 and plexin D1 and neuropilin-1 (NRP-1) and NRP-2 in directly extracted RNA from human NP cells and NP cells cultured in alginate for 2weeks before treatment for 48hours with interleukin (IL)-1, IL-6, or tumor necrosis factor α(TNFα) at 0 to 100ng/mL. Similarly, HDMEC-1 endothelial cells were treated with IL-1, IL-6, or TNFα at 0 and 10ng/mL for 48hours, after which mRNA expression was investigated by RT-PCR. Results Expression of semaphorins and their receptors were present in normal and degenerate disc samples. In human NP cells, IL-6 inhibited semaphorin expression, whereas IL-1 and TNFα significantly increased sema3D over 10-fold ( p≤0.05). IL-1 upregulated all plexins, conversely TNFα significantly decreased plexin A2. NRP-2 showed a significant increase in response to IL-1. While NRP-1 was significantly decreased in response to IL-1. In HDMEC-1 cells IL-1 significantly increased sema3C and sema3D while TNF significantly increased sema3C. TNF significantly increased NRP-2 expression, however, NRP-1 was decreased by all cytokines. Plexin receptor expression was increased upon IL-6 and TNF treatment whereas IL-1 caused significant decrease in plexin A3 expression. Conclusion Here, we have demonstrated that several cytokines known to be upregulated during disc degeneration and disc prolapse, regulate expression of certain members of class 3 semaphorins, as well as their receptors in human NP cells and HDMEC-1 cells. This data suggests that the presence of these factors within the degenerate disc may be responsible for modulating the ingrowth of blood vessels and promote nerve ingrowth leading to discogenic pain. Disclosure of Interest None declared

On and Off Retinal Circuit Assembly by Divergent Molecular Mechanisms

Direction-selective responses to motion can be to the onset (On) or cessation (Off) of illumination. Here, we show that the transmembrane protein semaphorin 6A and its receptor plexin A2 are critical for achieving radially symmetric arborization of On starburst amacrine cell (SAC) dendrites and normal SAC stratification in the mouse retina. Plexin A2 is expressed in both On and Off SACs; however, semaphorin 6A is expressed in On SACs. Specific On-Off bistratified direction-selective ganglion cells in semaphorin 6A(-/-) mutants exhibit decreased tuning of On directional motion responses. These results correlate the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion, shedding light on the development of visual pathways that use the same cell types for divergent outputs.

36. Blockade of the neuropilin-2/plexin A2 receptor – New small peptides found by phage display

The loss of sympathetic nerve fibers is a general principle in inflammatory diseases. Since sympathetic neurotransmitters exert anti-inflammatory effects at increased concentrations, their loss is reasonable to overcome infection but detrimental in chronic inflammatory autoimmune diseases. Semaphorins are major factors involved in axon guidance and repulsion mediated by a neuropilin-2/Plexin A2 receptor-complex on nerve endings. Antagonizing semaphorin might keep nerve fibers in the inflamed area and may be a new therapeutic principle in treatment of rheumatoid arthritis. We found very effective polyclonal antibodies neutralizing the effects of semaphorin 3F-induced nerve fiber repulsion. Based on this finding we generated peptides with high binding capacity to Plexin A2 and Semaphorin 3F. Phage display is a technique to study protein–peptide binding with high throughput of up to 109 different peptides on bacteriophages. On basis of the Phages, we found possible binding sites of Plexin A2 and Semaphorin 3F and created exactly fitting peptides to this hot spots on the proteins. We found different peptides to block the binding sites on Plexin A2, Neuropilin-2 and Semaphorin 3F. First in vitro tests showed nearly complete abrogation of nerve fiber repulsion at protein concentrations of 150 nmol/l. This study provided us with an important tool to manipulate sympathetic nerve fiber repulsion. Next step is testing the effect in an arthritis mouse model.

Identification of novel TMPRSS2:ERG mechanisms in prostate cancer metastasis: involvement of MMP9 and PLXNA2

Prostate cancer (PCa) is one of the major public health problems in Western countries. Recently, the TMPRSS2:ERG gene fusion, which results in the aberrant expression of the transcription factor ERG, has been shown to be the most common gene rearrangement in PCa. Previous studies have determined the contributions of this fusion in PCa disease initiation and/or progression in vitro and in vivo. In this study on TMPRSS2:ERG regulation in PCa, we used an androgen receptor and TMPRSS2:ERG fusion double-negative PCa cell model: PC3c. In three cell clones with different TMPRSS2:ERG expression levels, ectopic expression of the fusion resulted in significant induction of cell migration and invasion in a dose-dependent manner. In agreement with this phenotype, high-throughput microarray analysis revealed that a set of genes, functionally associated with cell motility and invasiveness, were deregulated in a dose-dependent manner in TMPRSS2:ERG-expressing cells. Importantly, we identified increased MMP9 (Metalloproteinase 9) and PLXNA2 (Plexin A2) expression in TMPRSS2:ERG-positive PCa samples, and their expression levels were significantly correlated with ERG expression in a PCa cohort. In line with these findings, there was evidence that TMPRSS2:ERG directly and positively regulates MMP9 and PLXNA2 expression in PC3c cells. Moreover, PLXNA2 upregulation contributed to TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. Furthermore, and importantly, PLXNA2 expression was upregulated in metastatic PCa tumors compared with localized primary PCa tumors. This study provides novel insights into the role of the TMPRSS2:ERG fusion in PCa metastasis.

Abstract 1412: Cross-species array comparative genomic hybridization identifies novel driver genes in embryonal rhabdomyosarcoma.

Abstract Human cancers are often associated with large chromosomal amplifications and deletions comprising megabases of sequence - making it challenging to identify drivers of cancer growth and progression. To bypass this obstacle, we have applied array comparative genomic hybridization (aCGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to identify novel collaborating oncogenes important to human disease. Because zebrafish ERMS predominantly contain small, focal amplifications that span only 50-100kb of sequence, candidate oncogenes were easily identified by cross-species comparisons with human ERMS. Remarkably, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, copy gains in human disease. Candidate oncogenes found in these regions were also over-expressed in a majority of human ERMS as assessed by microarray gene expression analysis and immunohistochemical stain of primary human ERMS and normal fetal muscle. siRNA and shRNA knockdown studies identified important roles for candidate ocogenes in regulating human ERMS growth. For example, knockdown of Cyclin D2, Homeobox Protein C6, and Plexin A1 (PLXNA1) resulted in reduced proliferation in both human RD and SMS-CTR cell lines. PLXNA1 knockdown also resulted in impaired migratory potential in scratch and transwell migration assays. Moreover, PLXNA1 loss resulted in increased numbers of terminally-differentiated myosin-expressing myocytes, suggesting that dysregulated expression of PLXNA1 plays a key role in the differentiation arrest of human ERMS. Finally, vascular endothelial growth factor (VEGF) was also amplified and over-expressed in a subset of human and zebrafish ERMS. Chemical inhibition of VEGF in ERMS-bearing zebrafish led to significantly reduced tumor growth which was associated with decreased tumor neovascularization. Analysis of human microarray gene expression data revealed that increased VEGFA expression correlated with poor clinical outcome in patients with ERMS independent of tumor grade and stage, indicating high VEGFA expression as an independent prognostic indicator and implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. In total, our results demonstrate the utility of aCGH and cross-species comparisons to rapidly identify candidate genes essential for the pathogenesis of human cancer. Citation Format: Eleanor Chen, Kimberly P. Dobrinski, Kim H. Brown, Ryan Clagg, Elena Edelman, Myron Ignatius, Jillian Brockmann, G. Petur Nielsen, Sridhar Ramaswamy, Charles Keller, Charles Lee, David M. Langenau. Cross-species array comparative genomic hybridization identifies novel driver genes in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2013-1412

Label‐free quantitative analysis of the membrane proteome of Bace1 protease knock‐out zebrafish brains

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β-secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter-aided sample preparation, strong-anion exchange fractionation, and label-free quantification. We used bace1-deficient zebrafish and quantified differences in protein levels between wild-type and bace1 -/- zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild-type and bace1 -/- zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty-four membrane proteins accumulated in the bace1 -/- brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican-1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock-out zebrafish tissue and demonstrates that combining gene knock-out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.

Blockade of the neuropilin-2/plexin A2 receptor – A new therapeutic approach towards rheumatoid arthritis?

The loss of sympathetic nerve fibers (SNF) is a general principle in inflammatory diseases. Since sympathetic neurotransmitters exert anti-inflammatory effects at increased concentrations, their loss in inflammed and infected tissue is reasonable to overcome infection. However, this mechanism is unfavourable in chronic inflammation like rheumatoid arthritis (RA). Semaphorins are major factors involved in axon guidance and repulsion mediated by a neuropilin-2/plexin A2 receptor complex on nerve endings. We hypothesize that antagonizing semaphorin binding to its receptor can keep nerve fibers in the inflamed area, and this may be a new therapeutic principle in the treatment of RA. In this study, we wanted to test the neutralizing effects of polyclonal antibodies to plexin A2 on semaphorin 3F-induced nerve fiber repulsion. For these investigations, a neurite outgrowth assay was used to study the behaviour of nerve fibers from sympathetic trunk ganglia of postnatal mice. Nerve repulsion caused by semaphorin 3F is about 35–50%. Repulsion can be completely abrogated by a polyclonal anti-plexin A2 antibody in a concentration of 157 nmol/l (to 0–9%; p This study provided us with a first important tool to manipulate sympathetic nerve fiber repulsion. We are testing these antibodies in an animal model of experimental arthritis.

Plexin A3 and Turnout Regulate Motor Axonal Branch Morphogenesis in Zebrafish

During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch morphogenesis by destabilizing membrane protrusions before the growth cone completes navigation into the synaptic target field.

Comparative analysis of semaphorin 3A in soleus and EDL muscle satellite cells in vitro toward understanding its role in modulating myogenin expression

Resident myogenic stem cells, satellite cells, up-regulate a secreted multi-functional modulator, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle-crush injury and treatment with hepatocyte growth factor (HGF) or basic fibroblast growth factor (FGF2). Here, we add evidence that the Sema3A expression and secretion induced by the growth factors is significantly higher in primary cultures from adult rat soleus than from the fast-twitch extensor digitorum longus (EDL) muscle. The higher Sema3A response, revealed by quantitative PCR and Western blotting of cell lysates and conditioned media, may account for the higher myogenin expression of soleus muscle satellite cells early in differentiation since addition of recombinant Sema3A stimulates myogenin expression in cultures. These experiments also showed that mRNA expression of plexin A2, which together with neuropilins, constitutes Sema3A composite-receptors, was higher in satellite cells from soleus than EDL with no difference in plexin A1 and A3 and neuropilin-1 and 2 levels. These comparative studies, therefore, highlight a possible Sema3A-plexin A2-myogenin signaling axis that may ensure promoting early differentiation by soleus muscle satellite cells.

Plexin A3 is involved in semaphorin 3F-mediated oligodendrocyte precursor cell migration

Class 3 semaphorins are expressed in the neurodevelopmental or damage repair phase of the central nervous system (CNS). They play an important role in guiding axon growth and directing cell migration, including the migration of oligodendrocyte precursor cells (OPCs). As co-receptors for semaphorin 3F(sema3F), the expression and role of neuropilin-2 (NRP2) and plexin A3 in OPC migration are unclear. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrated that primary OPCs and immature oligodendrocytes from neonatal rats express NRP2 and plexin A3. After transfection with NRP2 siRNA and plexin A3 siRNA, the number of migrating OPCs attracted to sema3F remarkably decreased. These results suggest that plexin A3 is expressed in OPCs and immature oligodendrocytes and is involved in OPC migration.

55. Blockade of the neuropilin-2/plexin A2 receptor – A new treatment of rheumatoid arthritis?

Background: The loss of sympathetic nerve fibers (SNF) is a general principle in inflammatory diseases. Since sympathetic neurotransmitters exert anti-inflammatory effects at increased concentrations, their loss in inflamed tissue is reasonable to overcome infection. However, this mechanism is unfavorable in chronic inflammation like rheumatoid arthritis (RA). Semaphorins are major factors involved in axon guidance and repulsion mediated by a neuropilin-2/ Plexin A2 receptor complex on nerve endings. We proposed that antagonizing semaphorin binding to its receptor can keep nerve fibers in the inflamed area and may be a new therapeutic principle in treatment of RA. In this study, we wanted to test the neutralizing effects of polyclonal antibodies to plexin A2 on semaphorin 3F-induced nerve fiber repulsion. Methods: A neurite outgrowth assay was used to investigate the behavior of nerve fibers from sympathetic trunk ganglia of postnatal mice. Results: Nerve repulsion caused by semaphorin 3F is about 35–50%. Repulsion can be completely abrogated by a polyclonal anti-plexin A2 antibody in a concentration of 157 nmol/l (to 0–9%; p

993 Development of a Therapeutic Peptide Targeting the Transmembrane Domain of Plexin A1

993 Development of a Therapeutic Peptide Targeting the Transmembrane Domain of Plexin A1

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.

Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F

Class 3 semaphorins (SEMA), SEMA3B and SEMA3F, are secreted proteins that regulate angiogenesis, tumor growth, and metastasis by binding to their transmembrane receptor complex consisting of plexins and neuropilins (NP). Expression of SEMAs and their receptors was assessed in tissue microarrays by immunohistochemistry. SEMA3B, SEMA3F, and plexin A3 were expressed strongly in normal endometrial tissues, whereas grade-dependent decreases were found in endometrial carcinomas. No change was observed in the expression of plexin A1, NP1, and NP2 in normal versus endometrial cancer tissues. Endometrial cancer cells showed decreased expression of SEMA3B, SEMA3F, and plexin A3 compared with their normal counterparts. Treatment of cancer cells with progesterone (P4) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] for a period of 72 hours induced a significant upregulation of SEMA3B and SEMA3F as well as inhibited growth of cancer cells by increasing caspase-3 activity. Cotreatment of cell lines with P4 or 1,25(OH)(2)D(3) and their respective antagonists confirmed the specificity of their actions. Transfection of siRNA-targeting SEMA3B and SEMA3F in endometrial cancer cells attenuated P4 or 1,25(OH)(2)D(3)-induced growth inhibition. Restoration of SEMA3B or SEMA3F expression in cancer cells caused growth inhibition, reduced soft agar colony formation, and cell invasiveness by inhibiting expression of matrix metalloproteinase-2 (MMP-2), MMP-9, integrin αvβ3, and proangiogenic genes and by upregulating antiangiogenic genes. Thus, we have identified two new P4 and 1,25(OH)(2)D(3)-regulated antitumor genes for endometrial cancer. These results suggest that the loss of SEMAs contribute to the malignant phenotype of endometrial cancer cells and that reexpression of SEMAs by ectopic expression or with anticancer agents P4 or 1,25(OH)(2)D(3) can be a promising therapeutic treatment against endometrial cancer.

Structural basis for semaphorin signalling through the plexin receptor

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.

Possible association of the semaphorin 3D gene ( SEMA3D) with schizophrenia

Semaphorins are ligands of plexins, and the plexin–semaphorin signaling system is widely involved in many neuronal events including axon guidance, cell migration, axon pruning, and synaptic plasticity. The plexin A2 gene ( PLXNA2) has been reported to be associated with schizophrenia. This finding prompted us to examine the possible association between the semaphorin 3D gene ( SEMA3D) and schizophrenia in a Japanese population. We genotyped 9 tagging single nucleotide polymorphisms (SNPs) of SEMA3D including a non-synonymous variation, Lys701Gln (rs7800072), in a sample of 506 patients with schizophrenia and 941 healthy control subjects. The Gln701 allele showed a significant protective effect against the development of schizophrenia ( p = 0.0069, odds ratio = 0.76, 95% confidence interval 0.63 to 0.93). Furthermore, the haplotype-based analyses revealed a significant association. The four-marker analysis (rs2190208–rs1029564–rs17159614–rs12176601), in particular, not including the Lys701Gln, revealed a highly significant association ( p = 0.00001, global permutation), suggesting that there may be other functional polymorphisms within SEMA3D. Our findings provide strong evidence that SEMA3D confers susceptibility to schizophrenia, which could contribute to the neurodevelopmental impairments in the disorder.

Multicenter, randomized, double-blind, active comparator and placebo-controlled trial of a corticotropin-releasing factor receptor-1 antagonist in generalized anxiety disorder

Antagonism of corticotropin-releasing factor (CRF) receptors has been hypothesized as a potential target for the development of novel anxiolytics. This study was designed to determine the safety and efficacy of pexacerfont, a selective CRF-1 receptor antagonist, in the treatment of generalized anxiety disorder (GAD). This was a multicenter, randomized, double-blind, placebo-controlled and active comparator trial. Two hundred and sixty patients were randomly assigned to pexacerfont 100 mg/day (after a 1 week loading dose of 300 mg/day), placebo or escitalopram 20 mg/day in a 2:2:1 ratio. The primary outcome was the mean change from baseline to end point (week 8) in the Hamilton Anxiety Scale total score. Pexacerfont 100 mg/day did not separate from placebo on the primary outcome measure. The half-powered active comparator arm, escitalopram 20 mg/day, demonstrated efficacy with significant separation from placebo at weeks 1, 2, 3, 6, and 8 (P<.02). Response rates for pexacerfont, placebo, and escitalopram were 42, 42, and 53%, respectively. Genetic and psychometric rating scale data was obtained in 175 randomized subjects. There was a significant association between a single nucleotide polymorphism (SNP) of the gene encoding plexin A2 (PLXNA2-2016) with the HAM-A psychic subscale score for the entire cohort at baseline (FDR-adjusted P=.015). Pexacerfont did not demonstrate efficacy compared to placebo for the treatment of GAD. Whether these findings are generalizable to this class of agents remains to be determined. Our preliminary genetic finding of an association between a SNP for the gene encoding plexin A2 and an anxiety phenotype in this study merits further exploration. The trial was registered at clinicaltrials.gov (NCT00481325) before enrollment.