Inhibition of branching and spine maturation by repulsive guidance molecule in cultured cortical neurons

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies in Xenopus and chick embryos revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos demonstrated its function in regulating cephalic neural tube closure. Moreover, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we demonstrate in vitro that RGMa, an RGM homolog, inhibits neurite growth and cortical neuron branching on mouse embryonic day 16. Further, exposure of cultured neurons to RGMa significantly reduced the number of colocalized immunoreactive clusters of synapsin 1 and PSD-95 in the spines. This RGMa-mediated inhibition of the assembly of presynaptic and postsynaptic components suggests a role of RGMa in inhibiting mature synapse formation. Thus, RGMa may negatively regulate neuronal network formation in cortical neurons.     

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

Overexpression of repulsive guidance molecule (RGM) a induces cell death through Neogenin in early vertebrate development

Repulsive guidance molecule (RGM) a is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein that has been implicated in chemorepulsive axon guidance. Although RGMa binds the transmembrane receptor Neogenin, the developmental events controlled by the RGMa-Neogenin interactions in vivo remain largely unknown. We have cloned full-length RGMa from Xenopus borealis for the first time and identified two homologous genes referred to as RGMa1 and RGMa2. Here we show RGMa1 overexpression at 2-cell-stage resulted in cell death, which lead to an early embryonic lethal phenotype of the embryos. Time-lapse photomicroscopy revealed that embryos began to show initial morphological defects from ∼5 h post-fertilization (hpf) which was then followed by extensive blastomere cell death at ∼11 hpf. This phenotype was rescued by simultaneous knock down of RGMa using translation blocking anti-sense morpholinos. Knock down of the RGMa1 receptor Neogenin in RGMa1 overexpressing embryos was also able to rescue the phenotype. Together these results indicated that RGMa1 was signalling through Neogenin to induce cell death in the early embryo. While previous studies have suggested that Neogenin is a dependence receptor that induces cell death in the absence of RGM, we have instead shown that Neogenin-RGM interactions induce cell death in the early embryo. The roles of RGMa1 and Neogenin appear to be context specific so that their co-ordinated and regulated expressions are essential for normal development of the vertebrate embryo.     

Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule

Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.     

Analysis of a repulsive axon guidance molecule, draxin, on ventrally directed axon projection in chick early embryonic midbrain

The mesencephalic V neurons and tectobulbar axons in chick embryo project over long distances that appear during the early development of the chick optic tectum. The mesencephalic V neuron and tectobulbar axonal growth begin at Hamburger and Hamilton stage 14 and stage 18, respectively. Both fibers proceed downward from the dorsal to the ventral side of the lateral wall of the optic tectum and then turn caudally and join the medial longitudinal fasciculus. Their axons appear in the most superficial layer of the tectum at early stages and do not cross the dorsal midline of the tectum. Here, we report the role of draxin, a recently identified axon guidance protein, in the formation of the ventrally directed tectum axonal tracts in chicken embryo. draxin is expressed in a high dorsal to low ventral gradient in chick optic tectum. In vitro experiments show that draxin repels neurite outgrowth from dorsal tectum explants. In vivo overexpression resulted in inhibition or misrouting of axon growth in the tectum. Therefore, draxin may be an important member of the collection of repulsive guidance molecules that regulate the formation of the ventrally directed tectum axon tracts.     

A stochastic model of neuronal growth cone guidance regulated by multiple sensors

Neuronal growth cones migrate directionally under the control of axon guidance molecules, thereby forming synapses in the developing brain. The signal transduction system by which a growth cone detects surrounding guidance molecules, analyzes the detected signals, and then determines the overall behavior remains undetermined. In this study, we describe a novel stochastic model of this behavior that utilizes multiple sensors on filopodia to respond to guidance molecules. Overall growth cone behavior is determined by using only the concentration gradients of guidance molecules in the immediate vicinity of each sensor. The detected signal at each sensor, which is treated as a vector quantity, is sent to the growth cone center and then integrated to determine axonal growth in the next step by means of a simple vector operation. We compared the results of computer simulations of axonal growth with observations of actual axonal growth from co-culture experiments using olfactory bulb and septum. The probabilistic distributions of axonal growth generated by the computer simulation were consistent with those obtained from the culture experiments, indicating that our model accurately simulates growth cone behavior. We believe that this model will be useful for elucidating the as yet unknown mechanisms responsible for axonal growth in vivo.     

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.     

Neogenin interacts with RGMa and netrin-1 to guide axons within the embryonic vertebrate forebrain

In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory.     

Substance P analogues act as broad-spectrum neuropeptide antagonists

Neuropeptides including bombesin, vasopressin and bradykinin are increasingly implicated in the control of cell proliferation. There is now considerable evidence that the growth of certain common cancers including small cell lung cancer (SCLC) can be stimulated by multiple neuropeptides which act in an autocrine/paracrine fashion. Consequently, the development of broad spectrum neuropeptide antagonists could be of therapeutic interest. Indeed, certain substance P (SP) analogues including (DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹)SP and (Arg⁶, DTrp^7,9, MePhe⁸)SP (6–11) inhibit the actions of multiple neuropeptides and block the growth of SCLC cells in vitro and in vivo. Moreover, one of these compounds is now in a phase I clinical study and so an understanding of the mechanism of action of these SP analogues is both of fundamental as well as clinical interest. We have found that the SP analogues coordinately and reversibly inhibit the downstream signals which emanate from neuropeptide receptors and competitively block the binding of neuropeptides to their respective receptors. These and other results using novel SP analogues which are reviewed here, suggest that the SP analogues act directly on the neuropeptide receptors to block neuropeptide action.     

Substance P analogues act as broad-spectrum neuropeptide antagonists

Summary Neuropeptides including bombesin, vasopressin and bradykinin are increasingly implicated in the control of cell proliferation. There is now considerable evidence that the growth of certain common cancers including small cell lung cancer (SCLC) can be stimulated by multiple neuropeptides which act in an autocrine/paracrine fashion. Consequently, the development of broad spectrum neuropeptide, antagonists could be of therapeutic interest. Indeed, certain substance P (SP) analogues including (DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹) SP and (Arg⁶, DTrp^7,9, MePhe⁸)SP (6–11) inhibit the actions of multiple neuropeptides and block the growth of SCLC cells in vitro and in vivo. Moreover, one of these compounds is now in a phase I clinical study and so an understanding of the mechanism of action of these SP analogues is both of fundamental as well as clinical interest. We have found that the SP analogues coordinately and reversibly inhibit the downstream signals which emanate from neuropeptide receptors and competitively block the binding of neuropeptides to their respective receptors. These and other results using novel SP analogues which are reviewed here, suggest that the SP analogues act directly on the neuropeptide receptors to block neuropeptide action.     

Proteoglycans as cues for axonal guidance in formation of retinotectal or retinocollicular projections

Understanding the formation of neuronal circuits has long been one of the basic problems in developmental neurobiology. Projections from the retina to their higher center, the optic tectum in nonmammalian vertebrates and the superior colliculus in mammals, are most amenable to experimental approaches; thus, much information has been accumulated about the mechanisms of axonal guidance. The retinal axons navigate along the appropriate pathway with the help of a series of guidance cues. Although much of the work has focused on proteinaceous factors, proteoglycans have been identified as playing important roles in retinal axon guidance. Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are involved in essential decisions of axon steering along the pathway. However, it has not been determined whether diversity of the carbohydrate chains results in differential effects and how their diversity is recognized by growth cones, which represent an important area of future research.     

Weak electric fields serve as guidance cues that direct retinal ganglion cell axons in vitro

Growing axons are directed by an extracellular electric field in a process known as galvanotropism. The electric field is a predominant guidance cue directing retinal ganglion cell (RGC) axons to the future optic disc during embryonic development. Specifically, the axons of newborn RGCs grow along the extracellular voltage gradient that exists endogenously in the embryonic retina (Yamashita, 2013 [8]). To investigate the molecular mechanisms underlying galvanotropic behaviour, the quantification of the electric effect on axon orientation must be examined. In the present study, a culture system was built to apply a constant, uniform direct current (DC) electric field by supplying an electrical current to the culture medium, and this system also continuously recorded the voltage difference between the two points in the medium. A negative feedback circuit was designed to regulate the supplied current to maintain the voltage difference at the desired value. A chick embryo retinal strip was placed between the two points and cultured for 24 h in an electric field in the opposite direction to the endogenous field, and growing axons were fluorescently labelled for live cell imaging (calcein-AM). The strength of the exogenous field varied from 0.0005 mV/mm to 10.0 mV/mm. The results showed that RGC axons grew in the reverse direction towards the cathode at voltage gradients of ≥0.0005 mV/mm, and straightforward extensions were found in fields of ≥0.2–0.5 mV/mm, which were far weaker than the endogenous voltage gradient (15 mV/mm). These findings suggest that the endogenous electric field is sufficient to guide RGC axons in vivo.     

Repulsive guidance molecule A (RGM A) and its receptor neogenin during neural and neural crest cell development of Xenopus laevis

Background information. RGM A (repulsive guidance molecule A) is a GPI (glycosylphosphatidylinositol)‐anchored glycoprotein which has repulsive properties on axons due to the interaction with its receptor neogenin. In addition, RGM A has been demonstrated to function as a BMP (bone morphogenetic protein) co‐receptor. Results. In the present study, we provide the first analysis of early RGM A and neogenin expression and function in Xenopus laevis neural development. Tissue‐specific RGM A expression starts at stage 12.5 in the anterior neural plate. Loss‐of‐function analyses suggest a function of RGM A and neogenin in regulating anterior neural marker genes, as well as eye development and neural crest cell migration. Furthermore, overexpression of RGM A leads to ectopic expression of neural crest cell marker genes. Conclusions. These data indicate that RGM A and neogenin have important functions during early neural development, in addition to their role during axonal guidance and synapse formation.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.     

Triterpenes from Alisma orientalis act as androgen receptor agonists,progesterone receptor antagonists,and glucocorticoid receptor antagonists

Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

Peptides derived from pyruvate dehydrogenase as substrates for pyruvate dehydrogenase kinase and phosphatase.

Evidence is presented that phosphopeptides produced by tryptic digestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.     

Structure-activity relationships of anthraquinone derivatives derived from bromaminic acid as inhibitors of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)

Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (K_i 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (K_i 0.328 μM) and NTPDase3 (K_i 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (K_i 1.5 μM).     

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK_1/2 phosphorylation and, to a lesser extent, IP₁ production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.