Neogenin mediates the action of repulsive guidance molecule

Repulsive guidance molecule (RGM) is a recently identified protein implicated in both axonal guidance and neural tube closure. The avoidance of chick RGM in the posterior optic tectum by growing temporal, but not nasal, retinal ganglion cell axons is thought to contribute to visual map formation. In contrast to ephrins, semaphorins, netrins and slits, no receptor mechanism for RGM action has been defined. Here, an expression cloning strategy identified neogenin as a binding site for RGM, with a sub-nanomolar affinity. Consistent with selective axonal responsiveness to RGM, neogenin is expressed in a gradient across the chick retina. Neogenin is known to be one of several netrin-binding proteins but only neogenin interacts with RGM. The avoidance of RGM by temporal retinal axons is blocked by the anti-neogenin antibody and the soluble neogenin ectodomain. Dorsal root ganglion axons are unresponsive to RGM but are converted to a responsive state by neogenin expression. Thus, neogenin functions as an RGM receptor.     

Neogenin regulates neuronal survival through DAP kinase

The repulsive guidance molecule (RGM) is a membrane-bound protein that has diverse functions in the developing central nervous system. Identification of neogenin as a receptor for RGM provided evidence of its cell death-inducing activity in the absence of RGM. Here, we show that the serine/threonine kinase death-associated protein kinase (DAPK) is involved in the signal transduction of neogenin. Neogenin interacts with DAPK and reduces DAPK autophosphorylation on Ser308 in vitro. Neogenin-induced cell death is abolished in the presence of RGM or by blocking DAPK. Although neogenin overexpression or RGM downregulation in the chick neural tube in vivo induces apoptosis, coexpression of the dominant-negative mutant or small-interference RNA of DAPK attenuates this proapoptotic activity. Thus, RGM/neogenin regulates cell fate by controlling the DAPK activity.     

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.     

Identification of the neogenin-binding site on the repulsive guidance molecule A

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the chick retinotectal system. RGMa, one of the 3 isoforms found in mammals, is involved in laminar patterning, cephalic neural tube closure, axon guidance, and inhibition of axonal regeneration. In addition to its roles in the nervous system, RGMa plays a role in enhancing helper T-cell activation. Binding of RGM to its receptor, neogenin, is considered necessary to transduce these signals; however, information on the binding of RGM to neogenin is limited. Using co-immunoprecipitation studies, we have identified that the RGMa region required for binding to neogenin contains amino acids (aa) 259-295. Synthesized peptide consisting of aa 284-293 directly binds to the extracellular domain (ECD) of recombinant neogenin, and addition of this peptide inhibits RGMa-induced growth cone collapse in mouse cortical neurons. Thus, we propose that this peptide is a promising lead in finding reagents capable of inhibiting RGMa signaling.     

Emerging roles for neogenin and its ligands in CNS development

It is now well established that the netrin guidance cues and their receptors comprise a major molecular guidance system driving axon pathfinding during nervous system development. One netrin receptor, neogenin, is now emerging as a key regulator of many developmental processes throughout the embryo. Unexpectedly, a new family of neogenin ligands, the repulsive guidance molecule (RGM) family, has recently been identified. The functional outcome of neogenin activation is dictated by both the nature of the ligand as well as the developmental context. Netrin-1–neogenin interactions mediate chemoattractive axon guidance, while RGMa–neogenin interactions repel axons. Neogenin is required for the establishment of the pseudostratified epithelium of the neural tube, probably by promoting cell adhesion. In addition, a role for RGMa and neogenin in neuronal differentiation has been demonstrated. While neogenin signaling cascades are poorly understood, the opposing responses of neogenin to RGMa and netrin-1 in the context of axon guidance indicates that neogenin signaling is complex and subject to tight spatiotemporal regulation. In summary, neogenin is a multifunctional receptor regulating diverse developmental processes. Thus, its contribution to neural development is proving to be considerably more extensive than originally predicted.     

Repulsive guidance molecule/neogenin: a novel ligand-receptor system playing multiple roles in neural development

The repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as an axon guidance molecule in the visual system. Recently, the transmembrane protein, neogenin, has been identified as the RGM receptor. In vitro analysis with retinal explants showed that RGM repels temporal retinal axons and collapses their growth cones through neogenin-mediated signaling. However, RGM and neogenin are also broadly expressed at the early embryonic stage, suggesting that they do not only control the guidance of visual axons. Gene expression perturbation experiments in chick embryos showed that neogenin induces cell death, and its ligand, RGM, blocks the pro-apoptotic activity of neogenin. Thus, RGM/neogenin is a novel dependence ligand/receptor couple as well as an axon guidance molecular complex.     

Neogenin and repulsive guidance molecule signaling in the central nervous system

The repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies have revealed that it has roles in axon guidance and laminar patterning in Xenopus and chick embryos, and in controlling cephalic neural tube closure in mouse embryos. The recent identification of neogenin as a receptor for RGM has provided evidence of the diverse functions of this ligand-receptor pair. Re-expression of RGM is observed after injury in the adult human and rat central nervous systems. Inhibition of RGM enhances growth of injured axons and promotes functional recovery after spinal cord injury in rats. Thus, re-expression of embryonic repulsive cues in adult tissues contributes to failure of axon regeneration in the central nervous system.     

Netrin‐1 is required for efficient neural tube closure

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell–cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin‐1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin‐1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin‐1 results in delayed neural fold apposition and neural tube closure. We further show that netrin‐1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin‐RGMa interactions, neogenin‐netrin‐1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin‐1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure. © 2012 Wiley Periodicals, Inc., 2013     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

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.     

Dragon (Repulsive Guidance Molecule RGMb) Inhibits E-cadherin Expression and Induces Apoptosis in Renal Tubular Epithelial Cells

Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45–66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo.     

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.     

Gene expression of the repulsive guidance molecules/neogenin in the developing and mature mouse visual system: C57BL/6J vs. the glaucoma model DBA/2J

We used in-situ hybridization to analyze the expression patterns of three known members (a, b and c) of the RGM ("repulsive guidance molecule") gene family and of the RGMa receptor neogenin in a glaucoma mouse model (DBA/2J strain) and the C57BL/6J strain, which served as a control. In order to understand the role of the RGMs and neogenin in glaucoma, we characterized their expression patterns in the developing and mature mouse retina and in the optic nerve. In all investigated stages from post-natal day (P) 0 to 15 months (M) RGMa, RGMb and neogenin expression was detected in the ganglion cell layer (GCL). From P10 to 15M, we found RGMa, RGMb and neogenin expression in the inner nuclear layer (INL) and the outer nuclear layer (ONL). In P10- and older mice, the expression patterns of RGMa and its receptor neogenin were similar, while that of RGMb differed from both. As expected, no specific retinal expression of RGMc was detected in any of the age groups investigated. C57BL/6J mice and DBA/2J mice displayed no differences in the expression pattern of RGMa, RGMb, RGMc and neogenin in the developing retina (gestational age 14.5 days (E14.5), P0 & P10). Interestingly, we found a higher expression of RGMa, RGMb and neogenin in the retinas of all glaucoma-affected mice than in the age-matched control strain. Furthermore, we detected a higher RGMa and RGMb expression in the optic nerves of glaucoma-affected DBA/2J-mice older than 11M than in C57BL/6J mice of the same age.     

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.     

Gene expression of the repulsive guidance molecules/neogenin in the developing and mature mouse visual system: C57BL/6J vs. the glaucoma model DBA/2J

We used in-situ hybridization to analyze the expression patterns of three known members (a, b and c) of the RGM (“repulsive guidance molecule”) gene family and of the RGMa receptor neogenin in a glaucoma mouse model (DBA/2J strain) and the C57BL/6J strain, which served as a control. In order to understand the role of the RGMs and neogenin in glaucoma, we characterized their expression patterns in the developing and mature mouse retina and in the optic nerve. In all investigated stages from post-natal day (P) 0 to 15 months (M) RGMa, RGMb and neogenin expression was detected in the ganglion cell layer (GCL). From P10 to 15 M, we found RGMa, RGMb and neogenin expression in the inner nuclear layer (INL) and the outer nuclear layer (ONL). In P10- and older mice, the expression patterns of RGMa and its receptor neogenin were similar, while that of RGMb differed from both. As expected, no specific retinal expression of RGMc was detected in any of the age groups investigated. C57BL/6J mice and DBA/2J mice displayed no differences in the expression pattern of RGMa, RGMb, RGMc and neogenin in the developing retina (gestational age 14.5 days (E14.5), P0 & P10). Interestingly, we found a higher expression of RGMa, RGMb and neogenin in the retinas of all glaucoma-affected mice than in the age-matched control strain. Furthermore, we detected a higher RGMa and RGMb expression in the optic nerves of glaucoma-affected DBA/2J-mice older than 11 M than in C57BL/6J mice of the same age.     

Peptides derived from repulsive guidance molecule act as antagonists

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system [T. Yamashita, B.K. Mueller, K. Hata, Neogenin and RGM signaling in the central nervous system, Curr. Opin. Neurobiol. 17 (2007) 29-34]. Functional studies in Xenopus and chick embryos have revealed the roles of RGM in axon guidance and laminar patterning, while those in mouse embryos have demonstrated its function in regulating the cephalic neural tube closure. Importantly, RGM inhibition enhanced the growth of injured axons and promoted functional recovery after spinal cord injury in rats. Here, we identified two RGMa-derived peptides that functioned as antagonists against RGMa. The peptides studied in vitro dose-dependently suppressed the neurite growth inhibition and growth cone collapse induced by RGMa. Thus, these peptides are promising reagents to treat injuries of the central nervous system.     

Repulsive guidance molecule A (RGMa): A molecule for all seasons

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.     

Neogenin: A multi-functional receptor regulating diverse developmental processes

Neogenin, a close relative of the axon guidance receptor Deleted in Colorectal Cancer (DCC), has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule (RGM) families. While Netrin-1-Neogenin interactions result in a chemoattractive axon guidance response, the interaction between Neogenin and RGMa induces a chemorepulsive response. Evidence is now accumulating that Neogenin is a multi-functional receptor regulating many diverse developmental processes, including neural tube and mammary gland formation, myogenesis and angiogenesis. Little is known of the function of Neogenin in the adult, however, a novel role in the regulation of iron homeostasis is now emerging. While the signal transduction pathways activated by Neogenin are poorly understood, it is clear that the functional outcome of Neogenin activation, at least in the embryo, depends on both the developmental context as well as the nature of the ligand.     

The axonal guidance receptor neogenin promotes acute inflammation

Neuronal guidance proteins (NGP) were originally described in the context of axonal growth and migration. Yet recent work has demonstrated that NGPs also serve as guidance cues for immune competent cells. A crucial target receptor for NGPs during embryonic development is the neogenin receptor, however its role during acute inflammation is unknown. We report here that neogenin is abundantly expressed outside the nervous system and that animals with endogenous repression of neogenin (Neo1(-/-)) demonstrate attenuated changes of acute inflammation. Studies using functional inhibition of neogenin resulted in a significant attenuation of inflammatory peritonitis. In studies employing bone marrow chimeric animals we found the hematopoietic presence of Neo1(-/-) to be responsible for the attenuated inflammatory response. Taken together our studies suggest that the guidance receptor neogenin holds crucial importance for the propagation of an acute inflammatory response and further define mechanisms shared between the nervous and the immune system.     

Repulsive guidance molecule A (RGMa)

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.