Activated microglia inhibit axonal growth through RGMa

By causing damage to neural networks, spinal cord injuries (SCI) often result in severe motor and sensory dysfunction. Functional recovery requires axonal regrowth and regeneration of neural network, processes that are quite limited in the adult central nervous system (CNS). Previous work has shown that SCI lesions contain an accumulation of activated microglia, which can have multiple pathophysiological influences. Here, we show that activated microglia inhibit axonal growth via repulsive guidance molecule a (RGMa). We found that microglia activated by lipopolysaccharide (LPS) inhibited neurite outgrowth and induced growth cone collapse of cortical neurons in vitro--a pattern that was only observed when there was direct contact between microglia and neurons. After microglia were activated by LPS, they increased expression of RGMa; however, treatment with RGMa-neutralizing antibodies or transfection of RGMa siRNA attenuated the inhibitory effects of microglia on axonal outgrowth. Furthermore, minocycline, an inhibitor of microglial activation, attenuated the effects of microglia and RGMa expression. Finally, we examined whether these in vitro patterns could also be observed in vivo. Indeed, in a mouse SCI model, minocycline treatment reduced the accumulation of microglia and decreased RGMa expression after SCI, leading to reduced dieback in injured corticospinal tracts. These results suggest that activated microglia play a major role in inhibiting axon regeneration via RGMa in the injured CNS.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.     

Neogenin-RGMa signaling at the growth cone is bone morphogenetic protein-independent and involves RhoA, ROCK, and PKC

The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin- and not BMP receptor-dependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) G?6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance.     

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.     

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.     

Silencing miR‐20a‐5p inhibits axonal growth and neuronal branching and prevents epileptogenesis through RGMa‐RhoA‐mediated synaptic plasticity

Epileptogenesis is a potential process. Mossy fibre sprouting (MFS) and synaptic plasticity promote epileptogenesis. Overexpression of repulsive guidance molecule a (RGMa) prevents epileptogenesis by inhibiting MFS. However, other aspects underlying the RGMa regulatory process of epileptogenesis have not been elucidated. We studied whether RGMa could be modulated by microRNAs and regulated RhoA in epileptogenesis. Using microRNA databases, we selected four miRNAs as potential candidates. We further experimentally confirmed miR‐20a‐5p as a RGMa upstream regulator. Then, in vitro, by manipulating miR‐20a‐5p and RGMa, we investigated the regulatory relationship between miR‐20a‐5p, RGMa and RhoA, and the effects of this pathway on neuronal morphology. Finally, in the epilepsy animal model, we determined whether the miR‐20a‐5p‐RGMa‐RhoA pathway influenced MFS and synaptic plasticity and then modified epileptogenesis. Our results showed that miR‐20a‐5p regulated RGMa and that RGMa regulated RhoA in vitro. Furthermore, in primary hippocampal neurons, the miR‐20a‐5p‐RGMa‐RhoA pathway regulated axonal growth and neuronal branching; in the PTZ‐induced epilepsy model, silencing miR‐20a‐5p prevented epileptogenesis through RGMa‐RhoA‐mediated synaptic plasticity but did not change MFS. Overall, we concluded that silencing miR‐20a‐5p inhibits axonal growth and neuronal branching and prevents epileptogenesis through RGMa‐RhoA‐mediated synaptic plasticity in the PTZ‐induced epilepsy model, thereby providing a possible strategy to prevent epileptogenesis.     

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 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.     

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.     

Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.     

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.     

The axonally secreted protein axonin-1 is a potent substratum for neurite growth

Axonin-1 is a neuronal glycoprotein occurring both as a membrane-bound and a secreted form. Membrane-bound axonin-1 is predominantly located in membranes of developing nerve fiber tracts and has recently been characterized as a cell adhesion molecule; the soluble form is secreted from axons and accumulates in the cerebrospinal fluid and the vitreous fluid of the eye. In the present study, we addressed the question as to whether secreted axonin-1 was released in a functionally competent form and we found that it strongly promotes neurite outgrowth when presented to neurons as an immobilized substratum. Neurite lengths elaborated by embryonic dorsal root ganglia neurons on axonin-1 were similar to those on the established neurite-promoting substrata L1 and laminin. Fab fragments of axonin-1 antibodies completely inhibited neurite growth on axonin-1, but not on other substrata. In soluble form, axonin-1 had an anti-adhesive effect, as revealed by perturbation of neurite fasciculation. In view of their structural similarity, we conclude that secreted and membrane-bound axonin-1 interact with the same growth-promoting neuritic receptor. The fact that secreted axonin-1 is functionally active, together with our previous findings that it is secreted from an internal cellular pool, suggests a functional dualism between membrane-bound and secreted axonin-1 at the site of secretion, which is most likely the growth cone. The secretion of adhesion molecules could represent a powerful and rapidly acting regulatory element of growth cone-neurite interactions in the control of neurite elongation, pathway selection, and possibly target recognition.     

Metalloproteases and γ‐secretase: new membrane partners regulating p75 neurotrophin receptor signaling?

Signaling by the p75 neurotrophin receptor (p75) has been implicated in diverse neuronal responses, including the control of neuronal survival versus death and axonal regeneration and growth cone collapse, involving p75 in different neuropathological conditions. There are different levels of complexity regulating p75-mediated signaling. First, p75 can interact with different ligands and co-receptors in the plasma membrane, forming tripartite complexes, whose activation result in different cellular outcomes. Moreover, it was recently described that trafficking capacities of p75 in neurons are regulating, in addition to p75 downstream interactions, also the sequential cleavage of p75. The proteolytical processing of p75 involves, first, a shedding event that releases a membrane-bound carboxiterminal fragment (p75-CTF), followed by a gamma-secretase mediated cleavage, generating a soluble intracellular domain (p75-ICD) with signaling capabilities. The first shedding event, generating a p75-CTF, is the key step to regulating the production of p75-ICD, and although the generation of p75-ICD is important for both p75-mediated control of neuronal survival and the control of neurite outgrowth, little is known how both cleavage events are regulated. In this review, we argue that both sheddases and gamma-secretase are key membrane components regulating p75-mediated signaling transduction; therefore, further attention should be paid to their roles as p75 signaling regulators.     

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.     

A homologue of the axonally secreted protein axonin-1 is an integral membrane protein of nerve fiber tracts involved in neurite fasciculation

Axonin-1 is a glycoprotein that is released from axons of cultured neurons (Stoeckli, E. T., P. F. Lemkin, T. B. Kuhn, M. A. Ruegg, M. Heller, and P. Sonderegger. 1989. Eur. J. Biochem. 180:249-258). It has recently been purified from the ocular vitreous fluid of the chicken embryo (Ruegg, M. A., E. T. Stoeckli, T. B. Kuhn, M. Heller, R. Zuellig, and P. Sonderegger. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:55-63). Immunohistochemistry localized axonin-1 prevalently in developing nerve fiber tracts. The presence of anti-axonin-1 Fab fragments during axon growth in vitro resulted in antibody binding to the axonal surfaces and in a marked perturbation of the fasciculation pattern. Hence, a fraction of axonin-1 is associated with axonal membranes and, by operational criteria, qualifies as a cell adhesion molecule. The major proportion of membrane-associated axonin-1 co-solubilized with the integral membrane proteins. By physico-chemical, immunological, and protein-chemical criteria, the integral membrane form was found to be highly similar to soluble axonin-1. In common with a number of other cell adhesion molecules, both soluble and membrane-bound axonin-1 express the L2/HNK-1 and the L3 epitopes. Radioactive pulse-chase and double-labeling experiments revealed that the released form was not derived from the membrane-bound form by shedding from the membrane surface, but directly secreted from an intracellular pool. Due to its high degree of similarity to the membrane-associated form and the presence of the L2/HNK-1 and L3 epitopes, reported to be ligands in adhesive cell interactions, adhesive properties are postulated for secreted axonin-1. As a soluble adhesive protein, it may function as a regulator of cell adhesion around its most likely site of secretion, the growth cone.     

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.     

The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.