Anti-repulsive guidance molecule: An antibody treatment in spinal cord injury

Abbreviations

BBB

blood brain barrier

BMP

bone morphogenetic protein

CNS

central nervous system

CST

corticospinal tract

DCC

deleted in colorectal cancer

GPI

glycosylphosphatidylinositol

MCAO

middle cerebral artery occlusion

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MS

multiple sclerosis

NMO

neuromyelitis optica

PD

Parkinson’s disease

RGC

retinal ganglion cell

RGM

repulsive guidance molecule

Rho GEF

Rho guanine nucleotide exchange factor

SCI

spinal cord injury

SN

substantia nigra

TBI

traumatic brain injury

UEMS

upper extremity motor score

Introduction

The repulsive guidance molecule (RGM) is a membrane-associated glycosylphosphatidylinositol (GPI)-anchored glycoprotein, which was identified as an axon guidance molecule in the retinotectal system ( ). By analyzing the development of the retinotectal projection system in chicks—a method frequently used to study the molecular basis of topographic projections, including axon guidance cues—Monnier et al. found that RGM is expressed in a spatial gradient manner along the anterior–posterior axis in the tectum ( Fig. 1 A ). Importantly, in growth cone collapse assays, temporal, but not nasal, retinal growth cones collapsed when confronted with RGM-decorated membranes ( Fig. 1 B). Similarly, in the stripe assay ( ), RGM guides axonal elongation only for temporal retinal axons ( Fig. 1 C). These findings strongly suggest that RGM can act as an axon-specific repulsive guidance molecule ( ).

$Fig. 1, RGM is an inhibitory axon guidance molecule. (A) Schematic illustration of retinotectal projections and expression patterns of RGMa and neogenin. Nasal retinal ganglion cell (RGC) axons project to the posterior part of the optic tectum, whereas temporal RGC axons project to the anterior part of the tectum. RGM is expressed in an increasing anterior–posterior gradient manner in the tectum, and neogenin is expressed along an elevating nasal–temporal gradient in the retina. (B) Representation of growth cone collapse assay. RGM exerts growth cone collapse effects only for temporal retinal axons. (C) Representation of stripe assay. The gray colored stripe indicates membrane fractions from RGM-transfected cells, whereas the white colored stripe represents membrane fractions from mock-transfected cells.$

However, the receptor mechanism for RGM function was not defined at that time. In 2004, Rajagopalan et al. and Matsunaga et al. reported that neogenin mediates the biological effects of RGM as its cognate receptor ( ; ). Strikingly, neogenin was revealed to be expressed in a temporal–nasal gradient in the retina ( ); thus, the repulsiveness of temporal, not nasal, retinal axons by RGM is well explained by RGM–neogenin interactions ( Fig. 1 A). RGM is also found to act as a co-receptor for bone morphogenetic proteins (BMPs) and modulates BMP signaling ( ).

Since then, RGM signaling in the central nervous system (CNS) has been extensively studied and a wide range of functions has been reported; RGM plays multiple roles in neuronal development [e.g., neural tube closure ( )], and is also involved in the pathophysiology of various CNS diseases, as discussed below. Moreover, external to the CNS, RGM has been shown to regulate endochondral bone formation ( ), iron homeostasis ( ), and inflammation ( ). In mammals, at least three homologues of RGM, RGMa, RGMb (also known as DRAGON), and RGMc (also known as hemojuvelin, HFE2), have been identified. Among them, RGMa is most closely related to chicken RGM (80% homology), and most of the critical findings in CNS development/disease are obtained from RGMa research. Thus, in this review, we mainly focus on the functions of RGMa.

Characteristics of RGMa

Ligand–receptor interactions and downstream signaling of RGMa

As mentioned above, RGM binds to neogenin, a transmembrane protein originally isolated from chick cerebellum as a homolog of deleted in colorectal cancer (DCC: a receptor for the axon guidance molecule netrin-1). RGM also binds to BMP morphogens as a co-receptor and modulates BMP signaling. On the other hand, neogenin is also known as a netrin-1 receptor. How these proteins interact with each other and how downstream signaling is regulated are not fully understood. However, recent studies, including crystal structure analysis ( ; ; ), provide direct insight into their interactions.

The major binding site of RGM for neogenin is the carboxyl-terminal domain of RGM (C-RGM) ( ; ). Interestingly, two C-RGMs act as a molecular staple bringing two neogenin receptors together, and this unique architecture is proposed to be important for the subsequent activation of downstream signaling ( ; ). On the other hand, the amino-terminal domain of RGM (N-RGM) has been shown to interact with BMP, and this binding is suggested to link BMP and neogenin signaling by comprising a BMP–RGM–neogenin complex ( ). RGM–BMP interactions have been thought to potentiate BMP signaling via the canonical SMAD pathway. However, a recent report revealed that RGM can compete with growth differentiation factor 5 (GDF5), a member of the BMP family, and thus act as an inhibitor of BMP signaling ( ). Therefore, how intracellular signaling is regulated through interactions involving N-RGM and BMPs is presumed to be context-dependent. Further studies are warranted to determine the physiological roles of RGM–BMP interactions in each developmental/pathological condition.

RGM is thought to transduce signals in both cis and trans manner. Its distinctive functions, such as inducing growth cone collapse or axon repulsion, are induced in trans , that is via cell-to-cell interactions. As the RGM extracellular domain is thought to be cleaved at, and secreted from, cell membranes, trans -signaling can be achieved by a gradient of soluble RGM ( Fig. 2 ). However, it is unknown whether long-range signal transduction is possible.

Fig. 2, RGM–neogenin interactions and downstream signaling. Schematic illustration of possible ligand-receptor interactions involving RGMa. The inhibitory effect for neurite outgrowth is brought about by trans -signaling; that is, neurogenin-expressing axons are repelled by cell-bound RGMa or via an RGMa gradient established by extracellular shedding. Neogenin localization to lipid rafts—membrane domains enriched in cholesterol, sphingolipids, and protein receptors, present a platform for cellular signaling events, including ligand:receptor interactions—has been proposed to be indispensable for signal transduction.

Although a detailed picture of RGM signaling has not yet been obtained, several key aspects have been uncovered. One of the most important downstream signaling pathways involves RhoA activation by RGM, because it is widely accepted that Rho-GTPases play key roles in axon guidance and neurite growth. Indeed, the inhibition of Rho kinase, a downstream effector of RhoA, abolishes the inhibitory effects of RGMa on neurite outgrowth ( ). These authors also revealed that Unc5B, a member of the netrin receptor family, interacts with neogenin as a co-receptor for RGMa and activates RhoA through LARG, a member of the Rho guanine nucleotide exchange factor (RhoGEF) subfamily ( ). In addition, the involvement of Ras activity was also evaluated because of its well-known functions as a mediator of growth cone collapse and neurite retraction ( ). As expected, RGMa–neogenin binding has been revealed to inactivate Ras activity, leading to growth cone collapse ( ).

More recently, detailed investigations unveiled more complex mechanisms surrounding RGM–neogenin signaling regulation, including the proteolytic processing of RGMa ( , well summarized in ), ectodomain shedding of neogenin ( ), and Υ-secretase cleavage of the neogenin intracellular domain ( ). Further research is needed to elucidate how these multiple signaling pathways are regulated and integrated in vivo.

RGMa in spinal cord injury (SCI)

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