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 ( ).
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.
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)
RGMa expression after SCI
Given that RGMa, as an inhibitory protein, is involved in neural circuit formation during development by modulating the pathfinding activity of growing axons, it is possible that RGMa exerts negative effects on axonal regeneration. To test this concept in vivo, RGMa expression in the spinal cord of adult animals under normal and SCI conditions has been addressed.
Immunohistochemical analysis of rat spinal cord using anti-C-RGMa antibody revealed its localization to neurons and oligodendrocytes under normal conditions ( ; ). After SCI, RGMa expression around the lesion site is upregulated; with expression by neurons, oligodendrocytes, certain reactive astrocytes, activated microglia, and infiltrating leukocytes ( ; ). These observations support the possibility that RGMa is an inhibitory protein against axonal regeneration after SCI; therefore, RGMa neutralization is considered to be a potential therapeutic strategy to attenuate neural deficits induced by SCI. An increase in RGMa around the SCI lesion site is also observed in primates ( ). In that study, RGMa was reported to be mainly expressed by Iba-1-positive microglia/macrophages.
Importantly, RGMa expression patterns in the human spinal cord have also been reported. In the uninjured spinal cord, RGMa is expressed at low levels by neurons and glial cells. However, immunoreactivity for RGMa in the spinal cord is significantly increased following SCI, and the localization of RGMa in neurons, axons, and glial cells has been confirmed ( ). On the other hand, the RGMa receptor neogenin is expressed by neurons in the spinal cord in both rats and humans ( ).
Taken together, RGMa upregulation induced by SCI is common across divergent species, suggesting the significance of RGMa in the pathophysiology of CNS injury.
RGMa inhibition promotes recovery after SCI in rodents
The first direct evidence of the therapeutic effects of RGMa inhibition on SCI was reported in 2006 ( ). To test the hypothesis that upregulated RGMa plays a negative role in axonal regeneration in vivo, Hata et al. generated an RGMa-neutralizing antibody (directed against RGM residues 309–322). After receiving Th9/10 dorsal transection, which resulted in both dorsal (main) and lateral corticospinal tract (CST) injury, rats were treated with the RGMa-neutralizing antibody via an osmotic mini-pump with catheters placed near the lesion site, and locomotor functions were monitored. As expected, the Basso–Beattie–Bresnahan (BBB) locomotion score ( ) was significantly improved by RGMa inhibition. The authors also performed anatomical tracing studies and observed a lesser degree of CST retraction, a greater number of collateral formations, and a significant amount of axon extension through the lesion site ( Fig. 3 A ). This enhanced anatomical regeneration temporally correlated with functional recovery.
Using the same experimental setting, Kyoto et al. tested the possibility that neural circuit plasticity might be enhanced by anti-RGMa antibody treatment—even in lesions distant from the injury site ( ). The results showed that antibody treatment facilitated synapse formation at the level of the cervical spinal cord ( Fig. 3 B), which may contribute to the rerouting of hind-limb CST connections through propriospinal neurons ( ; ; ). This observation may be explained by the notion that the adult CNS offers an inhibitory environment for adoptive reorganization (i.e., neural circuit remodeling) and RGMa is one of its major components; therefore, RGMa inhibition can contribute to the establishment of reorganization-permissive environments.
On the other hand, Tassew et al. explored strategies to inhibit neogenin ( ). During developmental stages, neogenin is known to act as a dependence receptor that induces cell death in the absence of RGM ( ). Therefore, targeting neogenin instead of RGMa may be beneficial, not only for neural circuit reorganization, but also for neuronal survival after SCI. Based on the observation that the receptor functions of neogenin require its localization in lipid rafts, the authors addressed whether blocking neogenin raft association improves SCI outcomes. They found that interactions involving the neogenin 4Ig domain and the N-terminal regions of RGMa (N-RGMa) are critical for the attraction of neogenin to lipid rafts, and recombinant 4Ig treatment effectively abolishes their interactions. Thus, they treated SCI rats with 4Ig (intravenous injection or local intrathecal infusion). As expected, axonal regeneration and functional recovery (as assessed by BBB scores and ladder walk tests) were significantly enhanced. Furthermore, this treatment attenuated neuronal loss in perilesional neurons.
Based on this evidence, humanized monoclonal antibodies specific for N-RGMa were generated ( ), and their therapeutic potential in impact-compression SCI was tested ( ). To provide a clinically applicable strategy, the authors examined the efficacy of RGMa inhibition by systemic administration of humanized anti-RGMa antibody and revealed that this treatment effectively enhanced functional recovery (as assessed by BBB scores, ladder walk tests, and footprint analysis). Moreover, by histological and anatomical analysis, it was also confirmed that anti-RGMa antibody treatment enhances neuronal survival and axonal regeneration. Interestingly, anti-RGMa antibody treatment also attenuated neuropathic pain after SCI. Although effects on microglia/macrophage were suggested, the precise mechanisms involved remain unknown. In a recent paper, Mothe et al. also reported that delayed administration (~ 3 h after SCI) of anti-N-RGMa antibody could still effectively enhance recovery of motor functions, with increased neuronal sparing and axonal plasticity ( ). Interestingly, the recovery of bladder function was also enhanced, indicating that anti-RGMa treatment exerts broad beneficial effects in terms of SCI convalescence.
Taken together, the effectiveness of the strategy for RGMa/neogenin signaling inhibition has been repeatedly confirmed in rodent SCI models ( Table 1 ). In the next chapter, evidence from primates is discussed.
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