Expression Of Repulsive Guidance Molecule A Research Articles

Increase in repulsive guidance molecule-a (RGMa) in lacunar and cortical stroke patients is related to the severity of the insult

Repulsive guidance molecule-a (RGMa) inhibits angiogenesis and increases inflammation. Animal models of cerebral ischemia have shown that an increased expression of RGMa leads to larger infarction and its inhibition attenuates effects of ischemia. We report on the relationship of RGMa to stroke types and severity. This is a prospective study in patients admitted to the stroke service in Qatar. We collected the clinical determinants, including NIHSS at admission, imaging and outcome at discharge and 90-days. RGMa levels were determined by measuring mRNA levels extracted from peripheral blood mononuclear cells (PBMCs) within 24 h of onset and at 5 days. There were 90 patients (lacunar: 64, cortical: 26) and 35 age-matched controls. RGMa mRNA levels were significantly higher in the stroke patients: day 1: 1.007 ± 0.13 versus 2.152 ± 0.19 [p < 0.001] and day-5: 3.939 ± 0.36 [p < 0.0001]) and significantly higher in patients with severe stroke (NIHSS ≥ 8) compared to milder symptoms (NIHSS < 8) at day 1 (NIHSS ≥ 8: 2.563 ± 0.36; NIHSS < 8: 1.947 ± 0.2) and day 5 (NIHSS ≥ 8: 5.25 ± 0.62; NIHSS < 8: 3.259 ± 0.419). Cortical stroke patients had marginally higher RGMa mRNA levels compared to lacunar stroke at day 1 (cortical stroke: 2.621 ± 0.46 vs lacunar stroke: 1.961 ± 0.19) and day 5 (cortical stroke: 4.295 ± 0.76 vs lacunar stroke: 3.774 ± 0.39). In conclusion, there is an increase in the level of RGMa mRNA in patients with acute stroke and seen in patients with lacunar and cortical stroke. The increase in RGMa mRNA levels is related to the severity of the stroke and increases over the initial 5 days. Further studies are required to determine the effects of the increase in RGMa on stroke recovery.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy

BackgroundThe repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported.ResultsRGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to ‘adherens junsctions’ and ‘extracellular-cell adhesion’, while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation.ConclusionsThese results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

RGMa regulates CCL5 expression via the BMP receptor in experimental autoimmune encephalomyelitis mice and endothelial cells.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Repulsive guidance molecule a (RGMa) has been indicated to act as a bone morphogenetic protein (BMP) co-receptor, enhancing BMP signalling activity. However, the role and downstream pathways of the BMP signalling pathway mediated by RGMa have yet to be fully elucidated. A recent study revealed that C-C motif chemokine ligand 5 (CCL5) has a major role in the pathogenesis of MS via the recruitment of macrophages and T-lymphocytes into the CNS. The present study aimed to evaluate whether RGMa regulates CCL5 via the BMP pathway in MS. The results demonstrated that RGMa regulated CCL5 expression in a BMP ligand-dependent manner in experimental autoimmune encephalomyelitis (EAE) mice in vivo and in endothelial cells in vitro. First, specific inhibition of the expression of RGMa via RNA interference led to a significant reduction of the expression of RGMa and this was associated with a significant delay of EAE, an alleviated disease course and downregulation of CCL5 expression at both the protein and mRNA levels. Furthermore, exogenous noggin, an extracellular antagonist of BMP ligand, abolished the induction effect of RGMa on CCL5 in endothelial cells. Taken together, these results suggested that RGMa is an important regulator of MS and inflammatory mediators such as CCL5, and the present results should prove to be useful in terms of further elucidating the RGMa-BMP receptor signalling pathway and the pathogenesis of RGMa on MS as far as the involvement of blood-brain barrier permeability is concerned.

Elevated RGMA Expression Predicts Poor Prognosis in Patients with Glioblastoma.

BackgroundGlioblastoma (GBM) is the most aggressive type of human brain tumor with a poor prognosis and a low survival rate. Secreted proteins from tumors are recently considered as important modulators to promote tumorigenesis by communicating with microenvironments. Repulsive guidance molecule A (RGMA) was initially characterized as an axon guidance molecule after secretion in the brain during embryogenesis but has not been studied in GBM. In this study, we investigated secreted gene expression patterns and the correlation between RGMA expression and prognosis in GBM using in silico analysis.MethodsRGMA mRNA levels in normal human astrocyte (NHA), human glioma cells, and GBM patient-derived glioma stem cells (GSCs) were assessed by qRT‐PCR. Patient survival analysis was performed with the Kaplan–Meier curve and univariate and multivariate analyses using publicly available datasets. The predictive roles of RGMA in progressive malignancy were evaluated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA).ResultsRGMA mRNA expression was elevated in glioma cells and GSCs compared with NHA and correlated with unfavorable prognosis in glioma patients. Thus, RGMA could serve as an independent predictive factor for GBM. Furthermore, the increased levels of RGMA expression and its putative receptor, neogenin (NEO1), were associated with poor patient survival rates in GBM.ConclusionWe identified RGMA as an independent prognostic biomarker for progressive malignancy in glioblastoma and address the possibilities to develop novel therapeutic strategies against glioblastoma.

Inhibition of repulsive guidance molecule-a protects dopaminergic neurons in a mouse model of Parkinson\u2019s disease

Repulsive guidance molecule-a (RGMa), a glycosylphosphatidylinositol-anchored membrane protein, has diverse functions in axon guidance, cell patterning, and cell survival. Inhibition of RGMa attenuates pathological dysfunction in animal models of central nervous system (CNS) diseases including spinal cord injury, multiple sclerosis, and neuromyelitis optica. Here, we examined whether antibody-based inhibition of RGMa had therapeutic effects in a mouse model of Parkinson’s disease (PD). We treated mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and found increased RGMa expression in the substantia nigra (SN). Intraventricular, as well as intravenous, administration of anti-RGMa antibodies reduced the loss of tyrosine hydroxylase (TH)-positive neurons and accumulation of Iba1-positive microglia/macrophages in the SN of MPTP-treated mice. Selective expression of RGMa in TH-positive neurons in the SN-induced neuronal loss/degeneration and inflammation, resulting in a progressive movement disorder. The pathogenic effects of RGMa overexpression were attenuated by treatment with minocycline, which inhibits microglia and macrophage activation. Increased RGMa expression upregulated pro-inflammatory cytokine expression in microglia. Our observations suggest that the upregulation of RGMa is associated with the PD pathology; furthermore, inhibitory RGMa antibodies are a potential therapeutic option.

The expression of repulsive guidance molecule a after traumatic brain injury: Time-course changes in gene expression in a murine model of controlled cortical impact.

Repulsive guidance molecule a (RGMa) is a key protein that negatively regulates neuronal regeneration as its inhibition enhances axonal growth and promotes functional recovery in animal models of spinal cord injury. However, the role of RGMa in traumatic brain injury (TBI) remains elusive. This study aimed to clarify TBI-responsive RGMa expression in a murine model. Adult male C57BL/6J mice were subjected to controlled cortical impact. Brains were extracted 6 hours and 1, 3, 7, 14 and 21 days after injury (n = 6 in each group). Changes in the messenger RNA (mRNA) expression of RGMa and its receptor, neogenin, were evaluated by quantitative polymerase chain reaction in the damaged area of the cortex and contralateral cortex, along with expression measurement of inflammation-related molecules. Neurological deficit was also assessed by the cylinder test. Neurological score was consistently lower in the TBI group compared to the sham group throughout the experimental period. The mRNA expressions of representative inflammatory cytokine TNF-α and chemokine receptor CCR2 were remarkably increased in the injured cortex on day 1 and gradually decreased over time, although remaining at higher values at least until day 14. The mRNA expressions of RGMa and neogenin were significantly suppressed in the damaged cortex until day 3. Interestingly, RGMa expression was suppressed most on day 1 and recovered over time. In the acute phase of TBI, gene expression of inflammatory cytokines significantly increased, and gene expressions of RGMa and neogenin significantly decreased in the inflammatory milieu of the damaged area. Despite the subsequent remission of inflammation, RGMa gene expression recovered to the normal level 1 week after TBI. Intrinsic regenerative response to acute brain injury might be hampered by the following recovery of RGMa expression, hinting at the possibility of functional RGMa inhibition as a new, effective maneuver against TBI.

MicroRNA-4472 Promotes Tumor Proliferation and Aggressiveness in Breast Cancer by Targeting RGMA and Inducing EMT

BackgroundBreast cancer is the most common cause of cancer-related death in women worldwide. MicroRNA (miRNA) ectopic expression has been reported to be involved in the regulation of gene expression in breast cancer. We screened several differentially expressed miRNAs associated with breast cancer chemoresistance, growth, and metastasis using a miRNA microarray. Increased expression of miR-4472 has been associated with larger breast tumors and chemoresistance. However, the biologic function of miR-4472 and its molecular mechanisms in cancer progression have not yet been reported. Materials and MethodsReal-time quantitative polymerase chain reaction was used to measure the expression of miR-4472 in breast cancer tissue and cell lines. The biologic functions of miR-4472 and its target gene were explored using Transwell, cell proliferation, and flow cytometry assays. Bioinformatics tools, dual-luciferase reporter assays, and Western blot were used to identify the target genes of miR-4472. Western blot was used to explain the participation of miR-4472 and target gene in epithelial-to-mesenchymal transition. ResultsmiR-4472 was significantly upregulated in highly metastatic breast cancer tissues, and its expression was positively associated with larger tumor size and advanced pTNM stage. miR-4472 promoted breast cancer cell metastasis and growth. Repulsive guidance molecule A (RGMA) was a direct target gene of miR-4472. RGMA was identified as a suppressor in cancer metastasis. miR-4472 downregulated expression of RGMA and promoted epithelial-to-mesenchymal transition by suppressing E-cadherin and initiating vimentin, β-catenin, and Slug. ConclusionsmiR-4472 contributes to the progression of breast cancer by regulating RGMA expression and inducing epithelial-to-mesenchymal transition, indicating that miR-4472/RGMA might serve as a therapeutic target for breast cancer.

Changes of fractional anisotropy and RGMa in crossed cerebellar diaschisis induced by middle cerebral artery occlusion.

Crossed cerebellar diaschisis (CCD) is the phenomenon of hypoperfusion and hypometabolism of the contralateral cerebellar hemisphere caused by dysfunction of the associated supratentorial region. The aim of the present study was to analyze the changes in fractional anisotropy (FA) in CCD induced by middle cerebral artery occlusion (MCAO) using magnetic resonance-diffusion tensor imaging (MR-DTI). Furthermore, the role of repulsive guidance molecule a (RGMa) in CCD was assessed by measuring RGMa expression using histochemical analysis. In the present study, the cerebellar hemisphere was serially scanned with T2-weighted, serial diffusion-weighted and diffusion tensor (DT) imaging using a 3.0T GE Signa HDxt Scanner to analyze the changes in FA over 72 h. Subsequently, immunohistochemistry analyses of the corresponding cerebellar hemisphere sections were performed to assess the expression of RGMa. Results indicated that FA of both sides of the cerebellar hemisphere, particularly that of the contralateral cerebellar hemisphere (right side) derived from DTI, was reduced during the 72-h time period following MCAO, and the decrease was maximal and statistically significant at 12 h (P<0.05). Immunohistochemistry analysis revealed a significant increase in the expression of RGMa protein in the affected region of the contralateral cerebellar hemisphere (right side) at 24 h following MCAO injury (P<0.05). Furthermore, the expression of RGMa and FA was negatively correlated in MCAO (P<0.05). The results suggest that MR-DTI is an important assessment to evaluate changes of FA in CCD induced by MCAO. Furthermore, the present results suggest that RGMa, which was negatively correlated with FA in MCAO rats, may serve an important role in CCD.

RGMa mediates reactive astrogliosis and glial scar formation through TGF\u03b21/Smad2/3 signaling after stroke

In response to stroke, astrocytes become reactive astrogliosis and are a major component of a glial scar. This results in the formation of both a physical and chemical (production of chondroitin sulfate proteoglycans) barrier, which prevent neurite regeneration that, in turn, interferes with functional recovery. However, the mechanisms of reactive astrogliosis and glial scar formation are poorly understood. In this work, we hypothesized that repulsive guidance molecule a (RGMa) regulate reactive astrogliosis and glial scar formation. We first found that RGMa was strongly expressed by reactive astrocytes in the glial scar in a rat model of middle cerebral artery occlusion/reperfusion. Genetic or pharmacologic inhibition of RGMa in vivo resulted in a strong reduction of reactive astrogliosis and glial scarring as well as in a pronounced improvement in functional recovery. Furthermore, we showed that transforming growth factor β1 (TGFβ1) stimulated RGMa expression through TGFβ1 receptor activin-like kinase 5 (ALK5) in primary cultured astrocytes. Knockdown of RGMa abrogated key steps of reactive astrogliosis and glial scar formation induced by TGFβ1, including cellular hypertrophy, glial fibrillary acidic protein upregulation, cell migration, and CSPGs secretion. Finally, we demonstrated that RGMa co-immunoprecipitated with ALK5 and Smad2/3. TGFβ1-induced ALK5-Smad2/3 interaction and subsequent phosphorylation of Smad2/3 were impaired by RGMa knockdown. Taken together, we identified that after stroke, RGMa promotes reactive astrogliosis and glial scar formation by forming a complex with ALK5 and Smad2/3 to promote ALK5-Smad2/3 interaction to facilitate TGFβ1/Smad2/3 signaling, thereby inhibiting neurological functional recovery. RGMa may be a new therapeutic target for stroke.

A novel role for RGMa in modulation of bone marrow-derived dendritic cells maturation induced by lipopolysaccharide

Repulsive guidance molecule a (RGMa) is known to mediate immune responses and has been indicated to modulates T cell activation and autoimmune diseases by dendritic cells (DCs), which hints its significant function in the latter cells. The aim of our study, therefore, was to evaluate the function of RGMa in DC maturation. We found that small interfering RNA (siRNA) successfully silenced the expression of RGMa in DCs. Even after LPS stimulation, RGMa-silenced DCs displayed an immature morphology, characterized by small, round cells with a few cell processes and organelles, and many pinocytotic vesicles. In the presence of LPS, RGMa siRNA transfection markedly reduced levels of CD80, CD86, CD40, and MHC II expression, as well as the secretion of IL-12p70 and TNF-α. With LPS treatment, RGMa siRNA-transfected DCs also showed increased levels of IL-10 and endocytosis. Moreover, in the presence of LPS, RGMa siRNA-transfected DCs displayed a low ability to induce T cell proliferation and differentiation, compared with negative control (NTi)-transfected or control DCs (p<0.05 for both). We conclude that after LPS stimulation, RGMa siRNA-transfected DCs show immunoregulatory and tolerogenic characteristics, which provides new insights into the immune system.

Lentiviral Vector-Induced Overexpression of RGMa in the Hippocampus Suppresses Seizures and Mossy Fiber Sprouting.

Repulsive guidance molecule a (RGMa) is a membrane-bound protein that inhibits axon outgrowth in the central nervous system. Temporal lobe epilepsy (TLE) is a common neurological disorder characterized by recurrent spontaneous seizures. To explore the role of RGMa in epilepsy, we investigated the expression of RGMa in patients with TLE, pilocarpine-induced rat model, and pentylenetetrazol kindling model of epilepsy, and then we performed behavioral, histological, and electrophysiological analysis by lentivirus-mediated overexpression of RGMa in the hippocampus of animal model. We found that RGMa was significantly decreased in TLE patients and in experimental rats from 6h to 60days after pilocarpine-induced seizures. In two types of epileptic animal models, pilocarpine-induced model and pentylenetetrazol kindling model, overexpression of RGMa in the hippocampus of rats exerted seizure-suppressant effects. The reduced spontaneous seizures were accompanied by attenuation of hippocampal mossy fiber sprouting. In addition, overexpression of RGMa inhibited hyperexcitability of hippocampal neurons via suppressing NMDAR-mediated currents in Mg2+-free-induced organotypic slice model. Collectively, these results demonstrate that overexpression of RGMa could be an alternative strategy for epilepsy therapy.

Potential roles of the RGMa-FAK-Ras pathway in hippocampal mossy fiber sprouting in the pentylenetetrazole kindling model.

Mossy fiber sprouting (MFS) is a unique feature of chronic epilepsy. However, the molecular signals underlying MFS are still unclear. The repulsive guidance molecule A (RGMa) appears to contribute to axon growth and axonal guidance, and may exert its biological effects by dephosphorylating focal adhesion kinase (FAK) at Tyr397, then regulating the activation of Ras. The objective of this study was to explore the expression patterns of RGMa, FAK (Tyr397) and Ras in epileptogenesis, and their correlation with MFS. The epileptic models were established by intraperitoneal pentylenetetrazole (PTZ) injection of Sprague-Dawley rats. At 3 days and at 1, 2, 4 and 6 weeks after the first PTZ injection, Timm staining was scored at different time points in the CA3 region of the hippocampus and dentate gyrus. The protein levels of RGMa, FAK (Tyr397) and Ras were analyzed at different time points in the CA3 region of the hippocampus using immunofluorescence, immunohistochemistry and western blot analysis. Compared with the control (saline-injected) group, the expression of RGMa in the CA3 area was significantly downregulated (P<0.05) from 3 days and still maintained the low expression at 6 weeks in the PTZ group. The expression of FAK (Tyr397) and Ras was upregulated (P<0.05) in the PTZ groups. The Timm score in the CA3 region was significantly higher than that in the control group at different time points and reached a peak at 4 weeks. In the CA3 region, no obvious distinction was observed at the different time points in the control group. To the best of our knowledge, these are the first results to indicate that the RGMa-FAK-Ras pathway may be involved in MFS and the development of temporal lobe epilepsy.

RGMa Regulates Cortical Interneuron Migration and Differentiation

The etiology of neuropsychiatric disorders, including schizophrenia and autism, has been linked to a failure to establish the intricate neural network comprising excitatory pyramidal and inhibitory interneurons during neocortex development. A large proportion of cortical inhibitory interneurons originate in the medial ganglionic eminence (MGE) of the ventral telencephalon and then migrate through the ventral subventricular zone, across the corticostriatal junction, into the embryonic cortex. Successful navigation of newborn interneurons through the complex environment of the ventral telencephalon is governed by spatiotemporally restricted deployment of both chemorepulsive and chemoattractive guidance cues which work in concert to create a migratory corridor. Despite the expanding list of interneuron guidance cues, cues responsible for preventing interneurons from re-entering the ventricular zone of the ganglionic eminences have not been well characterized. Here we provide evidence that the chemorepulsive axon guidance cue, RGMa (Repulsive Guidance Molecule a), may fulfill this function. The ventricular zone restricted expression of RGMa in the ganglionic eminences and the presence of its receptor, Neogenin, in the ventricular zone and on newborn and maturing MGE-derived interneurons implicates RGMa-Neogenin interactions in interneuron differentiation and migration. Using an in vitro approach, we show that RGMa promotes interneuron differentiation by potentiating neurite outgrowth. In addition, using in vitro explant and migration assays, we provide evidence that RGMa is a repulsive guidance cue for newborn interneurons migrating out of the ganglionic eminence ventricular zone. Intriguingly, the alternative Neogenin ligand, Netrin-1, had no effect on migration. However, we observed complete abrogation of RGMa-induced chemorepulsion when newborn interneurons were simultaneously exposed to RGMa and Netrin-1 gradients, suggesting a novel mechanism for the tight regulation of RGMa-guided interneuron migration. We propose that during peak neurogenesis, repulsive RGMa-Neogenin interactions drive interneurons into the migratory corridor and prevent re-entry into the ventricular zone of the ganglionic eminences.

CRMP-2 Is Involved in Axon Growth Inhibition Induced by RGMa In Vitro and In Vivo

Repulsive guidance molecule-a (RGMa) is associated with axon growth inhibition in different central nervous system (CNS) injuries, but its signaling pathways remain unclear. We examined the involvement of collapsin response mediator protein-2 (CRMP-2), a common downstream target of Rho-kinase and GSK-3β, in vitro by culturing neonatal rat primary cortical neurons with RGMa protein, Rho-kinase inhibitor (Y-27632), and GSK-3β inhibitor. We examined CRMP-2 in vivo by suppressing RGMa expression using recombinant adenovirus (rAd-shRGMa) in a rat MCAO/reperfusion model. RGMa induced neurite retraction and CRMP-2 phosphorylation in vitro, which were reversed by either Rho-kinase or GSK-3β inhibitors. After MCAO/reperfusion in rats, pCRMP-2 protein was greatly increased in the ischemic cortex, axons were damaged severely, Neurofilament-200 (NF-200) expression was significantly decreased, and neurological deficits were significant, which were all improved by down-regulating RGMa. We concluded RGMa inhibits axon growth by phosphorylating CRMP-2 via both Rho-kinase and GSK-3β signaling pathways.

RNA interference against repulsive guidance molecule A improves axon sprout and neural function recovery of rats after MCAO/reperfusion

Repulsive guidance molecule a (RGMa) is a neurite growth inhibitor that is of great interest in the study of CNS neuronal regeneration. We adopted RNA interference (RNAi) as a means of suppressing the expression of RGMa and observed the improvement in axonal regeneration and neurological function of rats after cerebral ischemic injury. Recombinant adenovirus rAd5-shRNA-RGMa was constructed and prepared for animal experimentation. RGMa and neurofilament protein 200 (NF200) in the ischemic cortex and ipsilateral hippocampus were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The ischemic regions were examined by triphenyltetrazolium chloride (TTC) staining and the newborn neurite branches by Biotinylated Dextran Amine (BDA) neuronal tracing. Behavior tests were adopted to evaluate neurologic function recovery. Results showed RGMa was down-regulated and axonal growth was improved in the RNAi treated group (P<0.01). The number of axonal sprouts of corticospinal tract from the uninjured side to the ischemic side in the RNAi treated group was increased (P<0.01). Behavior test scores in the RNAi treated group were significantly better than other groups after 6weeks (P<0.01). RGMa in rat brains after middle cerebral artery occlusion (MCAO) can be down-regulated by RNAi successfully, which may lead to improved axonal growth and neural anatomy plasticity, as well as neuron functional recovery.

Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol-anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain-of-function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell-autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation.

Fastigial Nucleus Electrostimulation Reduces the Expression of Repulsive Guidance Molecule, Improves Axonal Growth Following Focal Cerebral Ischemia

The role of repulsive guidance molecule A (RGMa) in cerebral ischemia remains unclear. In the study, we examined the expression of RGMa in ischemic brain tissues following focal cerebral ischemia/reperfusion (IR) in rats. An established middle cerebral artery suture occlusion model was employed. A dipolar electrode was placed into the cerebellum to stimulate the cerebellar fastigial nucleus for 1h at 2h after ischemia. Reverse transcription-polymerase chain reaction was used to measure the mRNA RGMa and its downstream mediator, Ras homolog A (RhoA). Immunohistochemistry was applied to detect RGMa and RhoA expressions and to evaluate axonal regeneration by optical density analysis of 200kDa neurofilaments. We found that both mRNA and protein levels of RGMa and RhoA were increased in the ischemic cortex and hippocampus 48h following cerebral IR and these elevated levels were maintained for 2weeks. Electrostimulation of the fastigial nucleus reduced the expression of RGMa and RhoA at 24h and 2weeks following cerebral IR. In addition, axonal growth was enhanced in the fastigial nucleus electrostimulated group compared to non-stimulated ischemic animals (P<0.05). RGMa/RhoA expression was negatively correlated with the growth of axons (P<0.05). Therefore, we concluded that RGMa and RhoA could be another key molecule and might inhibit axonal regeneration during cerebral IR injury. Electrostimulation of the fastigial nucleus enhances axonal growth, possibly by reducing the expression of RGMa and RhoA after cerebral IR.

Novel Roles of the Chemorepellent Axon Guidance Molecule RGMa in Cell Migration and Adhesion

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease.

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 and neogenin protein expression are influenced by lens injury following optic nerve crush in the rat retina

The death and the failure of neurons to regenerate their axons after lesion of the central nervous system in mammals, as in the case of spinal cord injury and optic nerve trauma, remain a challenge. In this study, we focused on the repulsive guidance molecule A (RGMA) and its receptor neogenin. Since it was reported that RGMA+ cells accumulate in lesioned areas after spinal cord injury, brain trauma, and optic nerve crush, and curiously, anti-apoptotic effects of RGMA were also described, we investigated the role of RGMA and neogenin in the retina after optic nerve crush (ONC). We evaluated the spatial and temporal protein pattern of RGMA and neogenin in the rat retina without (non-regenerating model) or with (regenerating model) lens injury (LI). We investigated the presence of RGMA, neogenin and other proteins at up to nine time points (6 h-20 days post-surgery) by performing immunohistochemistry and Western blots. Independent of the treatment, RGMA protein was present in the nuclear layers (NLs), plexiform layers (PLs), nerve fiber layer (NFL), and in retinal ganglion cells (RGCs) of the rat retina. RGC and nerve fibers were always RGMA+. Further RGMA+ cells in the retina were blood vessel endothelial cells, astrocytes, Müller cells, and some microglial cells. The RGMA pattern for the specific retinal cells resembled those of previously published data. The neogenin pattern was congruent to the RGMA pattern. Western blots of retinal tissue showed further RGMA+ products only in LI animals. Furthermore, a higher amount of RGMA was found in the retinae of ONC + LI rats compared to ONC rats. Although a difference in the localization of RGMA is not obvious, the difference in the amount of RGMA is striking, the higher amount of RGMA in the retinae of ONC + LI rats compared to ONC rats indicates a role for RGMA during degeneration/regeneration processes. Our results are consistent with several reported neuroprotective effects of RGMA. Our new data showing the upregulation of RGMA after ONC in our regenerating model (plus LI) confirm these findings conducted in different settings.