Repulsive Guidance Molecule A Research Articles

Abstract WP275: Longitudinal Biomarker Analysis in Patients With Ischemic Stroke: Exploratory Targeted and Omics Assessments From a Phase 2a Clinical Trial

Introduction: Elezanumab (EZB), a monoclonal antibody specific to repulsive guidance molecule A (RGMa), is under investigation to treat acute ischemic stroke. Biomarker evidence in ischemic stroke and for the RGMa pathway is limited. Methods: This blinded, in-study analysis from a 52-week phase 2a, multicenter, randomized (1:1; EZB:placebo) clinical trial (NCT04309474) included patients aged 30-90 years with an acute ischemic stroke (onset ≤ 24 hours before baseline), and a NIHSS score of 7-21. Blinded analyses did not distinguish between treatment arms. Healthy adult controls were procured outside this trial. Blood samples were taken post-stroke across 52 weeks, or 1 time (baseline) for healthy adults. Plasma concentrations of neurodegenerative markers neurofilament light chain protein (NfL) and glial fibrillary acidic protein (GFAP) were measured (validated Simoa Human Neurology 4-Plex A assay; Quanterix). Protein, mRNA, and miRNA were assessed with omics approaches. Results: This preliminary analysis included 34 patients with stroke (mean [SD] age 67.1 [12.3] years, 58.8% male) and 31 healthy adults (61.8 [8.0] years, 71.0% male). The mean (SD) baseline NIHSS total score was 10.9 (4.3). NfL and GFAP concentrations were elevated on day 1 in patients with stroke vs healthy adults (healthy adults are included for reference only). Mean concentrations of NfL were 48.7 pg/mL vs 13.9 pg/mL, respectively; mean concentrations of GFAP were 746.0 pg/mL vs 137.2 pg/mL, respectively. In patients with stroke, agnostic to treatment, peak NfL and GFAP elevations occurred at day 28 and days 2-4, respectively; NfL and GFAP concentrations were lower at week 52 than day 1. Conclusions: Preliminary blinded analysis of plasma markers of neurodegeneration confirm increased NfL and GFAP levels during acute stroke, which diminish over time with different temporal patterns. Final unblinded results will provide a robust natural time course of specific biomarkers of neurodegeneration including GAP43. Forthcoming analyses evaluate broad panels of proteins, mRNA, and miRNA markers. Identifying relevant post stroke biomarkers with directed and exploratory omics may allow development of validated assays to assess efficacy and support decision making in clinical trials.

Inhibition of repulsive guidance molecule-a ameliorates compromised blood–spinal cord barrier integrity associated with neuromyelitis optica in rats

The influx of pathogenic aquaporin-4 antibodies (AQP4-Abs) across the blood–spinal cord barrier (BSCB) is crucial for the development and exacerbation of neuromyelitis optica (NMO). We examined whether prophylactic intravenous administration of anti-repulsive guidance molecule-a antibodies (RGMa-Abs) has disease-modifying effects on BSCB dysfunction using an NMO model elicited by peripheral administration of AQP4-Abs to rats. RGMa-Ab treatment attenuated the acute exacerbation of perivascular astrocytopathy in the spinal cord and clinical symptoms, which were highly correlated with neurofilament light chain levels in both the cerebrospinal fluid (CSF) and serum. Additionally, RGMa-Ab treatment suppressed the expression of proinflammatory cytokines/chemokines and the infiltration of inflammatory cells into the spinal cord. CSF analysis of NMO rats revealed that RGMa-Ab treatment improved the CSF/serum albumin ratio and suppressed AQP4-Abs influx. RGMa inhibition using RGMa-Abs is suggested as a potential therapeutic option for BSCB dysfunction associated with NMO.

Repulsive guidance molecule A (RGMA) is widely expressed in the CNS of the sea lamprey Petromyzon marinus during early development.

RGM interactions with its receptor Neogenin play an important role in the regulation of axonal guidance or cell death in the developing central nervous system. The sea lamprey RGMA transcript has been recently identified. However, its expression has been only studied in the spinal cord of mature (premetamorphic) larval sea lampreys. Here, we report the expression of the sea lamprey RGMA transcript in developing embryos and prolarvae by means of in situ hybridization. Our data show that the RGMA transcript is broadly expressed in the central nervous system of embryos and prolarvae and with a rostro-caudal gradient of expression.

RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS.

Repulsive guidance molecule A (RGMa) was originally identified as a neuronal growth cone-collapsing factor. Previous reports have demonstrated the multifunctional roles of RGMa mediated by neogenin1. However, the pathogenic involvement of RGMa in amyotrophic lateral sclerosis (ALS) remains unclear. Here, we demonstrated that RGMa concentration was elevated in the cerebrospinal fluid of both patients with ALS and transgenic mice overexpressing the mutant human superoxide dismutase1 (mSOD1 mice). Treatment with humanized anti-RGMa monoclonal antibody ameliorated the clinical symptoms in mSOD1 mice. Histochemical analysis revealed that the anti-RGMa antibody significantly decreased mutant SOD1 protein accumulation in the motor neurons of mSOD1 mice via inhibition of actin depolymerization. In vitro analysis revealed that the anti-RGMa antibody inhibited the cellular uptake of the mutant SOD1 protein, presumably by reinforcing the neuronal actin barrier. Collectively, these data suggest that RGMa leads to the collapse of the neuronal actin barrier and promotes aberrant protein deposition, resulting in exacerbation of the ALS pathology.

RGMa and Neogenin control dendritic spine morphogenesis via WAVE Regulatory Complex-mediated actin remodeling.

Structural plasticity, the ability of dendritic spines to change their volume in response to synaptic stimulation, is an essential determinant of synaptic strength and long-term potentiation (LTP), the proposed cellular substrate for learning and memory. Branched actin polymerization is a major force driving spine enlargement and sustains structural plasticity. The WAVE Regulatory Complex (WRC), a pivotal branched actin regulator, controls spine morphology and therefore structural plasticity. However, the molecular mechanisms that govern WRC activation during spine enlargement are largely unknown. Here we identify a critical role for Neogenin and its ligand RGMa (Repulsive Guidance Molecule a) in promoting spine enlargement through the activation of WRC-mediated branched actin remodeling. We demonstrate that Neogenin regulates WRC activity by binding to the highly conserved Cyfip/Abi binding pocket within the WRC. We find that after Neogenin or RGMa depletion, the proportions of filopodia and immature thin spines are dramatically increased, and the number of mature mushroom spines concomitantly decreased. Wildtype Neogenin, but not Neogenin bearing mutations in the Cyfip/Abi binding motif, is able to rescue the spine enlargement defect. Furthermore, Neogenin depletion inhibits actin polymerization in the spine head, an effect that is not restored by the mutant. We conclude that RGMa and Neogenin are critical modulators of WRC-mediated branched actin polymerization promoting spine enlargement. This study also provides mechanistic insight into Neogenin's emerging role in LTP induction.

Netrin-1 and RGMa: Novel Regulators of Atherosclerosis-Related Diseases.

Neuronal guidance proteins (NGPs) havebeen demonstrated toguide the elongation of neuronal axonal growth cones in the developing central nervous system. Non-neuronal functions of NGPs have also been described, especially in relation to atherosclerosis. Netrin-1 and repulsive guidance molecule a (RGMa) are NGPs that have been shown to regulate endothelial cell adhesion and angiogenesis, macrophage migration and apoptosis, smooth muscle cells (SMCs) phenotypic dedifferentiation and mobility, chemokine activities, and inflammatory responses during atherosclerosis initiation and progression. However, mechanistic studies have generated controversy about the specific role of Netrin-1 in atherosclerosis due to the diversity of its structure, receptors and cell sources, and the actions of RGMa in atherosclerosis have not been reported in previous reviews. Therefore, the current work reviews the evidence for roles of Netrin-1 and RGMa in the initiation and progression of atherosclerosis and discusses potential therapeutic targets in the future.

Neutralizing RGMa with Elezanumab Promotes Cerebroprotection and Recovery in Rabbit Middle Cerebral Artery Occlusion.

Repulsive guidance molecule A (RGMa) is an inhibitor of neuronal growth and survival which is upregulated in the damaged central nervous system following acute spinal cord injury (SCI), traumatic brain injury, acute ischemic stroke (AIS), and other neuropathological conditions. Neutralization of RGMa is neuroprotective and promotes neuroplasticity in several preclinical models of neurodegeneration and injury including multiple sclerosis, AIS, and SCI. Given the limitations of current treatments for AIS due to narrow time windows to intervention (TTI), and restrictive patient selection criteria, there is significant unmet need for therapeutic agents that enable tissue survival and repair following acute ischemic damage for a broader population of stroke patients. In this preclinical study, we evaluated whether elezanumab, a human anti-RGMa monoclonal antibody, could improve neuromotor function and modulate neuroinflammatory cell activation following AIS with delayed intervention times up to 24h using a rabbit embolic permanent middle cerebral artery occlusion model (pMCAO). In two replicate 28-day pMCAO studies, weekly intravenous infusions of elezanumab, over a range of doses and TTIs of 6 and 24h after stroke, significantly improved neuromotor function in both pMCAO studies when first administered 6h after stroke. All elezanumab treatment groups, including the 24h TTI group, had significantly less neuroinflammation as assessed by microglial and astrocyte activation. The novel mechanism of action and potential for expanding TTI in human AIS make elezanumab distinct from current acute reperfusion therapies, and support evaluation in clinical trials of acute CNS damage to determine optimal dose and TTI in humans. A: Ramified/resting astrocytes and microglia in a normal, uninjured rabbit brain. B: Rabbit pMCAO brain illustrating lesion on right side of brain (red), surrounded by penumbra (pink) during acute phase post stroke, with minimal injury to left brain hemisphere. Penumbra characterized by activated astrocytes and microglia (region in crosshair within circle), with upregulation of free and bound RGMa. C: Elezanumab binds to both free and bound RGMa, preventing full activation of astrocytes and microglia. D: Elezanumab is efficacious in rabbit pMCAO with a 4 × larger TTI window vs. tPA (6 vs. 1.5 h, respectively). In human AIS, tPA is approved for a TTI of 3-4.5 h. Elezanumab is currently being evaluated in a clinical Ph2 study of AIS to determine the optimal dose and TTI (NCT04309474).

Knockdown of repulsive guidance molecule a promotes polarization of microglia into an anti-inflammatory phenotype after oxygen-glucose deprivation-reoxygenation in vitro

Repulsive guidance molecule a (RGMa) is a glycosylphosphatidylinositol-anchored glycoprotein that has been demonstrated to influence neuroinflammatory-related diseases in addition to regulating neuronal differentiation and survival during brain development. However, any function or mechanism of RGMa in the polarization of microglia after ischemic stroke remains unclear. In the current study, RGMa was found to be expressed at reduced levels in microglia after oxygen-glucose deprivation-reoxygenation (OGD/R) in vitro. RGMa overexpression induced HAPI microglia to predominantly polarize to the M1 phenotype, promoting the release of proinflammatory cytokines and knockdown induced the M2 phenotype, promoting the release of anti-inflammatory cytokines. RGMa overexpression also regulated the polarization of HAPI microglia by inhibiting the transportation of peroxisome proliferator-activated receptor γ (PPARγ) from the nucleus to cytoplasm. The opposite effect resulted from RGMa-knockdown and was reversed by the PPARγ antagonist, GW9662. In addition, RGMa-knockdown HAPI microglial conditioned medium improved the survival of oligodendrocytes after OGD/R in vitro. Thus, inhibition of RGMa may constitute a therapeutic strategy for reducing neuroinflammation after ischemic stroke.

Abstract TMP110: Elezanumab Decreases Neuronal Damage And Improves Functional Recovery In A Rabbit Model Of Permanent Middle Cerebral Artery Occlusion

Introduction: Current acute interventions for ischemic stroke aim to restore perfusion of affected tissue. There is an unmet need for therapies that protect neurons from ischemic damage and/or enhance natural repair processes to restore function. In humans, the neuronal growth and survival inhibitor RGMa (repulsive guidance molecule A) is upregulated following ischemic stroke. Elezanumab is a novel, humanized monoclonal antibody targeting RGMa. Preclinical evidence in traumatic and ischemic models of CNS damage suggest a protective and regenerative mechanisms for elezanumab following CNS damage. Methods: Elezanumab was evaluated in a rabbit permanent middle cerebral artery occlusion (MCAO) model of acute ischemic stroke by injection of an autologous blood clot. In replicate experiments, elezanumab (1, 10 and 40 mg/kg) or IgG control was administered 6 hours post occlusion or 24 hours post occlusion (10 mg/kg). Animals were dosed weekly through 28 days. Functional ability was assessed using the Neuromotor Score, a composite of sensorimotor performance tasks. Ex vivo MRI, histology, and fluid biomarker analyses were performed. Results: MCAO consistently reduced cerebral blood flow by ~60% and resulted in neuromotor dysfunction. All doses of elezanumab demonstrated statistically significantly improved neuromotor function and recovery rate versus controls when dosed 6 hours post occlusion. The 10 mg/kg group dosed 24 hours post occlusion did not show a statistically significant functional improvement versus controls. Infarct volumes, plasma neurofilament light chain levels, and histological evidence of microglial proliferation were all significantly reduced with elezanumab treatment versus controls, including in the delayed elezanumab treatment group. Conclusions: Elezanumab treatment was associated with improved functional recovery and reduced markers of neuronal damage. Collectively, these data support a neuroprotective role for elezanumab in this translational model of acute ischemic stroke. Elezanumab is now being tested in a Phase 2a clinical trial for acute ischemic stroke.

LncRNA RP11-551L14.4 suppresses breast cancer development by inhibiting the expression of miR-4472.

Previous studies have been reported that long non-coding RNA (lncRNA) can regulate the expression of genes which are involved in many important cellular processes The potential role of lncRNA RP11-551L14.4 in the development of breast cancer and the possible regulatory mechanisms was investigated. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to analyze RP11-551L14.4 expression in 36 paired breast cancer tissues and adjacent tissues. The expression of RP11-551L14.4 in multiple breast cancer cell lines was detected by qRT-PCR. Meanwhile, overexpression of RP11-551L14.4 models was established using lentivirus in BT474 and T47D breast cancer cells. Cell counting kit-8 (CCK-8), cell colony formation and cell cycle assays were performed to detect the effects of RP11-551L14.4 on the biological function of breast cancer cells. Besides, bioinformatics techniques, dual luciferase reporter gene assay and rescue experiments were used to investigate the potential mechanisms. RP11-551L14.4 expression was negatively associated with the advanced tumor stage. Breast cancer patients with low RP11-551L14.4 expression manifested a poorer prognosis. The results of qRT-PCR showed that RP11-551L14.4 expression in breast cancer tissues was significantly lower than in adjacent tissues. Meanwhile, overexpression of RP11-551L14.4 significantly decreased the cell proliferation and cell cycle. Bioinformatics technology showed that RP11-551L14.4 could complementarily bind to miR-4472. qRT-PCR results indicated that the expression levels of miR-4472 and RP11-551L14.4 in breast cancer were negatively correlated. Luciferase reporter gene assay showed that miR-4472 remarkably decreased the relative luciferase activity of the wild-type RP11-551L14.4 vector. miR-4472 is a direct target gene of RP11-551L14.4. miR-4472 levels were reduced, and repulsive guidance molecule A (RGMA) mRNA or protein levels were increased after overexpression of RP11-551L14.4 in the breast cancer cells. miR-4472 reversed the effects caused by RP11-551L14.4 in breast cancer cells. RP11-551L14.4 expression was remarkably decreased in breast cancer tissues and cells. RP11-551L14.4 may inhibit the malignant progression of breast cancer by regulating miR-4472 expression.

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.

Reduced plasma levels of RGM-A predict stroke-associated pneumonia in patients with acute ischemic stroke: A prospective clinical study.

BackgroundStroke-induced immunodepression syndrome is considered the major etiology of stroke-associated pneumonia (SAP). Repulsive guidance molecule A (RGM-A) is an immunomodulatory protein that is closely related to inflammation and immune responses. To explore the relationship between RGM-A and SAP and facilitate the early identification of patients at high risk of developing SAP, we investigated the predictive value of RGM-A in SAP.MethodsWe enrolled 178 patients with acute ischemic stroke (AIS) and finally analyzed 150 patients, among whom 69 had SAP and 81 had non-SAP. During the same period, 40 patients with community-acquired pneumonia and 40 healthy participants were included as controls. SAP was defined according to the modified US Centers for Disease Control and Prevention criteria. Blood samples were collected at 24 h, 48 h, 3 days, 4 to 7 days, and 8 to 14 days after stroke onset. An enzyme-linked immunosorbent assay was used to detect the plasma levels of RGM-A and interleukin-6.ResultsThe plasma RGM-A levels were significantly decreased in both patients with community-acquired pneumonia and those with AIS, and the decline was most pronounced in patients with SAP (P < 0.001). RGM-A started to decline within 24 h after stroke in the SAP group, and the lowest levels were detected on day 3 and days 4 to 7 (P < 0.001). The RGM-A levels in the SAP group were lower than those in the non-SAP group at all blood collection time points (P < 0.05). In the logistic regression analyses, RGM-A was a protective factor for SAP after adjusting for confounders (adjusted odds ratio = 0.22, 95% confidence interval = 0.091–0.538, P = 0.001). Receiver operating characteristic curve analysis showed that the area under the curve for RGM-A was 0.766 (0.091–0.538; P = 0.001), the cutoff value was 4.881 ng/mL, and the sensitivity and specificity were 80.00 and 76.36%, respectively.ConclusionsWe demonstrated that reduced plasma levels of RGM-A might help in the early identification of high-risk patients with SAP and predict the occurrence of SAP in patients with AIS. RGM-A might provide new clues to a potential alternative therapy for SAP.

RGMa promotes dedifferentiation of vascular smooth muscle cells into a macrophage-like phenotype in vivo and in vitro

Repulsive guidance molecule a (RGMa) is a glycosylphosphatidylinositol-anchored glycoprotein that has been demonstrated to influence inflammatory-related diseases in addition to regulating neuronal differentiation and survival during brain development. However, any function or mechanism of RGMa in dedifferentiation of contractile vascular smooth muscle cells (VSMCs) during inflammatory-related atherosclerosis is poorly understood. In the current study, we found that RGMa is expressed in VSMCs-derived macrophage-like cells from the fibrous cap of type V atherosclerotic plaques and the neointima of ligated carotid artery in ApoE−/− mice. We determined levels of RGMa mRNA and protein increased in oxidized LDL (ox-LDL)-induced VSMCs. Knockdown of RGMa, both in vivo and in vitro, inhibited the dedifferentiation of ox-LDL-induced VSMCs and their ability to proliferate and migrate, reduced the thickness of the neointima after ligation of the left common carotid artery in ApoE−/− mice. Additionally, we show RGMa promoted the dedifferentiation of VSMCs via enhancement of the role of transcription factor Slug. Slug knockdown reversed the dedifferentiation of ox-LDL-induced VSMCs promoted by RGMa overexpression. Thus, inhibition of RGMa may constitute a therapeutic strategy for atherosclerotic plaques prone to rupture and restenosis following mechanical injury.

Delayed administration of elezanumab, a human anti-RGMa neutralizing monoclonal antibody, promotes recovery following cervical spinal cord injury

Spinal cord injury (SCI) elicits a cascade of degenerative events including cell death, axonal degeneration, and the upregulation of inhibitory molecules which limit repair. Repulsive guidance molecule A (RGMa) is an axon growth inhibitor which is also involved in neuronal cell death and differentiation. SCI causes upregulation of RGMa in the injured rodent, non-human primate, and human spinal cord. Recently, we showed that delayed administration of elezanumab, a high affinity human RGMa-specific monoclonal antibody, promoted neuroprotective and regenerative effects following thoracic SCI. Since most human traumatic SCI is at the cervical level, and level-dependent anatomical and molecular differences may influence pathophysiological responses to injury and treatment, we examined the efficacy of elezanumab and its therapeutic time window of administration in a clinically relevant rat model of cervical impact-compression SCI. Pharmacokinetic analysis of plasma and spinal cord tissue lysate showed comparable levels of RGMa antibodies with delayed administration following cervical SCI. At 12w after SCI, elezanumab promoted long term benefits including perilesional sparing of motoneurons and increased neuroplasticity of key descending pathways involved in locomotion and fine motor function. Elezanumab also promoted growth of corticospinal axons into spinal cord gray matter and enhanced serotonergic innervation of the ventral horn to form synaptic connections caudal to the cervical lesion. Significant recovery in grip and trunk/core strength, locomotion and gait, and spontaneous voiding ability was found in rats treated with elezanumab either immediately post-injury or at 3 h post-SCI, and improvements in specific gait parameters were found when elezanumab was delayed to 24 h post-injury. We also developed a new locomotor score, the Cervical Locomotor Score, a simple and sensitive measure of trunk/core and limb strength and stability during dynamic locomotion.

Modulation of Microglia M2 Polarization and Alleviation of Hippocampal Neuron Injury By MiR-106b-5p/RGMa in a Mouse Model of Status Epilepticus.

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. The miRNA miR-106b-5p has been linked to epilepsy, but its specific role and mechanism of action remain unclear. This was investigated in the present study using a mouse model of pilocarpine-induced status epilepticus and an in vitro system of HT22 hippocampal cells treated with Mg2+-free solution and cocultured with BV2 microglia cells. We found that inhibiting miR-106b-5p expression promoted microglia M2 polarization, reduced the inflammatory response, and alleviated neuronal injury. These effects involved modulation of the repulsive guidance molecule A (RGMa)-Rac1-c-Jun N-terminal kinase (JNK)/p38-mitogen-activated protein kinase (MAPK) signaling axis. Our results suggest that therapeutic strategies targeting miR-106b-5p or downstream factors can be effective in preventing epileptogenesis or treating epilepsy.

RGMa Participates in the Blood-Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis.

The infiltration of inflammatory cells into the central nervous system (CNS) through the dysfunctional blood–brain barrier (BBB) was critical in the early stages of MS. However, the mechanisms underlying BBB dysfunction remain unknown. Repulsive guidance molecule-a (RGMa) is involved in the pathogenesis of multiple sclerosis (MS), but its role needs to be further explored. This study aimed to evaluate whether RMGa regulates BBB permeability in endothelial cells and MS, and if so, what mechanism may be involved. We created an experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice and a human brain microvascular endothelial cell (HBMEC) culture. The permeability of the BBB is measured in response to various interventions. Our results showed that RGMa is expressed in the endothelial cells in HBMECs and EAE mice. RGMa and its signaling counterpart, bone morphogenetic protein 2 (BMP2)/bone morphogenetic protein receptor type II (BMPRII), were gradually increased as the disease progressed. Moreover, as EAE progressed and the BBB was disrupted, the downstream effector, yes-associated protein (YAP), as well as the tight junctional proteins zonula occludens 1 (ZO-1) and claudin-5, decreased significantly. The permeability assay revealed that lentivirus-induced RGMa overexpression in HBMECs caused a significant breakdown of the BBB, whereas RGMa knockdown significantly strengthens the integrity of the BBB. Furthermore, specifically activating BMPR II or inhibiting YAP based on RGMa knockdown results in a significant decrease of ZO-1 and claudin-5 in vitro. On the contrary, inhibition of BMPR II or activation of YAP after upregulating RGMa prevents the downregulation of ZO-1 and claudin-5 in HBMECs. In addition, serum-soluble RGMa (sRGMa) levels were significantly higher in MS patients, particularly in MS patients with Gd+ lesions, indicating that the BBB has been disrupted. In conclusion, this study shows that RGMa causes BBB dysfunction in endothelial cells via BMP2/BMPR II/YAP, resulting in BBB integrity disruption in MS and that it could be a novel therapeutic target for BBB permeability in MS.

Transsynaptic cerebellin 4–neogenin 1 signaling mediates LTP in the mouse dentate gyrus

Five decades ago, long-term potentiation (LTP) of synaptic transmission was discovered at entorhinal cortex→dentate gyrus (EC→DG) synapses, but the molecular determinants of EC→DG LTP remain largely unknown. Here, we show that the presynaptic neurexin–ligand cerebellin-4 (Cbln4) is highly expressed in the entorhinal cortex and essential for LTP at EC→DG synapses, but dispensable for basal synaptic transmission at these synapses. Cbln4, when bound to cell-surface neurexins, forms transcellular complexes by interacting with postsynaptic DCC (deleted in colorectal cancer) or neogenin-1. DCC and neogenin-1 act as netrin and repulsive guidance molecule-a (RGMa) receptors that mediate axon guidance in the developing brain, but their binding to Cbln4 raised the possibility that they might additionally function in the mature brain as postsynaptic receptors for presynaptic neurexin/Cbln4 complexes, and that as such receptors, DCC or neogenin-1 might mediate EC→DG LTP that depends on Cbln4. Indeed, we observed that neogenin-1, but not DCC, is abundantly expressed in dentate gyrus granule cells, and that postsynaptic neogenin-1 deletions in dentate granule cells blocked EC→DG LTP, but again did not affect basal synaptic transmission similar to the presynaptic Cbln4 deletions. Thus, binding of presynaptic Cbln4 to postsynaptic neogenin-1 renders EC→DG synapses competent for LTP, but is not required for establishing these synapses or for otherwise enabling their function.

RGMa Signal in Macrophages Induces Neutrophil-Related Astrocytopathy in NMO.

Repulsive guidance molecule-a (RGMa) is a glycosylphosphatidylinositol-linked glycoprotein which has multiple functions including axon growth inhibition and immune regulation. However, its role in the pathophysiology of neuromyelitis optica (NMO) is poorly understood. Perivascular astrocytopathy, which is induced by the leakage of aquaporin-4 (AQP4)-specific IgG into the central nervous system parenchyma, is a key feature of NMO pathology. We investigated the RGMa involvement in the pathology of NMO astrocytopathy, and tested a therapeutic potential of humanized anti-RGMa monoclonal antibody (RGMa-mAb). Using a clinically relevant NMO rat model, we evaluated the therapeutic effect of a RGMa-mAb by behavioral testing, immunohistochemistry, and gene expression assay. We further performed in vitro experiments to address the RGMa-signaling in macrophages. In both NMO rats and an NMO-autopsied sample, RGMa was expressed by the spared neurons and astrocytes, whereas its receptor neogenin was expressed by infiltrating macrophages. AQP4-IgG-induced astrocytopathy and clinical exacerbation in NMO rats were ameliorated by RGMa-mAb treatment. RGMa-mAb treatment significantly suppressed neutrophil infiltration, and decreased the expression of neutrophil chemoattractants. Interestingly, neogenin-expressing macrophages accumulated in the lesion expressed CXCL2, a strong neutrophil chemoattractant, and further analysis revealed that RGMa directly regulated CXCL2 expression in macrophages. Finally, we found that our NMO rats developed neuropathic pain, and RGMa-mAb treatment effectively ameliorated the severity of neuropathic pain. RGMa signaling in infiltrated macrophages is a critical driver of neutrophil-related astrocytopathy in NMO lesions, and RGMa-mAb may provide an efficient therapeutic strategy for NMO-associated neuropathic pain and motor deficits in patients with NMO. ANN NEUROL 2022;91:532-547.

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.

Abstract TP7: Phase 2a Study Of Elezanumab In Patients With Acute Ischemic Stroke

Introduction: Treatment of acute ischemic stroke focuses on reperfusion but is limited by timing and other exclusions, and despite intervention, patients may sustain residual neurologic impairment. In addition to initial excitotoxic reactions following occlusion, upregulation of factors such as repulsive guidance molecule A (RGMa) inhibits cell survival and neuronal repair in infarcted regions. In vivo neutralization of RGMa is associated with decreased lesion size, reduced glial scarring, and enhanced functional recovery in animal models of cerebral artery occlusion; thus, RGMa suppression represents a novel therapeutic target due to its potential protective and restorative mechanisms. Elezanumab is an investigational monoclonal antibody that binds specifically to RGMa and is being evaluated for its function after ischemic stroke. Methods: Elezanumab in Acute Ischemic Stroke (EAISE) is a 52-week, phase 2a, randomized, double-blind, placebo-controlled study evaluating safety, efficacy, and pharmacokinetics of elezanumab in patients with acute ischemic stroke (NCT04309474). Patients will be randomized 1:1 within 23 hours since “last known normal” to receive elezanumab or placebo and every 4 weeks thereafter through week 48. Patients must be aged 30-90 years with a clinical diagnosis of acute ischemic stroke in anterior circulation and have a National Institutes of Health Stroke Scale (NIHSS) total score of 7 to 21. Patients who receive tissue plasminogen activator or endovascular therapy will be eligible if they continue to exhibit an NIHSS of 7 to 21 after treatment. Patients with evidence of seizure on stroke onset and history of repeated episodes of complex migraines will be excluded. Results: Projected enrollment is 120 patients across 45 sites in multiple countries. Primary endpoint is the NIHSS total score throughout the treatment period. Secondary endpoint is a favorable outcome based on the modified Rankin Scale, defined as a score of 0-2 at week 52. Exploratory outcomes include activities of daily living, cognition, mood, and fluid and imaging biomarkers. Conclusion: This phase 2 study will evaluate safety and efficacy of elezanumab, a novel therapeutic intervention to improve neurological recovery after acute ischemic stroke.