RESUMEN

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.

Asunto(s)

Neuromielitis Óptica , Animales , Ratas , Acuaporina 4 , Autoanticuerpos/metabolismo , Médula Espinal/patología

RESUMEN

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelin-associated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19 (that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the RhoA/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

RESUMEN

Background: Stroke-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. Methods: We 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. Results: The 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. Conclusions: We 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.

RESUMEN

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.

Asunto(s)

Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Barrera Hematoencefálica/metabolismo , Claudina-5/metabolismo , Células Endoteliales/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Proteínas del Tejido Nervioso/metabolismo

RESUMEN

INTRODUCTION: Current research paradigms on biomarkers for chronic neurodegenerative diseases, such as Parkinson's disease, focus on identification of reliable, easy-to-apply tools for diagnostic screening and progression assessment. AREAS COVERED: This perspective discusses possible misconceptions of biomarker research in chronic neurodegeneration from a clinician's view based on a not systematic literature search. Multifactorial disease triggers, heterogeneity of symptom and their progression are main reasons for the still missing availability of biomarkers. EXPERT OPINION: Onset of chronic neurodegenerative disease entities may probably result from a decompensated endogenous repair machinery in the central nervous system, for example the neogenin receptor associated repulsive guidance molecule pathway. Future clinical research is warranted on these repair structures and aim to identify markers for the imbalance between damage and repair, which hypothetically contributes to generation of disease. An assignment to a specific chronic neurodegenerative disease entity probably appears to be secondary. Decryption of probable molecular signals of an impaired repair potential will enable an earlier diagnosis, better monitoring of disease progress and of treatment response. This concept will hopefully provide better preconditions for prevention, cure or therapeutic beneficial disease modification. These unmet therapeutic needs may be achieved for example via antagonism of repulsive guidance molecule A.

Asunto(s)

Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Biomarcadores/metabolismo , Muerte Celular , Enfermedad Crónica , Progresión de la Enfermedad , Diagnóstico Precoz , Humanos , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/metabolismo

RESUMEN

Objectives: This study sought to explore the expression patterns of repulsive guidance molecules a (RGMa) in neuromyelitis optica spectrum disorders (NMOSD) and to explore the correlation between RGMa and the clinical features of NMOSD. Methods: A total of 83 NMOSD patients and 22 age-matched healthy controls (HCs) were enrolled in the study from October 2017 to November 2021. Clinical parameters, including Expanded Disability Status Scale (EDSS) score, degree of MRI enhancement, and AQP4 titer were collected. The expression of serum RGMa was measured by enzyme-linked immunosorbent assay (ELISA) and compared across the four patient groups. The correlation between serum RGMa levels and different clinical parameters was also assessed. Results: The average serum expression of RGMa in the NMOSD group was significantly higher than that in the HC group (p < 0.001). Among the patient groups, the acute phase group exhibited significantly higher serum RGMa levels than did the remission group (p < 0.001). A multivariate analysis revealed a significant positive correlation between RGMa expression and EDSS score at admission, degree of MRI enhancement, and segmental length of spinal cord lesions. There was a significant negative correlation between the expression of RGMa in NMOSD and the time from attack to sampling or delta EDSS. Conclusions: The current study suggests that RGMa may be considered a potential biomarker predicting the severity, disability, and clinical features of NMOSD.

Asunto(s)

Acuaporina 4/inmunología , Proteínas Ligadas a GPI/sangre , Proteínas del Tejido Nervioso/sangre , Neuromielitis Óptica/patología , Médula Espinal/diagnóstico por imagen , Adulto , Autoanticuerpos/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Evaluación de la Discapacidad , Femenino , Humanos , Modelos Lineales , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Análisis Multivariante , Neuromielitis Óptica/sangre , Neuromielitis Óptica/diagnóstico por imagen , Índice de Severidad de la Enfermedad , Médula Espinal/patología , Adulto Joven

RESUMEN

BACKGROUND: Cerebral edema and intracranial hypertension are major contributors to unfavorable prognosis in traumatic brain injury (TBI). Local epigenetic changes, particularly in DNA methylation, may influence gene expression and thus host response/secondary injury after TBI. It remains unknown whether DNA methylation in the central nervous system is associated with cerebral edema severity or intracranial hypertension post TBI. We sought to identify epigenome-wide DNA methylation patterns associated with these forms of secondary injury after TBI. METHODS: We obtained genome-wide DNA methylation profiles of DNA extracted from ventricular cerebrospinal fluid samples at three different postinjury time points from a prospective cohort of patients with severe TBI (n = 89 patients, 254 samples). Cerebral edema and intracranial pressure (ICP) measures were clustered to generate composite end points of cerebral edema and ICP severity. We performed an unbiased epigenome-wide association study (EWAS) to test associations between DNA methylation at 419,895 cytosine-phosphate-guanine (CpG) sites and cerebral edema/ICP severity categories. Given inflated p values, we conducted permutation tests for top CpG sites to filter out potential false discoveries. RESULTS: Our data-driven hierarchical clustering across six cerebral edema and ICP measures identified two groups differing significantly in ICP based on the EWAS-identified CpG site cg22111818 in RGMA (Repulsive guidance molecule A, permutation p = 4.20 × 10-8). At 3-4 days post TBI, patients with severe intracranial hypertension had significantly lower levels of methylation at cg22111818. CONCLUSIONS: We report a novel potential relationship between intracranial hypertension after TBI and an acute, nonsustained reduction in DNA methylation at cg22111818 in the RGMA gene. To our knowledge, this is the largest EWAS in severe TBI. Our findings are further strengthened by previous findings that RGMA modulates axonal repair in other central nervous system disorders, but a role in intracranial hypertension or TBI has not been previously identified. Additional work is warranted to validate and extend these findings, including assessment of its possible role in risk stratification, identification of novel druggable targets, and ultimately our ability to personalize therapy in TBI.

Asunto(s)

Edema Encefálico , Lesiones Traumáticas del Encéfalo , Hipertensión Intracraneal , Edema Encefálico/complicaciones , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/genética , Metilación de ADN , Epigenoma , Humanos , Hipertensión Intracraneal/complicaciones , Hipertensión Intracraneal/genética , Presión Intracraneal , Estudios Prospectivos

RESUMEN

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) co­receptor, 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 C­C motif chemokine ligand 5 (CCL5) has a major role in the pathogenesis of MS via the recruitment of macrophages and T­lymphocytes 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 ligand­dependent 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 RGMa­BMP receptor signalling pathway and the pathogenesis of RGMa on MS as far as the involvement of blood­brain barrier permeability is concerned.

Asunto(s)

Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Quimiocina CCL5/genética , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Células Endoteliales/metabolismo , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Animales , Biomarcadores , Células Cultivadas , Quimiocina CCL5/metabolismo , Susceptibilidad a Enfermedades , Encefalomielitis Autoinmune Experimental/diagnóstico , Femenino , Ratones , Interferencia de ARN , Índice de Severidad de la Enfermedad

RESUMEN

The term idiopathic Parkinson's disease describes an entity of various not well-characterized disorders resembling each other. They are characterized by chronic neuronal dying originating from various disease mechanisms. They result in the onset of motor and related non-motor features, both of which respond to administration of personalized drug combinations and surgical therapies. The unmet need is beneficial disease course modification with repair and neurogenesis. Objectives are to discuss the value of cell secretome based treatments including neuronal graft transplantation and to suggest as an alternative the stimulation of an endogenous available approach for neuronal repair. Chronic neurodegenerative processes result from different heterogeneous, but complementing metabolic, pathological cascade sequences. Accumulated evidence from experimental research suggested neuron transplantation, stem cell application and cell secretome-based therapies as a promising future treatment with cure as an ultimate goal. To date, clinical testing of disease-modifying treatments has focused on substitution or repair of the remaining dopamine synthesizing neurons following diagnosis. At diagnosis, many of the still surviving and functioning, but already affected neurons have lost most of their axons and are primed for cell death. A more promising therapeutic concept may be the stimulation of an existing, endogenous repair system in the peripheral and central nervous systems. The abundant protein repulsive guidance molecule A blocks restoration and neurogenesis, both of which are mediated via the neogenin receptor. Inhibition of the physiological effects of repulsive guidance molecule A is an endogenous available repair pathway in chronic neurodegeneration. Antagonism of this protein with antibodies or stimulation of the neogenin receptor should be considered as an initial repair step. It is an alternative to cell replacement, stem cell or associated cell secretome concepts.

RESUMEN

Symptomatic treatments are available for Parkinson's disease and Alzheimer's disease. An unmet need is cure or disease modification. This review discusses possible reasons for negative clinical study outcomes on disease modification following promising positive findings from experimental research. It scrutinizes current research paradigms for disease modification with antibodies against pathological protein enrichment, such as α-synuclein, amyloid or tau, based on post mortem findings. Instead a more uniform regenerative and reparative therapeutic approach for chronic neurodegenerative disease entities is proposed with stimulation of an endogenously existing repair system, which acts independent of specific disease mechanisms. The repulsive guidance molecule A pathway is involved in the regulation of peripheral and central neuronal restoration. Therapeutic antagonism of repulsive guidance molecule A reverses neurodegeneration according to experimental outcomes in numerous disease models in rodents and monkeys. Antibodies against repulsive guidance molecule A exist. First clinical studies in neurological conditions with an acute onset are under way. Future clinical trials with these antibodies should initially focus on well characterized uniform cohorts of patients. The efficiency of repulsive guidance molecule A antagonism and associated stimulation of neurogenesis should be demonstrated with objective assessment tools to counteract dilution of therapeutic effects by subjectivity and heterogeneity of chronic disease entities. Such a research concept will hopefully enhance clinical test strategies and improve the future therapeutic armamentarium for chronic neurodegeneration.

Asunto(s)

Enfermedades Neurodegenerativas/terapia , Enfermedad Crónica , Progresión de la Enfermedad , Humanos , Investigación Biomédica Traslacional

RESUMEN

BACKGROUND: In the event of middle cerebral artery occlusion (MCAO), leptomeningeal collaterals (LMCs) play a crucial role in determining the survival of brain tissue distal to occlusion. Previous findings indicated that genes controlling arteriogenesis can impact the extent of LMCs. Therefore, probe for potential genetic parameters correlating of arteriogenesis may be clinically useful in predicting LMCs status in MCAO. During the screening process, we focused on repulsive guidance molecule a (RGMa), which has been reported to play a negative role in angiogenesis after stroke by decreasing the proliferation, migration, and tube formation of endothelial cells (ECs) in vivo and in vitro. Indeed, endothelial function plays a main role in arteriogenesis and is essential in determining the LMCs status. Therefore, in present study, we aimed to testify the hypothesis that RGMa might be associated with LMCs status in MCAO. METHODS: We prospectively enrolled patients with acute M1 MCA +/- intracranial internal carotid artery (ICA) occlusions (n=96) and healthy controls (n=33). Status of LMCs was evaluated according to computed tomographic angiography (CTA) on admission. Baseline RGMa mRNA expression was quantified by using quantitative real-time PCR. RESULTS: Patients with poor LMCs showed significantly higher RGMa mRNA levels than patients with good LMCs status (P=0.001) as well as healthy controls (P=0.002), respectively; whereas good LMCs group showed similar baseline RGMa levels than controls (P=1.000). RGMa mRNA level and baseline NIHSS score were independent predictors for impaired LMCs. CONCLUSIONS: In MCAO patients, elevated PBMCs RGMa mRNA levels were associated with impaired LMCs status, indicating that measurement of RGMa mRNA expression in the early phase of stroke, together with other clinical approaches, was logically expected to be useful for predicting LMCs status. Moreover, a role for RGMa in leptomeningeal arteriogenesis following ischemic stroke can be hypothesized.

Asunto(s)

Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular , Células Endoteliales , Humanos , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/genética , Leucocitos Mononucleares , ARN Mensajero/genética , Accidente Cerebrovascular/genética

RESUMEN

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.

Asunto(s)

Proteínas Ligadas a GPI/fisiología , Proteínas de la Membrana/fisiología , MicroARNs/genética , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Convulsiones/prevención & control , Proteínas de Unión al GTP rho/fisiología , Regiones no Traducidas 3' , Animales , Axones/ultraestructura , Células Cultivadas , Convulsivantes/toxicidad , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Regulación de la Expresión Génica , Silenciador del Gen , Vectores Genéticos , Hipocampo/citología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , MicroARNs/antagonistas & inhibidores , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/genética , Neuronas/ultraestructura , Pentilenotetrazol/toxicidad , ARN/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/genética , Convulsiones/fisiopatología , Transducción de Señal , Proteínas de Unión al GTP rho/biosíntesis , Proteínas de Unión al GTP rho/genética

RESUMEN

Spinal cord injury (SCI) often results in permanent functional loss due to a series of degenerative events including cell death, axonal damage, and the upregulation of inhibitory proteins that impede regeneration. Repulsive Guidance Molecule A (RGMa) is a potent inhibitor of axonal growth that is rapidly upregulated following injury in both the rodent and human central nervous system (CNS). Previously, we showed that monoclonal antibodies that specifically block inhibitory RGMa signaling promote neuroprotective and regenerative effects when administered acutely in a clinically relevant rat model of thoracic SCI. However, it is unknown whether systemic administration of RGMa blocking antibodies are effective for SCI after delayed administration. Here, we administered elezanumab, a human monoclonal antibody targeting RGMa, intravenously either acutely or at 3 h or 24 h following thoracic clip impact-compression SCI. Rats treated with elezanumab acutely and at 3 h post-injury showed improvements in overground locomotion and fine motor function and gait. Rats treated 24 h post-SCI trended towards better recovery demonstrating significantly greater stride length and swing speed. Treated rats also showed greater tissue preservation with reduced lesion areas. As seen with acute treatment, delayed administration of elezanumab at 3 h post-SCI also increased perilesional neuronal sparing and serotonergic and corticospinal axonal plasticity. In addition, all elezanumab treated rats showed earlier spontaneous voiding ability and less post-trauma bladder wall hypertrophy. Together, our data demonstrate the therapeutic efficacy of delayed systemic administration of elezanumab in a rat model of SCI, and uncovers a new role for RGMa inhibition in bladder recovery following SCI.

Asunto(s)

Anticuerpos Monoclonales/administración & dosificación , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/fisiopatología , Animales , Femenino , Humanos , Ratas , Ratas Wistar , Micción/efectos de los fármacos

RESUMEN

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.

RESUMEN

Endothelial progenitor cells (EPCs) are multipotential stem cells considered to have immense clinical value for revascularization. However, the clinical application of EPCs has been hampered by their clinical potency in ischemic anoxic environments. This study aimed to explore the effect of microRNA-210 (miR-210) on EPCs under oxygen-glucose deprivation (OGD) conditions. We generated a model of EPCs cultured under OGD conditions to simulate ischemia and explore the expression of miR-210 in vitro. With longer exposure to hypoxia, we found that miR-210-3p expression was highly upregulated in OGD groups compared to that in controls from 4 to 24 h, but not miR-210-5p. We then transfected a miR-210-3p mimic and inhibitor into EPCs, and after 24 h, we exposed them to OGD conditions for 4 h to simulate ischemia. We detected miR-210 by real-time polymerase chain reaction (RT-PCR) and tested the proliferation, migration, and tube formation of normal EPCs and OGD-treated EPCs by CCK-8, transwell chamber, and Matrigel assays, respectively. The direct targets of miR-210-3p were predicted using miRWalk. Compared to that in normal EPCs, higher miR-210-3p expression was found in OGD-treated EPCs (p < 0.05). Moreover, upregulation of miR-210-3p was found to promote proliferation, migration, and tube formation in EPCs under normal and OGD conditions (p < 0.05), whereas down-regulation inhibited these abilities in OGD-treated EPCs (p < 0.05). Repulsive guidance molecule A (RGMA), a negative regulator of angiogenesis, was predicted to be a target of miR-210-3p. Accordingly, upregulation of miR-210-3p was found to inhibit its expression at the protein level in OGD-treated EPCs, whereas downregulation of miR-210-3p inhibited its expression (p < 0.05). A dual-luciferase reporter system confirmed that RGMA is a direct target of miR-210-3p. MicroRNA-210-3p overexpression enhances the angiogenic properties of OGD-treated EPCs by inhibiting RGMA.

RESUMEN

Damaged axons in the adult central nervous system (CNS) fail to regenerate spontaneously due to several intrinsic and extrinsic factors that inhibit axon elongation. An extrinsic inhibitory factor, repulsive guidance molecule-a (RGMa), is upregulated around spinal cord lesion sites. Inhibition of RGMa using an antibody promotes axon sprouting, regeneration, and motor recovery after spinal cord injury (SCI) in rodents and primates. Repetitive transcranial magnetic stimulation (rTMS) has been used as a form of rehabilitation, and accumulating studies have suggested that rTMS is able to modulate neural plasticity of the cortex. Here, we conducted rTMS with anti-RGMa antibody treatment in mice with SCI to investigate the potential synergistic effects on motor recovery. Although mice treated with anti-RGMa antibody and rTMS concomitantly did not show significant motor recovery, mice treated sequentially with anti-RGMa antibody followed by rTMS showed better motor performance than mice treated with anti-RGMa antibody alone. Moreover, we found that Ca2+/calmodulin-dependent kinase II (CaMKII) was upregulated in mice treated with anti-RGMa antibody and rTMS sequentially compared with mice received a single anti-RGMa antibody treatment. These results suggest that anti-RGMa antibody treatment and rTMS intervention have synergistic effects on motor recovery, and that the timing of rTMS intervention is a critical factor to maximize the effect of anti-RGMa antibody treatment. These interventions may provide new therapeutic strategies for promoting motor recovery after SCI.

Asunto(s)

Anticuerpos/administración & dosificación , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/terapia , Estimulación Transcraneal de Corriente Directa/métodos , Estimulación Magnética Transcraneal/métodos , Animales , Terapia Combinada/métodos , Femenino , Bombas de Infusión Implantables , Ratones , Ratones Endogámicos C57BL , Traumatismos de la Médula Espinal/fisiopatología

RESUMEN

Objective: To investigate the expression of repulsive guidance molecule A (RGMa) in polarized microglia after rat focal cerebral ischemia/reperfusion injury. Methods: Adult male Sprague-Dawley (SD) rats were randomly divided into control group, 7 days cerebral ischemia/reperfusion injury group (I/R), and 14 days I/R group. The transient middle cerebral occlusion (tMCAO) model was induced by ligation with nylon monofilament. Real-time PCR was used to test the mRNA expression of Ml and M2 microglia marker interleukin-1Î2 (IL-1Î2), inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and CD206 in ipsilateral cortex at day 7 and day 14. Double immunofluorescence staining was performed to investigate the co-expression of RGMa and Ibal in microglia. The microglia was incubated with lipopolysaccharides (LPS)or IL-4 in vitro. The mRNA expression of Ml and M2 microglia marker (IL-1Î2, iNOS, Arg-1, CD206) was tested by Real-time PCR. Western blotting was used to assess the proteins level of RGMa in Ml and M2 microglia. Results: M1 and M2 microglia marker mRNA level were increased in ipsilateral cortex at day 7 and day 14. RGMa was strongly expressed in reactive microglia at day 7 and day 14. LPS or IL-4 treatment polarized microglia to Ml or M2 in vitro. The expression of RGMa protein in Ml and M2 microglia was increased. Conclusion: RGMa was strongly expressed in reactive Ml and M2 microglia after ischemia/reperfusion injury. RGMa may play an important role in the process of microglia activation and polarization. RGMa may be a novel therapeutic target for stroke.

RESUMEN

Objective To investigate whether silencing repulsive guidance molecule A (KGMa) shows protective effects on blood brain barrier (BBB) and tight junction protein after the injury of cerebral ischemia and reperfusion (I/H) in rats. Methods Middle cerebral artery occlusion (MCAO)reperfusion was employed to establish the models in the male adult rats. Fourty male Sprague-Dawley rats were divided into blank control group (sh-con) and RGMa interference group (sh-RGMa). The effects of adenovirus on RGMa were observed at day 1 and day 3 after injection. The remaining 120 SD rats were randomly divided into sham group, I/R group, I/'R+sh-con group and I/R+sh-RGMa group. After reperfusion for 72 hours, the neurological recovery was evaluated in rats by neurological deficit score, the infarcted volume was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the permeability of BBB was performed through evans blue by tail vein injection. The expression of RGMa was detected by Western blotting and immunohistochemistry assay. The expression of claudin-5, matrix metalloprotein 9 (MMP-9)and zonula occludin l(ZO-l)were detected by Western blotting. Results Silencing RGMa could improve the permeability of BBB, the infarcted volume, down-regulate the expression of MMP-9, and up-regulate the expression of claudin-5 and Z0-1. Conclusion Silencing RGMa shows protective effects on BBB after I/R injury in rats.

RESUMEN

BACKGROUND: Repulsive guidance molecule A (RGMa) is implicated in focal cerebral ischemia-reperfusion (I/R) injury, but its mechanisms are still largely unknown. This work focused on the effects of RGMa on the blood-brain barrier (BBB) after focal cerebral I/R injury. METHODS: Sprague-Dawley (SD) rats were randomly divided into four groups: sham, middle cerebral artery occlusion (MCAO)/reperfusion (I/R), MCAO/reperfusion administered recombinant adenovirus expressing sh-con (I/R + sh-con) and MCAO/reperfusion administered recombinant adenovirus expressing sh-RGMa (I/R + sh-RGMa) groups. Infarct volume, brain edema and neurological scores were evaluated at 3 day after reperfusion. Evens blue leakage and transmission electron microscopy was performed. And the expression level of claudin-5 and ZO-1, CDC-42 and PAK-1, RGMa were detected by western blot. RESULTS: Compared with I/R or I/R + sh-con groups, I/R + sh-RGMa group showed smaller infarction volume, attenuated brain edema, improved neurological scores and better BBB integrity, such as reduced Evans blue leakage and ultra-structural change. We also observed improved BBB function followed by down-regulation of MMP-9 and up-regulation of claudin-5 and ZO-1 in the I/R + sh-RGMa group. In addition, up-regulation of the CDC-42 and PAK-1 in the I/R + sh-RGMa group was obtained. CONCLUSIONS: RGMa may be involved in I/R injury associated with BBB dysfunction via the CDC-42/PAK-1 signal pathway and may be a promising therapeutic target for I/R injury.

Asunto(s)

Barrera Hematoencefálica , Terapia Genética , Infarto de la Arteria Cerebral Media/terapia , Proteínas de la Membrana/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Daño por Reperfusión/terapia , Adenoviridae/genética , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Edema Encefálico/metabolismo , Edema Encefálico/patología , Edema Encefálico/terapia , Permeabilidad Capilar/fisiología , Proteínas Ligadas a GPI , Expresión Génica , Silenciador del Gen , Vectores Genéticos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Masculino , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Distribución Aleatoria , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología

RESUMEN

Repulsive guidance molecule a (RGMa) is a major neuron guidance factor in central nervous systems. We previously found that inhibition of RGMa could greatly enhance neural function rehabilitation in rats after MCAO/reperfusion. Neuron guidance factors are often regulators of angiogenesis. However, the effect of RGMa on angiogenesis and its mechanisms remain to be determined. Here, we investigated the effect of RGMa on endothelial cell (EC) proliferation, migration, tube formation, and cytoskeleton reassembly. The addition of recombinant RGMa significantly decreased the proliferation, migration, and tube formation of ECs. It also decreased the level of phosphorylated focal adhesion kinase (p-FAK Tyr397). Furthermore, the F-actin of the cytoskeleton assembly was obviously suppressed, with decreased filopodia and lamellipodia after the addition of RGMa. Knockout of neogenin or Unc5b significantly diminished RGMa's inhibition of EC migration, tube formation, and cytoskeleton reassembly. RGMa-induced p-FAK (Tyr397) decrease was also abolished by knockout of neogenin or Unc5b. These results indicate that RGMa may be a negative regulator of angiogenesis through downregulating VEGF and p-FAK (Tyr397) via neogenin and Unc5b in vitro.