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1.
Nat Commun ; 15(1): 1037, 2024 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-38310100

RESUMO

Liver failure causes breakdown of the Blood CNS Barrier (BCB) leading to damages of the Central-Nervous-System (CNS), however the mechanisms whereby the liver influences BCB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BCB-integrity. To study BCB-integrity, we developed light-sheet imaging for three-dimensional analysis. We show that liver- or muscle-specific knockout of Hfe2/Rgmc induces BCB-breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits in mice. Soluble HFE2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells, triggering BCB-disruption. HFE2 administration in female mice with experimental autoimmune encephalomyelitis, a model for multiple sclerosis, prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BCB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BCB-dysfunction.


Assuntos
Barreira Hematoencefálica , Encefalomielite Autoimune Experimental , Animais , Feminino , Camundongos , Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central/metabolismo , Células Endoteliais/metabolismo , Fígado/metabolismo , Músculos/metabolismo
2.
Neurobiol Dis ; 159: 105492, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34478849

RESUMO

Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.


Assuntos
Encefalomielite Autoimune Experimental , Proteínas Ligadas por GPI , Regeneração Nervosa , Proteínas do Tecido Nervoso , Neuroproteção , Traumatismos do Nervo Óptico , Nervo Óptico , Neurite Óptica , Recuperação de Função Fisiológica , Retina , Animais , Camundongos , Cuprizona/toxicidade , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/fisiopatologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Inibidores da Monoaminoxidase/toxicidade , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neuroproteção/efeitos dos fármacos , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/fisiologia , Traumatismos do Nervo Óptico/fisiopatologia , Neurite Óptica/fisiopatologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Retina/efeitos dos fármacos , Ressonância de Plasmônio de Superfície
3.
Neurobiol Dis ; 155: 105385, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33991647

RESUMO

Spinal cord injury (SCI) is a devastating condition characterized by loss of function, secondary to damaged spinal neurons, disrupted axonal connections, and myelin loss. Spontaneous recovery is limited, and there are no approved pharmaceutical treatments to reduce ongoing damage or promote repair. Repulsive guidance molecule A (RGMa) is upregulated following injury to the central nervous system (CNS), where it is believed to induce neuronal apoptosis and inhibit axonal growth and remyelination. We evaluated elezanumab, a human anti-RGMa monoclonal antibody, in a novel, newly characterized non-human primate (NHP) hemicompression model of thoracic SCI. Systemic intravenous (IV) administration of elezanumab over 6 months was well tolerated and associated with significant improvements in locomotor function. Treatment of animals for 16 weeks with a continuous intrathecal infusion of elezanumab below the lesion was not efficacious. IV elezanumab improved microstructural integrity of extralesional tissue as reflected by higher fractional anisotropy and magnetization transfer ratios in treated vs. untreated animals. IV elezanumab also reduced SCI-induced increases in soluble RGMa in cerebrospinal fluid, and membrane bound RGMa rostral and caudal to the lesion. Anterograde tracing of the corticospinal tract (CST) from the contralesional motor cortex following 20 weeks of IV elezanumab revealed a significant increase in the density of CST fibers emerging from the ipsilesional CST into the medial/ventral gray matter. There was a significant sprouting of serotonergic (5-HT) fibers rostral to the injury and in the ventral horn of lower thoracic regions. These data demonstrate that 6 months of intermittent IV administration of elezanumab, beginning within 24 h after a thoracic SCI, promotes neuroprotection and neuroplasticity of key descending pathways involved in locomotion. These findings emphasize the mechanisms leading to improved recovery of neuromotor functions with elezanumab in acute SCI in NHPs.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Plasticidade Neuronal/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Chlorocebus aethiops , Teste de Esforço/métodos , Humanos , Injeções Espinhais , Masculino , Plasticidade Neuronal/fisiologia , Neuroproteção/fisiologia , Primatas , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Vértebras Torácicas/lesões
4.
Neurobiol Dis ; 143: 104995, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32590037

RESUMO

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.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/fisiopatologia , Animais , Feminino , Humanos , Ratos , Ratos Wistar , Micção/efeitos dos fármacos
5.
J Med Chem ; 61(24): 11074-11100, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30384606

RESUMO

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Inibidores do Citocromo P-450 CYP2C9/química , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Meia-Vida , Humanos , Camundongos Endogâmicos C57BL , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Quinases Associadas a rho/química
6.
Sci Rep ; 7(1): 10529, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874746

RESUMO

Traumatic spinal cord injury (SCI) causes a cascade of degenerative events including cell death, axonal damage, and the upregulation of inhibitory molecules which prevent regeneration and limit recovery. Repulsive guidance molecule A (RGMa) is a potent neurite growth inhibitor in the central nervous system, exerting its repulsive activity by binding the Neogenin receptor. Here, we show for the first time that inhibitory RGMa is markedly upregulated in multiple cell types after clinically relevant impact-compression SCI in rats, and importantly, also in the injured human spinal cord. To neutralize inhibitory RGMa, clinically relevant human monoclonal antibodies were systemically administered after acute SCI, and were detected in serum, cerebrospinal fluid, and in the injured tissue. Rats treated with RGMa blocking antibodies showed significantly improved recovery of motor function and gait. Furthermore, RGMa blocking antibodies promoted neuronal survival, and enhanced the plasticity of descending serotonergic pathways and corticospinal tract axonal regeneration. RGMa antibody also attenuated neuropathic pain responses, which was associated with fewer activated microglia and reduced CGRP expression in the dorsal horn caudal to the lesion. These results show the therapeutic potential of the first human RGMa antibody for SCI and uncovers a new role for the RGMa/Neogenin pathway on neuropathic pain.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Neuralgia/terapia , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Células Cultivadas , Feminino , Proteínas Ligadas por GPI , Humanos , Camundongos , Plasticidade Neuronal , Ratos , Ratos Wistar
7.
Trends Cell Biol ; 27(5): 365-378, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28007423

RESUMO

Although originally discovered as neuronal growth cone-collapsing factors, repulsive guidance molecules (RGMs) are now known as key players in many fundamental processes, such as cell migration, differentiation, iron homeostasis, and apoptosis, during the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. Furthermore, three RGMs (RGMa, RGMb/DRAGON, and RGMc/hemojuvelin) have been linked to the pathogenesis of various disorders ranging from multiple sclerosis (MS) to cancer and juvenile hemochromatosis (JHH). While the molecular details of these (patho)biological effects and signaling modes have long remained unknown, recent studies unveil several exciting and novel aspects of RGM processing, ligand-receptor interactions, and downstream signaling. In this review, we highlight recent advances in the mechanisms-of-action and function of RGM proteins.


Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Humanos , Ligantes , Modelos Biológicos , Família Multigênica , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/metabolismo
9.
Toxicol Pathol ; 44(2): 259-66, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26839325

RESUMO

Hepcidin was originally detected as a liver peptide with antimicrobial activity and it functions as a central regulator in the systemic iron metabolism. Consequently suppression of hepcidin leads to iron accumulation in the liver. AbbVie developed a monoclonal antibody ([mAb]; repulsive guidance molecule [RGMa/c] mAb) that downregulates hepcidin expression by influencing the RGMc/bone morphogenetic protein (BMP)/neogenin receptor complex and causes iron deposition in the liver. In a dose range finding study with RGMa/c mAb, rats were treated with different dose levels for a total of 4 weekly doses. The results of this morphometric analysis in the liver showed that iron accumulation is not homogenous between liver lobes and the left lateral lobe was the most responsive lobe in the rat. Quantitative hepcidin messenger RNA analysis showed that the left lateral lobe was the most responsive lobe showing hepcidin downregulation with increasing antibody dose. In addition, the morphometric analysis had higher sensitivity than the chemical iron extraction and quantification using a colorimetric assay. In conclusion, the Prussian blue stain in combination with semi-quantitative and quantitative morphometric analysis is the most reliable method to demonstrate iron accumulation in the liver compared to direct measurement of iron in unfixed tissue using a colorimetric assay.


Assuntos
Hepcidinas/metabolismo , Ferro/análise , Ferro/metabolismo , Fígado/química , Fígado/metabolismo , Animais , Anticorpos Monoclonais , Relação Dose-Resposta a Droga , Feminino , Proteínas Ligadas por GPI , Hepcidinas/análise , Hepcidinas/genética , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Ratos , Ratos Sprague-Dawley
10.
J Neural Transm (Vienna) ; 123(4): 401-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26880022

RESUMO

Exposure to free radicals influences synthesis, degradation and function of proteins, such as repulsive guidance molecule A. Decay of this protein is essential for neuronal maintenance and recovery. Levodopa elevates oxidative stress. Therefore levodopa may impact repulsive guidance molecule A metabolism. Objectives were to investigate plasma concentrations of repulsive guidance molecule A, levodopa, cysteine and cysteinyl-glycine before and 1 h after levodopa application in patients with Parkinson's disease. Cysteine and cysteinyl-glycine as biomarkers for oxidative stress exposure decreased, repulsive guidance molecule A and levodopa rose. Repulsive guidance molecule A remained unchanged in levodopa naïve patients, but particularly went up in patients on a prior chronic levodopa regimen. Decay of cysteine specifically cysteinyl-glycine results from an elevated glutathione generation with rising cysteine consumption respectively from the alternative glutathione transformation to its oxidized form glutathione disulfide after free radical scavenging. Repulsive guidance molecule A rise may inhibit physiologic mechanisms for neuronal survival.


Assuntos
Carbidopa/efeitos adversos , Levodopa/sangue , Proteínas do Tecido Nervoso/sangue , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Idoso , Antiparkinsonianos/sangue , Antiparkinsonianos/uso terapêutico , Cromatografia Líquida de Alta Pressão , Cisteína/sangue , Dipeptídeos/sangue , Combinação de Medicamentos , Feminino , Proteínas Ligadas por GPI/sangue , Humanos , Levodopa/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Projetos Piloto
11.
AAPS J ; 17(4): 930-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25896304

RESUMO

High levels of hepcidin, the main regulator of systemic iron metabolism, lead to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies ABT-207 and h5F9-AM8 which inhibit hemojuvelin/repulsive guidance molecule C (RGMc) and downregulate hepcidin. We conducted single-application and dose response studies to understand the antibodies' mechanism and subchronic toxicology studies to exclude safety-related concerns. Investigation was carried out at different biological levels through qPCR, Affymetrix, liquid chromatography coupled with mass spectrometry (LC-MS/MS), histopathology, serum iron, unsaturated iron binding capacity (UIBC), and drug concentration measurements. After a single application of these antibodies, hepcidin expression in liver and its serum protein levels were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of approximately 80-fold higher than ABT-207. When hepcidin levels were downregulated, iron deposition in the liver was visible histologically 1 week post application. These antibody-mediated iron depositions were not associated with any adverse toxicologically relevant effect at the doses and time points evaluated. Iron depositions seen after 14 weekly treatments with ABT-207 were reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Hepcidinas/genética , Ferro/sangue , Proteínas de Membrana/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Cromatografia Líquida , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Feminino , Hepcidinas/sangue , Concentração Inibidora 50 , Macaca fascicularis , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Fatores de Tempo
12.
Cell Rep ; 10(11): 1887-98, 2015 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-25801027

RESUMO

Repulsive guidance molecule A (RGMa) is a potent inhibitor of neuronal regeneration and a regulator of cell death, and it plays a role in multiple sclerosis (MS). In autopsy material from progressive MS patients, RGMa was found in active and chronic lesions, as well as in normal-appearing gray and white matter, and was expressed by cellular meningeal infiltrates. Levels of soluble RGMa in the cerebrospinal fluid were decreased in progressive MS patients successfully treated with intrathecal corticosteroid triamcinolone acetonide (TCA), showing functional improvements. In vitro, RGMa monoclonal antibodies (mAbs) reversed RGMa-mediated neurite outgrowth inhibition and chemorepulsion. In animal models of CNS damage and MS, RGMa antibody stimulated regeneration and remyelination of damaged nerve fibers, accelerated functional recovery, and protected the retinal nerve fiber layer as measured by clinically relevant optic coherence tomography. These data suggest that targeting RGMa is a promising strategy to improve functional recovery in MS patients.


Assuntos
Glicoproteínas de Membrana/metabolismo , Esclerose Múltipla/tratamento farmacológico , Regeneração Nervosa , Proteínas do Tecido Nervoso/metabolismo , Adulto , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Feminino , Proteínas Ligadas por GPI , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/metabolismo , Nervo Óptico/fisiologia , Ratos , Ratos Wistar
13.
J Neural Transm (Vienna) ; 122(6): 841-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25209051

RESUMO

Repeated intrathecal application of the sustained release steroid triamcinolone acetonide is beneficial in progressive multiple sclerosis patients. Its putative regenerative effect may involve regulation of the repulsive guidance molecule A synthesis. This protein inhibits axonal regeneration and functional recovery. Objectives were to demonstrate the efficacy of four triamcinolone applications every other day in association with repulsive guidance molecule A levels in cerebrospinal fluid. Clinical evaluation was performed at baseline and on each day after a triamcinolone administration in 25 progressive multiple sclerosis patients. Repulsive guidance molecule A concentrations were determined before each triamcinolone application by western blot analysis with quantification. Clinical scores for multiple sclerosis improved, and the maximum walking distance and speed ameliorated in 17 patients. Repulsive guidance molecule A levels declined in these responders. The remaining patients showed no prompt clinical benefit and no decrease of repulsive guidance molecule A concentrations. Decline of repulsive guidance molecule A may reflect regeneration and functional recovery by triamcinolone in progressive multiple sclerosis patients.


Assuntos
Imunossupressores/uso terapêutico , Esclerose Múltipla Crônica Progressiva/líquido cefalorraquidiano , Esclerose Múltipla Crônica Progressiva/tratamento farmacológico , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Triancinolona Acetonida/uso terapêutico , Western Blotting , Teste de Esforço , Feminino , Proteínas Ligadas por GPI/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Resultado do Tratamento , Caminhada
14.
Glia ; 62(2): 217-32, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24311453

RESUMO

Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons (MN) and their axons, but is also influenced by neighboring cells such as astrocytes and microglial cells. The role of microglia in ALS is complex as it switches from an anti-inflammatory and neuroprotective phenotype in early disease to a proinflammatory and neurotoxic phenotype in later stages. Our previous studies in models of neurodegeneration identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression. Here, we examined the neuroprotective potential of the ROCK inhibitor Fasudil to target the central pathogenic features of ALS. Application of Fasudil to kainic acid-lesioned primary MN in vitro resulted in a strong prosurvival effect. In vivo, SOD1(G93A) mice benefited from oral treatment with Fasudil showing prolonged survival and improved motor function. These findings were correlated to an improved survival of motor neurons and a pronounced alteration of astroglial and microglial cell infiltration of the spinal cord under Fasudil treatment. Modeling a proinflammatory microglial phenotype by stimulation with LPS in vitro, Fasudil decreased the release of proinflammatory cytokines and chemokines TNFα, Il6, CCL2, CCL3, and CCL5 while CXCL1 release was only transiently suppressed. In sciatic nerve motor axons, neuromuscular junction remodeling processes were increased. In conclusion, we provide preclinical and neurobiological evidence that inhibition of ROCK by the clinically approved small molecule inhibitor Fasudil may be a novel therapeutic approach in ALS combining both neuroprotection and immunomodulation for the cure of this devastating disease.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Esclerose Lateral Amiotrófica/enzimologia , Microglia/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Axônios/efeitos dos fármacos , Axônios/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Medula Espinal/efeitos dos fármacos
15.
Neurosci Lett ; 510(1): 58-61, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22260793

RESUMO

The Nogo-66 receptor (NgR1) is part of a co-receptor complex on neurons that transmits a signal for inhibition of neurite outgrowth. In addition, NgR1 function has also been related to other disorders such as schizophrenia and Alzheimer's disease. Here, we studied the effect of life-long deletion of NgR1 (ngr(-/-)) in tests for cognition and positive symptoms of schizophrenia. In the water maze, ngr(-/-) mice learned to locate the hidden platform as well as wild type mice, although with slower acquisition. Deletion of NgR1 did not affect amphetamine- or phencyclidine (PCP)-induced hyperactivity, two models of positive symptoms of schizophrenia. Taken together, ngr(-/-) animals show slower acquisition of a spatial learning and memory task.


Assuntos
Aprendizagem em Labirinto , Memória , Proteínas da Mielina/deficiência , Doença de Alzheimer/tratamento farmacológico , Anfetamina/farmacologia , Animais , Ingestão de Alimentos , Feminino , Febre/etiologia , Hipercinese/induzido quimicamente , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina/genética , Plasticidade Neuronal , Proteínas Nogo , Fenciclidina/farmacologia , Restrição Física , Esquizofrenia/induzido quimicamente , Esquizofrenia/fisiopatologia , Fatores de Tempo
16.
Br J Pharmacol ; 157(5): 675-85, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19422372

RESUMO

Traumatic brain injury, a silent epidemic of modern societies, is a largely neglected area in drug development and no drug is currently available for the treatment of patients suffering from brain trauma. Despite this grim situation, much progress has been made over the last two decades in closely related medical indications, such as spinal cord injury, giving rise to a more optimistic approach to drug development in brain trauma. Fundamental insights have been gained with animal models of central nervous system (CNS) trauma and spinal cord injury. Neuroregenerative drug candidates have been identified and two of these have progressed to clinical development for spinal cord injury patients. If successful, these drug candidates may be used to treat brain trauma patients. Significant progress has also been made in understanding the fundamental molecular mechanism underlying irreversible axonal growth arrest in the injured CNS of higher mammals. From these studies, we have learned that the axonal retraction bulb, previously regarded as a marker for failure of regenerative growth, is not static but dynamic and, therefore, amenable to pharmacotherapeutic approaches. With the development of modified magnetic resonance imaging methods, fibre tracts can be visualised in the living human brain and such imaging methods will soon be used to evaluate the neuroregenerative potential of drug candidates. These significant advances are expected to fundamentally change the often hopeless situation of brain trauma patients and will be the first step towards overcoming the silent epidemic of brain injury.


Assuntos
Lesões Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Adulto , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Modelos Animais de Doenças , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/patologia , Inibidores do Crescimento/metabolismo , Humanos , Neurônios/metabolismo , Neurônios/patologia , Recuperação de Função Fisiológica , Transdução de Sinais/efeitos dos fármacos
17.
J Neurochem ; 107(2): 418-31, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18702663

RESUMO

Repulsive guidance molecule A (RGM A) was recently described as a potent inhibitor of neuroregeneration in a rat spinal cord injury model. The receptor mediating RGM A's repulsive activity was shown to be Neogenin, a member of the Deleted in Colorectal Cancer (DCC) family of netrin receptors. Binding of RGM A to Neogenin induces activation of the small GTPase RhoA and of its effector Rho-kinase by an unknown mechanism. Here we show, that the cytoplasmic tail of Neogenin interacts directly with the transcriptional coactivator LIM domain only 4 (LMO4) in human SH-SY5Y cells, human Ntera neurons, and in embryonic rat cortical neurons. RGM A binding to Neogenin but not binding of Netrin-1, induces release of LMO4 from Neogenin. Down-regulation of LMO4 neutralizes the repulsive activity of RGM A in neuronal cell lines and embryonic rat cortical neurons and prevents RhoA activation. These results show for the first time that an interaction of Neogenin with LMO4 is involved in the RGM A - Neogenin signal transduction pathway for RhoA activation.


Assuntos
Proteínas de Homeodomínio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Amidas/farmacologia , Análise de Variância , Animais , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Proteínas Ligadas por GPI , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Homeodomínio/biossíntese , Humanos , Proteínas com Domínio LIM , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Estrutura Terciária de Proteína , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Fatores de Transcrição/biossíntese , Transfecção/métodos , Tubulina (Proteína)/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
18.
J Neurochem ; 105(1): 113-26, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18005226

RESUMO

Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin - a specific myosin II inhibitor - and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.


Assuntos
Miosina Tipo II/fisiologia , Inibição Neural/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Cerebelo/citologia , Feminino , Proteínas Ligadas por GPI , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Imunoprecipitação/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Laminectomia/métodos , Glicoproteínas de Membrana/farmacologia , Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Inibição Neural/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Transfecção/métodos , Quinases Associadas a rho/metabolismo
19.
J Neurosci Res ; 86(3): 531-6, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17893923

RESUMO

Organotypic cultures of postnatal day 1 (P1) to P7 mouse cerebella are well-established models for studying cell survival. In the present work, we investigate the involvement of the Rho/ROCK intracellular pathway in Purkinje cell survival by using organotypic cultures of P3 Swiss mice. Specific inhibitors of Rho or ROCK were applied at different concentrations to the slice cultures, which were maintained for 5 days in vitro. We show that the bacterial exoenzyme C3 transferase, a specific inhibitor of the small GTPase Rho, increases Purkinje cell survival. There is a 4.5- and 2.5-fold increase in Purkinje cell survival when C3 intracellular uptake is promoted either by the PEP-1 peptide or by the C2IN carrier protein, respectively, and not with the commonly used TAT peptide. Moreover, treatment with Y27632 and H-1152, two specific inhibitors of the Rho kinase ROCK, also strongly reduces apoptotic cell death and results in 6.5- and 8.5-fold increases in cell survival, respectively. In immunohistochemical analysis, we also show that H-1152 did not change either glial fibrillary acidic protein or isolectin-B4 staining, indicating that this compound did not alter the cellular composition in our cultures. Thus, our data demonstrate that inhibition of Rho and its downstream effector ROCK may be used to enhance cell survival in neurodegenerative diseases.


Assuntos
Células de Purkinje/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , ADP Ribose Transferases/farmacologia , Amidas/farmacologia , Animais , Animais Recém-Nascidos , Toxinas Botulínicas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Lectinas/metabolismo , Camundongos , Fármacos Neuroprotetores/farmacologia , Técnicas de Cultura de Órgãos , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Piridinas/farmacologia , Coloração e Rotulagem
20.
J Neurochem ; 103(1): 181-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17608642

RESUMO

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/farmacologia , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Regeneração Nervosa/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Traumatismos do Nervo Óptico/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Humanos , Compressão Nervosa , Neuritos/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/enzimologia , Traumatismos do Nervo Óptico/patologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Quinases Associadas a rho
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