RESUMO
Endothelial blood-brain barrier (BBB) dysfunction is critical in the pathophysiology of brain injury. Rho-associated protein kinase (ROCK) activation disrupts BBB integrity in the injured brain. We aimed to test the efficacy of a novel ROCK2 inhibitor in preserving the BBB after acute brain injury. We characterized the molecular structure and pharmacodynamic and pharmacokinetic properties of a novel selective ROCK2 inhibitor, NRL-1049, and its first metabolite, 1-hydroxy-NRL-1049 (referred to as NRL-2017 hereon) and tested the efficacy of NRL-1049 on the BBB integrity in rodent models of acute brain injury. Our data show that NRL-1049 and NRL-2017 both inhibit ROCK activity and are 44-fold and 17-fold more selective towards ROCK2 than ROCK1, respectively. When tested in a mouse model of cortical cryoinjury, NRL-1049 significantly attenuated the increase in water content. Interestingly, 60% of the mice in the vehicle arm developed seizures within 2 hours after cryoinjury versus none in the NRL-1049 arm. In spontaneously hypertensive rats, NRL-1049 attenuated the dramatic surge in Evans Blue extravasation compared with the vehicle arm after transient middle cerebral artery occlusion. Hemorrhagic transformation was also reduced. We show that NRL-1049, a selective ROCK2 inhibitor, is a promising drug candidate to preserve the BBB after brain injury.
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The therapeutic use of RNAi has grown but often faces several hurdles related to delivery systems, compound stability, immune activation, and on-target/off-tissue effects. Self-delivering RNAi (sdRNA) molecules do not require delivery agents or excipients. Here we demonstrate the ability of sdRNA to reduce the expression of PTEN (phosphatase and tensin homolog) to stimulate regenerative axon regrowth in the injured adult CNS. PTEN-targeting sdRNA compounds were tested for efficacy in vivo by intravitreal injection after adult rat optic nerve injury. We describe critical steps in lead compound generation through the optimization of nucleotide modifications, enhancements for stability in biological matrices, and screening for off-target immunostimulatory activity. The data show that PTEN expression in vivo can be reduced using sdRNA and this enhances regeneration in adult CNS neurons after injury, raising the possibility that this method could be utilized for other clinically relevant nervous system indications.
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Acute traumatic spinal cord injury (SCI) can result in severe, lifelong neurological deficits. After SCI, Rho activation contributes to collapse of axonal growth cones, failure of axonal regeneration, and neuronal loss. This randomized, double-blind, placebo-controlled phase 2b/3 study evaluated the efficacy and safety of Rho inhibitor VX-210 (9 mg) in patients after acute traumatic cervical SCI. The study enrolled patients 14-75 years of age with acute traumatic cervical SCIs, C4-C7 (motor level) on each side, and American Spinal Injury Association Impairment Scale (AIS) Grade A or B who had spinal decompression/stabilization surgery commencing within 72 h after injury. Patients were randomized 1:1 with stratification by age (<30 vs. ≥30 years) and AIS grade (A vs. B with sacral pinprick preservation vs. B without sacral pinprick preservation). A single dose of VX-210 or placebo in fibrin sealant was administered topically onto the dura over the site of injury during decompression/stabilization surgery. Patients were evaluated for medical, neurological, and functional changes, and serum was collected for pharmacokinetics and immunological analyses. Patients were followed up for up to 12 months after treatment. A planned interim efficacy-based futility analysis was conducted after â¼33% of patients were enrolled. The pre-defined futility stopping rule was met, and the study was therefore ended prematurely. In the final analysis, the primary efficacy end-point was not met, with no statistically significant difference in change from baseline in upper-extremity motor score at 6 months after treatment between the VX-210 (9-mg) and placebo groups. This work opens the door to further improvements in the design and conduct of clinical trials in acute SCI.
Assuntos
Medula Cervical/lesões , Inibidores Enzimáticos/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/uso terapêutico , ADP Ribose Transferases , Adolescente , Adulto , Idoso , Toxinas Botulínicas , Vértebras Cervicais , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recuperação de Função Fisiológica , Resultado do Tratamento , Adulto JovemRESUMO
Cavernous angioma (CA) is a vascular pathology caused by loss of function in one of the 3 CA genes (CCM1, CCM2, and CCM3) that result in rho kinase (ROCK) activation. We investigated a novel ROCK2 selective inhibitor for the ability to reduce brain lesion formation, growth, and maturation. We used genetic methods to explore the use of a ROCK2-selective kinase inhibitor to reduce growth and hemorrhage of CAs. The role of ROCK2 in CA was investigated by crossing Rock1 or Rock2 hemizygous mice with Ccm1 or Ccm3 hemizygous mice, and we found reduced lesions in the Rock2 hemizygous mice. A ROCK2-selective inhibitor, BA-1049 was used to investigate efficacy in reducing CA lesions after oral administration to Ccm1+/- and Ccm3+/- mice that were bred into a mutator background. After assessing the dose range effective to target brain endothelial cells in an ischemic brain model, Ccm1+/- and Ccm3+/- transgenic mice were treated for 3 (Ccm3+/-) or 4 months (Ccm1+/-), concurrently, randomized to receive one of three doses of BA-1049 in drinking water, or placebo. Lesion volumes were assessed by micro-computed tomography. BA-1049 reduced activation of ROCK2 in Ccm3+/-Trp53-/- lesions. Ccm1+/-Msh2-/- (n=68) and Ccm3+/-Trp53-/- (n=71) mice treated with BA-1049 or placebo showed a significant dose-dependent reduction in lesion volume after treatment with BA-1049, and a reduction in hemorrhage (iron deposition) near lesions at all doses. These translational studies show that BA-1049 is a promising therapeutic agent for the treatment of CA, a disease with no current treatment except surgical removal of the brain lesions.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Hemangioma Cavernoso/tratamento farmacológico , Hemangioma Cavernoso/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Quinases Associadas a rho/antagonistas & inibidores , Administração Oral , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína KRIT1/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Supressora de Tumor p53/genética , Quinases Associadas a rho/genéticaRESUMO
Traumatic spinal cord injury (SCI) is associated with a lifetime of disability stemming from loss of motor, sensory, and autonomic functions; these losses, along with increased comorbid sequelae, negatively impact health outcomes and quality of life. Early decompression surgery post-SCI can enhance patient outcomes, but does not directly facilitate neural repair and regeneration. Currently, there are no U.S. Food and Drug Administration-approved pharmacological therapies to augment motor function and functional recovery in individuals with traumatic SCI. After an SCI, the enzyme, Rho, is activated by growth-inhibitory factors and regulates events that culminate in collapse of the neuronal growth cone, failure of axonal regeneration, and, ultimately, failure of motor and functional recovery. Inhibition of Rho activation is a potential treatment for injuries such as traumatic SCI. VX-210, an investigational agent, inhibits Rho. When administered extradurally after decompression (corpectomy or laminectomy) and stabilization surgery in a phase 1/2a study, VX-210 was well tolerated. Here, we describe the design of the SPRING trial, a multicenter, phase 2b/3, randomized, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of VX-210 (NCT02669849). A subset of patients with acute traumatic cervical SCI is currently being enrolled in the United States and Canada. Medical, neurological, and functional changes are evaluated at 6 weeks and at 3, 6, and 12 months after VX-210 administration. Efficacy will be assessed by the primary outcome measure, change in upper extremity motor score at 6 months post-treatment, and by secondary outcomes that include question-based and task-based evaluations of functional recovery.
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ADP Ribose Transferases/uso terapêutico , Toxinas Botulínicas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Projetos de Pesquisa , Traumatismos da Medula Espinal/tratamento farmacológico , Vértebras Cervicais , Método Duplo-Cego , Inibidores Enzimáticos/uso terapêutico , Humanos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
While neurons in the central nervous system (CNS) have the capacity to regenerate their axons after injury, they fail to do so, in part because regeneration is limited by growth inhibitory proteins present in CNS myelin. Myelin-associated glycoprotein (MAG) was the first myelin-derived growth inhibitory protein identified, and its inhibitory activity was initially elucidated in 1994 independently by the Filbin lab and the McKerracher lab using cell-based and biochemical techniques, respectively. Since that time we have gained a wealth of knowledge concerning the numerous growth inhibitory proteins that are present in myelin, and we also have dissected many of the neuronal signaling pathways that act as stop signs for axon regeneration. Here we give an overview of the early research efforts that led to the identification of myelin-derived growth inhibitory proteins, and the importance of this family of proteins for understanding neurotrauma and CNS diseases. We further provide an update on how this knowledge has been translated towards current clinical studies in regenerative medicine.
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The development of novel pharmaceutical treatments for disorders of the cerebral vasculature is a serious unmet medical need. These vascular disorders are typified by a disruption in the delicate Rho signaling equilibrium within the blood vessel wall. In particular, Rho kinase overactivation in the smooth muscle and endothelial layers of the vessel wall results in cytoskeletal modifications that lead to reduced vascular integrity and abnormal vascular growth. Rho kinase is thus a promising target for the treatment of cerebral vascular disorders. Indeed, preclinical studies indicate that Rho kinase inhibition may reduce the formation/growth/rupture of both intracranial aneurysms and cerebral cavernous malformations.
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Transtornos Cerebrovasculares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Transtornos Cerebrovasculares/enzimologia , Transtornos Cerebrovasculares/metabolismo , Humanos , Inibidores de Proteínas Quinases/química , Quinases Associadas a rho/metabolismoRESUMO
Cervical spinal cord injury (SCI) results in partial to full paralysis of the upper and lower extremities. Traditional primary endpoints for acute SCI clinical trials are too broad to assess functional recovery in cervical subjects, raising the possibility of false positive outcomes in trials for cervical SCI. Endpoints focused on the recovery of hand and arm control (e.g., upper extremity motor score, motor level change) show the most potential for use as primary outcomes in upcoming trials of cervical SCI. As the field moves forward, the most reliable way to ensure meaningful clinical testing in cervical subjects may be the development of a composite primary endpoint that measures both neurological recovery and functional improvement.
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The Cethrin™ clinical trial was an open-label Phase I/IIa trial undertaken to assess the safety, tolerability, and neurological status of patients with acute spinal cord injury (SCI) who were treated with escalating doses of Cethrin, a therapeutic protein drug that inactivates Rho. Recruitment, motor scores, and data on adverse events of treated patients have been reported (Fehlings et al.).(24) While determining efficacy in a small open-label study is not possible, it is important to understand if the results warrant further clinical study, because clinical trial failures have wide ranging impacts. We have examined the clinical data to analyze time to recruitment and sensory outcomes, and compared the outcomes with available registry and placebo data. The average time to recruitment was 52 h, and there was a trend to improved sensory scores in treated patients with thoracic injury. An analysis of patients who recovered two motor levels or more suggests that there may be a larger responder population in the Cethrin-treated participants than in historical controls. We examined time to recruitment and found that the extent of motor and sensory recovery could not be explained by early surgery, and the Cethrin-treated patients showed favorable trends compared with the Surgical Timing in Acute SCI data. The trends reported here indicate that further study of Cethrin is warranted and provide useful guidance to establish effective clinical protocols.
Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Botulínicas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Doença Aguda , Relação Dose-Resposta a Droga , Humanos , Resultado do TratamentoRESUMO
Spinal cord injury (SCI) often results in permanent paralysis because there is little spontaneous repair. Neuronal injury in the central nervous system (CNS) causes breakage of axonal connections, release of myelin, inflammation and cell death at the lesion site. Many factors contribute to the failure of spontaneous repair after SCI, including the presence of growth inhibitory proteins in myelin, the inflammatory environment of the injured CNS, and the resulting signaling cascades that result in over-activation of Rho, a signaling switch in neurons and axons. In this review, we provide a general overview of growth inhibition in the CNS, and show evidence that most growth inhibitory proteins signal through a common intracellular pathway. Rho is a convergent signal for growth inhibition, and also for signaling some of the secondary consequences of inflammation after SCI. We review the preclinical evidence that targeting Rho is an effective way to stimulate axon regeneration and functional recovery in preclinical animal models. In the last part of the review, we describe the creation of Cethrin, a new investigational drug, and summarize the results of the Phase I/IIa clinical study to examine the safety, tolerability and efficacy of Cethrin in patients with acute SCI. We conclude with some insight for future clinical studies.
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ADP Ribose Transferases/uso terapêutico , Toxinas Botulínicas/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , ADP Ribose Transferases/administração & dosagem , ADP Ribose Transferases/efeitos adversos , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Toxinas Botulínicas/administração & dosagem , Toxinas Botulínicas/efeitos adversos , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/efeitos adversos , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Multiple lines of evidence have validated the Rho pathway as important in controlling the neuronal response to growth inhibitory proteins after central nervous system (CNS) injury. A drug called BA-210 (trademarked as Cethrin(®)) blocks activation of Rho and has shown promise in pre-clinical animal studies in being used to treat spinal cord injury (SCI). This is a report of a Phase I/IIa clinical study designed to test the safety and tolerability of the drug, and the neurological status of patients following the administration of a single dose of BA-210 applied during surgery following acute SCI. Patients with thoracic (T2-T12) or cervical (C4-T1) SCI were sequentially recruited for this dose-ranging (0.3 mg to 9 mg Cethrin), multi-center study of 48 patients with complete American Spinal Injury Association assessment (ASIA) A. Vital signs; clinical laboratory tests; computed tomography (CT) scans of the spine, head, and abdomen; magnetic resonance imaging (MRI) of the spine, and ASIA assessment were performed in the pre-study period and in follow-up periods out to 1 year after treatment. The treatment-emergent adverse events that were reported were typical for a population of acute SCI patients, and no serious adverse events were attributed to the drug. The pharmacokinetic analysis showed low levels of systemic exposure to the drug, and there was high inter-patient variability. Changes in ASIA motor scores from baseline were low across all dose groups in thoracic patients (1.8±5.1) and larger in cervical patients (18.6±19.3). The largest change in motor score was observed in the cervical patients treated with 3 mg of Cethrin in whom a 27.3±13.3 point improvement in ASIA motor score at 12 months was observed. Approximately 6% of thoracic patients converted from ASIA A to ASIA C or D compared to 31% of cervical patients and 66% for the 3-mg cervical cohort. Although the patient numbers are small, the observed motor recovery in this open-label trial suggests that BA-210 may increase neurological recovery after complete SCI. Further clinical trials with Cethrin in SCI patients are planned, to establish evidence of efficacy.
Assuntos
ADP Ribose Transferases/uso terapêutico , Toxinas Botulínicas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , ADP Ribose Transferases/farmacocinética , Adulto , Toxinas Botulínicas/farmacocinética , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Masculino , Fármacos Neuroprotetores/farmacocinética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Recuperação de Função FisiológicaRESUMO
Spinal cord injury (SCI) leads to robust Rho activation at the lesion site. Here, we demonstrate that BA-210, a cell-permeable fusion protein derived from C3 transferase, formulated in fibrin sealant and delivered topically onto the dura matter, diffuses into the spinal cord and inactivates Rho in a dose-dependent manner. Treatment with BA-210 in rats with thoracic spinal cord contusion increased tissue sparing around the lesion area and led to significant improvement of locomotor function. In mice, BA-210 improved functional outcome when treatment was either applied at the time of injury or delayed by 24 h. In both rats and mice, treatment with BA-210 was well tolerated. Rats gained body weight normally, and BA-210 treatment had no impact on the development of allodynia. Inactivating Rho with BA-210 holds promise for treating patients with SCI.
Assuntos
Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Difusão , Relação Dose-Resposta a Droga , Dura-Máter , Escherichia coli/metabolismo , Feminino , Imuno-Histoquímica , Injeções , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
The failure of axons in the central nervous system (CNS) to regenerate has been considered the main factor limiting recovery from spinal cord injury (SCI). Impressive gains in identification of growth-inhibitory molecules in the CNS led to expectations that their neutralization would lead to functional regeneration. However, results of therapeutic approaches based on this assumption have been mixed. Recent data suggest that neurons differ in their ability to regenerate through similar extracellular environments, and moreover, they undergo a developmental loss of intrinsic regenerative ability. Factors mediating these intrinsic regenerative abilities include expression of (1) receptors for inhibitory molecules such as the myelin-associated growth inhibitors and developmental guidance molecules, (2) surface molecules that permit axon adhesion to cells in the path of growth, (3) cytoskeletal proteins that mediate the mechanics of axon growth, and (4) molecules in the intracellular signaling cascades that mediate responses to chemoattractive and chemorepulsive cues. In contrast to axon development, regeneration might involve internal protrusive forces generated by microtubules, either through their own elongation or by transporting other cytoskeletal elements such as neurofilaments into the axon tip. Because of the complexity of the regenerative program, one approach will probably be insufficient to achieve functional restoration of neuronal circuits. Combination treatments will be increasingly prominent. SCI is a debilitating and costly condition that compromises pursuit of activities usually associated with an independent and productive lifestyle. This article discusses recent advances in neurorehabilitation that can improve the life quality of individuals with SCI.
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Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/reabilitação , Cicatrização/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Humanos , Modelos Biológicos , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/metabolismoRESUMO
Rho is an important signaling target to promote repair following spinal cord injury (SCI). Myelin-derived inhibitory proteins, as well as other classes of known growth inhibitory proteins, block regeneration by signaling activation of Rho. Here, we review the molecular mechanisms of Rho activation after SCI and explain known Rho signaling antagonists. We review the data on use of Rho antagonists to promote axon regeneration, neuroprotection, and functional recovery after SCI. We report on efforts to translate the nonclincal studies on rodents to clinical trials in patients with acute SCI.
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Traumatismos da Medula Espinal/terapia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Sistemas de Liberação de Medicamentos , Ativação Enzimática , Inibidores Enzimáticos/uso terapêutico , Humanos , Regeneração Nervosa/fisiologia , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/fisiologia , Traumatismos da Medula Espinal/enzimologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidoresRESUMO
C3 transferase has been extensively used in the investigation of Rho signaling. It acts as a Rho antagonist by covalently attaching an ADP-ribose group to RhoA, B, and C proteins. Cell-permeable C3 fusion proteins have been developed so that lower doses of C3 can be used. We describe a simple and fast fluorescence-based assay to evaluate the enzymatic activity of cell-permeable C3 proteins purified from Escherichia coli. The assay measures glycohydrolase (GH) activity of C3 that cleaves NAD+ into ADP-ribose and nicotinamide. Results from the GH activity correlate with other tests carried out in tissue culture cells such as neurite outgrowth or ADP ribosylation of RhoA. This method provides reliable measurements of the activity of permeable C3 proteins or other C3-related proteins.
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ADP Ribose Transferases/metabolismo , Toxinas Botulínicas/metabolismo , ADP Ribose Transferases/genética , Animais , Toxinas Botulínicas/genética , NAD+ Nucleosidase/análise , Células PC12 , Permeabilidade , Mutação Puntual , Ratos , Espectrometria de Fluorescência , Células Tumorais CultivadasRESUMO
Traumatic brain injury (TBI) is characterized by a progressive cell loss and a lack of axonal regeneration. In the central nervous system (CNS), the Rho signaling pathway regulates the neuronal response to growth inhibitory proteins and regeneration of damaged axons, and Rho activation is also correlated with an increased susceptibility to apoptosis. To evaluate whether traumatic brain injury (TBI) results in changes in Rho activation in vulnerable regions of the brain, GTP-RhoA pull down assays were performed on rat cortical and hippocampal tissue homogenates obtained from 24 h to 3 days following lateral fluid percussion brain injury (FPI). Following FPI, a significantly increased RhoA activation was observed from 24 h to 3 days post-injury in the cortex and by 3 days in the hippocampus ipsilateral to the injury. We also detected activated RhoA in the cortex and hippocampus contralateral to the injury, without concomitant changes in total RhoA levels. To determine if immediate post-traumatic events such as seizures may activate Rho, we examined RhoA activation in the brains of rats with kainic acid-induced seizures. Severe seizures resulted in bilateral RhoA activation in the cortex and hippocampus. Together, these results indicate that RhoA is activated in vulnerable brain regions following traumatic and epileptic insults to the CNS.
Assuntos
Lesões Encefálicas/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Convulsões/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Anticonvulsivantes/farmacologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Diazepam/farmacologia , Modelos Animais de Doenças , Interações Medicamentosas , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Ácido Caínico , Masculino , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamenteRESUMO
Inactivation of Rho promotes neurite growth on inhibitory substrates and axon regeneration in vivo. Here, we compared axon growth when neuronal cell bodies or injured axons were treated with a cell-permeable Rho antagonist (C3-07) in vitro and in vivo. In neurons plated in compartmented cultures, application of C3-07 to either cell bodies or distal axons promoted axonal growth on myelin-associated glycoprotein substrates. In vivo, an injection of C3-07 into the eye promoted regeneration of retinal ganglion cell (RGC) axons in the optic nerve after microcrush lesion. Delayed application of C3-07 promoted RGC growth across the lesion scar. Application of C3-07 completely prevented RGC cell death for 1 week after axotomy. To investigate the mechanism by which Rho inactivation promotes RGC growth, we studied slow axonal transport. Reduction in slow transport of cytoskeletal proteins was observed after axotomy, but inactivation of Rho did not increase slow axonal transport rates. Together, our results indicate that application of a Rho antagonist at the cell body is neuroprotective and overcomes growth inhibition but does not fully prime RGCs for active growth.
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Regeneração Nervosa/efeitos dos fármacos , Neuritos/fisiologia , Fármacos Neuroprotetores/uso terapêutico , Traumatismos do Nervo Óptico/tratamento farmacológico , Nervo Óptico/fisiologia , Células Ganglionares da Retina/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , ADP Ribose Transferases/administração & dosagem , ADP Ribose Transferases/uso terapêutico , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Sobrevivência Celular , Células Cultivadas/efeitos dos fármacos , Cicatriz/patologia , Meios de Cultura , Proteínas do Citoesqueleto/metabolismo , Feminino , Injeções , Microcirurgia , Glicoproteína Associada a Mielina , Compressão Nervosa , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Nervo Óptico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/patologia , Gânglio Cervical Superior/citologia , Corpo Vítreo , Proteínas rho de Ligação ao GTP/fisiologiaAssuntos
Axônios/fisiologia , Proteínas da Mielina/metabolismo , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/história , Traumatismos da Medula Espinal/terapia , Anticorpos/metabolismo , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , História do Século XX , História do Século XXI , Humanos , Proteínas de Membrana , Proteínas da Mielina/genética , Glicoproteína Associada a Mielina/metabolismo , Proteínas do Tecido Nervoso , Proteínas Nogo , Receptores de Superfície Celular/metabolismoRESUMO
The influence of stereochemistry and alkyl side chain length on the bioactivity of the Rho kinase inhibitor Y-27632 [(+)-1, R=Me] was examined by the synthesis of (+)- and (-)-1, and two alkyl chain analogs (+)- and (-)-2 (R=n-propyl) and (-)-3 (R=n-octyl) as well as their evaluation in enzymatic and neurite outgrowth assays.
Assuntos
Amidas/síntese química , Inibidores Enzimáticos/síntese química , Neuritos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/síntese química , Amidas/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neuritos/fisiologia , Piridinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Quinases Associadas a rhoRESUMO
Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.