RESUMO
There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD) patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC). High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST), and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.
Assuntos
Encéfalo/citologia , Doença Granulomatosa Crônica/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , NADPH Oxidase 2/genética , Células-Tronco Neurais/citologia , Neurogênese , Animais , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Doença Granulomatosa Crônica/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , NADPH Oxidase 2/metabolismo , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive loss of motor neurons, gliosis, neuroinflammation and oxidative stress. The aim of this study was to evaluate the involvement of NADPH oxidases (NOX) in the oxidative damage and progression of ALS neuropathology. We examined the pattern of NOX expression in spinal cords of patients and mouse models of ALS and analyzed the impact of genetic deletion of the NOX1 and 2 isoforms as well as pharmacological NOX inhibition in the SOD1(G93A) ALS mouse model. A substantial (10-60 times) increase of NOX2 expression was detected in three etiologically different ALS mouse models while up-regulation of some other NOX isoforms was model-specific. In human spinal cord samples, high NOX2 expression was detected in microglia. In contrast to previous publications, survival of SOD1(G93A) mice was not modified upon breeding with constitutive NOX1 and NOX2 deficient mice. As genetic deficiency of a single NOX isoform is not necessarily predictive of a pharmacological intervention, we treated SOD1(G93A) mice with broad-spectrum NOX inhibitors perphenazine and thioridazine. Both compounds reached in vivo CNS concentrations compatible with NOX inhibition and thioridazine significantly decreased superoxide levels in the spinal cord of SOD1(G93A) mice in vivo. Yet, neither perphenazine nor thioridazine prolonged survival. Thioridazine, but not perphenazine, dampened the increase of microglia markers in SOD1(G93A) mice. Thioridazine induced an immediate and temporary enhancement of motor performance (rotarod) but its precise mode of action needs further investigation. Additional studies using specific NOX inhibitors will provide further evidence on the relevance of NOX as drug targets for ALS and other neurodegenerative disorders.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , NADPH Oxidase 1/genética , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Perfenazina/administração & dosagem , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Superóxido Dismutase-1/antagonistas & inibidores , Superóxido Dismutase-1/genética , Tioridazina/administração & dosagemRESUMO
Glioblastoma is a deadly malignant brain tumor and one of the most incurable forms of cancer in need of new therapeutic targets. As some cancers are known to be caused by a virus, the discovery of viruses could open the possibility to treat, and perhaps prevent, such a disease. Although an association with viruses such as cytomegalovirus or Simian virus 40 has been strongly suggested, involvement of these and other viruses in the initiation and/or propagation of glioblastoma remains vague, controversial and warrants elucidation. To exhaustively address the association of virus and glioblastoma, we developed and validated a robust metagenomic approach to analyze patient biopsies via high-throughput sequencing, a sensitive tool for virus screening. In addition to traditional clinical diagnostics, glioblastoma biopsies were deep-sequenced and analyzed with a multistage computational pipeline to identify known or potentially discover unknown viruses. In contrast to the studies reporting the presence of viral signatures in glioblastoma, no common or recurring active viruses were detected, despite finding an antiviral-like type I interferon response in some specimens. Our findings highlight a discrete and non-specific viral signature and uncharacterized short RNA sequences in glioblastoma. This study provides new insights into glioblastoma pathogenesis and defines a general methodology that can be used for high-resolution virus screening and discovery in human cancers.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virologia , Citomegalovirus/imunologia , Glioblastoma/genética , Glioblastoma/virologia , Interferon Tipo I/imunologia , Anticorpos Antivirais/sangue , Neoplasias Encefálicas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , MetagenômicaRESUMO
Glioblastoma is an aggressive brain tumor characterized by its high propensity for local invasion, formation of secondary foci within the brain, as well as areas of necrosis. This study aims to (i) provide a technical approach to reproduce features of the disease in vitro and (ii) characterize the tumor/host brain tissue interaction at the molecular level. Human engineered neural tissue (ENT) obtained from pluripotent stem cells was generated and co-cultured with human glioblastoma-initiating cells. Within two weeks, glioblastoma cells invaded the nervous tissue. This invasion displayed features of the disease in vivo: a primary tumor mass, diffuse migration of invading single cells into the nervous tissue, secondary foci, as well as peritumoral cell death. Through comparative molecular analyses, this model allowed the identification of more than 100 genes that are specifically induced and up-regulated by the nervous tissue/tumor interaction. Notably the type I interferon response, extracellular matrix-related genes were most highly represented and showed a significant correlation with patient survival. In conclusion, glioblastoma development within a nervous tissue can be engineered in vitro, providing a relevant model to study the disease and allows the identification of clinically-relevant genes induced by the tumor/host tissue interaction.