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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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (© 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.)

Significance: There is increasing evidence that the generation of reactive oxygen species (ROS) in the central nervous system (CNS) involves the NOX family of nicotinamide adenine dinucleotide phosphate oxidases. Controlled ROS generation appears necessary for optimal functioning of the CNS through fine-tuning of redox-sensitive signaling pathways, while overshooting ROS generation will lead to oxidative stress and CNS disease., Recent Advances: NOX enzymes are not only restricted to microglia (i.e. brain phagocytes) but also expressed in neurons, astrocytes, and the neurovascular system. NOX enzymes are involved in CNS development, neural stem cell biology, and the function of mature neurons. While NOX2 appears to be a major source of pathological oxidative stress in the CNS, other NOX isoforms might also be of importance, for example, NOX4 in stroke. Globally speaking, there is now convincing evidence for a role of NOX enzymes in various neurodegenerative diseases, cerebrovascular diseases, and psychosis-related disorders., Critical Issues: The relative importance of specific ROS sources (e.g., NOX enzymes vs. mitochondria; NOX2 vs. NOX4) in different pathological processes needs further investigation. The absence of specific inhibitors limits the possibility to investigate specific therapeutic strategies. The uncritical use of non-specific inhibitors (e.g., apocynin, diphenylene iodonium) and poorly validated antibodies may lead to misleading conclusions., Future Directions: Physiological and pathophysiological studies with cell-type-specific knock-out mice will be necessary to delineate the precise functions of NOX enzymes and their implications in pathomechanisms. The development of CNS-permeant, specific NOX inhibitors will be necessary to advance toward therapeutic applications.

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., (© 2013 Elsevier Ltd. All rights reserved.)