PLoS Biology: Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Christoph Kleinschnitz1*, Henrike Grund2, Kirstin Wingler2,3,4,5, Melanie E. Armitage3,5, Emma Jones3, Manish Mittal2, David Barit6, Tobias Schwarz1, Christian Geis1, Peter Kraft1, Konstanze Barthel7, Michael K. Schuhmann1,8, Alexander M. Herrmann1,8, Sven G. Meuth1,8, Guido Stoll1, Sabine Meurer3, Anja Schrewe9, Lore Becker9,10, Valérie Gailus-Durner9, Helmut Fuchs9, Thomas Klopstock10, Martin Hrabé de Angelis9,11, Karin Jandeleit-Dahm6, Ajay M. Shah12, Norbert Weissmann2, Harald H. H. W. Schmidt2,3,4,5*

1 Neurologische Klinik und Poliklinik, Universität Würzburg, Würzburg, Germany, 2 Rudolf-Buchheim-Institut für Pharmakologie & Medizinische Klinik, Justus-Liebig-Universität, Gießen, Germany, 3 Department of Pharmacology and Centre for Vascular Health, Monash University, Melbourne, Australia, 4 Department of Pharmacology and Toxicology and Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands, 5 National Stroke Research Institute, Florey Neuroscience Institutes, Melbourne, Australia, 6 Baker IDI Heart and Diabetes Institute, Juvenile Diabetes Research Foundation (JDRF) International Center for Diabetic Complications Research, Melbourne, Australia, 7 Abteilung Neurologie, Georg-August Universität Göttingen, Göttingen, Germany, 8 Universitätsklinik Münster, Klinik und Poliklinik für Neurologie—Entzündliche Erkrankungen des Nervensystems und Neuroonkologie, Münster, Germany, 9 Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany, 10 Friedrich-Baur-Institut an der Neurologischen Klinik, Klinikum der Ludwig-Maximilians-Universität München, München, Germany, 11 Lehrstuhl für Experimentelle Genetik, Technische Universität München, Freising-Weihenstephan, Germany, 12 King's College London School of Medicine, The James Black Centre, Cardiovascular Division, London, United Kingdom

Abstract

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4−/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4−/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.

Author Summary

Stroke is the second leading cause of death worldwide. Today, only one approved therapy exists—a drug that breaks down blood clots—the effectiveness of which is moderate, and it can only be used in about 10% of patients because of contraindications. New therapeutic strategies that are translatable to humans and more rigid thresholds of relevance in pre-clinical stroke models are needed. One candidate mechanism is oxidative stress, which is the damage caused by reactive oxygen species (ROS). Antioxidant approaches that specifically target ROS have thus far failed in clinical trials. For a more effective approach, we focus here on targeting ROS at its source by investigating an enzyme involved in generating ROS, known as NADPH oxidase type 4, or NOX4. We found that NOX4 causes oxidative stress and death of nerve cells after a stroke. Deletion of the NOX4-coding gene in mice, as well as inhibiting the ROS-generating activity of NOX with a pharmacological inhibitor, reduces brain damage and improves neurological function, even when given hours after a stroke. Importantly, neuroprotection was preserved in old male and female Nox4−/− mice as well as in Nox4−/− mice subjected to permanent ischemia. NOX4 thus represents a most promising new therapeutic target for reducing oxidative stress in general, and in brain injury due to stroke in particular.

Citation: Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, et al. (2010) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration. PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479

Academic Editor: Malcolm McLeod, University of Edinburgh, United Kingdom

Received: February 19, 2010; Accepted: July 28, 2010; Published: September 21, 2010

Copyright: © 2010 Kleinschnitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the NHMRC, Australia, the Deutsche Forschungsgemeinschaft (DFG), Germany (to HHHWS and CK), and by the Bundesministerium fur Bildung und Forschung within the framework of the NGFN-Plus and the European Commission (EUMODIC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: HHHWS and KW declare a potential competing interest as shareholder and previous employee, respectively, of Vasopharm GmbH, which develops NADPH oxidase inhibitors such as VAS2870. All authors declare that they adhere to all PLoS Biology policies on sharing data and materials as detailed in the PLoS Biology guide for authors.

Abbreviations: CISS, constructive interference in steady state; KO, knock out; pMCAO, permanent middle cerebral artery occlusion; ROS, reactive oxygen species; rt-PA, recombinant tissue plasminogen activator; tMCAO, transient middle cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium chloride; WT, wild type

* E-mail: h.schmidt@farmaco.unimaas.nl (HHHWS); christoph.kleinschnitz@mail.uni-wuerzbur​g.de (CK)

full-text:
PLoS Biology: Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration


NEUROLOGÍA
Identificada una enzima asociada a la muerte de células nerviosas tras un ictus
Actualidad Ultimas noticias - JANO.es
JANO.es · 27 Septiembre 2010 11:24

Se trata la enzima NOX4, identificada por investigadores alemanes, quienes también han creado un fármaco experimental capaz de inhibirla

Investigadores de las universidades de Maastricht y Würzburg (Alemania) han identificado una enzima asociada al ictus, hallazgo que podría ayudar en el tratamiento de la enfermedad. Los resultados de su trabajo se publican en PLoS Biology.

Los investigadores han descubierto una enzima responsable de la muerte de las células nerviosas después de un ictus. Se trata de la enzima NOX4, que produce peróxido de hidrógeno, molécula cáustica también utilizada en agentes blanqueadores. La inhibición de NOX4 mediante un nuevo fármaco experimental en ratones con ictus redujo en gran medida el daño cerebral y preservó las funciones cerebrales, incluso cuando se proporcionó horas después del ictus.

Según explica Christoph Kleinschnitz, el ictus isquémico es la segunda causa de mortalidad en todo el mundo. En la actualidad sólo existe una terapia aprobada y su eficacia es moderada y sólo puede utilizarse en el 10% de los pacientes debido a las contraindicaciones. El estudio propone una estrategia completamente nueva al inhibir la fuente importante de peróxido de hidrógeno y prevenir su formación.

La eliminación del gen NOX4 en los ratones no produjo anomalías en los animales y por ello no se esperan efectos secundarios obvios de un futuro fármaco inhibidor de NOX4.

Según los autores, la identificación de NOX4 como una enzima con un papel clave para eliminar las células nerviosas después de un ictus en ratones convierte la inhibición de NOX4 en un prometedor método terapéutico para el ictus en humanos.



PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479
PLoS Biology: Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration


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