Neuroprotection after stroke by targeting NOX4 as a source of oxidative stress

Abstract

Significance: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy.

Recent advances: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke.

Critical issues: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research.

Future directions: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy.

FIG. 1.

Cerebral expression pattern of NOX isoforms implicated in stroke. NOX2 is highly expressed in inflammatory cells such as resident microglia and peripheral neutrophils, whereas NOX4 is rather expressed in neurons. Both endothelial cells and astrocytes seem to express NOX homologues. Cerebral NOX activation and subsequent reactive oxygen species (ROS) generation contribute to blood-brain barrier (BBB) disruption, inflammation, and postischemic neuronal injury [adapted from Ref. (49)].

FIG. 2.

Schematic overview of preclinical stroke models. Rodent stroke models have been established with the aim of generating as reproducible infarct sizes as possible. Each stroke model tries to capture elements of human stroke, though with many well-recognized limits. The choice of the experimental stroke model is optional, but it is recommended to use at least one permanent and one transient ischemic stroke model. The use of mice presenting comorbidities is another recommended option.

FIG. 3.

Recommended quality criteria for preclinical stroke research. To date, animal stroke studies vary in quality and reliability. Thus, when performing preclinical stroke experiments, the quality criteria listed above should be followed. Adherence to these criteria can be used to assess the quality of experimental studies.

FIG. 4.

Existing NOX4 knockout (KO) models. The NOX4 protein is composed of six transmembrane domains and cytosolic binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) at the C-terminus. Several groups have generated NOX4 KO models by deleting different exons of the NOX4 gene. Our group's NOX4 KO mouse was generated by deleting exons 14 and 15, which correspond to the NADPH binding domain. This likely results in the expression of a nonfunctional enzyme (33). Another NOX4 KO mouse was generated by conditionally deleting exon 9 of NOX4 in cardiomyocytes, thereby deleting the FAD binding domain (35). The splice variants of NOX4 in different cell types and tissues are not known. However, independent of which residual protein would be expressed, these two KOs would be biochemically inactive. In a third NOX4 KO model, exons 1 and 2 were deleted in an attempt to delete the NOX4 protein completely (72). Another NOX4 KO model deleted exon 4 (9). Depending on alternative splicing, these may bear the risk of truncated splice variants being expressed, which contain the NADPH/FAD reductase domain. This domain is able to generate ROS independently of the double heme domain and may, thus, in some cells not eliminate NOX4-dependent ROS formation, target it to different subcellular compartments with different or no regulation. However, this is a hypothetical risk, as the splicing in wild-type and KO mice is not fully mapped. Alternatively, truncated NOX4 proteins, that is, without NADPH and/or FAD binding sites, may affect other NOX isoforms in a dominant negative fashion, for example, by scavenging p22^phox. However, there is no evidence as yet for this possibility (7).

FIG. 5.

NOX inhibitors assessed for their neuroprotective potential. The figure displays chemical structures of NOX-inhibiting compounds that have been tested as therapeutics in cerebral infarction. All of them have been shown to successfully reduce infarct size after ischemic stroke. Since apocynin and diphenylene iodonium (DPI) are not sufficiently specific for NADPH oxidases, their application in vivo is limited due to many off-target effects. VAS2870 seems to be more specific for NOX, although off-target effects cannot be excluded and due to poor solubility, the intrathecal mode of administration is used, which is not optimal.

FIG. 6.

Promising NOX inhibitors to be tested in preclinical stroke research. The VAS2870 derivate VAS3947 does not differ from VAS2870 in its NOX inhibition profile, but it may be better suited for in vivo application because of its better solubility. The peptidic inhibitor gp91ds-tat interferes with the assembly between NOX2 and p47^phox, which renders it NOX2 selective. An inhibition of NOX1 could also be imaginable, as the NOX1 activity also depends on subunit regulation by p47^phox. Another promising NOX inhibitor, GKT136901, has been reported to mainly inhibit NOX1 and NOX4.