Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons

Cell death induced by 6-hydroxydopamine (6-OHDA) is thought to be caused by reactive oxygen species (ROS) derived from 6-OHDA autooxidation and by a possible direct effect of 6-OHDA on the mitochondrial respiratory chain. However, the process has not been totally clarified. In rat primary mesencephalic cultures, we observed a significant increase in dopaminergic (DA) cell loss 24 h after administration of 6-OHDA (40 micromol/L) and a significant increase in NADPH subunit expression, microglial activation and superoxide anion/superoxide-derived ROS in DA cells that were decreased by the NADPH inhibitor apocynin. Low doses of 6-OHDA (10 micromol/L) did not induce a significant loss of DA cells or a significant increase in NADPH subunit expression, microglial activation or superoxide-derived ROS. However, treatment with the NADPH complex activator angiotensin II caused a significant increase in all the latter. Forty-eight hours after intrastriatal 6-OHDA injection in rats, there was still no loss of DA neurons although there was an increase in NADPH subunit expression and NADPH oxidase activity. The results suggest that in addition to the autooxidation-derived ROS and the inhibition of the mitochondrial respiratory chain, early microglial activation and NADPH oxidase-derived ROS act synergistically with 6-OHDA and constitute a relevant and early component of the 6-OHDA-induced cell death.     

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin-induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick-end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase-derived ROS production and pro-inflammatory cytokine expression, including interleukin-1beta and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.     

Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors

We report the synthesis of novel 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3, and their biological evaluation using NADPH oxidase (NOX) 1 and 4. Based on structural and pharmacophore analyses of known inhibitors such as hydroxypyrazole 2, we envisioned interesting 2-thiohydantoin compounds, 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3 that would be expected to well match the structural features in 2. Efficient synthesis of eighteen target compounds 3 were achieved through the synthetic pathway of 4 → 11 → 3, established after consideration of several plausible synthetic pathways. The inhibitory activities of compounds 3 against NOX 1 and 4 were measured, with some of the target compounds showing similar or higher activities compared with reference 2; in particular, compounds 3bz, 3cz, and 3ez were found to be promising inhibitors of both NOX 1 and 4 with modest isozyme selectivities, which highlights the significance of the 2-thiohydantoin substructure for inhibition of NOX 1 and 4. This marks the first time these compounds have been applied to the inhibition of NOX enzymes.     

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60 kDa accompanied by a broad band of 60–90 kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53 kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn¹⁶¹ and Asn²⁴¹ on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.     

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys³²⁵) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys³²⁵ to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta.     

The NADPH oxidase NOX4 inhibits hepatocyte proliferation and liver cancer progression

The NADPH oxidase NOX4 has emerged as an important source of reactive oxygen species in signal transduction, playing roles in physiological and pathological processes. NOX4 mediates transforming growth factor-β-induced intracellular signals that provoke liver fibrosis, and preclinical assays have suggested NOX4 inhibitors as useful tools to ameliorate this process. However, the potential consequences of sustained treatment of liver cells with NOX4 inhibitors are yet unknown. The aim of this work was to analyze whether NOX4 plays a role in regulating liver cell growth either under physiological conditions or during tumorigenesis. In vitro assays proved that stable knockdown of NOX4 expression in human liver tumor cells increased cell proliferation, which correlated with a higher percentage of cells in S/G2/M phases of the cell cycle, downregulation of p21(CIP1/WAF1), increase in cyclin D1 protein levels, and nuclear localization of β-catenin. Silencing of NOX4 in untransformed human and mouse hepatocytes also increased their in vitro proliferative capacity. In vivo analysis in mice revealed that NOX4 expression was downregulated under physiological proliferative situations of the liver, such as regeneration after partial hepatectomy, as well as during pathological proliferative conditions, such as diethylnitrosamine-induced hepatocarcinogenesis. Xenograft experiments in athymic mice indicated that NOX4 silencing conferred an advantage to human hepatocarcinoma cells, resulting in earlier onset of tumor formation and increase in tumor size. Interestingly, immunochemical analyses of NOX4 expression in human liver tumor cell lines and tissues revealed decreased NOX4 protein levels in liver tumorigenesis. Overall, results described here strongly suggest that NOX4 would play a growth-inhibitory role in liver cells.     

Systemic upregulation of NADPH oxidase in diet-induced obesity in rats

Abstract

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is upregulated in a variety of tissues in obesity. It is still unclear as to whether NADPH oxidase upregulation in a specific tissue is part of a systemic response. Here we analyzed the expression pattern of NADPH oxidase in vascular, adipose, and kidney tissues in a rat model of diet-induced obesity. After weaning, rats were fed either a normal or high-fat diet for 12 weeks. The high-fat diet resulted in 20% increased body weight. In the aorta, Nox4 expression was increased by three-fold in obese rats. Upregulations of p22phox and p47phox in adipose, and Nox4, p22phox, and p47phox in kidney were observed in obesity. Marked increases in plasma leptin and insulin were observed, with more modest changes in adiponectin in obese rats. The average systolic blood pressure in the obese group was 11 mmHg higher than that of lean rats (P < 0.005). There was a significant correlation between blood pressure and aortic Nox4 expression (P < 0.01). In cultured vascular smooth muscle cells, adiponectin reduced the expression of Nox4 in a protein kinase A-dependent manner. Our results suggest that upregulation of NADPH oxidase in multiple tissues during obesity appears to be a systemic response. At least in vitro, adiponectin may have a protective antioxidant role by suppressing vascular NADPH oxidase expression. The association between NADPH oxidase Nox4 expression in the vasculature and the elevated blood pressure in obesity requires further investigation.     

Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production

Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite.     

Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation

Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer's disease. We investigated the role of NADPH oxidase in microglial activation state using p47(phox) and gp91(phox) -deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or Aβ????. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47(phox) subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer's disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases.     

植物NADPH氧化酶研究进展

植物NADPH氧化酶又被称为Rboh(respiratory burst oxidase homologue),是动物巨噬细胞NADPH氧化酶主要功能亚基gp91phox的同源物。在受到外来信号的刺激时,该酶能通过自身的激活或失活迅速引起活性氧(reactive oxygen species,ROS)的升高或降低,进而在植物生长发育及应答生物或非生物胁迫中发挥重要作用。该文总结了近兼来植物中NADPH氧化酶的结构和功能,以及信号调节机制等方面的研究进展。     

The role of MAC1 in diesel exhaust particle‐induced microglial activation and loss of dopaminergic neuron function

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease‐like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron‐glia cultures were treated with either nanometer‐sized DEP (< 0.22 μM; 50 μg/mL), ultrafine carbon black (ufCB, 50 μg/mL), or DEP extracts (eDEP; from 50 μg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H₂O₂ production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP‐induced H₂O₂ production in microglia. However, pre‐treatment with the MAC1/CD11b inhibitor antibody blocked microglial H₂O₂ production in response to DEP. MAC1^?/? mesencephalic neuron‐glia cultures were protected from DEP‐induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP‐induced microglial H₂O₂ production and loss of DA neuron function.     

NOX family NADPH oxidases: not just in mammals

NOX family NADPH oxidases are enzymes whose biological function is electron transport and the generation of reactive oxygen species (ROS). NOX enzymes in mammalian organisms have received most attention. However, NOX enzymes are widely distributed in different kingdoms of life. While they are not found in prokaryotes and most unicellular eukaryotes, they are present in fungi, plants, and animals. The identity of the ancestral NOX is not known, but most likely it: (i) possessed the basic NOX structure consisting of 6 transmembrane domains (containing two assymmetrical hemes) and a long cytoplasmic C-terminal (containing the FAD and NADPH binding sites); and (ii) emerged before the divergence of life into fungi, plants, and animals. During evolution, acquisition of a Ca(2+)-binding EF hand domain by an ancestral NOX, led to NOX5-like isoforms. DUOX isoforms presumably developed from a NOX5-like isoform through the additional acquisition of a peroxidase homology domain. The expression pattern of NOX enzymes is specific to each kingdom of life. Fungi express only ancestral-type isoforms, and plants only NOX5-like isoforms. NOX expression patterns in animals are complex and ancestral NOXes, NOX5-like isoforms and DUOXes are generally found. But there are exceptions; for example rodents lack NOX5 and Caenorhabditis elegans expresses only DUOXes. Biological functions of NOX enzymes include, among others, host defense, post-translational modification of proteins, and regulation cell growth and differentiation. In summary, the invention of NOX enzymes early in the development of life was a success story: there is no evidence of multicellular life without NOX enzymes.     

Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach

Helicobacter pylori infection has been suggested to stimulate expression of the NADPH oxidase 1 (Nox1)-based oxidase system in guinea pig gastric epithelium, whereas Nox1 mRNA expression has not yet been documented in the human stomach. PCR of human stomach cDNA libraries showed that Nox1 and Nox organizer 1 (NOXO1) messages were absent from normal stomachs, while they were specifically coexpressed in intestinal- and diffuse-type adenocarcinomas including signet-ring cell carcinoma. Immunohistochemistry showed that Nox1 and NOXO1 proteins were absent from chronic atrophic gastritis (15 cases), adenomas (4 cases), or surrounding tissues of adenocarcinomas (45 cases). In contrast, Nox1 and its partner proteins were expressed in intestinal-type adenocarcinomas (19/21 cases), diffuse-type adenocarcinomas (15/15 cases), and signet-ring cell carcinomas (9/9 cases). Confocal microscopy revealed that Nox1, NOXO1, Nox activator 1, and p22^phox were predominantly associated with Golgi apparatus in these cancer cells, while diffuse-type adenocarcinomas also contained cancer cells having Nox1 and its partner proteins in their nuclei. Nox1-expressing cancer cells exhibited both gastric and intestinal phenotypes, as assessed by expression of mucin core polypeptides. Thus, the Nox1-base oxidase may be a potential marker of neoplastic transformation and play an important role in oxygen radical- and inflammation-dependent carcinogenesis in the human stomach.     

A roadmap for investigating the role of the prion protein in depression associated with neurodegenerative disease

The physiological properties of the native, endogenous prion protein (PrP^C) is a matter of concern, due to its pleiotropic functions and links to neurodegenerative disorders and cancer. In line with our hypothesis that the basic function of PrP^C is to serve as a cell surface scaffold for the assembly of signaling modules, multiple interactions have been identified of PrP^C with signaling molecules, including neurotransmitter receptors. We recently reported evidence that PrP^C may modulate monoaminergic neurotransmission, as well as depressive-like behavior in mice. Here, we discuss how those results, together with a number of other studies, including our previous demonstration that both inflammatory and behavioral stress modulate PrP^C content in neutrophils, suggest a distributed role of PrP^C in clinical depression and inflammation associated with neurodegenerative diseases. An overarching understanding of the multiple interventions of PrP^C upon physiological events may both shed light on the pathogenesis of, as well as help the identification of novel therapeutic targets for clinical depression, Prion and Alzheimer's Diseases.     

Activity of neutrophil NADPH oxidase in iron-deficient anemia

This study was designed to measure the effects of iron supplementation on respiratory burst in iron-deficient anemia. The performance of neutrophils was evaluated by measuring the activity of NADPH oxidase in 18 patients with iron-deficient anemia before and after body iron stores are saturated. The activity of NADPH oxidase was significantly lower in pretreatment patients relative to controls (p<0.05). The activity increased after iron supplementation to levels that had no significant differences relative to controls.