NADPH oxidase 4 (Nox4) deletion accelerates liver regeneration in mice.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-ß) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4-/-) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4-/- and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-ß pathway. In fact, levels of active TGF-ß1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-ß1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.

Decreased NOX2 expression in the brain of patients with bipolar disorder: association with valproic acid prescription and substance abuse.

Neuroinflammation and increased oxidative stress are believed to contribute to the development of psychiatric diseases. Animal studies have implicated NADPH oxidases (NOX) as relevant sources of reactive oxygen species in the brain. We have analyzed the expression of NOX isoforms in post-mortem brain samples from patients with psychiatric disorders (schizophrenia, bipolar disorder) and non-psychiatric subjects. Two collections from the Stanley Medical Research Institute were studied: the Array Collection (RNA, 35 individuals per group), and a neuropathology consortium collection (paraffin-embedded sections, 15 individuals per group). Quantitative PCR analysis revealed expression of NOX2 and NOX4 in prefrontal cortex. No impact of psychiatric disease on NOX4 levels was detected. Remarkably, the expression of NOX2 was specifically decreased in prefrontal and cingulate cortices of bipolar patients, as compared with controls and schizophrenic patients. NOX2 expression was not statistically associated with demographic parameters and post-mortem interval, but correlated with brain pH. Immunostaining demonstrated that NOX2 was predominantly expressed in microglia, which was corroborated by a decrease in the microglial markers CD68 and CD11b in the cingulate cortex of bipolar disorder patients. The analysis of potentially confounding parameters showed association of valproic acid prescription and heavy substance abuse with lower levels of NOX2. Taken together, we did not observe changes of NOX2 in schizophrenic patients, but a marked decrease of microglial markers and NOX2 in the brain of bipolar patients. This might be an underlying feature of bipolar disorder and/or a consequence of valproic acid treatment and substance abuse.

NADPH oxidases in oxidant production by microglia: activating receptors, pharmacology and association with disease.

Microglia are the resident immune cells of the CNS and constitute a self-sustaining population of CNS-adapted tissue macrophages. As mononuclear phagocytic cells, they express high levels of superoxide-producing NADPH oxidases (NOX). The sole function of the members of the NOX family is to generate reactive oxygen species (ROS) that are believed to be important in CNS host defence and in the redox signalling circuits that shape the different activation phenotypes of microglia. NOX are also important in pathological conditions, where over-generation of ROS contributes to neuronal loss via direct oxidative tissue damage or disruption of redox signalling circuits. In this review, we assess the evidence for involvement of NOX in CNS physiopathology, with particular emphasis on the most important surface receptors that lead to generation of NOX-derived ROS. We evaluate the potential significance of the subcellular distribution of NOX isoforms for redox signalling or release of ROS to the extracellular medium. Inhibitory mechanisms that have been reported to restrain NOX activity in microglia and macrophages in vivo are also discussed. We provide a critical appraisal of frequently used and recently developed NOX inhibitors. Finally, we review the recent literature on NOX and other sources of ROS that are involved in activation of the inflammasome and discuss the potential influence of microglia-derived oxidants on neurogenesis, neural differentiation and culling of surplus progenitor cells. The degree to which excessive, badly timed or misplaced NOX activation in microglia may affect neuronal homeostasis in physiological or pathological conditions certainly merits further investigation. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Microglia antioxidant systems and redox signalling.

For many years, microglia, the resident CNS macrophages, have been considered only in the context of pathology, but microglia are also glial cells with important physiological functions. Microglia-derived oxidant production by NADPH oxidase (NOX2) is implicated in many CNS disorders. Oxidants do not stand alone, however, and are not always pernicious. We discuss in general terms, and where available in microglia, GSH synthesis and relation to cystine import and glutamate export, and the thioredoxin system as the most important antioxidative defence mechanism, and further, we discuss in the context of protein thiolation of target redox proteins the necessity for tightly localized, timed and confined oxidant production to work in concert with antioxidant proteins to promote redox signalling. NOX2-mediated redox signalling modulates the acquisition of the classical or alternative microglia activation phenotypes by regulating major transcriptional programs mediated through NF-κB and Nrf2, major regulators of the inflammatory and antioxidant response respectively. As both antioxidants and NOX-derived oxidants are co-secreted, in some instances redox signalling may extend to neighboring cells through modification of surface or cytosolic target proteins. We consider a role for microglia NOX-derived oxidants in paracrine modification of synaptic function through long term depression and in the communication with the adaptive immune system. There is little doubt that a continued foray into the functions of the antioxidant response in microglia will reveal antioxidant proteins as dynamic players in redox signalling, which in concert with NOX-derived oxidants fulfil important roles in the autocrine or paracrine regulation of essential enzymes or transcriptional programs. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology.

Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47(phox) was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47(phox) were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations.

Ethanol-related adaptive changes and physical dependence in rats after exposure to ethanol.

Adaptive changes that occur after chronic exposure to ethanol are an important component in the development of physical dependence. We have focused our research on ethanol-induced changes in the expression of several genes that may be important in adaptation. In this article, we describe adaptive changes at the level of the N-methyl-D-aspartate receptor, in the protein expression and activity of the Egr transcription factors, and in the expression of a novel gene of unknown function.

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain.

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.

Analysis of cis-acting sequences from the myelin oligodendrocyte glycoprotein promoter.

Myelin oligodendrocyte glycoprotein (MOG), a minor component of the myelin sheath, appears to be implicated in the late events of CNS myelinogenesis. To investigate the transcriptional regulation of MOG, 657 bp of the 5'-flanking sequence of the murine MOG gene, previously shown to induce the highest level of transcription in an oligodendroglial cell line, was analyzed by in vitro footprinting and electrophoretic mobility shift assays. This region contains at least three sites that contact nuclear proteins in vitro. Each region described in this study binds specific nuclear proteins and enhances transcription in the OLN-93 glial cell line. More specifically, a region located at position -93 to -73 bp, which displays 100% homology in mouse and human MOG promoters, presents distinct binding affinities between brain and liver nuclear proteins. The results obtained by supershift assay and site-directed mutagenesis reveal that this region contains an essential positive element (TGACGTGG) related to the cyclic AMP-responsive element CREB-1 and are additional evidence for the involvement of the cyclic AMP transduction pathway in oligodendrocyte development.

An antisense transgenic strategy to inhibit the myelin oligodendrocyte glycoprotein synthesis.

To understand the function of the myelin oligodendrocyte glycoprotein (MOG), a myelin specific protein of the central nervous system, transgenic mice were produced. The transgene is a fusion gene containing 1.9 kb of murine myelin basic protein promoter, 430 bp of rat MOG cDNA in the reverse orientation and 4.5 kb of human proteolipid protein gene. In spite of high expression of antisense MOG mRNA in the oligodendrocytes, MOG synthesis was not inhibited in transgenic mice. This lack of inhibition of MOG underlines the difficulties encountered with antisense transgenic strategies.