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Free Radical Biology and Medicine (FRBM)

Copper accumulation in the brain causes the elevation of oxidative stress and less anxious behavior in Ts1Cje mice, a model of Down syndrome

Publication date: April 2019 Source: Free Radical Biology and Medicine, Volume 134 Author(s): Keiichi Ishihara, Eri Kawashita, Ryohei Shimizu, Kazuki Nagasawa, Hiroyuki Yasui, Haruhiko Sago, Kazuhiro Yamakawa, Satoshi Akiba AbstractElevated oxidative stress (OS) is widely accepted to be involved in the pathogenesis of Down syndrome (DS). However, the mechanisms underlying the elevation of OS in DS are poorly understood. Biometals, in particular copper and iron, play roles in OS. We therefore focused on biometals in the brain with DS. In this study, we analyzed the profile of elements, including biometals, in the brain of Ts1Cje mice, a widely used genetic model of DS. An inductively coupled plasma-mass spectrometry (ICP-MS)-based comparative metallomic/elementomic analysis of Ts1Cje mouse brain revealed a higher level of copper in the hippocampus and cerebral cortex, but not in the striatum, in comparison to wild-type littermates. The expression of the copper transporter CTR1, which is involved in the transport of copper into cells, was decreased in the ependymal cells of Ts1Cje mice, suggesting a decrease in the CTR1-mediated transport of copper into the ependymal cells, which excrete copper into the cerebrospinal fluid. To evaluate the pathological significance of the accumulation of copper in the brain of Ts1Cje mice, we examined the effects of a diet with a low copper content (LoCD) on the elevated lipid peroxidation, the accumulation of hyperphosphorylated tau, and some behavioral anomalies. Reducing the copper concentration in the brain by an LoCD restored the enhanced lipid peroxidation and phosphorylation of tau in the brain and reduced anxiety-like behavior, but not hyperactivity or impaired spatial leaning, in Ts1Cje mice. The findings highlight the reduction of accumulation of copper in the brain may be a novel therapeutic strategy for DS. Graphical abstract

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New red-shifted fluorescent biosensor for monitoring intracellular redox changes

Publication date: April 2019 Source: Free Radical Biology and Medicine, Volume 134 Author(s): Claudia Vanesa Piattoni, Florencia Sardi, Florencia Klein, Sergio Pantano, Mariela Bollati-Fogolin, Marcelo Comini AbstractMaintenance of intracellular redox homeostasis is critical for cell survival, proliferation, differentiation, and signaling. In this regard, major changes in the intracellular redox milieu may lead to cell death whereas subtle increases in the level of certain oxidizing species may act as signals that regulate a plethora of cellular processes. Redox-sensitive variants of green fluorescent proteins (roGFP2 and rxYFP) were developed and proved useful to monitor intracellular redox changes in a non-invasive and online manner. With the aim to extend the spectral range of the fluorescent redox biosensors, we here describe the generation, biochemical characterization and biological validation of a new redox reporter based on the red-shifted mRuby2 protein (rxmRuby2). Spectrofluorimetric analysis performed with the recombinant biosensor shows a reversible redox response produced by two redox-active cysteine residues predicted by molecular modeling. rxmRuby2 is highly selective for the couple glutathione/glutathione disulfide in the presence of the oxidoreductase glutaredoxin. The estimated redox potential of rxmRuby2 (E° −265 ± 22 mV) makes it suitable for its use in reducing subcellular compartments. Titration assays demonstrated the capacity of rxmRuby2 to monitor redox changes within a physiological pH range. rxmRuby2 responded sensitively and reversibly to different redox stimuli applied to HeLa and HEK293 cells expressing transiently and/or stable the biosensor. Fusing rxmRuby2 to the Clover fluorescent protein allowed normalization of the redox signal to the expression level of the reporter protein and/or to other factors that may affect fluorescence. The new red-shifted redox biosensor show promises for deep-tissue and in vivo imaging applications. Graphical abstract

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Interaction of methyl viologen-induced chloroplast and mitochondrial signalling in Arabidopsis

Publication date: April 2019 Source: Free Radical Biology and Medicine, Volume 134 Author(s): Fuqiang Cui, Mikael Brosché, Alexey Shapiguzov, Xin-Qiang He, Julia P. Vainonen, Johanna Leppälä, Andrea Trotta, Saijaliisa Kangasjärvi, Jarkko Salojärvi, Jaakko Kangasjärvi, Kirk Overmyer AbstractReactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. In plants, both the chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in the model plant Arabidopsis (Arabidopsis thaliana), we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies suggest that mitochondria are involved in the response to ROS induced by MV in plants. Graphical abstract

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Critical role of H2O2 in mediating sanguinarine-induced apoptosis in prostate cancer cells via facilitating ceramide generation, ERK1/2 phosphorylation, and Par-4 cleavage

Publication date: April 2019 Source: Free Radical Biology and Medicine, Volume 134 Author(s): Anees Rahman, Siraj Pallichankandy, Faisal Thayyullathil, Sehamuddin Galadari AbstractNatural products are a major source of potential anticancer agents, and in order to develop improved and more effective cancer treatments, there is an immense need in exploring and elucidating their mechanism of action. Sanguinarine (SNG), a quaternary benzophenanthridine alkaloid, has been shown to induce cytotoxicity in various human cancers and suppresses various pro-tumorigenic processes such as invasion, angiogenesis, and metastasis in different cancers. Lack of understanding the anticancer mechanism(s) of SNG has impeded the development of this molecule as a potential anticancer agent. Earlier, we have reported that SNG induces reactive oxygen species (ROS)-dependent ceramide (Cer) generation and Akt dephosphorylation, leading to the induction of apoptosis in human leukemic cells. In the present study, we demonstrate that SNG has potent anti-proliferative activity against prostate cancer cells. Our data suggest that SNG induces Cer generation via inhibiting acid ceramidase and glucosylceramide synthase, two important enzymes involved in Cer metabolism. Furthermore, we demonstrate that SNG induces ROS-depended extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation, and prostate apoptosis response-4 (Par-4) cleavage, leading to the induction of apoptosis in human prostate cancer cells. Overall, our findings provide molecular insight into the role of ROS signaling in the anticancer mechanism(s) of SNG. This may provide the basis for its use as a nontoxic and an effective therapeutic agent in the treatment of prostate cancer. Graphical abstract

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Hypochlorous acid-modified extracellular matrix contributes to the behavioral switching of human coronary artery smooth muscle cells

Publication date: April 2019 Source: Free Radical Biology and Medicine, Volume 134 Author(s): Huan Cai, Christine Y. Chuang, Siriluck Vanichkitrungruang, Clare L. Hawkins, Michael J. Davies AbstractThe extracellular matrix (ECM) influences the structure and function of the arterial wall and modulates the behavior of vascular cells through ECM-cell interactions. Alterations to the ECM have been implicated in multiple pathological processes, including atherosclerosis which is characterized by low-grade chronic inflammation and the infiltration and proliferation of smooth muscle cells during disease development. Considerable evidence has been presented for a role for inflammation-derived oxidation in atherogenesis, with enzymatically-active myeloperoxidase (MPO), elevated levels of 3-chlorotyrosine (a biomarker of MPO-catalyzed damage) and oxidized ECM materials detected in advanced human atherosclerotic lesions. Whether oxidant-modified ECM contributes to the altered behavior of smooth muscle cells is however unclear. This study therefore investigated the effects of hypochlorous acid (HOCl), a major MPO-derived oxidant, on the structure of the native ECM synthesized by human coronary artery smooth muscle cells (HCAMSCs) and whether modified ECM proteins affected HCASMC adhesion, proliferation and gene expression. Exposure of native HCASMC-derived ECM to reagent HOCl or a MPO-Cl--H2O2 system resulted in extensive ECM modifications as evidenced by the loss of antibody recognition of epitopes on type IV collagen, laminin, versican and fibronectin. Oxidation of HCASMC ECM markedly reduced HCASMC adhesion to matrix components, but facilitated subsequent proliferation in vitro. Multiple genes were upregulated in HCASMCs in response to HOCl-modified HCASMC-ECM including interleukin-6 (IL-6), fibronectin (FN1) and matrix-metalloproteinases (MMPs). These data reveal a mechanism through which inflammation-induced ECM-modification may contribute to the behavioral switching of HCASMCs, a key process in plaque formation during

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Redox Biology

Electrophilic nitro-oleic acid reverses obesity-induced hepatic steatosis

Publication date: April 2019 Source: Redox Biology, Volume 22 Author(s): Nicholas K.H. Khoo, Marco Fazzari, Dionysios V. Chartoumpekis, Lihua Li, Danielle Aparecida Guimaraes, Gavin E. Arteel, Sruti Shiva, Bruce A. Freeman AbstractNon-alcoholic fatty liver disease (NAFLD) is linked to obesity and insulin resistance and is the most prevalent chronic liver disease. During the development of obesity and NAFLD, mitochondria adapt to the increased lipid load in hepatocytes by increasing the rate of fatty acid oxidation. In concert with this, reactive species (RS) generation is increased, damaging hepatocytes and inducing inflammation. Hepatic mitochondrial dysfunction is central to the pathogenesis of NAFLD via undefined mechanisms. There are no FDA approved treatments for NAFLD other than weight loss and management of glucose tolerance. Electrophilic nitro-oleic acid (NO2-OA) displays anti-inflammatory and antioxidant signaling actions, thus mitochondrial dysfunction, RS production and inflammatory responses to NO2-OA and the insulin sensitizer rosiglitazone were evaluated in a murine model of insulin resistance and NAFLD. Mice on HFD for 20 wk displayed increased adiposity, insulin resistance and hepatic lipid accumulation (steatosis) compared to mice on normal chow (NC). The HFD mice had mitochondrial dysfunction characterized by lower hepatic mitochondrial complex I, IV and V activity compared to mice on NC. Treatment with NO2-OA or rosiglitazone for the last 42 days (out of 20 wk) abrogated HFD-mediated decreases in hepatic mitochondrial complex I, IV and V activity. Notably, NO2-OA treatment normalized hepatic triglyceride levels and significantly reversed hepatic steatosis. Despite the improved glucose tolerance observed upon rosiglitazone treatment, liver weight and hepatic triglycerides were significantly increased over vehicle-treated HFD mice. These observations support that the pleiotropic signaling actions of electrophilic fatty acids limit the complex hepatic and systemic pathogenic responses instigated

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Antenatal melatonin modulates an enhanced antioxidant/pro-oxidant ratio in pulmonary hypertensive newborn sheep

Publication date: April 2019 Source: Redox Biology, Volume 22 Author(s): Alejandro Gonzalez-Candia, Marcelino Veliz, Catalina Carrasco-Pozo, Rodrigo L. Castillo, J. Cesar Cárdenas, Germán Ebensperger, Roberto V. Reyes, Aníbal J. Llanos, Emilio A. Herrera AbstractChronic hypobaric hypoxia during fetal and neonatal life induces neonatal pulmonary hypertension. Hypoxia and oxidative stress are driving this condition, which implies an increase generation of reactive oxygen species (ROS) and/or decreased antioxidant capacity. Melatonin has antioxidant properties that decrease oxidative stress and improves pulmonary vascular function when administered postnatally. However, the effects of an antenatal treatment with melatonin in the neonatal pulmonary function and oxidative status are unknown. Therefore, we hypothesized that an antenatal therapy with melatonin improves the pulmonary arterial pressure and antioxidant status in high altitude pulmonary hypertensive neonates. Twelve ewes were bred at high altitude (3600 m); 6 of them were used as a control group (vehicle 1.4% ethanol) and 6 as a melatonin treated group (10 mg d-1 melatonin in vehicle). Treatments were given once daily during the last third of gestation (100–150 days). Lambs were born and raised with their mothers until 12 days old, and neonatal pulmonary arterial pressure and resistance, plasma antioxidant capacity and the lung oxidative status were determined. Furthermore, we measured the pulmonary expression and activity for the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase, and the oxidative stress markers 8-isoprostanes, 4HNE and nitrotyrosine. Finally, we assessed pulmonary pro-oxidant sources by the expression and function of NADPH oxidase, mitochondria and xanthine oxidase. Melatonin decreased the birth weight. However, melatonin enhanced the plasma antioxidant capacity and decreased the pulmonary antioxidant activity, associated with a diminished oxidative stress during postnatal life. Interestingly, melatonin also de

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CX3CR1-deficient microglia shows impaired signalling of the transcription factor NRF2: Implications in tauopathies

Publication date: April 2019 Source: Redox Biology, Volume 22 Author(s): Sara Castro-Sánchez, Ángel J. García-Yagüe, Sebastian Kügler, Isabel Lastres-Becker AbstractTAU protein aggregation is the main characteristic of neurodegenerative diseases known as tauopathies. Low-grade chronic inflammation is also another hallmark that indicates crosstalk between damaged neurons and glial cells. Previously, we have demonstrated that neurons overexpressing TAUP301L release CX3CL1, which activates the transcription factor NRF2 signalling to limit over-activation in microglial cells in vitro and in vivo. However, the connection between CX3CL1/CX3CR1 and NRF2 system and its functional implications in microglia are poorly described. We evaluated CX3CR1/NRF2 axis in the context of tauopathies and its implication in neuroinflammation. Regarding the molecular mechanisms that connect CX3CL1/CX3CR1 and NRF2 systems, we observed that in primary microglia from Cx3cr1-/- mice the mRNA levels of Nrf2 and its related genes were significantly decreased, establishing a direct linking between both systems. To determine functional relevance of CX3CR1, migration and phagocytosis assays were evaluated. CX3CR1-deficient microglia showed impaired cell migration and deficiency of phagocytosis, as previously described for NRF2-deficient microglia, reinforcing the idea of the relevance of the CX3CL1/CX3CR1 axis in these events. The importance of these findings was evident in a tauopathy mouse model where the effects of sulforaphane (SFN), an NRF2 inducer, were examined on neuroinflammation in Cx3cr1+/+ and Cx3cr1-/- mice. Interestingly, the treatment with SFN was able to modulate astrogliosis but failed to reduce microgliosis in Cx3cr1-/- mice. These findings suggest an essential role of the CX3CR1/NRF2 axis in microglial function and in tauopathies. Therefore, polymorphisms with loss of function in CX3CR1 or NRF2 have to be taken into account for the development of therapeutic strategies. Graphical abstract

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Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity

Publication date: April 2019 Source: Redox Biology, Volume 22 Author(s): Anita Carija, Francisca Pinheiro, Jordi Pujols, Inês C. Brás, Diana Fernandes Lázaro, Carlo Santambrogio, Rita Grandori, Tiago F. Outeiro, Susanna Navarro, Salvador Ventura AbstractThe aggregation of α-synuclein (α-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and innocuous α-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of α-syn has hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of this transition. The recent 3D structure of a pathogenic α-syn fibril provides a template for this kind of studies. The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of α-syn in different environments. The new covalent link biases the native structural ensemble of α-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early α-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD.

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Tumoral NOX4 recruits M2 tumor-associated macrophages via ROS/PI3K signaling-dependent various cytokine production to promote NSCLC growth

Publication date: April 2019 Source: Redox Biology, Volume 22 Author(s): Jiahao Zhang, Huachao Li, Qipeng Wu, Yueming Chen, Yanchao Deng, Zhicheng Yang, Luyong Zhang, Bing Liu AbstractM2-type tumor-associated macrophages (TAMs) infiltration contributes to cancer malignant progression. However, the mechanisms for controlling recruitment and M2 polarization of macrophages by cancer cells are largely unclear. NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and mediates cancer progression. NOXs are in close relation with cancer-related inflammation, nevertheless, whether tumoral NOXs influence microenvironmental macrophages remains undentified. This study found that there was a close association between NOX4 expression and macrophage chemotaxis in patients with NSCLC analyzed using TCGA RNA-sequencing data. NOX4 in NSCLC cells (A549 and Calu-1 cell lines) efficiently enhanced murine peritoneal macrophage migration and induces M2 polarization. Immunohistochemical analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206. The mechanical study revealed that tumoral NOX4-induced reactive oxygen species (ROS) stimulated various cytokine production, including CCL7, IL8, CSF-1 and VEGF-C, via PI3K/Akt signaling-dependent manner. Blockade of the function of these cytokines reversed NOX4 effect on macrophages. Specifically, the results showed that tumoral NOX4-educated M2 macrophages exhibited elevated JNK activity, expressed and released HB-EGF, thus facilitating NSCLC proliferation in vitro. Pretreatment of macrophages with JNK inhibitor blocked tumoral NOX4-induced HB-EGF production in M2 macrophages. Finally, in a xenograft mouse model, overexpression of NOX4 in A549 cells enhanced the tumor growth. Elimination of ROS by NAC or inhibition of NOX4 activity by GKT137831 suppressed tumor growth accompanied by reduction in macrophage infiltration and the percentage of M2 macrophages. In conclusion, our study indicates that tumoral NOX4 recruits M2 TAMs via

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