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Chemogenetic motoneuron excitation and astrocyte GPCR-Gi signaling restore blood–spinal cord barrier, disrupted in four ALS mouse models, revealing its role in disease progression but not initiation., Blood–spinal cord barrier (BSCB) disruption is thought to contribute to motoneuron (MN) loss in amyotrophic lateral sclerosis (ALS). It is currently unclear whether impairment of the BSCB is the cause or consequence of MN dysfunction and whether its restoration may be directly beneficial. We revealed that SOD1G93A, FUSΔNLS, TDP43G298S, and Tbk1+/− ALS mouse models commonly shared alterations in the BSCB, unrelated to motoneuron loss. We exploit PSAM/PSEM chemogenetics in SOD1G93A mice to demonstrate that the BSCB is rescued by increased MN firing, whereas inactivation worsens it. Moreover, we use DREADD chemogenetics, alone or in multiplexed form, to show that activation of Gi signaling in astrocytes restores BSCB integrity, independently of MN firing, with no effect on MN disease markers and dissociating them from BSCB disruption. We show that astrocytic levels of the BSCB stabilizers Wnt7a and Wnt5a are decreased in SOD1G93A mice and strongly enhanced by Gi signaling, although further decreased by MN inactivation. Thus, we demonstrate that BSCB impairment follows MN dysfunction in ALS pathogenesis but can be reversed by Gi-induced expression of astrocytic Wnt5a/7a.

Glucose, chief metabolic support for cancer cell survival and growth, is mainly imported into cells by facilitated glucose transporters (GLUTs). The increase in glucose uptake along with tumor progression is due to an increment of facilitative glucose transporters as GLUT1. GLUT1 prevents cell death of cancer cells caused by growth factors deprivation, but there is scarce information about its role on the damage caused by glucose deprivation, which usually occurs within the core of a growing tumor. In prostate cancer (PCa), GLUT1 is found in the most aggressive tumors, and it is regulated by androgens. To study the response of androgen-sensitive and insensitive PCa cells to glucose deprivation and the role of GLUT1 on survival mechanisms, androgen-sensitive LNCaP and castration-resistant LNCaP-R cells were employed. Results demonstrated that glucose deprivation induced a necrotic type of cell death which is prevented by antioxidants. Androgen-sensitive cells show a higher resistance to cell death triggered by glucose deprivation than castration-resistant cells. Glucose removal causes an increment of H2O2, an activation of androgen receptor (AR) and a stimulation of AMP-activated protein kinase activity. In addition, glucose removal increases GLUT1 production in androgen sensitive PCa cells. GLUT1 ectopic overexpression makes PCa cells more resistant to glucose deprivation and oxidative stress-induced cell death. Under glucose deprivation, GLUT1 overexpressing PCa cells sustains mitochondrial SOD2 activity, compromised after glucose removal, and significantly increases reduced glutathione (GSH). In conclusion, androgen-sensitive PCa cells are more resistant to glucose deprivation-induced cell death by a GLUT1 upregulation through an enhancement of reduced glutathione levels. Keywords: Glut1, Prostate cancer, Glucose deprivation, Androgen receptor, Glutathione, Oxidative stress

International audience; Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).

International audience; Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.

International audience; Recent metabolomic reports connect dysregulation of glycosphingolipids, particularly ceramide and glucosylceramide, to neurodegeneration and to motor unit dismantling in amyotrophic lateral sclerosis at late disease stage. We report here altered levels of gangliosides in the cerebrospinal fluid of amyotrophic lateral sclerosis patients in early disease stage. Conduritol B epoxide is an inhibitor of acid beta-glucosidase, and lowers glucosylceramide degradation. Glucosylceramide is the precursor for all of the more complex glycosphingolipids. In SOD1G86R mice, an animal model of amyotrophic lateral sclerosis, conduritol B epoxide preserved ganglioside distribution at the neuromuscular junction, delayed disease onset, improved motor function and preserved motor neurons as well as neuromuscular junctions from degeneration. Conduritol B epoxide mitigated gene dysregulation in the spinal cord and restored the expression of genes involved in signal transduction and axonal elongation. Inhibition of acid beta-glucosidase promoted faster axonal elongation in an in vitro model of neuromuscular junctions and hastened recovery after peripheral nerve injury in wild type mice. Here, we provide evidence that glycosphingolipids play an important role in muscle innervation, which degenerates in amyotrophic lateral sclerosis from the early disease stage. This is a first proof of concept study showing that modulating the catabolism of glucosylceramide may be a therapeutic target for this devastating disease.

International audience; The present study was undertaken to determine whether subacute treatment with aqueous extract of carob (Ceratonia siliqua L.) pods (AECPs) protects against ethanol (EtOH)-induced oxidative stress in rat liver. Animals were divided into four groups: control, carob, EtOH and EtOH+carob. Wistar rats were intraperitoneally pretreated with AECP (600mg/kg body weight (bw)) during 7days and intoxicated for 6h by acute oral administration of EtOH (6g/kg bw) 24h after the last injection. We found that acute administration of EtOH leads to hepatotoxicity as monitored by the increase in the levels of hepatic marker aspartate aminotransferase and alanine aminotransferase as well as hepatic tissue injury. EtOH also increased the formation of malondialdehyde in the liver, indicating an increase in lipid peroxidation and depletion of antioxidant enzyme activities as superoxide dismutase, catalase and glutathione peroxidase. Subacute carob pretreatment prevented all the alterations induced by EtOH and returned their levels to near normal. Importantly, we showed that acute alcohol increased hepatic and plasmatic hydrogen peroxide and free iron levels. The carob pretreatment reversed EtOH effects to near control levels. These data suggest that carob could have a beneficial effect in inhibiting the oxidative damage induced by acute EtOH administration and that its mode of action may involve an opposite effect on plasma and tissue-free iron accumulation. Indeed, carob can be offered as a food additive to protect against EtOH-induced oxidative damage.

10, 5 figures, 3 tables, 1 suppl figure and 1 suppl table; International audience; Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(*-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(*-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

International audience; Iron-induced oxidative stress promotes hepatic injury in hereditary hemochromatosis, which can be influenced by genetic traits affecting antioxidant enzymes. We assessed the influence of Ala16Val-superoxide dismutase 2, Pro198Leu-glutathione peroxidase 1, and -463G/A-myeloperoxidase genotypes (high activity for the Ala, Pro, and G alleles, respectively) on the risks of cirrhosis and hepatocellular carcinoma (HCC) in patients homozygous for the C282Y-hemochromatosis (HFE) gene mutation. Both the 2G-myeloperoxidase genotype and carriage of one or two copies of the Ala-superoxide dismutase 2 allele were more frequent in patients with cirrhosis or HCC. Patients cumulating these two genetic traits had higher rates of cirrhosis and HCC than other patients.

International audience; BACKGROUND & AIMS: Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration. METHODS: Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion. RESULTS: Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase. CONCLUSION: Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.

International audience; G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

International audience; Large cholinergic synaptic terminals known as C-boutons densely innervate the soma and proximal dendrites of motoneurons that are prone to neurodegeneration in amyotrophic lateral sclerosis (ALS). Studies using the Cu/Zn-superoxide dismutase (SOD1) mouse model of ALS have generated conflicting data regarding C-bouton alterations exhibited during ALS pathogenesis. In the present work, a longitudinal study combining immunohistochemistry, biochemical approaches and extra- and intra-cellular electrophysiological recordings revealed that the whole spinal cholinergic system is modified in the SOD1 mouse model of ALS compared to wild type (WT) mice as early as the second postnatal week. In WT motoneurons, both C-bouton terminals and associated M2 postsynaptic receptors presented a complex age-related dynamic that appeared completely disrupted in SOD1 motoneurons. Indeed, parallel to C-bouton morphological alterations, analysis of confocal images revealed a clustering process of M2 receptors during WT motoneuron development and maturation that was absent in SOD1 motoneurons. Our data demonstrated for the first time that the lamina X cholinergic interneurons, the neuronal source of C-boutons, are over-abundant in high lumbar segments in SOD1 mice and are subject to neurodegeneration in the SOD1 animal model. Finally, we showed that early C-bouton system alterations have no physiological impact on the cholinergic neuromodulation of newborn motoneurons. Altogether, these data suggest a complete reconfiguration of the spinal cholinergic system in SOD1 spinal networks that could be part of the compensatory mechanisms established during spinal development.

International audience; Amyotrophic lateral sclerosis, the most common adult-onset motor neuron disease, leads to death within 3 to 5 years after onset. Beyond progressive motor impairment, patients with amyotrophic lateral sclerosis suffer from major defects in energy metabolism, such as weight loss, which are well correlated with survival. Indeed, nutritional intervention targeting weight loss might improve survival of patients. However, the neural mechanisms underlying metabolic impairment in patients with amyotrophic lateral sclerosis remain elusive, in particular due to the lack of longitudinal studies. Here we took advantage of samples collected during the clinical trial of pioglitazone (GERP-ALS), and characterized longitudinally energy metabolism of patients with amyotrophic lateral sclerosis in response to pioglitazone, a drug with well-characterized metabolic effects. As expected, pioglitazone decreased glycaemia, decreased liver enzymes and increased circulating adiponectin in patients with amyotrophic lateral sclerosis, showing its efficacy in the periphery. However, pioglitazone did not increase body weight of patients with amyotrophic lateral sclerosis independently of bulbar involvement. As pioglitazone increases body weight through a direct inhibition of the hypothalamic melanocortin system, we studied hypothalamic neurons producing proopiomelanocortin (POMC) and the endogenous melanocortin inhibitor agouti-related peptide (AGRP), in mice expressing amyotrophic lateral sclerosis-linked mutant SOD1(G86R). We observed lower Pomc but higher Agrp mRNA levels in the hypothalamus of presymptomatic SOD1(G86R) mice. Consistently, numbers of POMC-positive neurons were decreased, whereas AGRP fibre density was elevated in the hypothalamic arcuate nucleus of SOD1(G86R) mice. Consistent with a defect in the hypothalamic melanocortin system, food intake after short term fasting was increased in SOD1(G86R) mice. Importantly, these findings were replicated in two other amyotrophic lateral sclerosis mouse models based on TDP-43 (Tardbp) and FUS mutations. Finally, we demonstrate that the melanocortin defect is primarily caused by serotonin loss in mutant SOD1(G86R) mice. Altogether, the current study combined clinical evidence and experimental studies in rodents to provide a mechanistic explanation for abnormalities in food intake and weight control observed in patients with amyotrophic lateral sclerosis. Importantly, these results also show that amyotrophic lateral sclerosis progression impairs responsiveness to classical drugs leading to weight gain. This has important implications for pharmacological management of weight loss in amyotrophic lateral sclerosis.

Oxidative stress has been suggested to be an important mediator of dopaminergic cell death in Parkinson's disease (PD). We investigated the neuroprotective potential of Cu/Zn superoxide dismutase (SOD1) overexpression in the rat substantia nigra (SN) following adenovirus-mediated gene transfer. Human dopaminergic SK-N-SH cells were transduced with adenoviral vectors expressing either human SOD1 (Ad-SOD1) or β-galactosidase (Ad-βgal) before exposure to 1 mM of the 1-methyl-4-phenylpyridinium ion (MPP+). A strong neuroprotective effect of SOD1 gene transfer was observed in the SK-N-SH cells exposed to MPP+ compared with controls. Adult rats were then given unilateral injections of either Ad-SOD1 or Ad-βgal into the striatum, and MPP+ was administered 8 days later at the same location. Strong transgene expression was detected in the SN dopaminergic neurons, a consequence of retrograde axonal transport of the adenoviral particles. The amphetamine-induced rotational behavior of the rats was markedly lower in Ad-SOD1-injected rats than in control animals. Also, behavioral recovery significantly correlated with the number of tyrosine hydrolase-expressing neurons in the SN of the treated rats. These results are consistent with oxidative stress contributing to the MPP+-induced neurodegenerative process. They also indicate that SOD1 gene transfer into the nigrostriatal system may be a potential neuroprotective strategy for treating PD. © 2005 Wiley-Liss, Inc.

International audience; The familial form of amyotrophic lateral sclerosis (FALS) has been linked in some cases to dominant mutations in the gene encoding the Cu/Zn superoxide dismutase (SOD1) mutation. Transgenic mice bearing the G93A SOD1 mutation develop clinical symptoms and pathological features similar to those described in the human disease and represent a good model to explore the potential benefit of therapeutic agents. Using this animal model, we tested the efficacy of morphine and tianeptine treatments, separately and in association, on both disease progression and survival. Acute injection of either of them, administered daily and before the onset of the disease, significantly prolonged the survival of the transgenic mice.

Synopsis Objective Polyphenols are strong antioxidant molecules allowing prevention of skin photo-ageing damages, but their use is limited due to low solubility and toxicity towards skin cells. We postulated that enzymatic glucosylation could improve their solubility, stability and, consequently, their efficacy. The aim of this work was to study changes induced by addition of a glucose moiety on two polyphenols displaying very different chemical structures [caffeic acid (CA), epigallocatechin-3-gallate (EGCG) and there glucosylated form, Glc-CA and Glc-EGCG] by assessing their cytotoxic properties and their antioxidant and anti-inflammatory activities. Methods Their antioxidant effect was assessed first by the classical DPPH radical-scavenging method. Then, a panel of human skin cells (keratinocytes, melanocytes, fibroblasts and endothelial cells) was used to evaluate their effect on cell toxicity and their antioxidant activities. With this aim, a photo-ageing model based on UV irradiation of skin cells was established. Molecule activity was assessed on reactive oxygen species (ROS) production, on superoxide dismutase (SOD) and catalase activities and, finally, on inflammatory factor production IL-6, IL-8 and IL-1β. Results In an acellular model, antioxidant activity assessed by DPPH method was strongly reduced for Glc-CA compared to CA, whereas it remained the same for Glc-EGCG compared to EGCG. Glucosylated derivatives did not display more toxic effect on various skin cells. Moreover, toxicity was even strongly reduced for caffeic acid upon glucosylation. The efficacy of glucosyl-compounds against UV-induced ROS production was preserved, both with pre- and post-UV treatments. Particularly, a better antioxidant efficacy was shown by Glc-EGCG, vs. EGCG, on keratinocytes. In addition, an induction of SOD and catalase activity was clearly observed for Glc-CA. Both glucosyl-polyphenols display the same activity as their parent molecule in decreasing inflammatory factor production. Conclusion Our results demonstrated that enzymatic glucosylation of CA and EGCG led to an improved or preserved antioxidant activity in a cellular model of UV-induced skin ageing, despite the decrease in instantaneous antioxidant properties observed for Glc-CA. Glc-EGCG is specifically more active on keratinocytes, suggesting a specific targeting. Such glucosylated polyphenols displaying improved physicochemical and biological properties should be better candidates than natural ones for use in food additives and cosmetics. Resume Objectifs Les polyphenols sont des molecules ayant un fort pouvoir antioxydant permettant de prevenir les dommages dus au photovieillissement de la peau mais leur utilisation est limitee de par leur faible solubilite et leur toxicite envers les cellules de la peau. Nous avons postule que la glucosylation enzymatique pourrait ameliorer leurs solubilite, stabilite et, en consequence, leur efficacite. Le but de ce travail est d'etudier les changements induits par l'addition d'un glucose sur deux polyphenols ayant une structure chimique tres differente [acide cafeique (CA), epigallocatechin-3-gallate (EGCG) et leurs formes glucosylees, Glc-CA et Glc-EGCG] en evaluant leurs proprietes cytotoxiques et leurs activites antioxydante et anti-inflammatoire. Methodes Leur pouvoir antioxydant a d'abord ete evalue par la methode classique de reduction du radical DPPH. Puis, un ensemble de cellules de peau humaine (keratinocytes, melanocytes, fibroblastes et cellules endotheliales) a ete utilise pour evaluer leur effet sur la toxicite cellulaire et leurs activites anti-oxydantes. Pour cela, un modele de photovieillissement base sur l'irradiation UV de cellules de peau a ete etabli. L'activite des molecules a ete evaluee sur la production d'especes reactives de l'oxygene (ROS), sur les activites superoxyde dismutase (SOD) et catalase et, enfin, sur la production de facteurs anti-inflammatoires IL-6, IL-8 et IL1β. Resultats Dans un modele acellulaire, l'activite antioxydante mesuree par la methode au DPPH etait fortement reduite pour le Glc-CA compare au CA, alors qu'elle restait la meme pour le Glc-EGCG compare a l'EGCG. Les derives glucosyles ne presentaient pas plus d'effet toxique sur les differentes cellules de peau. De plus, la toxicite etait meme fortement reduite pour l'acide cafeique suite a sa glucosylation. L'efficacite des composes glucosyles contre la production de ROS induite par les UV etait preservee, a la fois lors des traitements pre et post-UV. En particulier, une meilleure efficacite antioxydante a ete demontree avec le Glc-EGCG, vs. EGCG, sur les keratinocytes. Une augmentation des activites SOD et catalase a ete nettement observee pour le Glc-CA. Les deux polyphenols glucosyles montrent la meme activite que leur molecule parente en diminuant la production de facteurs anti-inflammatoires. Conclusions Nos resultats demontrent que la glucosylation enzymatique de CA et EGCG conduit a la preservation de leur activite antioxydante dans un modele cellulaire de vieillissement de la peau par les UV, en depit de la diminution de l'activite antioxydante instantanee observe pour Glc-CA. Glc-EGCC est specifiquement plus actif que l'EGCG sur les keratinocytes, suggerant un ciblage specifique. Ces antioxydants glucosyles presentant une amelioration de leurs proprietes physicochimiques et biologiques devraient etre de meilleurs candidats que les molecules naturelles pour etre utilises comme complements alimentaires ou en cosmetique.

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 104 M−1 s−1 and 4.3 ± 0.4 × 104 M−1 s−1 at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr35. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys83 mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys83 present in Fe-SODB acts as an electron donor that repairs Tyr35 radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells. Fil: Martinez, Alejandra. Universidad de la República; Uruguay Fil: Peluffo, Gonzalo. Universidad de la República; Uruguay Fil: Petruk, Ariel Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad Nacional de Tucumán; Argentina Fil: Hugo, Martín. Universidad de la República; Uruguay Fil: Piñeyro, Dolores. Universidad de la República; Uruguay Fil: Demicheli, Veronica. Universidad de la República; Uruguay Fil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina Fil: Lima, Analia Ethel. Instituto Pasteur de Montevideo; Uruguay Fil: Batthyány, Carlos. Instituto Pasteur de Montevideo; Uruguay. Universidad de la República; Uruguay Fil: Duran, Rosario. Instituto Pasteur de Montevideo; Uruguay Fil: Robledo, Carlos Walter. Universidad de la República; Uruguay Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Larrieux, Nicole. Instituto Pasteur de Montevideo; Uruguay Fil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; Uruguay. Instituto Pasteur; Francia Fil: Trujillo, Madia. Universidad de la República; Uruguay Fil: Radi, Rafael. Universidad de la República; Uruguay Fil: Piacenza, Lucia. Universidad de la República; Uruguay

10, 5 figures, 3 tables, 1 suppl figure and 1 suppl table; International audience; Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

10, 5 figures, 3 tables, 1 suppl figure and 1 suppl table; International audience; Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

International audience; Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance. Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR-mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics and markedly enhanced the efficacy of antibiotic treatment in experimental infections.

Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as “AbA cells”) from primary spinal cord cultures of symptomatic rats expressing the SOD1G93A mutation. Isolation was based on AbA cells’ marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100β protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1G93A astrocytes. AbA-like aberrant astrocytes expressing S100β and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression. Fil: Diaz Amarilla, Pablo. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay Fil: Olivera Bravo, Silvia. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay Fil: Trias, Emiliano. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay Fil: Cragnolini, Andrea Beatriz. Instituto Pasteur de Montevideo. Laboratorio de Neurodegeneracion; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Martinez Palma, Laura. Universidad de la República; Uruguay. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay Fil: Cassina, Patricia. Universidad de la República; Uruguay Fil: Beckman, Joseph. State University of Oregon; Estados Unidos. Environmental Health Sciences Center; Estados Unidos Fil: Barbeito, Luis. Instituto Pasteur de Montevideo. Laboratorio de Neurodegeneracion; Uruguay. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay

International audience; The German Shepherd Dog (GSD) is a popular working and companion breed for which over 50 hereditary diseases have been documented. Herein, SNP profiles for 197 GSDs were generated using the Affymetrix v2 canine SNP array for a genome-wide association study to identify loci associated with four diseases: pituitary dwarfism, degenerative myelopathy (DM), congenital megaesophagus (ME), and pancreatic acinar atrophy (PAA). A locus on Chr 9 is strongly associated with pituitary dwarfism and is proximal to a plausible candidate gene, LHX3. Results for DM confirm a major locus encompassing SOD1, in which an associated point mutation was previously identified, but do not suggest modifier loci. Several SNPs on Chr 12 are associated with ME and a 4.7 Mb haplotype block is present in affected dogs. Analysis of additional ME cases for a SNP within the haplotype provides further support for this association. Results for PAA indicate more complex genetic underpinnings. Several regions on multiple chromosomes reach genome-wide significance. However, no major locus is apparent and only two associated haplotype blocks, on Chrs 7 and 12 are observed. These data suggest that PAA may be governed by multiple loci with small effects, or it may be a heterogeneous disorder.

International audience; Oxidative stress, resulting from accumulation of reactive oxygen species (ROS), plays a critical role on astrocyte death associated with neurodegenerative diseases. Astroglial cells produce endozepines, a family of biologically active peptides that have been implicated in cell protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on hydrogen peroxide (H(2) O(2) )-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H(2) O(2) for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by lactate dehydrogenase assay. The cytotoxic effect of H(2) O(2) was associated with morphological modifications that were characteristic of apoptotic cell death. H(2) O(2) -treated cells exhibited high level of ROS associated with a reduction of both superoxide dismutases (SOD) and catalase activities. Pre-treatment of astrocytes with low concentrations of ODN dose-dependently prevented cell death induced by H(2) O(2) . This effect was accompanied by a marked attenuation of ROS accumulation, reduction of mitochondrial membrane potential and activation of caspase 3 activity. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H(2) O(2) -evoked inhibition of SOD and catalase activities. Blockers of SOD and catalase suppressed the effect of ODN on cell survival. Taken together, these data demonstrate for the first time that ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death.

International audience; Chromogranins interact with mutant forms of superoxide dismutase 1 (SOD1) responsible for a portion of familial amyotrophic lateral sclerosis (ALS). A particular variation (P413L) in the chromogranin B gene, CHGB, has been recently associated with an earlier age at onset in both familial and sporadic ALS. The aim of our study was to evaluate the P413L chromogranin variation in French patients with sporadic amyotrophic lateral sclerosis. We developed a High Resolution DNA Melting (HRM) protocol to analyse the P413L variation in the CHGB gene in 540 French patients with sporadic ALS and 504 controls. The clinical characteristics of patients were analysed in relation to their genotype. Results showed that our study on a large cohort of French-Caucasian patients with SALS and controls failed to confirm an increased frequency of the 413L variant in SALS patients. This frequency was 5.3% in the SALS population and 5.5% in the control group. Moreover, we did not observe a previous observation of a difference of age at onset between T-allele carriers and non-carriers (median age of onset 60.4 vs. 62.0 years of age, respectively). Thus, our findings do not support the 413L variant of rs742710 as a risk factor for sporadic ALS in the French population.

International audience; A comparative study of proteome maps from control and Cd-exposed rat liver was performed using a new technology of two-dimensional liquid chromatography separation method (PF-2D system, Beckman Coulter). Rats were fed for one month 0 or 100 μg Cd g(-1). The between-replicate and between-sample variations showed good repeatability and suitable reproducibility for the two dimensions of separation of proteins. In this complex mixture, PF-2D led to the separation of two major peaks which differed between control and Cd-exposed rat livers, one being identified by mass spectrometry as Cu/Zn superoxide dismutase (SOD), a well-known biomarker of Cd exposure, the other as phosphatidylethanolamine binding protein (PEBP). SOD content was decreased in Cd-exposed rat liver, compared to the control group which was corroborated by a significant decrease of SOD activity. PEBP content also tended to be decreased after Cd exposure. Present results demonstrate interest but also limitations of proteomic approach using PF-2D system to analyze effects of chemicals on organisms.

International audience; Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.

The aim of the current study was to investigate effects of temperature and a mixture of herbicides on the physiological status of the bivalve Mya arenaria. Bivalves acclimated to two temperatures (7 and 18°C) were exposed for 28 d to 0.01 mg/L of a pesticide formulation containing dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy) propionic acid (mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). At days 7, 14, and 28, mortality, immune parameters (hemocyte number, phagocytic activity, and efficiency), biomarkers of oxidative stress (catalase [CAT] and superoxide dismutase [SOD] activities and malondialdehyde [MDA] content), the metabolic enzyme cytochrome C oxidase (CCO), a biomarker of pesticide exposure (acetylcholinesterase [AChE]), and the activity of an enzyme related to gametogenesis (aspartate transcarbamylase [ATCase]) were monitored in clam tissues. Gonadosomatic index (GSI), condition factor (CF), and sex were also assessed. In clams acclimated to 7°C, exposure to pesticide enhanced CCO activity and CF and decreased MDA content, hemocyte number, CAT, and SOD activities. In clams kept at 18°C, pesticide effects appeared minor compared with samples kept at 7°C. In bivalves acclimated to 18°C, CCO, SOD, and ATCase activity and MDA content were enhanced, and hemocyte number, CAT, and AchE activities and phagocytosis were suppressed. In samples exposed to pesticides, increased temperature enhanced MDA content and CCO and SOD activity and suppressed hemocyte number and CAT and AchE activity. A gradual sexual maturation was observed in both sexes through experimental time, but females had a higher sensitivity to temperature and pesticides compared to males. Increased temperature altered the ability of the sentinel species Mya arenaria to respond to pesticide exposures. Further work is needed to understand the impacts of increasing temperature on the whole St. Lawrence estuary ecosystem. Environ. Toxicol. Chem. 2011;30:132–141. © 2010 SETAC

A growing body of evidence supports the therapeutic effects of blueberry in neurodegenerative disorders. Biotransformation of blueberry juice by Serratia vaccinii bacteria increases its phenolic content and antioxidant activity. In neuronal cell culture, biotransformed blueberry juice (BJ) significantly increased the activity of antioxidant enzymes, namely catalase and superoxide dismutase. Moreover, BJ protected neurons against H2O2-induced cell death in a dose-dependent manner. This associated with the upregulation of mitogen-activated protein kinase (MAPK) family enzymes p38 and c-Jun N-terminal kinase (JNK) activation, as well as with the protection of extracellular signal-regulated kinase (ERK1/2) and MAPK/ERK kinase (MEK1/2) activity loss induced by H2O2. The present studies demonstrate that BJ can protect neurons against oxidative stress possibly by increasing antioxidant enzyme activities and activating p38- and JNK-dependent survival pathways while blocking MEK1/2- and ERK1/2-mediated cell death. Thus, BJ may represent a novel approach to prevent and to treat neurodegenerative disorders, and it may represent a source of novel therapeutic agents against these diseases.

International audience; BACKGROUND: Mutations in SOD1, ANG, VAPB, TARDBP and FUS genes have been identified in amyotrophic lateral sclerosis (ALS). METHODS: The relative contributions of the different mutations to ALS were estimated by systematically screening a cohort of 162 families enrolled in France and 500 controls (1000 chromosomes) using molecular analysis techniques and performing phenotype-genotype correlations. RESULTS: 31 pathogenic missense mutations were found in 36 patients (20 SOD1, 1 ANG, 1 VAPB, 7 TARDBP and 7 FUS). Surprisingly two FUS mutation carriers also harboured ANG variants. One family of Japanese origin with the P56S VAPB mutation was identified. Seven novel mutations (three in SOD1, two in TARDBP, two in FUS) were found. None of them was detected in controls. Segregation of detected mutations with the disease was confirmed in 11 families including five pedigrees carrying the novel mutations. Clinical comparison of SOD1, TARDBP, FUS and other familial ALS patients (with no mutation in the screened genes) revealed differences in site of onset (predominantly lower limbs for SOD1 and upper limbs for TARDBP mutations), age of onset (younger with FUS mutations), and in lifespan (shorter for FUS carriers). One third of SOD1 patients survived more than 7 years: these patients had earlier disease onset than those presenting with a more typical course. Differences were also observed among FUS mutations, with the R521H FUS mutation being associated with longer disease duration. CONCLUSIONS: This study identifies new genetic associations with ALS and provides phenotype-genotype correlations with both previously reported and novel mutations.

Myocardial damages due to ischemia-reperfusion (I/R) are recognized to be the result of a complex interplay between genetic and environmental factors. Epidemiological studies suggested that, among environmental factors, carbon monoxide (CO) urban pollution can be linked to cardiac diseases and mortality. The aim of this work was to evaluate the impact of exposure to CO pollution on cardiac sensitivity to I/R. Regional myocardial I/R was performed on isolated perfused hearts from rats exposed for 4 wk to air enriched with CO (30–100 ppm). Functional variables, reperfusion ventricular arrhythmias (VA) and cellular damages (infarct size, lactate dehydrogenase release) were assessed. Sarcomere length shortening and Ca2+handling were evaluated in intact isolated cardiomyocytes during a cellular anoxia-reoxygenation protocol. The major results show that prolonged CO exposure worsens myocardial I/R injuries, resulting in increased severity of postischemic VA, impaired recovery of myocardial function, and increased infarct size (60 ± 5 vs. 33 ± 2% of ischemic zone). The aggravating effects of CO exposure on I/R could be explained by a reduced myocardial enzymatic antioxidant status (superoxide dismutase −45%; glutathione peroxidase −49%) associated with impaired intracellular Ca2+handling. In conclusion, our results are consistent with the idea that chronic CO pollution dramatically increases the severity of myocardial I/R injuries.

International audience; Manganese (Mn) is an essential element, acting as cofactor in numerous enzymes. In particular, a Mn cluster is indispensable for the function of the oxygen-evolving complex of photosystem II. Metal transporters of the Natural Resistance-Associated Macrophage Protein (NRAMP) family have the ability to transport both iron and Mn. AtNRAMP3 and AtNRAMP4 are required for iron mobilization in germinating seeds. The results reported here show that, in adult Arabidopsis (Arabidopsis thaliana) plants, AtNRAMP3 and AtNRAMP4 have an important role in Mn homeostasis. Vacuolar Mn accumulation in mesophyll cells of rosette leaves of adult nramp3nramp4 double mutant plants was dramatically increased when compared with the wild type. This suggests that a considerable proportion of the cellular Mn pool passes through the vacuole and is retrieved in an AtNRAMP3/AtNRAMP4-dependent manner. The impaired Mn release from mesophyll vacuoles of nramp3nramp4 double mutant plants is associated with reduced growth under Mn deficiency. However, leaf AtNRAMP3 and AtNRAMP4 protein levels are unaffected by Mn supply. Under Mn deficiency, nramp3nramp4 plants contain less functional photosystem II than the wild type. These data are consistent with a shortage of Mn to produce functional photosystem II, whereas mitochondrial Mn-dependent superoxide dismutase activity is maintained under Mn deficiency in both genotypes. The results presented here suggest an important role for AtNRAMP3/AtNRAMP4-dependent Mn transit through the vacuole prior to the import into chloroplasts of mesophyll cells.

International audience; Alcohol consumption increases reactive oxygen species (ROS) formation, which can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. To test whether manganese superoxide dismutase (MnSOD) modulates acute alcohol-induced mitochondrial alterations, transgenic MnSOD-overexpressing (MnSOD(+++)) mice, heterozygous knockout (MnSOD(+/-)) mice, and wild-type (WT) littermates were sacrificed 2 or 24 h after intragastric ethanol administration (5 g/kg). Alcohol administration further increased MnSOD activity in MnSOD(+++) mice, but further decreased it in MnSOD(+/-) mice. In WT mice, alcohol administration transiently increased mitochondrial ROS formation, decreased mitochondrial glutathione, depleted and damaged mtDNA, and decreased complex I and V activities; alcohol durably increased inducible nitric-oxide synthase (NOS) expression, plasma nitrites/nitrates, and the nitration of tyrosine residues in complex V proteins. These effects were prevented in MnSOD(+++) mice and prolonged in MnSOD(+/-) mice. In alcoholized WT or MnSOD(+/-) mice, mtDNA depletion and the nitration of tyrosine residues in complex I and V proteins were prevented or attenuated by cotreatment with tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a superoxide scavenger; N(omega)-nitro-l-arginine methyl ester and N-[3-(aminomethyl)benzyl]acetamidine (1,400W), two NOS inhibitors; or uric acid, a peroxynitrite scavenger. In conclusion, MnSOD overexpression prevents, and MnSOD deficiency prolongs, mtDNA depletion after an acute alcohol binge in mice. The protective effects of MnSOD, tempol, NOS inhibitors, and uric acid point out a role of the superoxide anion reacting with NO to form mtDNA-damaging peroxynitrite.

International audience; RATIONALE: Epidemiologic studies associate atmospheric carbon monoxide (CO) pollution with adverse cardiovascular outcomes and increased cardiac mortality risk. However, there is a lack of data regarding cellular mechanisms in healthy individuals. OBJECTIVES: To investigate the chronic effects of environmentally relevant CO levels on cardiac function in a well-standardized healthy animal model. METHODS: Wistar rats were exposed for 4 weeks to filtered air (CO < 1 ppm) or air enriched with CO (30 ppm with five peaks of 100 ppm per 24-h period), consistent with urban pollution. Myocardial function was assessed by echocardiography and analysis of surface ECG and in vitro by measuring the excitation-contraction coupling of single left ventricular cardiomyocytes. MEASUREMENTS AND MAIN RESULTS: Chronic CO pollution promoted left ventricular interstitial and perivascular fibrosis, with no change in cardiomyocyte size, and had weak, yet significant, effects on in vivo cardiac function. However, both contraction and relaxation of single cardiomyocytes were markedly altered. Several changes occurred, including decreased Ca(2+) transient amplitude and Ca(2+) sensitivity of myofilaments and increased diastolic intracellular Ca(2+) subsequent to decreased SERCA-2a expression and impaired Ca(2+) reuptake. CO pollution increased the number of arrhythmic events. Hyperphosphorylation of Ca(2+)-handling and sarcomeric proteins, and reduced responses to beta-adrenergic challenge were obtained, suggestive of moderate CO-induced hyperadrenergic state. CONCLUSIONS: Chronic CO exposure promotes a pathological phenotype of cardiomyocytes in the absence of underlying cardiomyopathy. The less severe phenotype in vivo suggests a role for compensatory mechanisms. Arrhythmia propensity may derive from intracellular Ca(2+) overload.

Background During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes. Methods We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation. Results Repeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation. Conclusions Here we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.

International audience; This paper deals with the effects of exercise training on oxygen consumption (MO(2)) and ROS metabolism in the red muscle of trained and untrained female silver eels. Their critical swimming speed (U(crit)) was determined before and after a 4-day training (10h of swimming at 70% of U(crit) and 14 h at 50%, every day). The U(crit) of trained eels increased significantly (by about 7%). The in vitro MO(2) and ROS production by the red fibres were higher (not significant) in trained than in untrained eels, but the ROS production/MO(2) ratio was alike in both groups. The antioxidant-enzyme activities and lipoperoxidation index in trained eels were both lower than those of the untrained ones. These biochemical changes related to the increase in U(crit) suggest that such a training session could maintained or even increased aerobic power of the red muscle without deleterious impact by ROS. These regulations could play a role in the eel's swimming performance efficiency.

International audience; Reactive oxygen species have been related to the aetiology of cancer as they are known to be mitogenic and therefore capable of tumour promotion. The aim of this study was to assess the role of common variation in three polymorphic genes (MnSOD Ala-9Val, GPX1 Pro198Leu and CAT -262 C > T) coding for antioxidant defence enzymes in modulating individual susceptibility to hepatocellular carcinoma (HCC) using a case-control study (cases = 96 and controls = 222). PCR-RFLP and sequencing methods were used to determine the genotype. Overall, there were no associations between genotypes GPX1 and HCC risk (OR, 1.16; 95% CI, 0.56-2.42; p = 0.685). The MnSOD Ala/Ala and CAT TT genotypes were more frequent in HCC than in control (p = 0.001 and p = 0.072, respectively). Further analyses stratified by gender or HCV infection revealed that men and HCV-infected patients carrying CAT TT genotype had a higher risk to develop HCC when compared with controls (OR = 15.94; 95% CI, 3.48-72.92; p < 0.000001 and 12.01; 95% CI, 0.64-223.63, p = 0.056, respectively). Combined MnSOD Ala/Ala and GPx1 Leu/Leu had a synergistic effect on HCC risk, with an OR of 3.84 (p = 0.029). Furthermore an even more pronounced risk was observed when we combined MnSOD Ala/Ala and CAT TT (OR = 13.60, p = 0.023). It appears that variants in MnSOD, CAT or GPX1 have an influence on HCC risk in this cohort. Furthermore, it is possible that cumulative defects in protection from oxidative stress may result in increased risk of liver cancer in the Moroccan population.

International audience; Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which targets motor neurons of the cortex, brainstem and spinal cord. About 5-10% of all amyotrophic lateral sclerosis cases are familial (FALS), and 15-20% of FALS cases are caused by mutations in the zinc-copper superoxide dismutase gene (SOD1). We identified a large family from France with ten members affected with ALS. Linkage was established to the SOD1 locus on chromosome 21 and genomic and cDNA sequencing was performed for the SOD1 gene. This revealed an activated pseudoexon between exons 4 and 5 that was present in two tested members of the family. Translation of this 43 base pair exon results in the introduction of seven amino acids before a stop codon is present, leading to a prematurely truncated SOD1 protein product of 125 amino acids. Sequencing intron 4 in a patient revealed a eterozygous change 304 bp before exon 5 (c.358 - 304C > G), but only 5 bp after the cryptic exon, thus causing this alternative splice product. This mutation segregated in all affected individuals of the family. This adds an additional genetic mechanism for developing OD1-linked ALS and is one which can be more readily targeted by gene therapy.

International audience; OBJECTIVE: To determine the effects of a dried aqueous extract of cinnamon on antioxidant status of people with impaired fasting glucose that are overweight or obese. METHODS: Twenty-two subjects, with impaired fasting blood glucose with BMI ranging from 25 to 45, were enrolled in a double-blind placebo-controlled trial. Subjects were given capsules containing either a placebo or 250 mg of an aqueous extract of cinnamon (Cinnulin PF) two times per day for 12 weeks. Plasma malondialdehyde (MDA) concentrations were assessed using high performance liquid chromatography and plasma antioxidant status was evaluated using ferric reducing antioxidant power (FRAP) assay. Erythrocyte Cu-Zn superoxide (Cu-Zn SOD) activity was measured after hemoglobin precipitation by monitoring the auto-oxidation of pyrogallol and erythrocyte glutathione peroxidase (GPx) activity by established methods. RESULTS: FRAP and plasma thiol (SH) groups increased, while plasma MDA levels decreased in subjects receiving the cinnamon extract. Effects were larger after 12 than 6 weeks. There was also a positive correlation (r = 0.74; p = 0.014) between MDA and plasma glucose. CONCLUSION: This study supports the hypothesis that the inclusion of water soluble cinnamon compounds in the diet could reduce risk factors associated with diabetes and cardiovascular disease.

International audience; The continuous recycling of haem iron following phagocytosis and catabolism of senescent and damaged red blood cells by macrophages is a crucial process in the maintenance of systemic iron homoeostasis. However, little is known about macrophage iron handling in haemolytic states resulting from a deficiency in antioxidant defences. Our observations indicate that the recently described chronic, but moderate regenerative, haemolytic anaemia of aged SOD1 (superoxide dismutase 1)-knockout mice is associated with red blood cell modifications and sensitivity to both intra- and extra-vascular haemolysis. In the present study, we have characterized the molecular pathways of iron turnover in the liver of Sod1-deficient mice. Despite iron accumulation in liver macrophages, namely Kupffer cells, we did not measure any significant change in non-haem liver iron. Interestingly, in Kupffer cells, expression of the rate-limiting enzyme in haem degradation, haem oxygenase-1, and expression of the iron exporter ferroportin were both up-regulated, whereas the hepcidin mRNA level in the liver was decreased in Sod1-/- mice. These results suggest that concerted changes in the hepatic expression of iron- and haem-related genes in response to haemolytic anaemia in Sod1-/- mice act to reduce toxic iron accumulation in the liver and respond to the needs of erythropoiesis.

International audience; Chelation therapy is thought to not only remove contaminating metals but also to decrease free radical production. However, in standard ethylene diamine tetracetic acid (EDTA) chelation therapy, high doses of vitamin C with potential pro-oxidant effects are often added to the chelation solution. The authors demonstrated previously that the intravenous administration of the standard chelation cocktail, containing high amounts of vitamin C, resulted in an acute transitory pro-oxidant burst that should be avoided in the treatment of pathologies at risk of increased oxidative stress such as diabetes and cardiovascular disease. The current study was designed to determine the acute and chronic biochemical effects of chelation therapy on accepted clinical, antioxidant variables. An EDTA chelation cocktail not containing ascorbic acid was administered to six adult patients for five weeks (10 sessions of chelation therapy); antioxidant indicators were monitored. Immediately after the initial chelation session, in contrast with the data previously reported with the standard cocktail containing high doses of vitamin C, none of the oxidative stress markers were adversely modified. After five weeks, plasma peroxide levels, monitored by malondialdehyde, decreased by 20 percent, and DNA damage, monitored by formamidopyrimidine-DNA glycosylase (Fpg) sensitive sites, decreased by 22 percent. Remaining antioxidant-related variables did not change. In summary, this study demonstrates that multiple sessions of EDTA chelation therapy in combination with vitamins and minerals, but without added ascorbic acid, decreases oxidative stress. These results should be beneficial in the treatment of diseases associated with increased oxidative stress such as diabetes and cardiovascular diseases.

International audience; OBJECTIVE: The aim of the study was to investigate whether zinc supplementation affects antioxidant status in European middle-aged and elderly people. DESIGN: Multicentre prospective intervention study, randomized, double-blind, placebo-control. SETTING: France (Clermont-Ferrand/Theix, and Grenoble), Italy (Rome), Northern Ireland (Coleraine). SUBJECTS: A total of 387 healthy middle-aged (55-70 yrs) and free-living older aged (70-85 yrs) subjects were randomly allocated to three groups: 0, 15 or 30 mg zinc gluconate/d in addition to usual dietary intake during 6 months. METHODS: Oxidative stress status was evaluated by measurement of protein oxidation (plasma thiol groups), lipid peroxidation (plasma thio-barbituric acid reactants, TBARS), whole blood glutathione levels, erythrocyte copper/zinc superoxide dismutase activity and plasma antioxidant status (ferric reducing antioxidant power assay), at baseline and after 3 and 6 months. RESULTS: Zinc supplementation did not alter oxidative stress markers and antioxidant defenses in elderly, after 3 or 6 months, except an increase in Cu/Zn superoxide dismutase activity. CONCLUSIONS: In apparently healthy free living elderly people, a single zinc supplementation had no effects on oxidative stress status.

International audience; We recently reported that in vitro Cognac polyphenolic compounds (CPC) induce NO-dependent vasorelaxant effects and stimulate cardiac function. In the present study, we aim to investigate the effect of CPC on both nitric oxide (NO) and superoxide anions (O(2)(-)) production in cultured human endothelial cells. In addition, its effect on the bradykinin (BK)-induced NO production was also tested. The role and sources of O(2)(-) in the concomitant effect of BK plus CPC were pharmacologically determined. NO and O(2)(-) signals were measured using electron paramagnetic resonance technique using specific spin trappings. Both, CPC and BK induced an increase in NO production in human endothelial cells. The combination of both further enhanced NO release. The capacity of CPC plus BK to increase NO signal was blunted by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and was enhanced in the presence either of superoxide dismutase or catalase. Moreover, CPC plus BK response was greater after inhibition of either NADPH oxidase by apocynin or xanthine oxidase by allopurinol but it was not affected by rotenone. CPC did not affect O(2)(-) level either alone or after its increase upon lipopolysaccharide treatment. Finally, the capacity of BK alone to increase NO was enhanced either by apocynin or allopurinol. Altogether, these data demonstrate that CPC is able to directly increase NO production without affecting O(2)(-) and enhances the BK-induced NO production in human endothelial cells. The data highlight the ability of BK to stimulate not only NADPH oxidase- but also xanthine oxidase-inhibitor sensitive mechanisms that reduce its efficiency in increasing NO either alone or in the presence of CPC. These results bring pharmacological evidence for vascular protection by CPC via its potentiating effect of BK response in terms of endothelial NO release.

International audience; A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond.

International audience; Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Assigning functions to every gene in a living organism is the next challenge for functional genomics. In fact, 85–90% of the 19,000 genes of the nematode Caenorhabditis elegans genome do not produce any visible phenotype when inactivated, which hampers determining their function, especially when they do not belong to previously characterized gene families. We used 1 H high-resolution magic angle spinning NMR spectroscopy ( 1 H HRMAS-NMR) to reveal the latent phenotype associated to superoxide dismutase ( sod -1) and catalase ( ctl -1) C. elegans mutations, both involved in the elimination of radical oxidative species. These two silent mutations are significantly discriminated from the wild-type strain and from each other. We identify a metabotype significantly associated with these mutations involving a general reduction of fatty acyl resonances from triglycerides, unsaturated lipids being known targets of free radicals. This work opens up perspectives for the use of 1 H HRMAS-NMR as a molecular phenotyping device for model organisms. Because it is amenable to high throughput and is shown to be highly informative, this approach may rapidly lead to a functional and integrated metabonomic mapping of the C. elegans genome at the systems biology level.

Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an alpha-nNOS knockout mutant (nNOSalpha(-/-)) to reduce the production of NO. Analysis of the double mutants showed that blocking alpha-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluRdelta2(+/Lc):nNOS(-/-) double mutant Purkinje cells, which suggests that the failure to rescue GluRdelta2(+/Lc):nNOS(-/-) Purkinje cells may be explained by the induction of alternative nNOS isoforms.

International audience; Aging triggers several abnormalities in muscle glycolytic fibers including increased proteolysis, reactive oxygen species (ROS) production and apoptosis. Since the mitochondria are the main site of substrate oxidation, ROS production and programmed cell death, we tried to know whether the cellular disorders encountered in sarcopenia are due to abnormal mitochondrial functioning. Gastrocnemius mitochondria were extracted from adult (6 months) and aged (21 months) male Wistar rats. Respiration parameters, opening of the permeability transition pore and ROS production, with either glutamate (amino acid metabolism) or pyruvate (glucose metabolism) as a respiration substrate, were evaluated at different matrix calcium concentrations. Pyruvate dehydrogenase and respiratory complex activities as well as their contents measured by Western blotting analysis were determined. Furthermore, the fatty acid profile of mitochondrial phospholipids was also measured. At physiological calcium concentration, state III respiration rate was lowered by aging in pyruvate conditions (-22%), but not with glutamate. The reduction of pyruvate oxidation resulted from a calcium-dependent inactivation of the pyruvate dehydrogenase system and could provide for the well-known proteolysis encountered during sarcopenia. Matrix calcium loading and aging increased ROS production. They also reduced the oxidative phosphorylation. This was associated with lower calcium retention capacities, suggesting that sarcopenic fibers are more prone to programmed cell death. Aging was also associated with a reduced mitochondrial superoxide dismutase activity, which does not intervene in toxic ROS overproduction but could explain the lower calcium retention capacities. Despite a lower content, cytochrome c oxidase displayed an increased activity associated with an increased n-6/n-3 polyunsaturated fatty acid ratio of mitochondrial phospholipids. In conclusion, we propose that mitochondria obtained from aged muscle fibers display several functional abnormalities explaining the increased proteolysis, ROS overproduction and vulnerability to apoptosis exhibited by sarcopenic muscle. These changes appear to be related to modifications of the fatty acid profile of mitochondrial lipids.

Amyotrophic lateral sclerosis is a lethal, adult-onset disease characterized by progressive degeneration of motoneurons. Recent data have suggested that the disease could be linked to abnormal development of the motor nervous system. Therefore, we investigated the electrical properties of lumbar motoneurons in an in-vitro neonatal spinal cord preparation isolated from SOD1(G85R) mice, which is a transgenic model of amyotrophic lateral sclerosis. The study was performed on young animals at the beginning of their second week, between postnatal days 6 and 10. Measurements of resting membrane potential and action potential characteristics of motoneurons were similar in wild-type and SOD1(G85R) mice. However, the input resistance of motoneurons from transgenic mice was significantly lower than that of wild-type animals, whereas their membrane capacitance was increased, strongly suggesting larger SOD1(G85R) motoneurons. Furthermore, the slope of the frequency-intensity curve was steeper in motoneurons from wild-type pups. Interestingly, the input resistance as well as the slope of the frequency-intensity curves of other spinal neurons did not show such differences. Finally, the amplitude of dorsal root-evoked potentials following high-intensity stimulation was significantly smaller in SOD1(G85R) motoneurons. The superoxide dismutase 1 mutation thus induces specific alterations of the functional properties of motoneurons early in development.

International audience; A mitochondrial matrix copper ligand (CuL) complex, conserved in mammalian cells, is the likely source of copper for assembly of cytochrome c oxidase (CcO) and superoxide dismutase 1 (Sod1) within the intermembrane space (IMS) in yeast. Targeting the copper-binding proteins human Sod1 and Crs5 to the mitochondrial matrix results in growth impairment on non-fermentable medium caused by decreased levels of CcO. This effect is reversed by copper supplementation. Matrix-targeted Crs5 diminished Sod1 protein within the IMS and impaired activity of an inner membrane tethered human Sod1. Copper binding by the matrix-targeted proteins attenuates levels of the CuL complex without affecting total mitochondrial copper. These data suggest that attenuation of the matrix CuL complex via heterologous competitors limits available copper for metallation of CcO and Sod1 within the IMS. The ligand also exists in the cytoplasm in an apparent metal-free state.