Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.     

Life extension through neurofibromin mitochondrial regulation and antioxidant therapy for neurofibromatosis-1 in Drosophila melanogaster

We investigated the pathophysiology of neurofibromatosis-1 (NF1) in Drosophila melanogaster by inactivation or overexpression of the NF1 gene. NF1 gene mutants had shortened life spans and increased vulnerability to heat and oxidative stress in association with reduced mitochondrial respiration and elevated reactive oxygen species (ROS) production. Flies overexpressing NF1 had increased life spans, improved reproductive fitness, increased resistance to oxidative and heat stress in association with increased mitochondrial respiration and a 60% reduction in ROS production. These phenotypic effects proved to be modulated by the adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A pathway, not the Ras/Raf pathway. Treatment of wild-type D. melanogaster with cAMP analogs increased their life span, and treatment of NF1 mutants with metalloporphyrin catalytic antioxidant compounds restored their life span. Thus, neurofibromin regulates longevity and stress resistance through cAMP regulation of mitochondrial respiration and ROS production, and NF1 may be treatable using catalytic antioxidants.     

NAD(P)H:quinone oxidoreductase 1 NQO1*2 genotype (P187S) is a strong prognostic and predictive factor in breast cancer

NQO1 guards against oxidative stress and carcinogenesis and stabilizes p53. We find that a homozygous common missense variant (NQO1(*)2, rs1800566(T), NM_000903.2:c.558C>T) that disables NQO1 strongly predicts poor survival among two independent series of women with breast cancer (P = 0.002, N = 1,005; P = 0.005, N = 1,162), an effect particularly evident after anthracycline-based adjuvant chemotherapy with epirubicin (P = 7.52 x 10(-6)) and in p53-aberrant tumors (P = 6.15 x 10(-5)). Survival after metastasis was reduced among NQO1(*)2 homozygotes, further implicating NQO1 deficiency in cancer progression and treatment resistance. Consistently, response to epirubicin was impaired in NQO1(*)2-homozygous breast carcinoma cells in vitro, reflecting both p53-linked and p53-independent roles of NQO1. We propose a model of defective anthracycline response in NQO1-deficient breast tumors, along with increased genomic instability promoted by elevated reactive oxygen species (ROS), and suggest that the NQO1 genotype is a prognostic and predictive marker for breast cancer.     

Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint

During electron transport, the mitochondrion generates ATP and reactive oxygen species (ROS), a group of partially reduced and highly reactive metabolites of oxygen. In this in vivo genetic analysis in Drosophila melanogaster, we establish that disruption of complex I of the mitochondrial electron transport chain specifically retards the cell cycle during the G1-S transition. The mechanism involves a specific signaling cascade initiated by ROS and transduced by ASK-1, JNK, FOXO and the Drosophila p27 homolog, Dacapo. On the basis of our data combined with previous analyses of the system, we conclude that mitochondrial dysfunction activates at least two retrograde signals to specifically enforce a G1-S cell cycle checkpoint. One such signal involves an increase in AMP production and downregulation of cyclin E protein; another independent pathway involves increased ROS and upregulation of Dacapo. Thus, our results indicate that the mitochondrion can use AMP and ROS at sublethal concentrations as independent signaling molecules to modulate cell cycle progression.     

Cidea-deficient mice have lean phenotype and are resistant to obesity

The thermogenic activity of brown adipose tissue (BAT), important for adaptive thermogenesis and energy expenditure, is mediated by the mitochondrial uncoupling protein1 (Ucp1) that uncouples ATP generation and dissipates the energy as heat. We show here that Cidea, a protein of unknown function sharing sequence similarity with the N-terminal region of DNA fragmentation factors Dffb and Dffa, is expressed at high levels in BAT. Cidea-null mice had higher metabolic rate, lipolysis in BAT and core body temperature when subjected to cold treatment. Notably, Cidea-null mice are lean and resistant to diet-induced obesity and diabetes. Furthermore, we provide evidence that the role of Cidea in regulating thermogenesis, lipolysis and obesity may be mediated in part through its direct suppression of Ucp1 activity. Our data thus indicate a role for Cidea in regulating energy balance and adiposity.     

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2. These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2-/- mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2-/- mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2-/- mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2-/- mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2-/- mice following Ucn, but Crhr2-/- mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2-/- mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2-/- mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.     

Loss of collagenase-2 confers increased skin tumor susceptibility to male mice

Matrix metalloproteinases (MMPs) have fundamental roles in tumor progression, but most clinical trials with MMP inhibitors have not shown improvements in individuals with cancer. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8(-/-)mice. Female Mmp8(-/-)mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.     

Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana

Plant immune responses are usually accompanied by the production of extracellular superoxide at and surrounding infection sites. Extracellular reactive oxygen intermediates (ROIs) in plants were proposed to drive programmed cell death correlated with disease resistance (the hypersensitive response). ROIs derived from this oxidative burst are generated by plasma membrane NADPH oxidases, anchored by gp91(phox) proteins related to those responsible for the respiratory oxidative burst activated in mammalian neutrophils during infection. Mutation of Arabidopsis thaliana respiratory burst oxidase (Atrboh) genes eliminated pathogen-induced ROI production but had only a modest effect on the hypersensitive response. We show that Atrboh function can be activated by exogenous ROIs. Unexpectedly, the subsequent oxidative burst can suppress cell death in cells surrounding sites of NADPH oxidase activation. This cell death requires salicylic acid, a plant immune system activator. Thus, ROIs generated by Atrboh proteins can antagonize salicylic acid-dependent pro-death signals. These results have implications for understanding how salicylic acid activates defense signaling in cells spatially removed from infection sites without causing cell death.     

Apport de l'étude de l'avidité des IgG spécifiques pour l'exclusion d'une toxoplasmose évolutive: adaptation à l'automate alpha 4®

The study of anti-Toxoplasma gondii IgG avidity of 108 sera from patients affected by toxoplasmosis and 85 sequential sera from pregnant women presenting an acute infection allows to confirm the interest and the pertinence of the Toxo IgG Avidity test (SFRI Laboratoire), entirely automatised on the Alpha 4^® (SFRI Laboratoire). The avidity test of IgG SFRI^® has been compared to a method to date the infection based on a report of titers obtained between 2 different IgG determinations. (Abbott Diagnostic). Sequentials sera have also been studied by determining specific IgG, IgM, and IgA. The results obtained confirm that an Avidity Index (AI) higher or equal to 0.6 corresponds to a toxoplasmosis acquired more than one year before, an AI between 0.3 and 0.6 is characteristic to a toxoplasmosis acquired more than three months and less than one year before and that an AI higher than 0.3 exludes a recent infection of less than 3 months in 97 % of cases. The interest of the study of IgG avidity to exclude a recent infection, notably when a first sample reveals the presence of anti-Toxoplasma gondii IgM, is confirmed.     

Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation

In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF). The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response. We generated Cdc25b-deficient (Cdc25b-/-) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b-/- mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b-/- mice were sterile, and Cdc25b-/- oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b-/- oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b-/- female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.     

Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.     

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus

Aicardi-Goutières syndrome (AGS) presents as a severe neurological brain disease and is a genetic mimic of the sequelae of transplacentally acquired viral infection. Evidence exists for a perturbation of innate immunity as a primary pathogenic event in the disease phenotype. Here, we show that TREX1, encoding the major mammalian 3' --> 5' DNA exonuclease, is the AGS1 gene, and AGS-causing mutations result in abrogation of TREX1 enzyme activity. Similar loss of function in the Trex1(-/-) mouse leads to an inflammatory phenotype. Our findings suggest an unanticipated role for TREX1 in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response.     

Lifespan and mitochondrial control of neurodegeneration

We examine the allometric (comparative scaling) relationships between rates of neurodegeneration resulting from equivalent mutations in a diverse group of genes from five mammalian species with different maximum lifespan potentials. In both retina and brain, rates of neurodegeneration vary by as much as two orders of magnitude and are strongly correlated with maximum lifespan potential and rates of formation of mitochondrial reactive oxygen and nitrogen species (RONS). Cell death in these disorders is directly or indirectly regulated by the intrinsic mitochondrial cell death pathway. Mitochondria are the main source of RONS production and integrate cellular stress signals to coordinate the intrinsic pathway. We propose that these two functions are intimately related and that steady-state RONS-mediated signaling or damage to the mitochondrial stress-integration machinery is the principal factor setting the probability of cell death in response to a diverse range of cellular stressors. This provides a new and unifying framework for investigating neurodegenerative disorders.     

Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTPsigma

The LAR-family protein tyrosine phosphatase sigma (PTPsigma, encoded by the gene Ptprs) consists of a cell adhesion-like extracellular domain composed of immunoglobulin and fibronectin type-III repeats, a single transmembrane domain and two intracellular catalytic domains. It was previously shown to be expressed in neuronal and lung epithelial tissues in a developmentally regulated manner. To study the role of PTPsigma in mouse development, we inactivated Ptprs by gene targeting. All Ptprs+/- mice developed normally, whereas 60% of Ptprs-/- mice died within 48 hours after birth. The surviving Ptprs-/- mice demonstrated stunted growth, developmental delays and severe neurological defects including spastic movements, tremor, ataxic gait, abnormal limb flexion and defective proprioception. Histopathology of brain sections revealed reduction and hypocellularity of the posterior pituitary of Ptprs-/- mice, as well as a reduction of approximately 50-75% in the number of choline acetyl transferase-positive cells in the forebrain. Moreover, peripheral nerve electrophysiological analysis revealed slower conduction velocity in Ptprs-/- mice relative to wild-type or heterozygous animals, associated with an increased proportion of slowly conducting, small-diameter myelinated fibres and relative hypomyelination. By approximately three weeks of age, most remaining Ptprs-/- mice died from a wasting syndrome with atrophic intestinal villi. These results suggest that PTPsigma has a role in neuronal and epithelial development in mice.     

Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity

Neuropathy target esterase (NTE) is involved in neural development and is the target for neurodegeneration induced by selected organophosphorus pesticides and chemical warfare agents. We generated mice with disruptions in Nte, the gene encoding NTE. Nte(-/-) mice die after embryonic day 8, and Nte(+/-) mice have lower activity of Nte in the brain and higher mortality when exposed to the Nte-inhibiting compound ethyl octylphosphonofluoridate (EOPF) than do wild-type mice. Nte(+/-) and wild-type mice treated with 1 mg per kg of body weight of EOPF have elevated motor activity, showing that even minor reduction of Nte activity leads to hyperactivity. These studies show that genetic or chemical reduction of Nte activity results in a neurological phenotype of hyperactivity in mammals and indicate that EOPF toxicity occurs directly through inhibition of Nte without the requirement for Nte gain of function or aging.