Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Post-transcriptional regulation of circadian gene expression by miRNAs

La grande majorité des organismes vivants ont développé un système d'horloges biologiques internes, appelées aussi horloges circadiennes, contrôlant l'expression de gênes impliqués dans de nombreux processus moléculaires et comportementaux. Au cours de la dernière décennie, des analyses « microarray » et séquençages à haut débit sur divers tissus de mammifères, indiquent que jusqu'à 20% du transcriptome serait sous contrôle circadien. Il était jusqu'à présent admis que la majorité des ARNm ayant une accumulation rythmique était générée par une transcription qui était elle-même rythmique. Toutefois, de récentes études ont suggéré qu'une proportion considérable des ARNm cycliques serait en fait générée par des mécanismes post-transcriptionnelles, incluant une régulation par micro-ARN (miARN). Lorsque j'ai débuté mon travail de thèse, l'influence des miARN sur l'expression des gènes circadiens, au niveau pangénomique, était encore méconnue. Par l'utilisation d'un modèle murin, dont la biogenèse des miARN a été spécifiquement désactivée au niveau des cellules hépatiques (knockout conditionnel pour Dicer), je me suis donc intéressée au rôle que jouaient ces molécules régulatrices sur la rythmicité de l'expression génique dans le foie. Des séquençages sur l'ensemble du transcriptome révèlent que l'horloge interne du foie est étonnement résistante à la perte totale des miARN. Nous avons cependant trouvé que les miARN agissent de façon importante sur la régulation de l'expression des gènes contrôlés par l'horloge moléculaire. La corégulation par les miARN, affectant jusqu'à 30% des gènes transcrits de façon rythmiques, conduit ainsi à une modulation de phase et d'amplitude du rythme de l'abondance des ARNm. En revanche, seuls peu de transcrits dépendent uniquement des miARN pour la rythmicité de leur accumulation. Enfin, mon travail met en évidence plusieurs miARN spécifiques, qui semblent préférentiellement moduler l'expression des gènes cycliques et permet l'identification de voies hépatiques particulièrement sujettes à une double régulation par les miARN et l'horloge biologique interne. La première masse d'analyses a essentiellement porté sur le rôle que jouent les miARN au niveau de l'expression des gènes contrôlés par l'horloge interne. Dans deux études de suivi, je me suis penchée sur deux aspects supplémentaires et complémentaires de la manière dont les miARN et l'oscillation de l'expression des gènes interagissent. Dans les hépatocytes murins, spécifiquement privés de Dicer, je me suis demandée si un phénotype horloge avait pu être masqué, dû à un entraînement stable de l'horloge du foie par l'horloge maîtresse du cerveau. J'ai donc commencé une série d'expériences ambitieuses (impliquant la mesure de la rythmicité du foie in vivo, chez l'animal vivant) afin de déséquilibrer l'entrainement de l'horloge hépatique via l'utilisation d'un protocole nutritionnel spécifique. Les premiers résultats suggèrent que dans des conditions où l'animal subit une restriction alimentaire pendant la journée, les miARN sont importants dans la cinétique d'adaptation des organes périphériques à un nouvel horaire de sustentation. Dans une deuxième ligne de recherche, j'ai plus profondément étudié quels seraient les miARN responsables des rythmes post-transcriptionnels des ARNm, en utilisant le séquençage de « small » ARN sur 24h. L'analyse est en cours et se poursuivra après l'obtention de mon diplôme. De façon générale, mon travail révèle d'importants et nouveaux rôles des miARN dans la modulation de l'expression circadienne des gènes hépatiques. De plus, le set de données générées dans l'étude déjà publiée, peut dorénavant servir de ressource valable pour de prochaines investigations sur le rôle physiologique que les miARN jouent au niveau du foie. -- Most living organisms have developed internal timing systems, called circadian clocks, to drive the rhythmic expression of genes involved in many molecular and behavioral processes. Over the last decade, microarray analyses and high- throughput sequencing from various mammalian tissues have indicated that up to 20% of the transcriptome are under circadian control. It was generally assumed that the majority of rhythmic mRNA accumulation is generated by rhythmic transcription. However, recent studies have suggested that a considerable proportion of mRNA cycling may actually be generated by post-transcriptional mechanisms, including by microRNAs. When I started my thesis work, it was still unknown how miRNAs influence circadian gene expression in a genome-wide fashion. Using a mouse model in which miRNA biogenesis can be inactivated in hepatocytes (conditional Dicer knockout mouse), I have thus addressed the role that these regulatory molecules play in rhythmic gene expression in the liver. Whole transcriptome sequencing revealed that the hepatic core clock was surprisingly resilient to total miRNA loss. However, we found that miRNAs acted as important regulators of clock-controlled gene expression. Co- regulation by miRNAs, which affected up to 30% of rhythmically transcribed genes, thus led to the modulation of phases and amplitudes of mRNA abundance rhythms. By contrast, only very few transcripts were strictly dependent on miRNAs for their rhythmic accumulation. Finally, my work highlights several specific miRNAs that appear to preferentially modulate cyclic gene expression, and identifies pathways in the liver that are particularly prone to dual regulation through miRNAs and the clock. The first bulk of analyses mainly dealt with the role that miRNAs play at the level of rhythmic clock output gene expression. In two follow-up studies I further delved into two additional, complementary aspects of how miRNAs and gene expression oscillations interact. First, I addressed whether a core clock phenotype in the hepatocyte-specific Dicer knockout could have been masked due to the stable entrainment of the liver clock by the animals' master clock in the brain. I thus started a series of ambitious experiments (involving the in vivo recording of liver rhythms in live animals) to bring the stable entrainment of the liver clock out of equilibrium using specific feeding protocols. My first results suggest that under conditions when animals are challenged by food restriction to daytime, miRNAs are important for the kinetics of adapting to unusual mealtime in peripheral tissue. In a second line of research, I have more carefully investigated which miRNAs are responsible for post- transcriptional mRNA rhythms using small RNA sequencing around-the-clock. The analyses are ongoing and will be continued after my graduation. Overall, my work uncovered important and novel roles of miRNA activity in shaping hepatic circadian gene expression; moreover, the datasets collect in the published studies can serve as a valuable resource for further investigations into the physiological roles that miRNAs play in liver. -- L'alternance du jour et de la nuit dirige depuis longtemps la vie quotidienne des êtres humains et de la plupart des organismes sur terre. Ce cycle de 24 heures façonne beaucoup de changements comportementaux et physiologiques tels que la vigilance, la température corporelle et le sommeil. Les rythmes journaliers, appelés rythmes circadiens, sont dirigés par des horloges biologiques tournant dans presque chaque cellule du corps. Une structure dans le cerveau agit en tant qu'horloge maitresse pour synchroniser les horloges internes entre elles et en fonction des signaux de jour/nuit extérieurs. Dans les cellules "les gènes de l'horloge" sont activés et désactivés une fois par jour ce qui déclenche des cycles dans lesquels des protéines sont produites de manière circadienne. Ces rythmes protéiques sont spécialisés pour chaque tissu ou organe et peuvent les aider à réaliser leurs tâches quotidiennes. Les rythmes circadiens peuvent être générés d'autres manières n'impliquant pas directement les composants des gènes de l'horloge. Les ARN messagers (ARNm) sont des molécules intermédiaires dans la production de protéines à partir d'ADN. Dans le foie des souris jusqu'à 20% des molécules d'ARNm sont produites suivant des rythmes circadiens. Le foie réalise des tâches essentielles dans le contrôle du métabolisme incluant celui des hydrates de carbone, des graisses et du cholestérol. Un timing précis est important afin de traiter les substances nutritives correctement lors des repas il en résulte une variation des quantités de certains ARNm et protéines coïncidant avec les repas. Les microARNs constituent une autre classe de molécules ARN de très petite taille qui régulent l'efficacité de traduction des ARNm en protéines et la stabilité des ARNm. Lors de mon travail de thèse, j'ai exploré de manière approfondie l'influence de ces petits régulateurs sur les rythmes circadiens du foie de souris. Ces expériences qui impliquaient le "Knock-out" d'un gène essentiel à la production de microARNs montrent qu'au lieu de générer les rythmes des ARNm, les microARNs les ajustent pour répondre aux besoins spécifiques du foie comme assurer leur pic au bon moment de la journée. Le ciblage de microARNs spécifiques peut révéler de nouvelles stratégies pour rectifier ces rythmes lorsque par exemple les fonctions métaboliques ne fonctionnent plus normalement. -- The rising and setting of the sun have long driven the daily schedules of humans and most organisms on the earth. This 24-hr cycle shapes many behavioural and physiological changes, such as alertness, body temperature, and sleep. These daily rhythms, which are called circadian rhythms, are dictated by biological clocks that are ticking in almost every single cell of the body. A region in the brain acts as a master clock to synchronize the internal clocks with each other and with the outside light/dark cycles. In cells, "core clock genes" are turned on and off once per day, which triggers cycles that cause some proteins to be produced in a circadian manner. The protein rhythms are specialized to a particular tissue or organ, and may help them to carry out their designated daily tasks. However, circadian rhythms might also be produced by other ways that do not involve these core clock components. Messenger RNAs (mRNAs) are intermediate molecules in the production of proteins from DNA. In the mouse liver, up to 20% of mRNA molecules are produced in circadian cycles. The liver performs essential tasks that control metabolism-including that of carbohydrates, fats, and cholesterol. Precisely timing when certain mRNAs and proteins reach peaks and troughs in their activities to coincide with mealtimes is important for nutrients to be properly processed. Other RNA molecules called microRNAs, i.e. RNAs of very small size, regulate at which rate mRNA molecules are translated into proteins. In my thesis work, I have explored at the influence of these small regulators on circadian rhythms in the mouse liver in greater detail. These experiments, which involved "knocking out" a gene that is essential for the production of microRNAs, show that rather than generating the mRNA rhythms, the microRNAs appear to adjust them to meet the specific needs of the liver, such as ensuring that they peak at the right time-of-day. Targeting specific microRNA molecules may reveal new strategies to tweak these rhythms, which could help to improve conditions when metabolic functions go wrong.

Targeted γ-Secretase Inhibition To Control the Notch Pathway in Renal Diseases.

Notch is a membrane inserted protein activated by the membrane-inserted γ-secretase proteolytic complex. The Notch pathway is a potential therapeutic target for the treatment of renal diseases but also controls the function of other cells, requiring cell-targeting of Notch antagonists. Toward selective targeting, we have developed the γ-secretase inhibitor-based prodrugs 13a and 15a as substrates for γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT) as well as aminopeptidase A (APA), which are overexpressed in renal diseases, and have evaluated them in experimental in vitro and in vivo models. In nondiseased mice, the cleavage product from Ac-γ-Glu-γ-secretase inhibitor prodrug 13a (γ-GT-targeting and γ-GCT-targeting) but not from Ac-α-Glu-γ-secretase inhibitor prodrug 15a (APA-targeting) accumulated in kidneys when compared to blood and liver. Potential nephroprotective effects of the γ-secretase inhibitor targeted prodrugs were investigated in vivo in a mouse model of acute kidney injury, demonstrating that the expression of Notch1 and cleaved Notch1 could be selectively down-regulated upon treatment with the Ac-γ-Glu-γ-secretase-inhibitor 13a.

OLFM4 : a neutrophil shield against autoimmunity?

Neutrophil extracellular traps (NETs) formation is a cell death mechanism characterized by the extrusion of DNA fibers associated to antimicrobial peptides such as LL37. Beside their antimicrobial role, NETs are highly immunogenic by their ability to activate plasmacytoid dendritic cells (pDCs). In this context, LL37 binds to NET-DNA, leading to endosomal Toll¬like-receptor (TLR) 9 binding, resulting in Interferon alpha (IFNa) production by pDCs. Uncontrolled pDC activation by NETs is an important player in the pathogenesis of autoimmune disease such as Lupus Erythematosus (LE); however the regulation of NET- driven pDC activation is poorly characterized. Olfactomedin 4 (OLFM4) is a granule protein present in a subset of circulating neutrophils and was shown to bear anti-inflammatory properties in a mouse model, raising the possibility that it may regulate neutrophil-induced inflammation. Therefore, in this project, we aimed at deciphering the mechanism by which OLFM4 may regulate inflammation induced by NET-activated pDC and its relevance in the pathogenesis of Lupus Erythematosus (LE). First, we show that OLFM4 directly interacted with LL37 in neutrophils, impairing LL37/DNA complexes formation and pDC activation to produce IFNa. Then, by using an in vivo model of acute inflammation depending on NET- driven activation of pDCs, we observed that the absence of Olfm4 led to uncontrolled type I IFN production, confirming the regulatory role of neutrophil-derived OLFM4. Beyond controlling NET-induced inflammation, we also show that OLFM4 could inhibit pDC activation mediated by DNA-containing immune complexes (ICs), suggesting that OLFM4 holds anti¬inflammatory properties in the context of LE. Of note, we identified a previously unknown population of OLFM4hi9h neutrophils in healthy individuals that may belong to the immunosuppressive subset of granulocytic myeloid-derived suppressor cells (g-MDSCs). Strikingly, we observed a decreased frequency of OLFM4h'9h cells among inflammatory Low density granulocytes (LDGs) neutrophils in LE patients, suggesting that a disequilibrium between pro- and anti-inflammatory neutrophils may participate to the disease pathogenesis. Altogether, this study demonstrates that OLFM4 is involved in the resolution of inflammation. -- La NETose (formation de Neutrophil Extracellular Traps, NETs) est une réponse à un stimulus inflammatoire caractérisée par l'expulsion de l'ADN lié à des peptides antimicrobiens comme le LL37, induisant la mort de la cellule. Les NETs possèdent des propriétés antibactériennes et sont pro-inflammatoires via leur capacité à activer les cellules dendritiques plasmacytoïdes (pDCs). Dans ce contexte, les complexes ADN/LL37 libérés lient le récepteur Toll-like 9 des pDCs, induisant la production d'Interféron alpha (IFNa). La production incontrôlée d'IFNa par les pDCs est impliquée dans la pathogenèse du Lupus Erythemateux (LE), cependant la régulation de l'activation des pDCs reste mal connue. L'Oflactomédine 4 (OLFM4) est une protéine produite par une sous-population de neutrophiles, avec des propriétés anti-inflammatoires possibles. Le but de ce projet était d'identifier les mécanismes par lesquels l'OLFM4 pourrait réguler l'inflammation induite par les NETs et sa relevance dans la pathogenèse du LE. Tout d'abord, nous avons montré que l'OLFM4 interagissait avec le LL37, empêchant la production des complexes ADN/LL37 qui activent les pDCs. Nous avons vérifié notre hypothèse in vivo en utilisant un modèle murin d'inflammation locale dépendant des pDCs et des NETs. Dans ce contexte, le déficit en Olfm4 était associé à une production accrue d'IFNa, confirmant le rôle de l'OLFM4 dans le contrôle de l'inflammation. De plus, l'OLFM4 pouvait également inhiber l'activation des pDCs induite par des complexes immuns, suggérant que l'OLFM4 serait aussi anti-inflammatoire dans le contexte du LE. Ensuite, nous avons identifié une nouvelle population de neutrophiles OLFM4h'9h chez les sujets sains qui pourraient appartenir au sous-type anti¬inflammatoire des g-MDSCs (granulocytic myeloid-derived suppressor cells). Nous avons observé une diminution de ces cellules parmi les neutrophiles pro-inflammatoires LDGs (Low Density Granulocytes) dans le LE suggérant qu'un déséquilibre entre les sous-types de neutrophiles pourrait participer à l'inflammation excessive de cette maladie. Ces travaux mettent en évidence l'implication de l'OLFM4 dans la résolution de l'inflammation et suggèrent qu'une expression altérée de l'OLFM4 pourrait participer à la pathogenèse du LE. -- Les neutrophils constituent la majorité des globules blancs circulants et sont rapidement mobilisés depuis le sang dans un organe lésé en cas d'infection ou de blessure. Ils représentent la première ligne de défense du système immunitaire. Ils sont indispensables dans la défense contre les infections par leur capacité à tuer les bactéries, par exemple en produisant des peptides antimicrobiens (AMPs) qui fonctionnent comme des antibiotiques naturels. De plus, les neutrophiles recrutent les autres membres du système immunitaire qui sont nécessaires à l'éradication complète des microbes et à la réparation des tissus. Les nombreux outils permettant aux neutrophiles de contrôler les infections ne sont cependant pas sans danger pour les tissus. En effet, diverses molécules comme les AMPs peuvent induire des dommages tissulaires substantiels en participant au développement d'une inflammation chronique. Ceci est particulièrement le cas lorsque les neutrophiles meurent par un processus nommé NETose. Dans ce contexte, la cellule subit une dissolution de sa membrane suivie de l'expulsion de son ADN associé à des AMPs. Ces complexes formés d'ADN et d'AMPs induisent la production de cytokines pro-inflammatoires dont l'Interféron alpha (IFNa). Certaines maladies auto-immunes comme le lupus érythémateux sont associées à un excès de NETose produit par les neutrophiles et à un excès d'IFNa qui participe au développement de la maladie. Dans cette thèse, nous avons montré que l'Olfactomédine 4 (OLFM4), une protéine produite par les neutrophiles eux-mêmes, est un inhibiteur de cette inflammation. Nous avons démontré que TOLFM4 empêchait la formation des complexes ADN/AMPs, réduisant par là la production d'IFNa in vitro et in vivo. Finalement, nos recherches ont suggéré que l'OLFM4 pourrait être insuffisamment produite chez les patients souffrant de lupus, ce qui pourrait participer à l'inflammation chronique associée à la maladie.

Targeting iron-mediated retinal degeneration by local delivery of transferrin.

Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.

Nrf2 Activation Promotes Keratinocyte Survival during Early Skin Carcinogenesis via Metabolic Alterations.

Pharmacologic activation of the transcription factor NRF2 has been suggested to offer a strategy for cancer prevention. In this study, we present evidence from murine tumorigenesis experiments suggesting there may be limitations to this possibility, based on tumorigenic effects of Nrf2 in murine keratinocytes that have not been described previously. In this setting, Nrf2 expression conferred metabolic alterations in keratinocytes that were protumorigenic in nature, affecting enzymes involved in glutathione biosynthesis or in the oxidative pentose phosphate pathway and other NADPH-producing enzymes. Under stress conditions, coordinate increases in NADPH, purine, and glutathione levels promoted the survival of keratinocytes harboring oncogenic mutations, thereby promoting tumor development. The protumorigenic activity of Nrf2 in keratinocytes was particularly significant in a mouse model of skin tumorigenesis that did not rely upon chemical carcinogenesis. In exploring the clinical relevance of our findings, we confirm that NRF2 and protumorigenic NRF2 target genes were activated in some actinic keratoses, the major precancerous lesion in human skin. Overall, our results reveal an unexpected tumor-promoting activity of activated NRF2 during early phases of skin tumorigenesis. Cancer Res; 75(22); 4817-29. ©2015 AACR.

Redox dysregulation in schizophrenia : effect on myelination of cortical structures and connectivity

Cette thèse traite du rôle qu'un facteur de risque génétique développé chez les patients souffrant de schizophrénie, à savoir un déficit de la synthèse du glutathion, peut jouer dans les anomalies de la connectivité cérébrale trouvées chez ces patients. L'essentiel du travail a été consacré à évaluer la structure de la substance blanche dans l'ensemble du cerveau chez un modèle animal par une méthode similaire à celle utilisée en recherche clinique avec l'imagerie par résonance magnétique (IRM). Cette approche de translation inverse chez la souris knock-out de glutamate-cystéine ligase modulateur sous-unité (Gclm KO), avait l'objectif d'étudier l'effet des défenses redox déficientes sur le développement des connexions cérébrales, tout en excluant celui des facteurs non liés au génotype. Après avoir établi le protocole de recherche, l'influence d'une manipulation environnementale a également été étudiée. Pour effectuer une analyse statistique fiable des données d'IRM obtenues, nous .avons d'abord créé un atlas du cerveau de la souris afin de l'utiliser comme modèle pour une segmentation précise des différentes régions du cerveau sur les images IRM obtenues in vivo. Les données provenant de chaque région d'intérêt ont ensuite été étudiées séparément. La qualité de cette méthode a été évaluée dans une expérience de simulation pour déduire la puissance statistique réalisable dans chaque région en fonction du nombre d'animaux utilisés. Ces outils d'analyse nous ont permis d'évaluer l'intégrité de la substance blanche dans le cerveau des souris durant le développement grâce à une expérience longitudinale, en utilisant l'imagerie du tenseur de diffusion (DTI). Nous avons ainsi observé des anomalies dans les paramètres dérivés du tenseur (diffusivité et anisotropie) dans la Commissure Antérieure et le Fimbria/Fornix des souris Gclm KO, par rapport aux animaux contrôles. Ces résultats suggèrent une substance blanche endommagée dans ces régions. Dans une expérience électrophysiologique, Pascal Steullet a montré que ces anomalies ont des conséquences fonctionnelles caractérisées par une réduction de la vitesse de conduction dans les fibres nerveuses. Ces données renforcent les conclusions des analyses d'imagerie. Le mécanisme par lequel une dérégulation redox affecte la structure de la substance blanche reste encore à définir, car une analyse immunohistochimique des protéines constituantes de la couche de myéline des fibres concernées n'a pas donné de résultats concluants. Nous avons également constaté un élargissement des ventricules dans les jeunes souris Gclm KO, mais pas chez les adultes et des anomalies neurochimiques déjà connues chez ces animaux (Duarte et al. 2011), à savoir une réduction du Glutathion et une augmentation de l'acide N-acétylaspartique, de l'Alanine et du ratio Glutamine/Glutamate. Nous avons ensuite testé l'effet d'un stress environnemental supplémentaire, l'élevage en isolement social, sur le phénotype. Ce stress n'a eu aucun effet sur la structure de la substance blanche évaluée par DTI, mais a réduit la concentration de myo-Inositol et augmenté le ratio de Glutamine/Glutamate dans le cortex frontal. Nous avons aussi reproduit dans ce groupe indépendant d'animaux les effets du génotype sur le profil neurochimique, sur la taille des ventricules et aussi sur les paramètres dérivés du tenseur de diffusion dans le Fimbria/Fornix, mais pas dans la Commissure Antérieure. Nos résultats montrent qu'une dérégulation redox d'origine génétique perturbe la structure et la fonction de la substance blanche dans des régions spécifiques, causant ainsi l'élargissement des ventricules. Ces phénotypes rassemblent certaines caractéristiques neuro-anatomiques de la schizophrénie, mais les mécanismes qui en sont responsables demeurent encore inconnus. L'isolement social n'a pas d'effet sur la structure de la substance blanche évaluée par DTI, alors qu'il est prouvé qu'il affecte la maturation des oligodendrocytes. La neurochimie corticale et en particulier le rapport Glutamine/Glutamate a été affecté par le dérèglement redox ainsi que par l'isolement social. En conséquence, ce ratio représente un indice prometteur dans la recherche sur l'interaction du stress environnemental avec le déséquilibre redox dans le domaine de la schizophrénie. -- The present doctoral thesis is concerned with the role that a genetic risk factor for the development of schizophrenia, namely a deficit in Glutathione synthesis, may play in the anomalies of brain connectivity found in patients. Most of the effort was devoted to perform a whole-brain assessment of white matter structure in the Glutamate-Cysteine ligase modulatory knockout mouse model (Gclm KO) using Magnetic Resonance Imaging (MRI) techniques similar to those used in state-of-the-art clinical research. Such reverse translational approach taking brain imaging from the bedside to the bench aimed to investigate the role that deficient redox defenses may play in the development of brain connections while excluding all influencing factors beside the genotype. After establishing the protocol, the influence of further environmental manipulations was also studied. Analysis of MRI images acquired in vivo was one of the main challenges of the project. Our strategy consisted in creating an atlas of the mouse brain to use as segmentation guide and then analyze the data from each region of interest separately. The quality of the method was assessed in a simulation experiment by calculating the statistical power achievable in each brain region at different sample sizes. This analysis tool enabled us to assess white matter integrity in the mouse brain along development in a longitudinal experiment using Diffusion Tensor Imaging (DTI). We discovered anomalies in diffusivity parameters derived from the tensor in the Anterior Commissure and Fimbria/Fornix of Gclm KO mice when compared to wild-type animals, which suggest that the structure of these tracts is compromised in the KO mice. In an elegant electrophysiological experiment, Pascal Steullet has provided evidence that these anomalies have functional consequences in form of reduced conduction velocity in the concerned tracts, thus supporting the DTI findings. The mechanism by which redox dysregulation affects WM structure remains unknown, for the immunohistochemical analysis of myelin constituent proteins in the concerned tracts produced inconclusive results. Our experiments also detected an enlargement of the lateral ventricles in young but not adult Gclm KO mice and confirmed neurochemical anomalies already known to affect this animals (Duarte et al. 2011), namely a reduction in Glutathione and an increase in Glutamine/Glutamate ratio, N-acetylaspartate and Alanine. Using the same methods, we tested the effect of an additional environmental stress on the observed phenotype: rearing in social isolation had no effect on white matter structure as assessed by DTI, but it reduced the concentration of myo-Inositol and increased the Glutamine/Glutamate ratio in the frontal cortex. We could also replicate in this separate group of animals the effects of genotype on the frontal neurochemical profile, ventricular size and diffusivity parameters in the Fimbria/Fornix but not in the Anterior Commissure. Our data show that a redox dysregulation of genetic origin may disrupt white matter structure and function in specific tracts and cause a ventricular enlargement, phenotypes that resemble some neuroanatomical features of schizophrenia. The mechanism responsible remains however unknown. We have also demonstrated that environmental stress in form of social isolation does not affect white matter structure as assessed by DTI even though it is known to affect oligodendrocyte maturation. Cortical neurochemistry, and specifically the Glutamine to Glutamate balance was affected both by redox dysregulation and social isolation, and is thus a good target for further research on the interaction of redox imbalance and environmental stress in schizophrenia.

Chlamydiaceae and Chlamydia-like organisms in the koala (Phascolarctos cinereus)--organ distribution and histopathological findings.

Chlamydial infections in koalas can cause life-threatening diseases leading to blindness and sterility. However, little is known about the systemic spread of chlamydiae in the inner organs of the koala, and data concerning related pathological organ lesions are limited. The aim of this study was to perform a thorough investigation of organs from 23 koalas and to correlate their histopathological lesions to molecular chlamydial detection. To reach this goal, 246 formalin-fixed and paraffin embedded organ samples from 23 koalas were investigated by histopathology, Chlamydiaceae real-time PCR and immunohistochemistry, ArrayTube Microarray for Chlamydiaceae species identification as well as Chlamydiales real-time PCR and sequencing. By PCR, two koalas were positive for Chlamydia pecorum whereas immunohistochemical labelling for Chlamydiaceae was detected in 10 tissues out of nine koalas. The majority of these (n=6) had positive labelling in the urogenital tract related to histopathological lesions such as cystitis, endometritis, pyelonephritis and prostatitis. Somehow unexpected was the positive labelling in the gastrointestinal tract including the cloaca as well as in lung and spleen indicating systemic spread of infection. Uncultured Chlamydiales were detected in several organs of seven koalas by PCR, and four of these suffered from plasmacytic enteritis of unknown aetiology. Whether the finding of Chlamydia-like organisms in the gastrointestinal tract is linked to plasmacytic enteritis is unclear and remains speculative. However, as recently shown in a mouse model, the gastrointestinal tract might play a role being the site for persistent chlamydial infections and being a source for reinfection of the genital tract.

Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury.

Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.

Regulating Mechanisms of Gonadal and Pituitary Tumorigenesis in Mice Producing Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are structurally and functionally similar glycoprotein hormones acting through the same luteinizing hormone chorionic gonadotropin receptor (LHCGR). The functions of LH in reproduction and hCG in pregnancy are well known. Recently, the expression of LHCGR has been found in many nongonadal tissues and cancers, and this has raised the question of whether LH/hCG could affect the function or tumorigenesis of these nongonadal tissues. We have also previously generated an hCG expressing mouse model presenting nongonadal phenotypes. Using this model it is possible to improve our understanding of nongonadal action of highly elevated LH/hCG. In the current study, we analyzed the effect of moderately and highly elevated hCG levels on male reproductive development and function. The main finding was the appearance of fetal Leydig cell (FLC) adenomas in prepubertal males. However, the development and differentiation of FLCs were not significantly affected. We also show that the function of hCG is different in FLCs and in adult Leydig cells (ALC), because in the latter cells hCG was not able to induce tumorigenesis. In FLCs, LHCGR is not desensitized or downregulated upon ligand binding. In this study, we found that the testicular expression of two G protein-coupled receptor kinases responsible for receptor desensitization or downregulation is increased in adult testis. Results suggest that the lack of LHCGR desensitization or downregulation in FLCs protect testosterone (Te) synthesis, but also predispose FLCs for LH/hCG induced adenomas. However, all the hCG induced nongonadal changes observed in male mice were possible to explain by the elevated Te level found in these males. Our findings indicate that the direct nongonadal effects of elevated LH/hCG in males are not pathophysiologically significant. In female mice, we showed that an elevated hCG level was able to induce gonadal tumorigenesis. hCG also induced the formation of pituitary adenomas (PA), but the mechanism was indirect. Furthermore, we found two new potential risk factors and a novel hormonally induced mechanism for PAs. Increased progesterone (P) levels in the presence of physiological estradiol (E2) levels induced the formation of PAs in female mice. E2 and P induced the expression and nuclear localization of a known cell-cycle regulator, cyclin D1. A calorie restricted diet was also able to prevent the formation of PAs, suggesting that obesity is able to promote the formation of PAs. Hormone replacement therapy after gonadectomy and hormone antagonist therapy showed that the nongonadal phenotypes observed in hCG expressing female mice were due to ovarian hyperstimulation. A slight adrenal phenotype was evident even after gonadectomy in hCG expressing females, but E2 and P replacement was able to induce a similar phenotype in WT females without elevated LH/hCG action. In conclusion, we showed that the direct effects of elevated hCG/LH action are limited only to the gonads of both sexes. The nongonadal phenotypes observed in hCG expressing mice were due to the indirect, gonadal hormone mediated effects of elevated hCG. Therefore, the gonads are the only physiologically significant direct targets of LHCGR signalling.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Epigenetic alterations in hippocampus of SAMP8 senescent mice and modulation by voluntary physical exercise.

The senescence-accelerated SAMP8 mouse model displays features of cognitive decline and Alzheimer's disease. With the purpose of identifying potential epigenetic markers involved in aging and neurodegeneration, here we analyzed the expression of 84 mature miRNAs, the expression of histone-acetylation regulatory genes and the global histone acetylation in the hippocampus of 8-month-old SAMP8 mice, using SAMR1 mice as control. We also examined the modulation of these parameters by 8 weeks of voluntary exercise. Twenty-one miRNAs were differentially expressed between sedentary SAMP8 and SAMR1 mice and seven miRNAs were responsive to exercise in both strains. SAMP8 mice showed alterations in genes involved in protein acetylation homeostasis such as Sirt1 and Hdac6 and modulation of Hdac3 and Hdac5 gene expression by exercise. Global histone H3 acetylation levels were reduced in SAMP8 compared with SAMR1 mice and reached control levels in response to exercise. In sum, data presented here provide new candidate epigenetic markers for aging and neurodegeneration and suggest that exercise training may prevent or delay some epigenetic alterations associated with accelerated aging.

Epigenetic alterations in hippocampus of SAMP8 senescent mice and modulation by voluntary physical exercise.

The senescence-accelerated SAMP8 mouse model displays features of cognitive decline and Alzheimer's disease. With the purpose of identifying potential epigenetic markers involved in aging and neurodegeneration, here we analyzed the expression of 84 mature miRNAs, the expression of histone-acetylation regulatory genes and the global histone acetylation in the hippocampus of 8-month-old SAMP8 mice, using SAMR1 mice as control. We also examined the modulation of these parameters by 8 weeks of voluntary exercise. Twenty-one miRNAs were differentially expressed between sedentary SAMP8 and SAMR1 mice and seven miRNAs were responsive to exercise in both strains. SAMP8 mice showed alterations in genes involved in protein acetylation homeostasis such as Sirt1 and Hdac6 and modulation of Hdac3 and Hdac5 gene expression by exercise. Global histone H3 acetylation levels were reduced in SAMP8 compared with SAMR1 mice and reached control levels in response to exercise. In sum, data presented here provide new candidate epigenetic markers for aging and neurodegeneration and suggest that exercise training may prevent or delay some epigenetic alterations associated with accelerated aging.

Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.