Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

The role of AUF1 in AU-rich interleukin-6 mRNA regulation

Abstract: The AU-rich elements (AREs) consisting of repeated AUUUA motifs confer rapid degradation to many cellular mRNAs when present in the 3' untranslated region (3'UTR). We have studied the instability of interleukin-6 mRNA by grafting its 3' untranslated region to a stable green fluorescent protein mRNA. Subsequent scanning mutagenesis identified two conserved elements, which taken together account for most of the instability. The first corresponds to a short non-canonical AU-rich element. The other comprises a sequence predicted to form astern-loop structure. Both elements need to be present in order to confer full instability (Paschoud et al. 2006). Destabilization of ARE-containing mRNAs is thought to involve ARE-binding proteins such as AUF1. We tested whether AUF1 binding to interleukin-6 mRNA correlates with decreased mRNA stability. Overexpression of myc-tagged p37AUFl and p42AUF1 as well as suppression of all four AUF1 isoforms by RNA interference stabilized the interleukin-6 mRNA. Furthermore, the interleukin-6 mRNA co-immunoprecipitated specifically with myc-tagged p37AUF1 and p42AUF1 in cell extracts. Both the stabilization and AUF1-binding required the non-canonical AU-rich sequence. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes interleukin6 mRNA degradation. The combination of mRNA co-immunoprecipitation with microarray technology revealed that at least 500 cellular mRNAs associate with AUF1. Résumé: "La présence d'éléments riches en A et U (ARE), en particulier les motifs répétés d'AUUUA dans la région 3' non traduite, confère une dégradation rapide à beaucoup d'ARN cellulaires. Nous avons étudié l'instabilité de l'ARN codant pour l'interleukine 6 en greffant sa région 3' non traduite à un ARN stable codant pour la protéine fluorescente verte. La mutagenèse systématique des séquences non traduites a permis l'identification de deux éléments conservés qui confèrent l'instabilité à l'ARN. Le premier correspond à un élément AU-riche non canonique court. Le second comporte une structure en 'épingle à cheveux'. Tous les deux éléments doivent être présents afin de conférer une instabilité complète (Paschoud et al. 2006). On pense que des protéines telles que AUF1, pouvant se lier aux éléments ARE, sont impliquées dans la dégradation des ARN messagers. Nous avons examiné si la liaison de AUFl sur l'ARN de l'interleukine 6 corrèle avec une stabilité diminuée. La surexpression des protéines p37AUF1 et de p42AUF1 myc-étiquetées ainsi que la suppression de chacun des quatre isoformes de AUF1 par interférence d'ARN a stabilisé l'ARN messager d'interleukine 6. En outre, cet ARN co-immunoprécipite spécifiquement avec p37AUF1 et p42AUF1 dans des extraits cellulaires. La présence de l'élément AUriche non canonique est nécessaire pour la stabilisation de l'ARN et sa liaison avec AUFI. Ces résultats indiquent qu'AUF1 se lie à l'élément AU-riche in vivo et favorise la dégradation de l'ARN messager d'interleukine 6. La combinaison des techniques de coimmunoprécipitation des ARN messagers et des analyses par `microarray' indique qu'au moins 500 ARN cellulaires s'associent à AUF1.

Genetic regulation of colony social organization in fire ants: an integrative overview

Expression of colony social organization in fire ants appears to be under the control of a single Mendelian factor of large effect. Variation in colony queen number in Solenopsis invicta and its relatives is associated with allelic variation at the gene Gp-9, but not with variation at other unlinked genes; workers regulate queen identity and number on the basis of Gp-9 genotypic compatibility. Nongeneticfactors, such as prior social experience, queen reproductive status, and local environment, have negligible effects on queen number which illustrates the nearly complete penetrance of Gp-9. As predicted, queen number can be manipulated experimentally by altering worker Gp-9 genotype frequencies. The Gp-9 allele lineage associated with polygyny in South American fire? ants has been retained across multiple speciation events, which may signal the action of balancing selection to maintain social polymorphism in these species. Moreover positive selection is implicated in driving the molecular evolution of Gp-9 in association with the origin of polygyny. The identity of the product of Gp-9 as an odorant-binding protein suggests plausible scenarios for its direct involvement in the regulation of queen number via a role in chemical communication. While these and other lines of evidence show that Gp-9 represents a legitimate candidate gene of major effect, studies aimed at determining (i) the biochemical pathways in which GP-9 functions; (ii) the phenotypic effects of molecular variation at Gp-9 and other pathway genes; and (iii) the potential involvement of genes in linkage disequilibrium with Gp-9 are needed to elucidate the genetic architecture underlying social organization in fire ants. Information that reveals the links between molecular variation, individual phenotype, and colony-level behaviors, combined with behavioral models that incorporate details of the chemical communication involved in regulating queen number will yield a novel integrated view of the evolutionary changes underlying a key social adaptation.

Endogenous and exogenous regulation of monocyte responses

Mononuclear phagocytes are essential for the innate response to pathogens and for the repair of injured tissue. The cells - which can be broadly divided into circulating monocytes and tissue-resident macrophages and dendritic cells - are selectively equipped to protect the host by mediating pleiotropic and tissue-specific functions. The properties of some mononuclear phagocytes, however, also contribute to the development and the progression of inflammatory diseases. Consequently, current research investigates mononuclear phagocytes into greater detail with the aim to clarify their contributions to pathophysiologic inflammation. Recent studies indicate that circulating monocytes can be divided into distinct populations, which differ in their tissue tropism and functional commitment. Also, tissue macrophages and dendritic cells have been found to adopt context-dependent phenotypes, which can range from "pro-" to "anti-" inflammatory. These findings have markedly contributed to our understanding of the functional heterogeneity of mononuclear phagocyte populations. Yet, in many cases, the factors that control the quantity and/or quality of phagocyte responses in vivo remain largely unknown. The goal of this thesis was to identify cell endogenous and cell exogenous factors that dictate the fate of mononuclear phagocyte populations. To this end we made use of the recent identification of phenotypic markers, which permit to track mononuclear cell types and their lineage precursors. A main approach consisted to define candidate regulatory factors of certain types of mononuclear phagocytes and then to manipulate the expression of these factors in mice so as to address their functions and causal contributions on mononuclear phagocyte lineages in vivo. Human patient material was further used to validate findings. First, we investigated a microRNA and a transcription factor as candidate cell endogenous co- regulators of monocyte subset responses. Second, we studied a tumor-derived hormone as a candidate exogenous factor that amplifies the production of a population of mononuclear phagocytes with tumor-promoting functions. The endogenous and exogenous factors identified in this research appear to act as effective regulators of mononuclear phagocyte responses in vivo and thus may be exploited in future therapeutic approaches to regulate disease-associated inflammation. - Les phagocytes mononucléaires sont essentiels pour la réponse innée aux pathogènes et pour la réparation des tissus lésés. Ces cellules - qui peuvent être largement divisées en deux groupes, les monocytes circulant dans le sang et les macrophages et cellules dendritiques résidant dans les tissus - sont capables de protéger l'hôte en exerçant des fonctions pléiotropiques. Cependant, les propriétés de certains phagocytes mononucléaires contribuent également au développement et à la progression des maladies inflammatoires. Par conséquent, la recherche actuelle étudie les phagocytes mononucléaires plus en détail afin de clarifier leurs contributions à l'inflammation pathophysiologique. Des études récentes indiquent que les monocytes circulants peuvent être divisés en populations distinctes, qui diffèrent dans leur tropisme tissulaire et dans leurs fonctions biologiques. En outre, les macrophages et les cellules dendritiques peuvent adopter des phénotypes dépendants de l'environnement dans lequel ils se trouvent; ces phénotypes peuvent aller du type "pro-" au type "anti-" inflammatoire. Ces récentes découvertes ont contribué à notre compréhension sur l'hétérogénéité fonctionnelle des phagocytes mononucléaires. Pourtant, dans de nombreux cas, les facteurs qui contrôlent la quantité et/ou la qualité des réponses produites par ces cellules restent encore largement inconnus. L'objectif de cette thèse a consisté à identifier de nouveaux facteurs (endogènes ou exogènes) qui contrôlent les phagocytes mononucléaires. Dans ce but, nous avons fait usage de l'identification récente de marqueurs qui permettent d'identifier différents types de phagocytes mononucléaires ainsi que des cellules (souches) dont ils sont issus. Notre approche a consisté à définir des facteurs candidats qui pourraient contrôler certains phagocytes mononucléaires, puis à manipuler l'expression de ces facteurs chez la souris de manière à tester leurs fonctions et leur contributions in vivo. Nous avons également utilisé des échantillons biologiques de patients pour vérifier nos résultats chez l'homme. Tout d'abord, nous avons étudié un microARN et un facteur de transcription pour déterminer si ces deux facteurs opèrent en tant que co-régulateurs d'un certain type de monocytes. Deuxièmement, nous avons considéré une hormone produite par certaines tumeurs afin d'examiner son rôle dans la production d'une population de macrophages qui favorisent la progression des tumeurs. Les facteurs endogènes et exogènes identifiés dans cette recherche semblent agir comme régulateurs dominants de réponses produites par certains phagocytes mononucléaires et pourraient donc être exploités dans de futures approches thérapeutiques afin de contrôler les réponses immunitaires inflammatoires associées a certaines maladies.

Regulation of Nav1.7 and Nav1.8 voltage-gated sodium channels by Nedd4-2 and β-subunits and its implication in neuropathic pain

In mammals, the presence of excitable cells in muscles, heart and nervous system is crucial and allows fast conduction of numerous biological information over long distances through the generation of action potentials (AP). Voltage-gated sodium channels (Navs) are key players in the generation and propagation of AP as they are responsible for the rising phase of the AP. Navs are heteromeric proteins composed of a large pore-forming a-subunit (Nav) and smaller ß-auxiliary subunits. There are ten genes encoding for Navl.l to Nav1.9 and NaX channels, each possessing its own specific biophysical properties. The excitable cells express differential combinations of Navs isoforms, generating a distinct electrophysiological signature. Noteworthy, only when anchored at the membrane are Navs functional and are participating in sodium conductance. In addition to the intrinsic properties of Navs, numerous regulatory proteins influence the sodium current. Some proteins will enhance stabilization of membrane Navs while others will favour internalization. Maintaining equilibrium between the two is of crucial importance for controlling cellular excitability. The E3 ubiquitin ligase Nedd4-2 is a well-characterized enzyme that negatively regulates the turnover of many membrane proteins including Navs. On the other hand, ß-subunits are known since long to stabilize Navs membrane anchoring. Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of Navs expressed in dorsal root ganglion (DRG) sensory neurons as highlighted in different animal models of neuropathic pain. Among Navs, Nav1.7 and Nav1.8 are abundantly and specifically expressed in DRG sensory neurons and have been recurrently incriminated in nociception and neuropathic pain development. Using the spared nerve injury (SNI) experimental model of neuropathic pain in mice, I observed a specific reduction of Nedd4-2 in DRG sensory neurons. This decrease subsequently led to an upregulation of Nav1.7 and Nav1.8 protein and current, in the axon and the DRG neurons, respectively, and was sufficient to generate neuropathic pain-associated hyperexcitability. Knocking out Nedd4-2 specifically in nociceptive neurons led to the same increase of Nav1.7 and Nav1.8 concomitantly with an increased thermal sensitivity in mice. Conversely, rescuing Nedd4-2 downregulation using viral vector transfer attenuated neuropathic pain mechanical hypersensitivity. This study demonstrates the significant role of Nedd4-2 in regulating cellular excitability in vivo and its involvement in neuropathic pain development. The role of ß-subunits in neuropathic pain was already demonstrated in our research group. Because of their stabilization role, the increase of ßl, ß2 and ß3 subunits in DRGs after SNI led to increased Navs anchored at the membrane. Here, I report a novel mechanism of regulation of a-subunits by ß- subunits in vitro; ßl and ß3-subunits modulate the glycosylation pattern of Nav1.7, which might account for stabilization of its membrane expression. This opens new perspectives for investigation Navs state of glycosylation in ß-subunits dependent diseases, such as in neuropathic pain. - Chez les mammifères, la présence de cellules excitables dans les muscles, le coeur et le système nerveux est cruciale; elle permet la conduction rapide de nombreuses informations sur de longues distances grâce à la génération de potentiels d'action (PA). Les canaux sodiques voltage-dépendants (Navs) sont des participants importants dans la génération et la propagation des PA car ils sont responsables de la phase initiale de dépolarisation du PA. Les Navs sont des protéines hétéromériques composées d'une grande sous-unité a (formant le pore du canal) et de petites sous-unités ß accompagnatrices. Il existe dix gènes qui codent pour les canaux sodiques, du Nav 1.1 au Nav 1.9 ainsi que NaX, chacun possédant des propriétés biophysiques spécifiques. Les cellules excitables expriment différentes combinaisons des différents isoformes de Navs, qui engendrent une signature électrophysiologique distincte. Les Navs ne sont fonctionnels et ne participent à la conductibilité du Na+, que s'ils sont ancrés à la membrane plasmique. En plus des propriétés intrinsèques des Navs, de nombreuses protéines régulatrices influencent également le courant sodique. Certaines protéines vont favoriser l'ancrage et la stabilisation des Navs exprimés à la membrane, alors que d'autres vont plutôt favoriser leur internalisation. Maintenir l'équilibre des deux processus est crucial pour contrôler l'excitabilité cellulaire. Dans ce contexte, Nedd4-2, de la famille des E3 ubiquitin ligase, est une enzyme bien caractérisée qui régule l'internalisation de nombreuses protéines, notamment celle des Navs. Inversement, les sous-unités ß sont connues depuis longtemps pour stabiliser l'ancrage des Navs à la membrane. La douleur neuropathique périphérique est une condition débilitante résultant d'une atteinte à un nerf. Elle est caractérisée par la dérégulation des Navs exprimés dans les neurones sensoriels du ganglion spinal (DRG). Ceci a été démontré à de multiples occasions dans divers modèles animaux de douleur neuropathique. Parmi les Navs, Nav1.7 et Nav1.8 sont abondamment et spécifiquement exprimés dans les neurones sensoriels des DRG et ont été impliqués de façon récurrente dans le développement de la douleur neuropathique. En utilisant le modèle animal de douleur neuropathique d'épargne du nerf sural (spared nerve injury, SNI) chez la souris, j'ai observé une réduction spécifique des Nedd4-2 dans les neurones sensoriels du DRG. Cette diminution avait pour conséquence l'augmentation de l'expression des protéines et des courants de Nav 1.7 et Nav 1.8, respectivement dans l'axone et les neurones du DRG, et était donc suffisante pour créer l'hyperexcitabilité associée à la douleur neuropathique. L'invalidation pour le gène codant pour Nedd4-2 dans une lignée de souris génétiquement modifiées a conduit à de similaires augmentations de Nav1.7 et Nav1.8, parallèlement à une augmentation à la sensibilité thermique. A l'opposé, rétablir une expression normale de Nedd4-2 en utilisant un vecteur viral a eu pour effet de contrecarrer le développement de l'hypersensibilité mécanique lié à ce modèle de douleur neuropathique. Cette étude démontre le rôle important de Nedd4-2 dans la régulation de l'excitabilité cellulaire in vivo et son implication dans le développement des douleurs neuropathiques. Le rôle des sous-unités ß dans les douleurs neuropathiques a déjà été démontré dans notre groupe de recherche. A cause de leur rôle stabilisateur, l'augmentation des sous-unités ßl, ß2 et ß3 dans les DRG après SNI, conduit à une augmentation des Navs ancrés à la membrane. Dans mon travail de thèse, j'ai observé un nouveau mécanisme de régulation des sous-unités a par les sous-unités ß in vitro. Les sous-unités ßl et ß3 régulent l'état de glycosylation du canal Nav1.7, et stabilisent son expression membranaire. Ceci ouvre de nouvelles perspectives dans l'investigation de l'état de glycosylation des Navs dans des maladies impliquant les sous-unités ß, notamment les douleurs neuropathiques.

Regulation and glucocorticoid-independent induction of lipocortin I in cultured astrocytes.

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in the prefrontal cortex of human opioid addicts.

The neurofilament (NF) proteins (NF-H, NF-M, and NF-L for high, medium, and low molecular weights) play a crucial role in the organization of neuronal shape and function. In a preliminary study, the abundance of total NF-L was shown to be decreased in brains of opioid addicts. Because of the potential relevance of NF abnormalities in opioid addiction, we quantitated nonphosphorylated and phosphorylated NF in postmortem brains from 12 well-defined opioid abusers who had died of an opiate overdose (heroin or methadone). Levels of NF were assessed by immunoblotting techniques using phospho-independent and phospho-dependent antibodies, and the relative (% changes in immunoreactivity) and absolute (changes in ng NF/microg total protein) amounts of NF were calculated. Decreased levels of nonphosphorylated NF-H (42-32%), NF-M (14-9%) and NF-L (30-29%) were found in the prefrontal cortex of opioid addicts compared with sex, age, and postmortem delay-matched controls. In contrast, increased levels of phosphorylated NF-H (58-41%) and NF-M (56-28%) were found in the same brains of opioid addicts. The ratio of phosphorylated to nonphosphorylated NF-H in opioid addicts (3.4) was greater than that in control subjects (1.6). In the same brains of opioid addicts, the levels of protein phosphatase of the type 2A were found unchanged, which indicated that the hyperphosphorylation of NF-H is not the result of a reduced dephosphorylation process. The immunodensities of GFAP (the specific glial cytoskeletol protein), alpha-internexin (a neuronal filament related to NF-L) and synaptophysin (a synapse-specific protein) were found unchanged, suggesting a lack of gross changes in glial reaction, other intermediate filaments of the neuronal cytoskeletol, and synaptic density in the prefrontal cortex of opioid addicts. These marked reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts may play a significant role in the cellular mechanisms of opioid addiction.

Regulation of the integration of newly generated neurons in the adult hippocampus

Hippocampal adult neurogenesis results in the continuous formation of new neurons in the adult hippocampus, which participate to learning and memory. Manipulations increasing adult neurogenesis have a huge clinical potential in pathologies involving memory loss. Intringuingly, most of the newborn neurons die during their maturation. Thus, increasing newborn neuron survival during their maturation may be a powerful way to increase overall adult neurogenesis. The factors governing this neuronal death are yet poorly known. In my PhD project, we made the hypothesis that synaptogenesis and synaptic activity play a role in the survival of newborn hippocampal neurons. We studied three factors potentially involved in the regulation of the synaptic integration of adult-born neurons. First, we used propofol anesthesia to provoke a global increase in GABAergic activity of the network, and we evaluated the outcome on newborn neuron synaptic integration, morphological development and survival. Propofol anesthesia impaired the dendritic maturation and survival of adult-born neurons in an age-dependent manner. Next, we examined the development of astrocytic ensheathment on the synapses formed by newborn neurons, as we hypothesized that astrocytes are involved in their synaptic integration. Astrocytic processes ensheathed the synapses of newborn neurons very early in their development, and the processes modulated synaptic transmission on these cells. Finally, we studied the cell-autonomous effects of the overexpression of synaptic adhesion molecules on the development, synaptic integration and survival of newborn neurons, and we found that manipulating of a single adhesion molecule was sufficient to modify synaptogenesis and/or synapse function, and to modify newborn neuron survival. Together, these results suggest that the activity of the neuronal network, the modulation of glutamate transport by astrocytes, and the synapse formation and activity of the neuron itself may regulate the survival of newborn neurons. Thus, the survival of newborn neurons may depend on their ability to communicate with the network. This knowledge is crucial for finding ways to increase neurogenesis in patients. More generally, understanding how the neurogenic niche works and which factors are important for the generation, maturation and survival of neurons is fundamental to be able to maybe, one day, replace neurons in any region of the brain.

Type I IFN-mediated regulation of IL-1 production in inflammatory disorders.

Although contributing to inflammatory responses and to the development of certain autoimmune pathologies, type I interferons (IFNs) are used for the treatment of viral, malignant, and even inflammatory diseases. Interleukin-1 (IL-1) is a strongly pyrogenic cytokine and its importance in the development of several inflammatory diseases is clearly established. While the therapeutic use of IL-1 blocking agents is particularly successful in the treatment of innate-driven inflammatory disorders, IFN treatment has mostly been appreciated in the management of multiple sclerosis. Interestingly, type I IFNs exert multifaceted immunomodulatory effects, including the reduction of IL-1 production, an outcome that could contribute to its efficacy in the treatment of inflammatory diseases. In this review, we summarize the current knowledge on IL-1 and IFN effects in different inflammatory disorders, the influence of IFNs on IL-1 production, and discuss possible therapeutic avenues based on these observations.

Calcineurin-Mediated Regulation of Hyphal Growth, Septation, and Virulence in Aspergillus fumigatus.

Calcineurin is a heterodimeric protein phosphatase complex composed of catalytic (CnaA) and regulatory (CnaB) subunits and plays diverse roles in regulating fungal stress responses, morphogenesis, and pathogenesis. Fungal pathogens utilize the calcineurin pathway to survive in the host environment and cause life-threatening infections. The immunosuppressive calcineurin inhibitors (FK506 and cyclosporine A) are active against fungi, making calcineurin a promising antifungal drug target. Here, we review novel findings on calcineurin localization and functions in Aspergillus fumigatus hyphal growth and septum formation through regulation of proteins involved in cell wall biosynthesis. Extensive mutational analysis in the functional domains of A. fumigatus CnaA has led to an understanding of the relevance of these domains for the localization and function of CnaA at the hyphal septum. An evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi was found to be responsible for virulence in A. fumigatus. This finding of a filamentous fungal-specific mechanism controlling hyphal growth and virulence represents a potential target for antifungal therapy.

How to keep the brain awake? : pharmacogenomics and aging effects on sleep-wake regulation in inbred mice

ABSTRACT : Genetic approach in the sleep field is at the beginning of its wide expansion. Transitions between sleep and wakefulness, and the maintenance of these states are driven by complex neurobiologic mechanisms with reciprocal interactions. Impairment in both transitions and maintenance of behavioral states leads to debilitating conditions. The major symptom being excessive daytime sleepiness, characterizing most sleep disorders but also a wide variety of psychiatric and neurologic disorders, as well as the elderly. Until now, most wake-promoting drugs available directly (e.g., amphetamines and possibly modafinil) or indirectly (e.g., caffeine) provokes dopamine release which is believed to influence the abuse potential of these drugs. The effects of genetic components were assessed here, on drug-induced wakefulness and age-related sleep changes in three inbred mouse strains [AKR/J, C57BL/6J, DBA/2J] that differ in their major sleep phenotypes. Three wake-promoting drugs were used; d-amphetamine, a classical stimulant, modafinil, the most widely-prescribed stimulant, and YKP-10A, a novel wake-promoting agent with antidepressant proprieties. Electrical activity (Electroencephalogram) and gene expression of the brain were assessed and indicate a highly genotype-dependant response to wake promotion and subsequent recovery sleep. Aging effects on sleep-wake regulation were also strongly influenced by genetic determinants. By assessing the age-dependant effects at several time points (from 3 months to 2 years old mice), we found a strong genetic effect on vigilance states. These studies demonstrate a critical role for genetic factors neglected till now in the fields of pharmacology and aging effects on vigilance states.

Life-history evolution and the polyphenic regulation of somatic maintenance and survival.

Here we discuss life-history evolution from the perspective of adaptive phenotypic plasticity, with a focus on polyphenisms for somatic maintenance and survival. Polyphenisms are adaptive discrete alternative phenotypes that develop in response to changes in the environment. We suggest that dauer larval diapause and its associated adult phenotypes in the nematode (Caenorhabditis elegans), reproductive dormancy in the fruit fly (Drosophila melanogaster) and other insects, and the worker castes of the honey bee (Apis mellifera) are examples of what may be viewed as the polyphenic regulation of somatic maintenance and survival. In these and other cases, the same genotype can--depending upon its environment--express either of two alternative sets of life-history phenotypes that differ markedly with respect to somatic maintenance, survival ability, and thus life span. This plastic modulation of somatic maintenance and survival has traditionally been underappreciated by researchers working on aging and life history. We review the current evidence for such adaptive life-history switches and their molecular regulation and suggest that they are caused by temporally and/or spatially varying, stressful environments that impose diversifying selection, thereby favoring the evolution of plasticity of somatic maintenance and survival under strong regulatory control. By considering somatic maintenance and survivorship from the perspective of adaptive life-history switches, we may gain novel insights into the mechanisms and evolution of aging.

Cellular localization, expression and regulation of neuropeptide Y in kidneys of hypertensive rats.

Neuropeptide Y (NPY) is a key modulator of the autonomic nervous system playing pivotal roles in cardiovascular and neuronal functions. In this study, we assessed the cellular localization and gene expression of NPY in rat kidneys. We also examined the relationship between NPY gene expression and renin in two rat models of hypertension (two-kidney, one-clip renal hypertension (2K1C), and deoxycorticosterone-salt-induced hypertension (DOCA-salt)) characterized by a similar blood pressure elevation. In situ hybridization and immunohistochemistry, using anti-NPY or anti-C-flanking peptide of NPY (CPON) antibodies, showed that NPY transcript and protein were colocalized in the tubules of rat kidneys. During experimental hypertension, NPY mRNA was decreased in both kidneys of the 2K1C animals, but not in the kidney of DOCA-salt rats. In 2K1C rats, renal NPY content was also decreased. The difference in NPY gene expression between 2K1C rats (a high renin model of hypertension) and DOCA-salt rats (a low renin model of hypertension) suggests that circulating angiotensin II plays a role in local renal NPY gene expression and that the elevated blood pressure per se is not the primary factor responsible for the control of NPY gene expression in the kidney.

Epigenetic regulation of histone H3 serine 10 phosphorylation status by HCF-1 proteins in C. elegans and mammalian cells.

BACKGROUND: The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status. CONCLUSIONS/SIGNIFICANCE: These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation.