Consequences of modified lipid and glucose metabolism on peripheral nervous system structure and function

284 million people worldwide suffered from type 2 diabetes mellitus (T2DM) in 2010, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy (DPN). Although DPN is the most common complication of diabetes mellitus and the leading cause of non-traumatic amputations its pathophysiology is still poorly understood. To get more insight into the molecular mechanism underlying DPN in T2DM, I used a rodent model of T2DM, the db/db mice.¦ln vivo electrophysiological recordings of diabetic animals indicated that in addition to reduced nerve conduction velocity db/db mice also present increased nerve excitability. Further ex vivo evaluation of the electrophysiological properties of db/db nerves clearly established a presence of the peripheral nerve hyperexcitability (PNH) phenotype in diabetic animals. Using pharmacological inhibitors we demonstrated that PNH is mostly mediated by the decreased activity of Kv1 channels. ln agreement with these data 1 observed that the diabetic condition led to a reduced presence of the Kv1.2 subunits in juxtaparanodal regions of db/db peripheral nerves whereas its mANA and protein expression levels were not affected. Lmportantly, I confirmed a loss of juxtaparanodal Kv1.2 subunits in nerve biopsies from type 2 diabetic patients. Together these observations indicate that the type 2 diabetic condition leads to potassium-channel mediated changes of nerve excitability thus identifying them as potential drug targets to treat sorne of the DPN related symptoms.¦Schwann cells ensheath and isolate peripheral axons by the production of myelin, which consists of lipids and proteins in a ratio of 2:1. Peripheral myelin protein 2 (= P2, Pmp2 or FABP8) was originally described as one of the most abundant myelin proteins in the peripheral nervous system. P2, which is a member of the fatty acid binding protein (FABP) family, is a 14.8 kDa cytosolic protein expressed on the cytoplasmic side of compact myelin membranes. As indicated by their name, the principal role of FABPs is thought to be the binding and transport of fatty acids.¦To study its role in myelinating glial cells I have recently generated a complete P2 knockout mouse model (P2-/-). I confirmed the loss of P2 in the sciatic nerve of P2-/- mice at the mRNA and protein level. Electrophysiological analysis of the adult (P56) mutant mice revealed a mild but significant reduction in the motor nerve conduction velocity. lnterestingly, this functional change was not accompanied by any detectable alterations in general myelin structure. However, I have observed significant alterations in the mRNA expression level of other FABPs, predominantly FABP9, in the PNS of P2-/- mice as compared to age-matched P2+/+ mice indicating a role of P2 in the glial myelin lipid metabolism.¦Le diabète de type 2 touche 284 million de personnes dans le monde en 2010 et son évolution conduit dans la moitié des cas à une neuropathie périphérique diabétique. Bien que la neuropathie périphérique soit la complication la plus courante du diabète pouvant conduire jusqu'à l'amputation, sa physiopathologie est aujourd'hui encore mal comprise. Dans le but d'améliorer les connaissances moléculaires expliquant les mécanismes de la neuropathie liée au diabète de type 2, j'ai utilisé un modèle murin du diabète de type 2, les souris db/db.¦ln vivo, les enregistrements éléctrophysiologiques des animaux diabétiques montrent qu'en plus d'une diminution de la vitesse de conduction nerveuse, les souris db/db présentent également une augmentation de l'excitabilité nerveuse. Des mesures menées Ex­ vivo ont montré l'existence d'un phénotype d'hyperexcitabilité sur les nerfs périphériques isolés d'animaux diabétiques. Grâce à l'utilisation d'inhibiteurs pharmacologiques, nous avons pu démontrer que l'hyperexcitabilité démontrée était due à une réduction d'activité des canaux Kv1. En accord avec ces données, j'ai observé qu'une situation de diabète conduisait à une diminution des canaux Kv1.2 aux régions juxta-paranodales des nerfs périphériques db/db, alors que l'expression du transcrit et de la protéine restait stable. J'ai également confirmé l'absence de canaux Kv1.2 aux juxta-paranoeuds de biopsies de nerfs de patients diabétiques. L'ensemble de ces observations montrent que les nerfs périphériques chez les patients atteints de diabète de type 2 est due à une diminution des canaux potassiques rapides juxtaparanodaux les identifiant ainsi comme des cibles thérapeutiques potentielles.¦Les cellules de Schwann enveloppent et isolent les axones périphériques d'une membrane spécialisée, la myéline, composée de deux fois plus de lipides que de protéines. La protéine P2 (Pmp2 "peripheral myelin protein 2" ou FABP8 "fatty acid binding protein") est l'une des protéines les plus abondantes au système nerveux périphérique. P2 appartient à la famille de protéines FABP liant et transportant les acides gras et est une protéine cytosolique de 14,8 kDa exprimée du côté cytoplasmique de la myéline compacte.¦Afin d'étudier le rôle de P2 dans les cellules de Schwann myélinisantes, j'ai généré une souris knockout (P2-/-). Après avoir validé l'absence de transcrit et de protéine P2 dans les nerfs sciatiques P2-/-, des mesures électrophysiologiques ont montré une réduction modérée mais significative de la vitesse de conduction du nerf moteur périphérique. Il est important de noter que ces changements fonctionnels n'ont pas pu être associés à quelconque changement dans la structure de la myéline. Cependant, j'ai observé dans les nerfs périphériques P2-/-, une altération significative du niveau d'expression d'ARNm d'autres FABPs et en particulier FABP9. Ce dernier résultat démontre l'importance du rôle de la protéine P2 dans le métabolisme lipidique de la myéline.

Glial and axonal body fluid biomarkers are related to infarct volume, severity, and outcome.

Body fluid biomarkers of central nervous system damage may help improve the prognostic and diagnostic accuracy in ischemic stroke. We studied 53 patients. Stroke severity and outcome was rated using the National Institutes of Health Stroke Scale and modified Rankin scale. Ferritin, S100B, and NfH were measured in cerebrospinal fluid (CSF) and serum. Infarct volume was calculated from T2W images. CSF S100B (median 1.00 ng/mL) and CSF ferritin (10.0 ng/mL) levels were elevated in patients with stroke compared with control subjects (0.62 ng/mL, P < .0001; 2.34 ng/mL, P < .0001). Serum S100B (0.09 ng/mL) was higher in patients with stroke compared with control subjects (0.01 ng/mL). CSF S100B levels were higher in patients with a cardioembolic stroke (2.88 ng/mL) than in those with small-vessel disease (0.89 ng/mL, P < .05). CSF S100B levels correlated with the National Institutes of Health Stroke Scale score on admission (R = 0.56, P < .01) and the stroke volume (R = 0.44, P = .01). CSF S100B and NfH-SMI35 levels correlated with outcome on the modified Rankin scale. CSF S100B levels were related to stroke severity and infarct volume and highest in cardioembolic stroke.

Varicelle pendant la grossesse: quelles conséquences pour la mère et l'enfant [Varicella during pregnancy: consequences for the mother and the newborn].

In our area, varicella is a frequent and essentially benign childhood disease. In contrast, the disease course is likely to be more severe or complicated in the adult, particularly so in the pregnant woman. There is a definite risk of congenital varicella syndrome when the chickenpox occurs during the first 20 weeks of pregnancy. This syndrome predominantly affects the skin, the subcutaneous tissue, muscles and bones, as well as the central nervous system, and can bring about major functional sequellae. In case of chickenpox occurring at the very end of pregnancy, transplacental transfer of the virus may result in a perinatal varicella disease. We propose a approach of each of these different situations.

Restricted transgene expression in the brain with cell-type specific neuronal promoters.

Tissue-targeted expression is of major interest for studying the contribution of cellular subpopulations to neurodegenerative diseases. However, in vivo methods to investigate this issue are limited. Here, we report an analysis of the cell specificity of expression of fluorescent reporter genes driven by six neuronal promoters, with the ubiquitous phosphoglycerate kinase 1 (PGK) promoter used as a reference. Quantitative analysis of AcGFPnuc expression in the striatum and hippocampus of rodents showed that all lentiviral vectors (LV) exhibited a neuronal tropism; however, there was substantial diversity of transcriptional activity and cell-type specificity of expression. The promoters with the highest activity were those of the 67 kDa glutamic acid decarboxylase (GAD67), homeobox Dlx5/6, glutamate receptor 1 (GluR1), and preprotachykinin 1 (Tac1) genes. Neuron-specific enolase (NSE) and dopaminergic receptor 1 (Drd1a) promoters showed weak activity, but the integration of an amplification system into the LV overcame this limitation. In the striatum, the expression profiles of Tac1 and Drd1a were not limited to the striatonigral pathway, whereas in the hippocampus, Drd1a and Dlx5/6 showed the expected restricted pattern of expression. Regulation of the Dlx5/6 promoter was observed in a disease condition, whereas Tac1 activity was unaffected. These vectors provide safe tools that are more selective than others available, for the administration of therapeutic molecules in the central nervous system (CNS). Nevertheless, additional characterization of regulatory elements in neuronal promoters is still required.

SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the peripheral nervous system.

Charcot-Marie-Tooth disease type 4C (CMT4C) is an early-onset, autosomal recessive form of demyelinating neuropathy. The clinical manifestations include progressive scoliosis, delayed age of walking, muscular atrophy, distal weakness, and reduced nerve conduction velocity. The gene mutated in CMT4C disease, SH3TC2/KIAA1985, was recently identified; however, the function of the protein it encodes remains unknown. We have generated knockout mice where the first exon of the Sh3tc2 gene is replaced with an enhanced GFP cassette. The Sh3tc2(DeltaEx1/DeltaEx1) knockout animals develop progressive peripheral neuropathy manifested by decreased motor and sensory nerve conduction velocity and hypomyelination. We show that Sh3tc2 is specifically expressed in Schwann cells and localizes to the plasma membrane and to the perinuclear endocytic recycling compartment, concordant with its possible function in myelination and/or in regions of axoglial interactions. Concomitantly, transcriptional profiling performed on the endoneurial compartment of peripheral nerves isolated from control and Sh3tc2(DeltaEx1/DeltaEx1) animals uncovered changes in transcripts encoding genes involved in myelination and cell adhesion. Finally, detailed analyses of the structures composed of compact and noncompact myelin in the peripheral nerve of Sh3tc2(DeltaEx1/DeltaEx1) animals revealed abnormal organization of the node of Ranvier, a phenotype that we confirmed in CMT4C patient nerve biopsies. The generated Sh3tc2 knockout mice thus present a reliable model of CMT4C neuropathy that was instrumental in establishing a role for Sh3tc2 in myelination and in the integrity of the node of Ranvier, a morphological phenotype that can be used as an additional CMT4C diagnostic marker.

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1alpha or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca(2+)-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1alpha/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca(2+) from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.

Réplication cérébrale du VIH malgré une thérapie antirétrovirale efficace: impact clinique et prise en charge [Intracerebral HIV replication despite an efficient antiretroviral treatment: clinical impact and management].

Various neurological and neuropsychological manifestations are still relatively frequently reported in HIV infected patients in the highly active antiretroviral therapy era. A fraction of them could be related to HIV replication in the central nervous system (CNS) despite adequate peripheral viral suppression. This hypothesis is supported by numerous reports of detectable HIV RNA in the cerebrospinal fluid in the context of a low or undetectable viremia in association with neurological or neuropsychological complaints. In addition, some antiviral molecules may not achieve adequate levels in the CNS, thus potentially favoring intracerebral HIV replication and even antiretroviral resistance. Neurologic manifestations in the presence of CNS HIV replication often decrease after antiretroviral treatment CNS penetration optimization.

Central control of glucose homeostasis: the brain--endocrine pancreas axis.

A large body of data gathered over the last decades has delineated the neuronal pathways that link the central nervous system with the autonomic innervation of the endocrine pancreas, which controls alpha- and beta-cell secretion activity and mass. These are important regulatory functions that are certainly keys for preserving the capacity of the endocrine pancreas to control glucose homeostasis over a lifetime. Identifying the cells involved in controlling the autonomic innervation of the endocrine pancreas, in response to nutrient, hormonal and environmental cues and how these cues are detected to activate neuronal activity are important goals of current research. Elucidation of these questions may possibly lead to new means for preserving or restoring defects in insulin and glucagon secretion associated with type 2 diabetes.

Prévalence des maladies infectieuses au Centre hospitalier universitaire vaudois [Prevalence of infectious diseases at the Vaud University Hospital Center].

The prevalence of infectious diseases at our hospital (Centre hospitalier universitaire vaudois, Lausanne [CHUV], 900 beds) was studied retrospectively over a two years period (1980-1981). The medical diagnosis of 30203 patients recorded in the computerized medical archives, representing 93% of the patients admitted during the period of observation, was reviewed. To assess the reliability of the computerized data, quality control was carried out through detailed analysis of all the histologically proven appendicitis recorded during 1981. 88% of the histologically proven appendicitis were registered in the computer and the diagnosis was specific in 87% of cases. An infectious disease was the primary reason for admission in 12.8% of the patients (3873) during the study period. Altogether, 20.2% of patients presented with an infection during their hospital stay. Because of the retrospective nature of the study it was not possible to determine whether these additional infections were nosocomially acquired. The organ systems most frequently infected were the respiratory tract (28.5% of all infections), the digestive tract (20.5%), the skin and osteoarticular system (16%) and the urogenital tract (11.6%). An infection was the primary reason for admission of 40.2% of the patients hospitalized in the dermatology service, of 19.7% of patients admitted in internal medicine, of 15-17% of the patients admitted in pediatrics, ENT and general surgery, and of 1-2% of the patients admitted in neurosurgery and radiotherapy. These observations highlight the continuing importance of infectious diseases in a modern hospital, in spite of high socio-economic levels, stringent hygiene and epidemiologic measures, and modern antibiotic availability.

Les aquaporines 4 et 9 dans le système nerveux central : expression lors de la différenciation cellulaire et implication dans le métabolisme énergétique astrocytaire "in vitro"

RESUME : Les aquaporines (AQPs) sont des protéines membranaires perméables à l'eau (aquaporines strictes) et, pour certaines d'entre elles, également au glycérol (aquaglycéroporines). Ces protéines sont présentes dans les bactéries, les plantes et les différents organes des mammifères. Dans le cerveau, la moindre augmentation de volume hydrique peut avoir de graves conséquences sur son fonctionnement, d'où l'importance de la régulation de l'homéostasie de l'eau grâce aux AQPs. L'AQP4, une aquaporine stricte, est présente dans les astrocytes et est impliquée dans la formation et la résorption des oedèmes cérébraux. En revanche, l'AQP9 est une aquaglycéroporine, qui est localisée non seulement dans les astrocytes mais également dans les neurones catécholaminergiques. Bien que la distribution de l'AQP4 dans le cerveau soit clairement établie, la présence de l'AQP9 est toujours une donnée controversée et son rôle fonctionnel dans le système nerveux central n'est pas connu. Par ailleurs, aucune donnée n'existe sur l'expression des AQP4 et 9 lors de la différenciation de cellules souches neurales foetales (CSNf) en astrocytes ou en neurones catécholaminergiques. Dans la première partie de ce travail, un protocole a été mis au point permettant de différencier des CSNf de souris en astrocytes et neurones, dont des neurones catécholaminergiques. La caractérisation des cultures de CSNf et des cultures mixtes par immunofluorescence a permis de montrer que l'immunomarquage AQP9 est présent dans les CSNf et est conservé lors de leur différenciation en astrocytes ou en neurones catécholaminergiques. Les résultats obtenus ont mis en évidence une très bonne corrélation entre l'expression de la TH (tyrosine hydroxylase: enzyme limitante de la synthèse des catécholamines) et celle de l'AQP9 lors de la différenciation des CSNf en neurones catécholaminergiques. Par contre, l'immunomarquage AQP4 n'est pas présent dans les CSNf alors qu'il est observé dans les astrocytes. De plus, aucun immunomarquage AQP4 ou AQP9 n'a été observé dans les neurones NIAP2-positifs. Dans la deuxième partie de ce travail, l'expression des AQP4 et 9 a été quantifiée dans les CSNf ainsi que dans trois populations d'astrocytes présentant des propriétés métaboliques différentes. Ces trois populations astrocytaires sont issues de la différenciation des CSNf par le CNTF, le LIF ou le sérum de veau foetal. Les analyses par RTPCR quantitative et western blot ont montré une augmentation de l'expression de l'AQP9 et de l'AQP4 corrélée à l'acquisition de propriétés métaboliques spécifiques des astrocytes matures. Dans la dernière partie, la technique d'ARN interférents a permis d'étudier le rôle fonctionnel de l'AQP9 dans le modèle de culture pure d'astrocytes différenciés par le sérum. L'inhibition de l'expression d'AQP9 entraîne une diminution de la perméabilité au glycérol et une augmentation de l'utilisation de glucose, corrélée à une stimulation du métabolisme oxydatif astrocytaire. En revanche, 1a baisse d'expression d'AQP9 n'a aucun effet sur la glycolyse anaérobie ni sur la libération du lactate. En conclusion, dans ce modèle in vitro, seule l'AQP9 est exprimée dans les CSNf et les neurones catécholaminergiques alors que dans Ies astrocytes, à la fois l'AQP9 et l'AQP4 sont exprimées. Cette distribution est identique à celle observée in vivo et confirme la localisation spécifique de l'AQP9 dans les neurones catécholaminergiques. De plus, ces résultats montrent, pour la première fois, l'implication de l'AQP9 dans la perméabilité des astrocytes au glycérol et son implication dans le métabolisme énergétique astrocytaire. ABSTACT : Aquaporins (AQPs) are membrane proteins permeable to water (orthodoxes aquaporins) and some of them are also permeable to glycerol (aquaglyceroporins). These proteins are widely expressed in bacteria, plants and mammals. AQP water homeostasis regulation in brain is of primary importance as the brain volume cannot increase. AQP4, an orthodoxe aquaporin, is present in astrocytes and seems to be involved in edema formation and resorption. On the other hand, AQP9 is an aquaglyceroporin which is localised not only in astrocytes but also in catecholaminergic neurons. Although AQP4 distribution in brain is clearly established, the presence of AQP9 is still a discussed data and its functional role in the central nervous system is unknown. In addition, no data exists on AQP4 or AQP9 expression during fetal neural stem cells (fNSC) differentiation into astrocytes or catecholaminergic neurons. In the first part of this work, a protocol was developed to differentiate mouse fNSC into astrocytes and neurons, with the aim to obtain catecholaminergic neurons. By immunefluorescence, we have shown that AQP9 is expressed in fNSC cultures and also in astrocytes and catecholaminergic neurons in mixt cultures. The results obtained highlighted a very good correlation between TH expression (tyrosin hydroxylase being a limiting enzyme of catecholamines synthesis) and AQP9 in fNSC and all along their differentiation into catecholaminergic neurons. On the other hand, AQP4 immunolabelling is not observed in fNSC whereas it is in astrocytes. Moreover, neitheir AQP4, nor AQP9 immunoreactivity was observed in MAP2-positive neurons. In the second part of this work, AQP4 and AQP9 expression was quantified in fNSC and in three populations of astrocytes presenting different metabolic properties. These three astrocyte populations result from fNSC differentiation by addition of CNTF, LIF or fetal calf serum. Quantitative RT-PCR and western blot analyses have shown an increase in both AQP4 and AQP9 expression, correlated with the acquisition of specific metabolic properties of mature astrocytes. In the last part, siRNA were used to study the functional role of AQP9 in the pure astrocyte culture model differentiated by addition of fetal calf serum. Inhibition of AQP9 expression leads to a decrease of glycerol uptake and to an increase of glucose uptake, correlated with a stimulation of the astrocyte oxydative metabolism. On the other hand, inhibition of AQP9 expression does not have any effect on anaerobic glycolysis nor on lactate release. In conclusion, in this in vitro model, only AQP9 is expressed in fNSC and in catecholaminergic neurons whereas in astrocytes, both AQP9 and AQP4 are expressed. This distribution is identical to that observed in vivo and confirms the specific AQP9 localization in catecholaminergic neurons. IVloreover, these results show, for the first time, that AQP9 is implicated in glycerol uptake and in astrocyte energetic metabolism. Résumé large public : Les aquaporines, des protéines localisées dans les membranes cellulaires sont, comme leur nom l'indique, des canaux à eau. Pendant longtemps, il a été considéré que l'eau diffusait librement dans et à travers les cellules; la caractérisation des AQPs a révolutionné la vision des scientifiques concernant les mouvements d'eau entre les différents compartiments infra et extracellulaires, et a d'ailleurs valu le Prix Nobel à Peter Agre en 1992. Certaines AQPs, dites "strictes", laissent passer uniquement l'eau et participent au contrôle du volume hydrique. Ce contrôle est particulièrement important pour le bon fonctionnement du cerveau en raison de la présence de la boîte crânienne qui limite les variations de volume. D'autres AQPs, les aquaglycéroporines, sont perméables non seulement à l'eau mais également à d'autres molécules comme le glycérol. Elles facilitent, par exemple, la sortie du glycérol des cellules graisseuses et sa capture par les cellules du foie afin de produire du glucose en période de jeûne. Le cerveau est principalement composé de deux types de cellules: les neurones et les cellules gliales, majoritairement des astrocytes. L'AQP4, une AQP stricte, est présente dans les astrocytes et joue un rôle dans la formation et la résorption des oedèmes cérébraux. L'AQP9, une aquaglycéroporine, est également présente dans les astrocytes et dans une population spécifique de neurones, les neurones catécholaminergiques, touchés dans la maladie de Parkinson. A ce jour, la présence de l'AQP9 dans le cerveau est une donnée controversée et son rôle fonctionnel est inconnu. Ce travail de thèse a permis de montrer que l'AQP9 est bien présente d'une part dans les cellules souches neurales foetales et d'autre ,part dans les astrocytes et neurones catécholaminergiques issus de leur différenciation. De plus, ces expériences ont mis en évidence un rôle de l'AQP9 dans l'entrée du glycérol dans les astrocytes, ce qui pourrait être bénéfique dans des conditions d'ischémie. Enfin, les .résultats de cette étude suggèrent également un rôle de l'AQP9 dans le métabolisme énergétique des astrocytes. L'ensemble de ces travaux démontre le rôle important de l'AQP9 dans le cerveau et ouvre de nouvelles perspectives quant aux rôles des AQPs dans des situations pathologiques telles que l'ischémie cérébrale ou encore la maladie de Parkinson.

Genetic studies of body mass index yield new insights for obesity biology

Obesity is heritable and predisposes to many diseases. To understand the genetic basis of obesity better, here we conduct a genome-wide association study and Metabochip meta-analysis of body mass index (BMI), a measure commonly used to define obesity and assess adiposity, in up to 339,224 individuals. This analysis identifies 97 BMI-associated loci (P < 5 × 10(-8)), 56 of which are novel. Five loci demonstrate clear evidence of several independent association signals, and many loci have significant effects on other metabolic phenotypes. The 97 loci account for ∼2.7% of BMI variation, and genome-wide estimates suggest that common variation accounts for >20% of BMI variation. Pathway analyses provide strong support for a role of the central nervous system in obesity susceptibility and implicate new genes and pathways, including those related to synaptic function, glutamate signalling, insulin secretion/action, energy metabolism, lipid biology and adipogenesis.

Associated anomalies in multi-malformed infants with cleft lip and palate: An epidemiologic study of nearly 6 million births in 23 EUROCAT registries.

We studied 5,449 cases of cleft lip (CL) with or without cleft palate (CL/P) identified between 1980 and 2000 from the EUROCAT network of 23 registers (nearly 6 million births) in 14 European countries. We investigated specific types of defects associated with clefts. Among CL/P cases (prevalence = 9.1 per 10,000), 1,996 (36.6%) affected only the lip (CL) and 3,453 (63.4%) involved CL and palate (CLP). A total of 3,860 CL/P cases (70.8%) occurred as isolated anomalies and 1,589 (29.2%) were associated with other defects such as multiple congenital anomalies of unknown origin (970), chromosomal (455) and recognized syndromes (164). Associated malformations were more frequent in infants who had CLP (34.0%) than in infants with CL only (20.8%). Among multi-malformed infants, 2 unrelated anomalies were found in 351 cases, 3 in 242 cases, and 4 or more in 377 cases. Among 5,449 CL/P cases, 4,719 were live births (LB) (86.6%), 203 stillbirths (SB) (3.7%), while 508 (9.3%) were terminations of pregnancy (ToP). CL/P occurred significantly more frequently in males (M/F = 1.70), especially among total isolated cases (M/F = 1.87) and CLP isolated cases (M/F = 1.92). The study confirmed that musculoskeletal, cardiovascular, and central nervous system defects are frequently associated with CL/P. An association with reduction anomalies of the brain was found. This association suggests that clinicians should seek to identify structural brain anomalies in these patients with CL/P as the potential functional consequences may be important for rehabilitation and clinical management.