Contribution à l étude fonctionnelle des effets du PACAP sur l ontogénèse du cervelet chez la souris in vivo

Le cervelet adulte, constitue, chez les mammifères, la région la plus volumineuse de l encéphale après les hémisphères cérébraux et représente avant tout un centre moteur qui contribue au contrôle des contractions des muscles squelettiques nécessaires à la coordination motrice, au maintien de la posture et de l équilibre. Toutefois, il a été démontré que cette structure intervient également dans l'apprentissage et la mémorisation. Le cervelet est constitué de deux lobes flocculo-nodulaires et de deux hémisphères encadrant la région médiane du vermis. L'ensemble est recouvert par une structure laminaire, appelée cortex cérébelleux, qui est constituée de quatre couches distinctes, à savoir la couche granulaire externe (CGE), la couche moléculaire (CMol), la couche des cellules de Purkinje (CP) et la couche granulaire interne (CGI). Chez la souris, pour laquelle l apprentissage de la marche s effectue graduellement au cours de la période postnatale, le cervelet est immature à la naissance et son ontogenèse se poursuit pendant les premières semaines de la vie. Au cours de cette phase développementale, le cortex cérébelleux est sous l influence de divers facteurs. Parmi ceux-ci, le pituitary adenylate cyclase-activating polypeptide (PACAP) s'est révélé être un neuropeptide neurotrophique important. En effet, une forte expression de PACAP et de son récepteur PAC-1 a été observée dans les différentes couches cérébelleuses au cours des périodes pré- et postnatales. De plus, de nombreuses études réalisées in vivo et in vitro ont démontré que le PACAP exerce des effets neurotrophiques sur les neurones en grain, favorisant notamment la différenciation, l'élongation neuritique et la survie des cellules en grain. Enfin, il est maintenant bien établi que le PACAP exerce un puissant effet anti-apoptotique sur les cellules granulaires du cervelet en culture en inhibant la voie mitochondriale et la caspase-3. L objectif de cette thèse a tout d abord été de vérifier le rôle physiologique du PACAP in vivo dans le cervelet des mammifères en caractérisant le phénotype du cervelet de souris invalidées pour le gène PACAP (PACAP-/-). Cette étude a été réalisée sur des animaux âgés de 4 et 7 jours postnataux (P4 et P7), deux stades correspondant à des étapes clés de la genèse des cellules en grain. L'organisation générale des structures cérébelleuses n est pas sensiblement affectée par l'absence de PACAP. Toutefois, une analyse morphométrique fine a mis en évidence une réduction significative de l épaisseur des couches granulaires externe à P4 et interne à P7 chez les souris invalidées, suggérant une perte neuronale dans le cortex cérébelleux. L étude de la densité cellulaire a révélé que les variations d épaisseur observées sont effectivement dues à une réduction du nombre de cellules. Par ailleurs, nous avons pu démontrer par western blot et PCR quantitative que, chez les souris PACAP-/-, l expression de la nestine, un marqueur de précurseurs neuraux, n est pas modifiée alors que celle de la synaptophysine, un marqueur de neurones différenciés, est réduite à P4 et P7. Enfin, nous avons montré que le nombre de cellules exprimant la caspase-3 clivée, enzyme effectrice de l apoptose, ainsi que l activité biologique de cette enzyme sont significativement plus importants chez les souris PACAP-/- à P4 et P7. L'ensemble de ces résultats suggère que le PACAP exerce un rôle neurotrophique in vivo, d'une part, sur la différenciation des neurones en grain en modifiant l expression de protéines associées à la maturité neuronale et, d'autre part, sur la survie cellulaire en inhibant l'activité de la caspase-3 pro-apoptotique. Il est maintenant bien établi que l'ajustement de la population neuronale au cours du développement cérébral dépend d'un équilibre entre les facteurs neurotrophiques et les facteurs de mort cellulaire. Nous avons donc émis l'hypothèse que l'absence de PACAP chez les souris PACAP-/- provoquerait un déséquilibre en faveur de l'apoptose entraînant une perte des cellules en grain matures de la CGI. Le second volet de cette thèse a donc consisté à étudier les effets du facteur Fas ligand (FasL) sur le développement du cervelet de souris sauvages et PACAP-/- afin de déterminer si le neuropeptide est en mesure de bloquer la mort cellulaire induite par FasL. En effet, il a été démontré que FasL intervient dans l induction de la mort cellulaire programmée lors de la mise en place de diverses populations cellulaires et que son effet pro-apoptotique implique l activation de la caspase-3. Avant toute étude physiologique, nous avons vérifié par immunohistochimie la présence de Fas, le récepteur du FasL, dans le cervelet, démontrant ainsi pour la première fois sa localisation sur les cellules en grain et les cellules de Purkinje de souris. Nous avons ensuite réalisé des expériences ex vivo sur des tranches organotypiques de cervelet de souriceaux âgés de 7 jours qui ont révélé que le FasL (5 ng/ml) induit une augmentation significative de l activité de la caspase-3 et que cet effet est inhibé par le PACAP (10-7 M). Ces données montrent que le PACAP est capable de s'opposer aux actions pro-apoptotiques du FasL via la caspase-3. Afin de transposer ce travail dans des conditions in vivo, nous avons effectué des injections sous-durales de FasL (0.1 g/ l) en l absence ou en présence de PACAP (0.3 g/ l) au niveau du cortex cérébelleux chez des souris sauvages et PACAP-/-. Les résultats indiquent que les animaux traités avec le FasL seul présentent une diminution de l épaisseur de la CGI à P8. Il semble donc que, malgré la présence du récepteur Fas sur les neurones en grain et les cellules de Purkinje, le FasL agisse spécifiquement sur les cellules granulaires. Cette hypothèse est étayée par une étude immunohistochimique qui a révélé que l'administration de FasL dans le cervelet augmente le nombre de cellules exprimant la caspase-3 clivée dans la CGI chez les animaux sauvages et PACAP-/-. Par ailleurs, nos résultats démontrent que la co-injection de PACAP prévient la réduction de la CGI, partiellement chez les souris sauvages et totalement chez les animaux PACAP-/-. Les tests comportementaux réalisés sur des souris sauvages traitées par le FasL et le PACAP n'ont pas révélé de déficits comportementaux significatifs si ce n est un défaut d équilibre transitoire à P8 chez les souris traitées simultanément par le FasL et le PACAP. Par ailleurs, aucune altération morphologique du cervelet et aucun trouble moteur n ont été observés entre les différents groupes chez l adulte, suggérant la mise en place de mécanismes de compensation au cours du temps. En résumé, cette étude réalisée sur un modèle de souris invalidées démontre pour la première fois que le PACAP exerce in vivo des effets neurotrophiques et anti-apoptotiques. Ainsi, nous avons observé que le PACAP stimule l expression de protéines associées à la maturité neuronale telle que la synaptophysine et inhibe l'activité de la caspase-3 dans les cellules en grain matures de la CGI de souris. De plus, le PACAP est capable de s'opposer aux effets pro-apoptotiques de FasL, indiquant que sa présence au cours de l'ontogenèse du cervelet participe à l'équilibre entre les facteurs neurotrophiques et les facteurs de mort cellulaire. Enfin, l'administration de FasL chez des souriceaux n'affecte pas le comportement moteur à l'âge adulte, suggérant l'implication d'autres facteurs dans la mise en place d'un mécanisme de compensation au cours de la croissance.The adult cerebellum represents, in mammals, the most voluminous brain region after the cerebral hemispheres and constitutes mainly a motor centre, implicated in the control of skeletal muscle contractions, which is required for motor coordination, posture and equilibrium. In addition, it has been shown that the cerebellum plays a role in learning and memory. The cerebellum is composed of two flocculo-nodular lobes and two hemispheres flanking the median region called the vermis. The entire structure is covered by a laminar coating named cerebellar cortex which is organized in four different layers, namely the external granule cell layer (EGL), molecular cell layer (Mol), Purkinje cell layer (PL) and internal granule cell layer (IGL). In mouse, in which walking ability appears gradually during the postnatal period, the cerebellum is immature at birth and its ontogenesis carries on during the first weeks of life. During this developmental phase, the cerebellar cortex is under the influence of various factors. Among those, pituitary adenylate cyclase-activating polypeptide (PACAP) has been identified as an important trophic neuropeptide. High concentrations of PACAP and its receptor PAC-1 have been actually detected in different layers of the cerebellar cortex during the pre- and postnatal periods. Moreover, several in vivo and in vitro studies have shown that PACAP exerts neurotrophic effects on granule neurons, notably in promoting differentiation, neurite outgrowth and survival. Finally, it is now well established that PACAP exerts a potent anti-apoptotic effect on cultured cerebellar granule cells through inhibition of the mitochondrial pathway and caspase 3. The aim of this Ph D thesis was first to confirm the physiological role of PACAP in vivo in the cerebellum of mammals by characterizing the neuroanatomical and neurochemical alterations of the cerebellum in PACAP knockout (PACAP-/-) mice. This study was carried out on 4- and 7-day-old animals (P4 and P7), two stages corresponding to key periods of granule cell genesis. The global organization of the cerebellar cortex was not affected by the absence of PACAP. However, detailed morphometric analysis revealed a significant reduction of the thickness of the EGL and IGL, respectively, at P4 and P7 in knockout animals, suggesting neuronal loss in the cerebellar cortex. The study of cellular density indicated that the alteration of the thickness of the cortical layers could be ascribed to a decrease in cell number. In addition, western blot and qPCR experiments revealed that the expression of nestin, a neural marker, was not modified in PACAP-/- mice whereas the expression of synaptophysin, a marker of differentiated neurons, was reduced at P4 and P7 in knockout animals. Finally, we have shown that the number of cells expressing cleaved caspase-3, the effector enzyme of apoptosis, as well as the biologic activity of this enzyme are significantly elevated in PACAP-/- mice at P4 and P7. Taken together, these data suggest that PACAP exerts a neurotrophic role in vivo, both on neuronal differentiation by modifying the expression of proteins associated with neuronal maturity and on cell survival by inhibiting the activity of the pro-apoptotic enzyme caspase-3. It is now clearly established that the regulation of neuronal population during brain development depends on a balance between neurotrophic and cell death factors. We thus hypothesized that the lack of PACAP in knockout animals could alter the equilibrium to favour apoptosis leading to a loss of granular cells in the IGL. So, the second part of this work consisted in studying the effect of Fas ligand (FasL) on cerebellar development in wild-type and PACAP-/- mice in order to determine if the neuropeptide PACAP is able to block cell death induced by FasL. Indeed, it has been previously shown that FasL interferes in the induction of cell death during the adjustment of various cellular populations and that this pro-apoptotic effect involves the activation of caspase-3. Here, we have used an immunohistochemical approach to investigate the presence of FasL receptor, called Fas, in the cerebellum and we observed, for the first time, the occurrence of Fas on granule and Purkinje cells in mice. Then, we performed ex vivo experiments on organotypic slices of the cerebellum from 7-day-old mice which revealed that FasL (5 ng/ml) induces a significant increase of caspase-3 activity and that this effect is inhibited by PACAP (10-7 M). These data show that PACAP is able to reverse the pro-apoptotic actions of FasL via caspase-3. In order to transpose our work to in vivo conditions, we administered FasL (0.1 g/ l) in the absence or presence of PACAP (0.3 g/ l) in the subarachnoid space of the cerebellar cortex in wild-type and PACAP-/- mice. The data indicate that animals treated with FasL alone display a decrease of IGL thickness at P8, suggesting that, in spite of the presence of Fas on granule and Purkinje cells, FasL specifically affects granule neurons. This hypothesis is supported by an immunohistochemical study showing that FasL administration at the cerebellar surface increases the number of cells expressing caspase-3 in the IGL in wild-type and PACAP-/- mice. In addition, we found that co-injection of PACAP prevents FasL-induced IGL atrophy partially in wild-type mice and totally in PACAP-/- animals. Behavioral tests carried out on wild-type mice treated with FasL and/or PACAP did not reveal any locomotor alteration except a transient impairment of equilibrium at P8 in animals treated simultaneously with FasL and PACAP. Moreover, no locomotor defect or morphological alteration of the cerebellum was observed between the different groups in adult mice, suggesting the occurrence of a compensation mechanism in adulthood. To summarize, this Ph D thesis conducted on a model of knockout mice demonstrates for the first time that PACAP exerts neurotrophic and anti-apoptotic effects in vivo. Thus, we observed that PACAP stimulates the expression of neuronal differentiation markers such as synaptophysin and inhibits the activity of caspase-3 in mature granule cells in the mouse IGL. Moreover, PACAP is able to block the pro-apoptotic effect of FasL, indicating that its presence participates in the equilibrium between neurotrophic and cell death factors during ontogenesis of the cerebellum. Finally, FasL administration in young mice did not affect motor behaviour at adult stage, suggesting the implication of other factors in the setting of a compensatory mechanisms.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Multi-view interferometric out-of-focus imaging for the tomography of irregular rough particles in a flow

International audienceMulti-view interferometric out-of-focus imaging of particles is realized to determine 3D-characteristics of irregular particles in a flow. Using two angles of view, we determine the global 3D-shape, the dimension, and the 3D-orientation of particles that belong to some families of irregular rough particles as sticks, crosses or plates

Intermittent hypoxia in a mouse model of apnea of prematurity leads to a retardation of cerebellar development and long-term functional deficits

International audienceBackground: Apnea of prematurity (AOP) is caused by respiratory control immaturity and affects nearly 50% of premature newborns. This pathology induces perinatal intermittent hypoxia (IH), which leads to neurodevelopmental disorders. The impact on the brain has been well investigated. However, despite its functional importance and immaturity at birth, the involvement of the cerebellum remains poorly understood. Therefore, this study aims to identify the effects of IH on cerebellar development using a mouse model of AOP consisting of repeated 2-min cycles of hypoxia and reoxygenation over 6 h and for 10 days starting on postnatal day 2 (P2).Results At P12, IH-mice cerebella present higher oxidative stress associated with delayed maturation of the cerebellar cortex and decreased dendritic arborization of Purkinje cells. Moreover, mice present with growth retardation and motor disorders. In response to hypoxia, the developing cerebellum triggers compensatory mechanisms resulting in the unaltered organization of the cortical layers from P21 onwards. Nevertheless, some abnormalities remain in adult Purkinje cells, such as the dendritic densification, the increase in afferent innervation, and axon hypomyelination. Moreover, this compensation seems insufficient to allow locomotor recovery because adult mice still show motor impairment and significant disorders in spatial learning.Conclusions All these findings indicate that the cerebellum is a target of intermittent hypoxia through alterations of developmental mechanisms leading to long-term functional deficits. Thus, the cerebellum could contribute, like others brain structures, to explaining the pathophysiology of AOP

Neuroprotective effects of PACAP against ethanol-induced toxicity in the developing rat cerebellum.

International audienceThe developing rat cerebellum is particularly sensitive to alcohol at the end of the first postnatal week, a period of intense neurogenesis. The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) has previously been shown to prevent the death of cultured neurons in vitro. We have thus investigated the capacity of PACAP to counteract ethanol toxicity in 8-day-old rats. Behavioral studies revealed that PACAP reduces the deleterious action of alcohol in the negative geotaxis test. Administration of ethanol induced a transient increase of the expression of pro-apoptotic genes including c-jun or caspase-3 , which could be partially blocked by PACAP. Alcohol inhibited the expression of the α6 GABA ( A ) subunit while PACAP increased neuroD2 mRNA level, two markers of neuronal differentiation. Although gene regulations occurred rapidly, a third injection of ethanol was required to strongly reduce the number of granule cells in the internal granule cell layer, an effect which was totally blocked by PACAP. The action of PACAP was mimicked by D-JNKi1 and Z-VAD-FMK, indicating the involvement of the jun and caspase-3 pathways in alcohol toxicity. The present data demonstrate that PACAP can counteract in vivo the deleterious effect of ethanol. The beneficial action of PACAP on locomotor activity precedes its activity on cell survival, indicating that PACAP can block the detrimental action of ethanol on cell differentiation

New Introductions of Enterovirus 71 Subgenogroup C4 Strains, France, 2012

In France during 2012, human enterovirus 71 (EV-A71) subgenogroup C4 strains were detected in 4 children hospitalized for neonatal fever or meningitis. Phylogenetic analysis showed novel and independent EV-A71 introductions, presumably from China, and suggested circulation of C4 strains throughout France. This observation emphasizes the need for monitoring EV-A71 infections in Europe

Multiplexed infectious protein microarray immunoassay suitable for the study of the specificity of monoclonal immunoglobulins

International audienceEnzyme-linked immunosorbent assays (ELISAs) used to detect antibodies specific for common infectious agents such as Epstein–Barr virus (EBV), cytomegalovirus (CMV), Toxoplasma gondii (T. gondii), and hepatitis C virus (HCV) are time-consuming and require large volumes of samples, which restrict their use. We propose a new assay based on a multiplexed infectious protein (MIP) microarray combining different epitopes representative of the four germs. Antigens and lysates were printed on nitrocellulose slides to constitute the microarray. First, the microarray was incubated with human serum samples. Then, the suitability of the microarray for analysis of the specificity of purified monoclonal immunoglobulin (mc Ig) was assessed using serum and mc Ig of HCV-positive patients. Bound human immunoglobulin G (IgG) was detected using fluorescently labeled secondary antibodies, and the signals were quantified. Results obtained in serum samples with the new MIP microarray immunoassay were compared with ELI-SAs; we observed concordances of 95% for EBV, 93% for CMV, 91% for T. gondii, and 100% for HCV. Regarding purified mc Ig of HCV-positive patients, 3 of 3 recognized antigens printed on the microarray. Hence, the novel EBV/CMV/T. gondii/HCV MIP microarray allows simultaneous diagnosis of polyclonal and monoclonal immune response to infectious diseases using very small volume samples

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

International audiencePituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally alpha-amidated peptide that was first isolated 20 years ago from an ovine hypothalamic extract on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al., 1989. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-secretin-growth hormone-releasing hormone-glucagon superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordates to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide, the activity of which remains unknown. Two types of PACAP binding sites have been characterized: type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1-R, which is coupled to several transduction systems, and the PACAP/VIP-indifferent VPAC1-R and VPAC2-R, which are primarily coupled to adenylyl cyclase. PAC1-Rs are particularly abundant in the brain, the pituitary and the adrenal gland, whereas VPAC receptors are expressed mainly in lung, liver, and testis. The development of transgenic animal models and specific PACAP receptor ligands has strongly contributed to deciphering the various actions of PACAP. Consistent with the wide distribution of PACAP and its receptors, the peptide has now been shown to exert a large array of pharmacological effects and biological functions. The present report reviews the current knowledge concerning the pleiotropic actions of PACAP and discusses its possible use for future therapeutic applications

Neurosteroid biosynthesis: Enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides

International audienceNeuroactive steroids synthesized in neuronal tissue, referred to as neurosteroids, are implicated in proliferation, differentiation, activity and survival of nerve cells. Neurosteroids are also involved in the control of a number of behavioral, neuroendocrine and metabolic processes such as regulation of food intake, locomotor activity, sexual activity, aggressiveness, anxiety, depression, body temperature and blood pressure. In this article, we summarize the current knowledge regarding the existence, neuroanatomical distribution and biological activity of the enzymes responsible for the biosynthesis of neurosteroids in the brain of vertebrates, and we review the neuronal mechanisms that control the activity of these enzymes. The observation that the activity of key steroidogenic enzymes is finely tuned by various neurotransmitters and neuropeptides strongly suggests that some of the central effects of these neuromodulators may be mediated via the regulation of neurosteroid production

The two glycolytic markers GLUT1 and MCT1 correlate with tumor grade and survival in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. Although ccRCC is characterized by common recurrent genetic abnormalities, including inactivation of the von Hippel-Lindau (vhl) tumor suppressor gene resulting in stabilization of hypoxia-inducible factors (HIFs), the tumor aggressiveness and outcome of ccRCC is variable. New biomarkers are thus required to improve ccRCC diagnosis, prognosis and therapeutic options. This work aims to investigate the expression of HIF and proteins involved in metabolism and pH regulation. Their correlation to histoprognostic parameters and survival was analyzed.ccRCC of 45 patients were analyzed. HIF-1α, HIF-2α, HAF, GLUT1, MCT1, MCT4, CAIX and CAXII expression was assessed by immunohistochemistry in a semi-quantitative and qualitative manner. The GLUT1, MCT1, MCT4, CAIX and CAXII mRNA levels were analyzed in an independent cohort of 43 patients.A significant correlation was observed between increased GLUT1, MCT1, CAXII protein expression and a high Fuhrman grade in ccRCC patients. Moreover, while HIF-1α, HIF-2α and HAF expression was heterogenous within tumors, we observed and confirmed that HIF-2α co-localized with HAF. We confirmed, in an independent cohort, that GLUT1, MCT1 and CAXII mRNA levels correlated with the Fuhrman grade. Moreover, we demonstrated that the high mRNA level of both MCT1 and GLUT1 correlated with poor prognosis.This study demonstrates for the first time a link between the aggressiveness of high- Fuhrman grade ccRCC and metabolic reprogramming. It also confirms the role of HIF-2α and HAF in tumor invasiveness. Finally, these results demonstrate that MCT1 and GLUT1 are strong prognostic markers and promising therapeutic targets

Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides. Hormones and the Brain. Research and Perspectives in Endocrinology, pp 99-109

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