Leptin modulation of locomotor and emotional behaviors : the role of STAT3 signaling in dopamine neurons

La leptine circule en proportion de la masse graisseuse du corps et la transduction de son signal à travers la forme longue de son récepteur via un certain nombre de voies neurales , y compris MAPK, PI3-K ,AMPK et JAK2 - STAT3 . Il faut noter que STAT3 constitue une voie clée au récepteur de la leptine par laquelle la leptine module l'expression des gènes impliqués dans la régulation du bilan énergétique. La plupart des recherches ont porté sur la fonction du récepteur de la leptine au sein de l' hypothalamus, en particulier la fonction du récepteur de la leptine dans le noyau arqué. Toutefois, les récepteurs de la leptine sont également exprimés sur les neurones dopaminergiques de l'aire tégmentale ventrale et la leptine agit sur cette région du cerveau pour influencer la prise alimentaire, la motivation, la locomotion, l'anxiété et la transmission de la dopamine. De plus, la leptine active la STAT3 dans les dopaminergiques et GABAergiques populations neuronales. Bien que ces résultats contribuent à notre compréhension des multiples actions de la leptine dans le système nerveux central, il reste à résoudre les cellules et la signalisation du récepteur de la leptine qui sont responsables des effets neurocomportementaux de la leptine dans le mésencéphale. Visant à déterminer la contribution de la voie de signalisation STAT3 dans les neurones dopaminergiques du mésencéphale, nous avons généré une lignée de souris knockout conditionnel dans lequel l'activation du gène de STAT3 sur son résidu tyrosine 705 ( Tyr 705 ) est absent spécifiquement dans les neurones dopaminergiques. Avec l'utilisation de ce modèle de souris génétique, nous avons évalué l'impact de l'ablation de la signalisation STAT3 dans les neurones dopaminergiques sur un certain nombre de fonctions liées à la dopamine, y compris l'alimentation, la locomotion, les comportements liés à la récompense, l'émotion et la libération de dopamine dans le noyau accumbens. Fait intéressant, nous avons observé un dimorphisme sexuel dans le phénotype des souris STAT3DAT-KO. L'activation de la voie de signalisation STAT3 dans les neurones dopaminergiques est responsable de l'action de la leptine dans la réduction de la locomotion, récompense liée à l'activité physique, et de l'augmentation de la libération et de la disponibilité de la dopamine chez les souris mâles. Cependant, il ne module pas le comportement émotionnel. D'autre part, les souris femelles STAT3DAT-KO augmentent les niveaux d'anxiété et les niveaux plasmatiques de corticostérone, sans provoquer de changements de la dépression. Cependant, la perte d'activation de STAT3 dans les neurones dopaminergiques ne module pas le comportement locomoteur chez les souris femelles. Notamment, les actions de la leptine dans le mésencéphale pour influencer le comportement alimentaire ne sont pas médiées par l'activation de STAT3 dans les neurones dopaminergiques, considérant que les souris mâles et femelles ont un comportement alimentaire normal. Nos résultats démontrent que la voie de signalisation STAT3 dans les neurones dopaminergiques est responsable des effets anxiolytiques de la leptine, et soutient l'hypothèse que la leptine communique l'état d'énergie du corps (i.e. la relation entre la dépense et les apports énergétiques) pour les régions mésolimbiques pour atténuer les effets de motivation et de récompense de plusieurs comportements qui servent à réhabiliter ou à épuiser les réserves d'énergie. En outre, ce travail souligne l'importance d'étudier la modulation de la signalisation de la leptine dans différente types de cellules, afin d'identifier les voies de signalisation et les mécanismes cellulaires impliqués dans les différentes fonctions neuro-comportementales de la leptine.

Feedback regulation of Gab2-dependent signaling by the Ras/MAPK pathway

La voie de signalisation des Récepteurs Tyrosine Kinase (RTK) occupe un rôle central dans la régulation de la croissance cellulaire, la prolifération, la différentiation et la motilité. Une régulation anormale des RTKs mène à plusieurs maladies humaines telles que le cancer du sein, la seconde cause de mortalité chez les femmes à cause de l’amplification et la mutation fréquente de la protéine tyrosine kinase HER2 (ERBB2). Grb2-associated binder (Gab) 2 est une protéine adaptatrice qui agit en aval de plusieurs RTKs, y compris HER2, pour réguler de multiples voies de signalisation. En réponse à la stimulation par de nombreux facteurs de croissances et cytokines, Gab2 est recruté à la membrane plasmique où il potentialise l’activation des voies de signalisation Ras/mitogen-activated protein kinase (MAPK) et PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B). En plus d’occuper un rôle essentiel durant le développement du système hématopoïétique, Gab2 est souvent amplifié dans les cancers, notamment le cancer du sein et les mélanomes. Cependant, les mécanismes moléculaires qui régulent le fonctionnement de Gab2 sont peu connus. Il est établi que lors de l’activation des RTKs, Gab2 est phosphorylé au niveau de plusieurs résidus Tyrosine, menant à l’association de différentes protéines comme p85 et Shp2. En plus de la phosphorylation en Tyrosine, notre laboratoire ainsi que d’autres groupes de recherche avons identifié que Gab2 est aussi phosphorylé au niveau de résidus Ser/Thr suite à l’activation de la voie de signalisation MAPK. Cependant, la régulation des fonctions de Gab2 par ces modifications post-traductionnelles est encore peu connue. Dans le but de comprendre comment Gab2 est régulé par la voie de signalisation MAPK, nous avons utilisé différentes approches. Dans la première partie de ma thèse, nous avons déterminé un nouveau mécanisme démontrant que la voie de signalisation Ras/MAPK, par le biais des protéines kinases RSK (p90 ribosomal S6 kinase), phosphoryle Gab2. Ce phénomène se produit à la fois in vivo et in vitro au niveau de trois résidus Ser/Thr conservés. Des mutations au niveau de ces sites de phosphorylation entrainent le recrutement de Shp2 menant à l’augmentation de la motilité cellulaire, ce qui suggère que les protéines RSK restreignent les fonctions dépendantes de Gab2. Ce phénomène est le résultat de la participation de RSK dans la boucle de rétroaction négative de la voie de signalisation MAPK. Dans la seconde partie de ma thèse, nous avons démontré que les protéines ERK1/2 phosphorylent Gab2 au niveau de plusieurs résidus pS/T-P à la fois in vitro et in vivo, entrainant l’inhibition du recrutement de p85. De plus, nous avons établi pour la première fois que Gab2 interagit physiquement avec ERK1/2 dans des cellules lors de l’activation de la voie de signalisation MAPK. Par ailleurs, nous avons montré un nouveau domaine d’attache du module ERK1/2 sur Gab2. Des mutations sur les résidus essentiels de ce domaine d’attache n’entrainent pas seulement la dissociation de ERK1/2 avec Gab2, mais diminuent également la phosphorylation dépendante de ERK1/2 sur Gab2. Ainsi, nos données montrent que la voie de signalisation MAPK régule les fonctions de la protéine Gab2 par le biais des kinases RSK et ERK1/2. Cette thèse élabore par ailleurs un schéma complet des fonctions de Gab2 dépendantes de la voie de signalisation MAPK dans le développement de nombreux cancers.

CXCR3 biased signaling, heteromerization and decoy properties

Le récepteur de chimiokine CXCR3 est un récepteur couplé à la protéine G (RCPG) exprimé, entre autre, sur les cellules T activées lors d’une réponse immune. CXCR3 est activé par trois ligands inductibles par l’interféron-γ (CXCL9, 10, 11) et, plus récemment, il a été découvert que CXCL4 liait CXCR3. Nous savons que CXCR3 joue un rôle dans la chimiotaxie des leucocytes, mais peu d’attention a été portée sur la signalisation biaisée induite par ces quatre ligands. Alors que l’homodimérisation entre récepteurs de chimiokine est un concept grandement observé, l’hétéromérisation entre deux récepteurs reste un domaine de recherche active. La signalisation biaisée et l’hétéromérisation ont été testées grâce à la technique de bioluminescene resonance energy transfer (BRET) dans des cellules HEK293E. Nous présentons une caractérisation pharmacologique des quatre ligands de CXCR3 et démontrons l’hétéromérisation de CXCR3 avec CXCR4 et avec CXCR7. Nos résultats suggèrent que les ligands de CXCR3 n’agissent pas de manière redondante.

Unveiling the components of the signaling pathway between endosomes and the phagocytic uptake machinery in Dictyostelium discoideum

The soil amoebae Dictyostelium discoideum take up particles from their environment in order to obtain nutrition. The particle transits through the cell within a phagosome that fuses with organelles of different molecular compositions, undergoing a gradual degradation by different sets of hydrolytic enzymes. Griffiths’ concept of “phagosome individuality” predicts signaling from phagosomes into the cytoplasm, which might regulate many aspects of cell physiology. The finding that Dictyostelium cells depleted of the lysozyme AlyA or over-expressing the esterase Gp70 exhibit increased uptake of food particles, led to the postulation of a signaling cascade between endocytic compartments and the cytoskeletal uptake machinery at the plasma membrane. Assuming that Gp70 acts downstream of AlyA, gene-expression profiling of both mutants revealed different and overlapping sets of misregulated genes that might participate in this signaling cascade. Based on these results, we analyzed the effects of the artificial misregulation of six candidate genes by over-expression or negative genetic interference, in order to reconstruct at least part of the signaling pathway. SSB420 and SSL793 were chosen as candidates for the first signaling step, as they were up-regulated in AlyA-null cells and remained unaltered in the Gp70 over-expressing cells. The over-expression of SSB420 enhanced phagocytosis and raised the expression levels of Gp70, supporting its involvement in the signaling pathway between AlyA and Gp70 as a positive regulator of phagocytosis. However, this was not the case of cells over-expressing SSL793, as this mutation had no effects on phagocytosis. For the signaling downstream of Gp70, we studied four commonly misregulated genes in AlyA-depleted and Gp70 over-expressing cells. The expression levels of SLB350, SSB389 and TipD were lower in both mutants and therefore these were assumed as possible candidates for the negative regulation of phagocytosis. Cells depleted of SLB350 exhibited an increased phagocytic activity and no effect on Gp70 expression, proving its participation in the signaling pathway downstream of Gp70. Unlike SLB350, the disruption of the genes coding for SSB389 and TipD had no effects on particle uptake, excluding them from the pathway. The fourth candidate was Yipf1, the only gene that was commonly up-regulated in both mutants. Yet, the artificial over-expression of this protein had no effects on phagocytosis, so this candidate is also not included in the signaling pathway. Furthermore, localizing the products of the candidate genes within the cell helped unveiling several cellular organelles that receive signals from the phagosome and transduce them towards the uptake machinery.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41Δ, sap190Δ, ppm1Δ, rrd1Δ) and U34 modification defects (elp3Δ, kti12Δ, urm1Δ, ncs2Δ) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3Δ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3Δ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3Δ, urm1Δ) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TORsensitive NCR branch via Gln3 misregulation.

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca2+ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca2+ baseline concentration and frequency of oscillating Ca2+ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca2+ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca2+ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K+ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K+ and Na+ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.

Differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis in brown adipocytes

Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-¹³C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-¹³C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine’s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin).

Suburban immigrants to wildlands disrupt honest signaling in ultra-violet plumage

Urbanization changes habitat in a multitude of ways, including altering food availability. Access to human-provided food can change the relationship between body condition and honest advertisements of fitness, which may result in changes to behavior, demography, and metapopulation dynamics. We compared plumage color, its relationship with body condition and feather growth, and use as signal of dominance between a suburban and a wildland population of Florida Scrub-Jay (Aphelocoma coerulescens). Although plumage color was not related to body condition at either site, suburban birds had plumage with a greater proportion of total reflectance in the ultra-violet (UV) and peak reflectance at shorter wavelengths. Despite the use of plumage reflectance as a signal of dominance among individuals in the wildlands, we found no evidence of status signaling at the suburban site. However, birds emigrating from the suburban site to the wildland site tended to be more successful at acquiring breeder status but less successful at reproducing than were immigrants from an adjacent wildland site, suggesting that signaled and realized quality differ. These differences in signaling content among populations could have demographic effects at metapopulation scales and may represent an evolutionary trap whereby suburban immigrants are preferred as mates even though their reproductive success relative to effort is lower.

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

-Aminobutyric acid type A (GABAA) receptors, a family of Cl-permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for -aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated byGABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2 influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2 with Ba2, or Ca2/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2 channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

Short communication: Suppressor of cytokine signaling-2 mRNA increases after parturition in the liver of dairy cows

After parturition, the somatotropic axis of the dairy cow is uncoupled, partly because of reduced concentration of liver-specific GH receptor (GHR) 1A. Estradiol-17 beta (E-2) concentrations increase at parturition and E-2 upregulates suppressors of cytokine signaling-2 (SOCS-2) mRNA expression, potentially inhibiting GH signaling. Therefore, we hypothesized that SOCS-2 mRNA is upregulated after parturition. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition and fed diets to meet nutrient requirements at ad libitum or limited dry matter intake during the dry period. All cows were fed the same diet ad libitum from calving until 4 wk after parturition. Blood samples were collected weekly and more frequently near parturition. Liver biopsies obtained at -21, -7, 2, and 28 d relative to parturition were assessed for SOCS-2 and GHR 1A mRNA by quantitative real-time reverse-transcription PCR. The relative amount of SOCS-2 mRNA increased after parturition with both treatments and was greater on d 2 for cows limit-fed during the dry period compared with cows fed at ad libitum dry matter intake. Plasma E2 concentrations increased on d -13, -5 and 1 relative to parturition and the increases were greater in limit-fed cows. Plasma GH concentration was greater for limit-fed cows and increased after parturition in all cows. The amount of GHR 1A mRNA did not differ between diets but decreased on d 2. In addition to reduced GHR 1A, increased SOCS-2 mRNA after parturition, perhaps because of increased E-2, may further uncouple GH signaling in the liver of the transition dairy cow.

PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

The stimulation of mitogenic signaling pathways by N-POMC peptides

The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC1-28 Stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC1-28 and N-POMC1-49 in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5 min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC1-49 stimulated the phosphorylation of Akt more robustly than N-POMC1-28. We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal. (C) 2008 Elsevier Ireland Ltd. All rights reserved.