Caractérisation du système 26Rfa/GPR103 et recherche du mécanisme d action du 26Rfa dans le contrôle hypotalamique du comportement alimentaire

Le 26RFa est un nouveau membre de la famille des peptides RFamides qui a été initialement isolé dans le cerveau de la grenouille et ensuite caractérisé chez les mammifères. Parallèlement, le 26RFa a été identifié comme le ligand endogène d un récepteur jusqu alors orphelin, le GPR103. Chez les rongeurs, les ARNm du 26RFa sont essentiellement exprimés dans le noyau hypothalamique ventromédian (VMH) et l aire hypothalamique latérale (LH), deux structures bien connues pour être impliquées dans le contrôle du comportement alimentaire, et il a été montré que l injection intracérébroventriculaire (icv) du neuropeptide provoque une augmentation dose-dépendante de la consommation de nourriture. Au cours de cette thèse, nous nous sommes intéressés, dans un premier temps, au système à 26RFa chez l homme. Ainsi, nous avons montré que le 26RFa est effectivement produit dans le cerveau humain, mais aussi sa forme allongée, le 43RFa. De plus, nos résultats indiquent que le 26RFa est contenu dans des populations neuronales localisées dans le VMH et le noyau paraventriculaire (PVN), suggérant que le neuropeptide pourrait être également impliqué dans le contrôle hypothalamique du comportement alimentaire chez l homme. Dans un second temps, nous avons établi une cartographie détaillée des neurones exprimant le récepteur du 26RFa dans le cerveau du rat afin d identifier les régions cibles du neuropeptide au niveau central et notamment d orienter nos recherches sur le mécanisme d action du 26RFa dans le contrôle du comportement alimentaire. Nos observations indiquent que le GPR103 est fortement exprimé dans de nombreux noyaux du cerveau impliqués dans le contrôle du comportement alimentaire tels que le VMH, le noyau hypothalamique dorsomédian, le PVN, la LH et le noyau arqué (Arc) dans l hypothalamus mais aussi le cortex piriforme ou encore le noyau du tractus solitaire dans le tronc cérébral, renforçant donc l idée que le système à 26RFa joue un rôle important dans le contrôle central du comportement alimentaire chez les mammifères. Nous avons ensuite recherché par quel mécanisme d action le 26RFa contrôlait la prise alimentaire. La forte densité d ARNm du GPR103 dans le Arc suggérait que le 26RFa pouvait exercer son activité orexigène en régulant l activité des neurones à NPY et à POMC de ce noyau. Nous montrons que le 26RFa régule de façon opposée les deux systèmes neuropeptidergiques puisqu il stimule l activité des neurones à NPY et inhibe celle des neurones à POMC. Notre étude a aussi mis en évidence l absence de GPR103 sur les neurones à POMC et la présence du récepteur sur les neurones à NPY, et a révélé que l effet du 26RFa sur l activité des neurones à POMC ainsi que sur la prise alimentaire est totalement bloqué par des antagonistes des récepteurs Y1-R et Y5-R du NPY. L ensemble de ces derniers résultats démontre que le 26RFa exerce son activité orexigène en stimulant la libération du NPY dans le Arc qui va ensuite inhiber l activité des neurones à POMC via l activation des récepteurs Y1-R et Y5-R. En conclusion, l ensemble de mes travaux de thèse apportent de nouveaux éléments d informations sur la présence et le rôle du système 26RFa/GPR103 chez les mammifères et, plus particulièrement, sur son mécanisme d action dans le contrôle hypothalamique du comportement alimentaire.26Rfa is an Rfamide peptide initially isolated from a frog brain extract and it has been subqequently characterized in mammals. This neuropeptide has been identified as the endogenous ligand of an orphan receptor, GPR103.In rodents, 26Rfa is essentially expressed in hypothalamicx nuclei which are involved in control of feeding behavior and it has been shown that the neuropeptide stimulated the food consumption. Our data reveal that 26Rfa is produced in human brain and notably in hypothalamus supporting therefore the view that the neuropeptide may also be involved in central regulation of food intake in human. We have next investigated GPR103 distribution in rat brain in order to identify target nuclei of 26Rfa with regard to the control of food intake. Our results indicate that 26Rfa increase food consumption by regulating the activity of hypothalamic neurons.ROUEN-BU Sciences (764512102) / SudocROUEN-BU Sciences Madrillet (765752101) / SudocSudocFranceF

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

<div><p>Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events.</p></div

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat

International audienceThe novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors

N- and C-terminal truncation mutants of ASPP2 can still promote Ras nanoclustering.

<p>(<b>A</b>) Schematic of full-length ASPP2, as well as ASPP2(1–360) and ASPP2(123–1128) truncation mutants. ASPP2 domains from left to right: Ubl, ubiquitin-like domain; α-helical domain; Pro, proline-rich domain; Ank, Ankyrin repeats; SH3, SRC homology 3 domain. (<b>B</b>) Confocal microscopic images of HEK cells cotransfected with mGFP-H-rasG12V (green) and full-length or truncated ASPP2 (red). (<b>C-E</b>) Nanoclustering-FRET analysis of HEK cells coexpressing mGFP- and mCherry-tagged (<b>C</b>) H-rasG12V, (<b>D</b>) K-rasG12V or (<b>E</b>) N-rasG12V. Cells were analysed after overexpression of Gal-1, full-length ASPP2 or its truncation mutants. (<b>C-E</b>) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM, n = 3; ns, not significant; ****, p< 0.0001). (<b>F</b>) Western blot of anti-GFP immunoprecipitation samples probed with anti-ASPP2- (top) or anti-GFP- (bottom) antibodies. Samples were lysates prepared from mGFP-H-rasG12V transfected HEK cells that were cotransfected with full-length ASPP2 or its truncation mutants or an empty plasmid (control), as indicated. In, input; Ft, flow-through; W1, wash; E, elution. Red boxes indicate the immunoprecipitated ASPP2 fragments.</p

ASPP2 dominates over Gal-1 thus robustly inducing senescence and abrogating mammosphere formation.

<p>(<b>A</b>) SA-β-gal assay of MCF-7 cells transfected with plasmids encoding H-rasG12V, ASPP2, Gal-1 or the combination of the latter two, as indicated. Cells were stained 7 days after transfection. On the left, percentages of SA-β-gal positive cells are shown in the graph (mean ± SEM, n = 3). On the right, representative images from the assay. (<b>B</b>) Mammosphere formation assay with MCF-7, MDA-MB-231 or HS-578T breast cancer cell lines. Mammospheres were transfected with Gal-1, ASPP2, or both (1:1 ratio) and cells were then grown under non-adherent conditions for 9 days. On the right, representative images of mammospheres are shown as indicated. (<b>A, B</b>) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM n≥3; ns, not significant; ****, p<0.0001).</p

Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction.

International audienceUrotensin II (UII) and UII-related peptide (URP) are paralog neuropeptides whose existence and distribution in mouse have not yet been investigated. In this study, we showed by HPLC/RIA analysis that the UII-immunoreactive molecule in the mouse brain corresponds to a new UII(17) isoform. Moreover, calcium mobilization assays indicated that UII(17) and URP were equally potent in stimulating UII receptor (UT receptor). Quantitative RT-PCR and in situ hybridization analysis revealed that in the CNS UII and URP mRNAs were predominantly expressed in brainstem and spinal motoneurons. Besides, they were differentially expressed in the medial vestibular nucleus, locus coeruleus and the ventral medulla. In periphery, both mRNAs were expressed in skeletal muscle, testis, vagina, stomach, and gall bladder, whereas only URP mRNA could be detected in the seminal vesicle, heart, colon, and thymus. By contrast, the UT receptor mRNA was widely expressed, and notably, very high amounts of transcript occurred in skeletal muscle and prostate. In the biceps femoris muscle, UII-like immunoreactivity was shown to coexist with synaptophysin in muscle motor end plate regions. Altogether these results suggest that (i) UII and URP may have many redundant biological effects, especially at the neuromuscular junction; (ii) URP may more specifically participate to autonomic, cardiovascular and reproductive functions

Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord

International audience26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis