Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane.

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

Effects of supplementation with essential amino acids on intrahepatic lipid concentrations during fructose overfeeding in humans.

BACKGROUND: A high dietary protein intake has been shown to blunt the deposition of intrahepatic lipids in high-fat- and high-carbohydrate-fed rodents and humans. OBJECTIVE: The aim of this study was to evaluate the effect of essential amino acid supplementation on the increase in hepatic fat content induced by a high-fructose diet in healthy subjects. DESIGN: Nine healthy male volunteers were studied on 3 occasions in a randomized, crossover design after 6 d of dietary intervention. Dietary conditions consisted of a weight-maintenance balanced diet (control) or the same balanced diet supplemented with 3 g fructose · kg(-1) · d(-1) and 6.77 g of a mixture of 5 essential amino acids 3 times/d (leucine, isoleucine, valine, lysine, and threonine) (HFrAA) or with 3 g fructose · kg(-1) · d(-1) and a maltodextrin placebo 3 times/d (HFr); there was a washout period of 4 to 10 wk between each condition. For each condition, the intrahepatocellular lipid (IHCL) concentration, VLDL-triglyceride concentration, and VLDL-[(13)C]palmitate production were measured after oral loading with [(13)C]fructose. RESULTS: HFr increased the IHCL content (1.27 ± 0.31 compared with 2.74 ± 0.55 vol %; P < 0.05) and VLDL-triglyceride (0.55 ± 0.06 compared with 1.40 ± 0.15 mmol/L; P < 0.05). HFr also enhanced VLDL-[(13)C]palmitate production. HFrAA significantly decreased IHCL compared with HFr (to 2.30 ± 0.43 vol%; P < 0.05) but did not change VLDL-triglyceride concentrations or VLDL-[(13)C]palmitate production. CONCLUSIONS: Supplementation with essential amino acids blunts the fructose-induced increase in IHCL but not hypertriglyceridemia. This is not because of inhibition of VLDL-[(13)C]palmitate production. This trial was registered at www.clinicaltrials.gov as NCT01119989.

Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation.

Engagement of TNF receptor 1 by TNFalpha activates the transcription factor NF-kappaB but can also induce apoptosis. Here we show that upon TNFalpha binding, TNFR1 translocates to cholesterol- and sphingolipid-enriched membrane microdomains, termed lipid rafts, where it associates with the Ser/Thr kinase RIP and the adaptor proteins TRADD and TRAF2, forming a signaling complex. In lipid rafts, TNFR1 and RIP are ubiquitylated. Furthermore, we provide evidence that translocation to lipid rafts precedes ubiquitylation, which leads to the degradation via the proteasome pathway. Interfering with lipid raft organization not only abolishes ubiquitylation but switches TNFalpha signaling from NF-kappaB activation to apoptosis. We suggest that lipid rafts are crucial for the outcome of TNFalpha-activated signaling pathways.

Interaction of Fas(Apo-1/CD95) with proteins implicated in the ubiquitination pathway.

Fas(Apo-1/CD95), a receptor belonging to the tumor necrosis factor receptor family, induces apoptosis when triggered by Fas ligand. Upon its activation, the cytoplasmic domain of Fas binds several proteins which transmit the death signal. We used the yeast two-hybrid screen to isolate Fas-associated proteins. Here we report that the ubiquitin-conjugating enzyme UBC9 binds to Fas at the interface between the death domain and the membrane-proximal region of Fas. This interaction is also seen in vivo. UBC9 transiently expressed in HeLa cells bound to the co-expressed cytoplasmic segment of Fas. FAF1, a Fas-associated protein that potentiates apoptosis (Chu et al. (1996) Proc. Natl. Acad. Sci. USA 92, 11894-11898), was found to contain sequences similar to ubiquitin. These results suggest that proteins related to the ubiquitination pathway may modulate the Fas signaling pathway.

IB1, a JIP-1-related nuclear protein present in insulin-secreting cells.

JIP-1 is a cytoplasmic inhibitor of the c-Jun amino-terminal kinase activated pathway recently cloned from a mouse brain cDNA library. We report herein the expression cloning of a rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic beta-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was isolated by its ability to bind to GTII, a cis-regulatory element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino acid protein, which differs from JIP-1 by the insertion of 47 amino acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative helix-loop-helix motif. Recombinant IB1 (amino acids 1-714 and 280-714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells transfected with an expression vector encoding IB1. Using a heterologous GAL4 system, we localized an activation domain of IB1 within the first 280 amino acids of the protein. These data demonstrate that IB1 is a DNA-binding protein related to JIP-1, which is highly expressed in pancreatic beta-cells where it functions as a transactivator of the GLUT2 gene.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Interaction of GAG trinucleotide repeat and C-129T polymorphisms impairs expression of the glutamate-cysteine ligase catalytic subunit gene.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the de novo synthesis of glutathione (GSH). The catalytic subunit (GCLC) of GCL contains a GAG trinucleotide-repeat (TNR) polymorphism within the 5'-untranslated region (5'-UTR) that has been associated with various human disorders. Although several studies suggest that this variation influences GSH content, its implication for GCLC expression remains unknown. To better characterize its functional significance, we performed reporter gene assays with constructs containing the complete GCLC 5'-UTR upstream of a luciferase gene. Transfection of these vectors into various human cell lines did not reveal any significant differences between 7, 8, 9, or 10 GAG repeats, under either basal or oxidative stress conditions. To correlate these results with the previously described down-regulation induced by the C-129T GCLC promoter polymorphism, combinations of both variations were tested. Interestingly, the -129T allele down-regulates gene expression when combined with 7 GAG but not with 8, 9, or 10 GAG TNRs. This observation was confirmed in primary fibroblast cells, in which the combination of GAG TNR 7/7 and -129C/T genotypes decreased the GCLC protein level. These results provide evidence that interaction of the two variations can efficiently impair GCLC expression and thus suggest its involvement in the pathogenesis of diseases related to GSH metabolism.

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ‟closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

Study of protein complexes

Summary : A lot of information can be obtained on proteins when proteomics methods are used. In our study, we aimed to characterize complexes containing pro-apoptotic proteins by different proteomics methods and finally focused on PIDD (p53-induced protein with a death domain), for which the most interesting results were obtained. PIDD has been shown to function as a molecular switch between genotoxic stress-induced apoptotis and genotoxic stress-induced cell survival through NF-κB activation. To exert these two functions, PIDD forms alternate complexes respectively with caspase2 and CRADD on one hand and RIP 1 and NEMO on the other hand. The first part of our study focuses on the processing of PIDD. PIDD full length (FL) is constitutively cleaved into three fragments, an N-terminal one (PIDD-N) and two fragments containing the C-terminus (PIDD-C and PIDD-CC). Localization of the two PIDD cleavage sites by mass spectrometry (MS) allowed to understand that PIDD is probably not cleaved by proteases but is subject to protein (self-)splicing and also to map the PIDD-N, PIDD-C and PIDD-CC fragments exactly. Further characterization of these three fragments by Tinel et al. (Tinel et al., 2007) showed that PIDD-C is involved in activation of an apoptotic pathway while PIDD-CC is involved in NF-κB activation. We also found that PIDD is subject to proline-directed phosphorylation at two serine residues in PIDD-N, the regulatory fragment of PIDD. The second part of the study aimed at identifying by proteomics techniques proteins that co-purify with PIDD and therefore are putative cellular interaction partners. In this respect we analyzed samples obtained in different conditions or with different PIDD constructs corresponding to processed fragments. This allowed us to identify a large number of potential interactors for PIDD. For example, by comparing data obtained from PIDD-C and PIDD-FL affinity purifications, we found that the Hsp90 chaperone system interacts strongly with PIDD-N. In the third part of this study, we developed methods to selectively and rapidly quantify by MS proteins of interest in PIDD affinity purifications or negative controls. Using these tools we detected significant changes in PIDD-FL-copurifying proteins treated by heat shock. Overall, our studies provide informative data on the processing of PIDD and its possible involvement in several molecular pathways.

n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.

We elucidated the mechanisms of action of two n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in Jurkat T-cells. Both DHA and EPA were principally incorporated into phospholipids in the following order: phosphatidylcholine < phosphatidylethanolamine < phosphatidylinositol/phosphatidylserine. Furthermore, two isoforms of phospholipase A(2) (i.e., calcium-dependent and calcium-independent) were implicated in the release of DHA and EPA, respectively, during activation of these cells. The two fatty acids inhibited the phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane translocation of protein kinase C (PKC)-alpha and -epsilon. The two n-3 PUFAs also inhibited the nuclear translocation of nuclear factor kappaB (NF-kappaB) and the transcription of the interleukin-2 (IL-2) gene in PMA-activated Jurkat T-cells. Together, these results demonstrate that DHA and EPA, being released by two isoforms of phospholipase A(2), modulate IL-2 gene expression by exerting their action on two PKC isoforms and NF-kappaB in Jurkat T-cells.

Adiponectin and C-reactive protein are positively associated with microalbuminuria in an adult Caucasian population

Objective: Microalbuminuria (MAU) is a marker of early kidney injury and cardiovascular risk. We assessed the association of MAU with plasma adiponectin, leptin, and hsCRP as inflammatory marker, accounting for hypertension, diabetes and obesity. Design and Methods: Population based, cross-sectional study in Caucasian subjects aged 35 to 75 years in Lausanne, Switzerland. MAU, measured by quantitative immunonephelometry on spot morning urine, was used either as a continuous (MAU) or dichotomized variable (MA defined as MAU > 2.5 and >3.5 mg/mmol creatinine in men and women, respectively). Results: The 2955 women (age 53.3_10.7, mean_SD years) had mean body mass index (BMI) 24.9_4.5 kg/m. The 2479 men (age 53.1_10.8 years) hadmean BMI 27.0_3.9 kg/m2.Median hsCRP was 1.3 and 1.3 mg/L, median adiponectin 6.2 and 10.6mg/mL in men and women, respectively. MA prevalence was 4.9% in women and 9.8% in men. In multivariate regression analysis adjusting for potential confounders (age, sex, hypertension, diabetes, eGFR, BMI, percent fat mass, insulin and smoking), logtransformed MAU was positively associated with hsCRP (P<0.001) and adiponectin (P¼0.002), but not with leptin. The association of adiponectin with MAU was stronger in subjects with low hsCRP, and vice versa (P interaction<0.001). Conclusion: Adiponectin and hsCRP are significant positive determinants of MAU, independently of diabetes, hypertension and fat mass. A negative interaction between hsCRP and adiponectin was found for their effect on MAU. Whether hyperadiponectinemia represents an adequate protective response to vascular stress or has negative causal impact on the development of MAU should be assessed in further studies.

Gliotransmission in the hippocampus, mechanisms and interaction with synaptic functions

SUMMARYAstrocytes represent the largest cell population in the human brain. In addition to a well established role as metabolic support for neuronal activity, in the last years these cells have been found to accomplish other important and, sometimes, unexpected functions. The tight enwrapping of synapses by astrocytic processes and the predominant expression of glutamate uptake carriers in the astrocytic rather than neuronal plasma membranes brought to the definition of a critical involvement of astrocytes in the clearance of glutamate from synaptic junctions. Moreover, several publications showed that astrocytes are able to release chemical transmitters (gliotransmitters) suggesting their active implication in the control of synaptic functions. Among gliotransmitters, the best characterized is glutamate, which has been proposed to be released from astrocytes in a Ca2+ dependent manner via exocytosis of synaptic-like microvesicles.In my thesis I present results leading to substantial advancement of the understanding of the mechanisms by which astrocytes modulate synaptic activity in the hippocampus, notably at excitatory synapses on dentate granule cells. I show that tumor necrosis factor- alpha (TNFa), a molecule that is generally involved in immune system functions, critically controls astrocyte-to-synapse communication (gliotransmission) in the brain. With constitutive levels of TNFa present, activation of purinergic G protein-coupled receptors in astrocytes, called P2Y1 receptors, induces localized intracellular calcium ([Ca2+]j) elevation in astrocytic processes (measured by two-photon microscopy) followed by glutamate release and activation of pre-synaptic NMDA receptors resulting in synaptic potentiation. In preparations lacking TNFa, astrocytes respond with identical [Ca2+]i elevations but fail to induce neuromodulation. I find that TNFa specifically controls the glutamate release step of gliotransmission. Addition of very low (picomolar) TNFa concentrations to preparations lacking the cytokine, promptly reconstitutes both normal exocytosis in cultured astrocytes and gliotransmission in hippocampal slices. These data provide the first demonstration that gliotransmission and its synaptic effects are controlled not only by astrocyte [Ca2+]i elevations but also by permissive/homeostatic factors like TNFa.In addition, I find that higher and presumably pathological TNFa concentrations do not act just permissively but instead become direct and potent triggers of glutamate release from astrocytes, leading to a strong enhancement of excitatory synaptic activity. The TNFa action, like the one observed upon P2Y1R activation, is mediated by pre-synaptic NMDA receptors, but in this case the effect is long-lasting, and not reversible. Moreover, I report that a necessary molecular target for this action of TNFa is TNFR1, one of the two specific receptors for the cytokine, as I found that TNFa was unable to induce synaptic potentiation when applied in slices from TNFR1 knock-out (Tnfrlv") mice. I then created a double transgenic mouse model where TNFR1 is knocked out in all cells but can be re-expressed selectively in astrocytes and I report that activation of the receptors in these cells is sufficient to reestablish TNFa-dependent long-lasting potentiation of synaptic activity in the TNFR1 knock-out mice.I therefore discovered that TNFa is a primary molecule displaying both permissive and instructive roles on gliotransmission controlling synaptic functions. These reports might have profound implications for the understanding of both physiological and pathological processes associated to TNFa production, including inflammatory processes in the brain.RÉSUMÉLes astrocytes sont les cellules les plus abondantes du cerveau humain. Outre leur rôle bien établi dans le support métabolique de l'activité neuronale, d'autres fonctions importantes, et parfois inattendues de ces cellules ont été mises en lumière au cours de ces dernières années. Les astrocytes entourent étroitement les synapses de leurs fins processus qui expriment fortement les transporteurs du glutamate et permettent ainsi aux astrocytes de jouer un rôle critique dans l'élimination du glutamate de la fente synaptique. Néanmoins, les astrocytes semblent être capables de jouer un rôle plus intégratif en modulant l'activité synaptique, notamment par la libération de transmetteurs (gliotransmetteurs). Le gliotransmetteur le plus étudié est le glutamate qui est libéré par l'exocytose régulée de petites vésicules ressemblant aux vésicules synaptiques (SLMVs) via un mécanisme dépendant du calcium.Les résultats présentés dans cette thèse permettent une avancée significative dans la compréhension du mode de communication de ces cellules et de leur implication dans la transmission de l'information synaptique dans l'hippocampe, notamment des synapses excitatrices des cellules granulaires du gyrus dentelé. J'ai pu montrer que le « facteur de nécrose tumorale alpha » (TNFa), une cytokine communément associée au système immunitaire, est aussi fondamentale pour la communication entre astrocyte et synapse. Lorsqu'un niveau constitutif très bas de TNFa est présent, l'activation des récepteurs purinergiques P2Y1 (des récepteurs couplés à protéine G) produit une augmentation locale de calcium (mesurée en microscopie bi-photonique) dans l'astrocyte. Cette dernière déclenche ensuite une libération de glutamate par les astrocytes conduisant à l'activation de récepteurs NMDA présynaptiques et à une augmentation de l'activité synaptique. En revanche, dans la souris TNFa knock-out cette modulation de l'activité synaptique par les astrocytes n'est pas bien qu'ils présentent toujours une excitabilité calcique normale. Nous avons démontré que le TNFa contrôle spécifiquement l'exocytose régulée des SLMVs astrocytaires en permettant la fusion synchrone de ces vésicules et la libération de glutamate à destination des récepteurs neuronaux. Ainsi, nous avons, pour la première fois, prouvé que la modulation de l'activité synaptique par l'astrocyte nécessite, pour fonctionner correctement, des facteurs « permissifs » comme le TNFa, agissant sur le mode de sécrétion du glutamate astrocytaire.J'ai pu, en outre, démontrer que le TNFa, à des concentrations plus élevées (celles que l'on peut observer lors de conditions pathologiques) provoque une très forte augmentation de l'activité synaptique, agissant non plus comme simple facteur permissif mais bien comme déclencheur de la gliotransmission. Le TNFa provoque 1'activation des récepteurs NMD A pré-synaptiques (comme dans le cas des P2Y1R) mais son effet est à long terme et irréversible. J'ai découvert que le TNFa active le récepteur TNFR1, un des deux récepteurs spécifiques pour le TNFa. Ainsi, l'application de cette cytokine sur une tranche de cerveau de souris TNFR1 knock-out ne produit aucune modification de l'activité synaptique. Pour vérifier l'implication des astrocytes dans ce processus, j'ai ensuite mis au point un modèle animal doublement transgénique qui exprime le TNFR1 uniquement dans les astrocytes. Ce dernier m'a permis de prouver que l'activation des récepteurs TNFR1 astrocytaires est suffisante pour induire une augmentation de l'activité synaptique de manière durable.Nous avons donc découvert que le TNFa possède un double rôle, à la fois un rôle permissif et actif, dans le contrôle de la gliotransmission et, par conséquent, dans la modulation de l'activité synaptique. Cette découverte peut potentiellement être d'une extrême importance pour la compréhension des mécanismes physiologiques et pathologiques associés à la production du TNFa, en particulier lors de conditions inflammatoires.RÉSUMÉ GRAND PUBLICLes astrocytes représentent la population la plus nombreuse de cellules dans le cerveau humain. On sait, néanmoins, très peu de choses sur leurs fonctions. Pendant très longtemps, les astrocytes ont uniquement été considérés comme la colle du cerveau, un substrat inerte permettant seulement de lier les cellules neuronales entre elles. Il n'y a que depuis peu que l'on a découvert de nouvelles implications de ces cellules dans le fonctionnement cérébral, comme, entre autres, une fonction de support métabolique de l'activité neuronale et un rôle dans la modulation de la neurotransmission. C'est ce dernier aspect qui fait l'objet de mon projet de thèse.Nous avons découvert que l'activité des synapses (régions qui permettent la communication d'un neurone à un autre) qui peut être potentialisée par la libération du glutamate par les astrocytes, ne peut l'être que dans des conditions astrocytaires très particulières. Nous avons, en particulier, identifié une molécule, le facteur de nécrose tumorale alpha (TNFa) qui joue un rôle critique dans cette libération de glutamate astrocytaire.Le TNFa est surtout connu pour son rôle dans le système immunitaire et le fait qu'il est massivement libéré lors de processus inflammatoires. Nous avons découvert qu'en concentration minime, correspondant à sa concentration basale, le TNFa peut néanmoins exercer un rôle indispensable en permettant la communication entre l'astrocyte et le neurone. Ce mode de fonctionnement est assez probablement représentatif d'un processus physiologique qui permet d'intégrer la communication astrocyte/neurone au fonctionnement général du cerveau. Par ailleurs, nous avons également démontré qu'en quantité plus importante, le TNFa change son mode de fonctionnement et agit comme un stimulateur direct de la libération de glutamate par l'astrocyte et induit une activation persistante de l'activité synaptique. Ce mode de fonctionnement est assez probablement représentatif d'un processus pathologique.Nous sommes également arrivés à ces conclusions grâce à la mise en place d'une nouvelle souche de souris doublement transgéniques dans lesquelles seuls les astrocytes (etnon les neurones ou les autres cellules cérébrales) sont capables d'être activés par le TNFa.

Whole-body protein turnover and resting energy expenditure in obese, prepubertal children.

BACKGROUND: Obesity is becoming more frequent in children; understanding the extent to which this condition affects not only carbohydrate and lipid metabolism but also protein metabolism is of paramount importance. OBJECTIVE: We evaluated the kinetics of protein metabolism in obese, prepubertal children in the static phase of obesity. DESIGN: In this cross-sectional study, 9 obese children (x +/- SE: 44+/-4 kg, 30.9+/-1.5% body fat) were compared with 8 lean (28+/-2 kg ,16.8+/-1.2% body fat), age-matched (8.5+/-0.2 y) control children. Whole-body nitrogen flux, protein synthesis, and protein breakdown were calculated postprandially over 9 h from 15N abundance in urinary ammonia by using a single oral dose of [15N]glycine; resting energy expenditure (REE) was assessed by indirect calorimetry (canopy) and body composition by multiple skinfold-thickness measurements. RESULTS: Absolute rates of protein synthesis and breakdown were significantly greater in obese children than in control children (x +/- SE: 208+/-24 compared with 137+/-14 g/d, P < 0.05, and 149+/-20 compared with 89+/-13 g/d, P < 0.05, respectively). When these variables were adjusted for fat-free mass by analysis of covariance, however, the differences between groups disappeared. There was a significant relation between protein synthesis and fat-free mass (r = 0.83, P < 0.001) as well as between protein synthesis and REE (r = 0.79, P < 0.005). CONCLUSIONS: Obesity in prepubertal children is associated with an absolute increase in whole-body protein turnover that is consistent with an absolute increase in fat-free mass, both of which contribute to explaining the greater absolute REE in obese children than in control children.