MP2RAGE, a self bias-field corrected sequence for improved segmentation and T1-mapping at high field.

The large spatial inhomogeneity in transmit B(1) field (B(1)(+)) observable in human MR images at high static magnetic fields (B(0)) severely impairs image quality. To overcome this effect in brain T(1)-weighted images, the MPRAGE sequence was modified to generate two different images at different inversion times, MP2RAGE. By combining the two images in a novel fashion, it was possible to create T(1)-weighted images where the result image was free of proton density contrast, T(2) contrast, reception bias field, and, to first order, transmit field inhomogeneity. MP2RAGE sequence parameters were optimized using Bloch equations to maximize contrast-to-noise ratio per unit of time between brain tissues and minimize the effect of B(1)(+) variations through space. Images of high anatomical quality and excellent brain tissue differentiation suitable for applications such as segmentation and voxel-based morphometry were obtained at 3 and 7 T. From such T(1)-weighted images, acquired within 12 min, high-resolution 3D T(1) maps were routinely calculated at 7 T with sub-millimeter voxel resolution (0.65-0.85 mm isotropic). T(1) maps were validated in phantom experiments. In humans, the T(1) values obtained at 7 T were 1.15+/-0.06 s for white matter (WM) and 1.92+/-0.16 s for grey matter (GM), in good agreement with literature values obtained at lower spatial resolution. At 3 T, where whole-brain acquisitions with 1 mm isotropic voxels were acquired in 8 min, the T(1) values obtained (0.81+/-0.03 s for WM and 1.35+/-0.05 for GM) were once again found to be in very good agreement with values in the literature.

Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites.

Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.

Formation of the long range Dpp morphogen gradient.

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.

The chemokine receptor CXCR4 strongly promotes neuroblastoma primary tumour and metastatic growth, but not invasion.

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.

A dystroglycan mutation associated with limb-girdle muscular dystrophy.

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

Experimental support for the make-up hypothesis in nestling tawny owls (Strix aluco)

Body condition can affect coloration of traits used in sexual selection and parent-offspring communication by inducing rapid internal changes in pigment concentration or aggregation, thickness of collagen arrays, or blood flux. The recent "makeup hypothesis" proposes an alternative honesty-reinforcing mechanism, with behaviorally mediated deposition of substances on body surfaces ("cosmetics") generating covariation between body condition and coloration. In birds, the uropygial gland wax is actively spread on feathers using the bill and changes in its deposition rate may cause rapid changes in bill and plumage coloration. Using tawny owl nestlings, we tested 3 predictions of the makeup hypothesis, namely that 1) quantity of preen wax deposited accounts for variation in bill coloration, 2) an immune stimulation (induced by injection of a lipopolysaccharide [LPS]) impairs uropygial gland wax production, and 3) different intensities of immune stimulations (strong vs. weak stimulations induced by injections of either LPS or phytohemagglutinin [PHA], respectively) and high versus low food availabilities result in different bill colorations. We found that 1) preen wax reduced bill brightness, 2) a challenge with LPS impaired uropygial gland development, and 3) nestlings challenged with LPS had a brighter bill than PHA-injected nestlings, whereas diet manipulation had no significant effect. Altogether, these results suggest that a strong immune challenge may decrease preen wax deposition rate on the bill of nestling birds, at least by impairing gland wax production, which causes a change in bill coloration. Our study therefore highlights that cosmetic colors might signal short-term variation in immunological status.

ARE PPARS NOVEL THERAPEUTIC TARGETS TO IMPROVE MELANOMA TREATMENT ?

Cutaneous melanoma is an aggressive malignant tumor of melanocytes, the pigment- producing cells of the epidermis, with a high incidence in developed countries. Despite some major clinical breakthroughs in the last few years, efficient therapies for metastatic melanoma, which portends a very bad prognosis, are still lacking. Among the potential therapeutic targets that have been attracting at-tention in melanoma are the peroxisome proliferator-activated receptors (PPARs). These members - a, ß and 7 - of the nuclear hormone receptor family, which are ligand-gated transcription factors endowed with a multitude of functions besides metabolism homeostasis, have displayed promising antitumor properties in a wide range of cancer cells, including melanoma. However, our knowledge of PPARs' functions in this skin cancer is far from complete, making the usefulness of any of the a, ß or 7 isotype as a therapeutic target uncertain. In this work, we showed that all three PPAR isotypes are expressed in normal melanocytes, in most melanoma cell lines and in primary and metastatic melanomas, and that PPAR/3 and 7 display transcriptional activity in normal melanocytes and melanoma cells. We also showed that the PPAR7 agonist rosiglitazone had anti-melanoma properties largely independent of PPAR7 expression, which was widely varying across the different cell lines and melanoma biopsies we evaluated and was not correlated with cell line stage. Consistent with the general view of PPAR7 as a tumor suppressor gene, we found that, in human samples, PPAR7 was less expressed in melanoma than in normal skin. Transcriptornic profiling of metastatic melanoma cells in which PPAR7 was pharmacologically modulated revealed an association with epithelial-to-mesenchymal transition, though the functional relevance of this finding remains to be determined. Collectively, our results suggests that PPAR7 activity in melanoma is highly complex and that a straightforward picture of PPAR7's role in this skin cancer is difficult to draw. In this study, we also provided compelling evidence that thioredoxin interacting protein (TXNIP) is, in melanoma, a bona fide PPAR7 target gene, the expression of which is repressed by PPAR7 activation. Although TXNIP is mostly known as an inhibitor of the major antioxidant thioredoxin, it has demonstrated a range of biological functions and is generally considered as a tumor suppressor gene. Consistently, we found that TXNIP expression is associated with growth arrest of melanoma cells in vitro and that forced expression of TXNIP strongly impairs cell proliferation. Interestingly, we also discovered that TXNIP favors melanoma cell migration while it diminishes their adhesion. Finally, we provided several lines of evidence that TXNIP may regulate these processes at the transcriptional level as well as by direct protein-protein interactions in the plasma membrane. Altogether, our findings suggest that the PPAR7 target TXNIP may be a double-edged sword in melanoma, hindering tumor growth but promoting invasion and dissemination. Experiments to evaluate the net biological outcome of TXNIP modulation in vivo are ongoing. -- Le mélanome cutané est une tumeur maligne agressive des mélanocytes, cellules de l'épiderme qui produisent la mélanine. Ce cancer présente un taux d'incidence élevé dans les pays développés et est grevé d'un pronostic très sombre une fois qu'il a disséminé. Malgré les importants progrès réalisés ces dernières années, aucune thérapie lie s'est encore montrée véritablement efficace contre le mélanome métastatique. Parmi les cibles thérapeutiques potentielles, nombre de groupes de recherche se sont penchés sur les peroxisome proliferator-activated receptors (PPARs). Ces récepteurs - a, ß et 7 - font partie de la famille des récepteurs nucléaires aux hormones, des facteurs de transcription activés par des ligands et dotés d'une multitude de fonctions en sus de la régulation du métabolisme. Ces protéines ont démontré des propriétés anti-tumorales prometteuses dans une large gamme de cellules cancéreuses, y compris le mélanome. Cependant, nous connaissons encore très mal les fonctions des PPARs dans ce cancer de la peau, rendant l'utilité thérapeutique de l'un des isotypes a, ß ou 7 incertaine. Dans ce travail, nous avons montré que les trois isotypes sont exprimés dans les mélanocytes normaux, dans la plupart des lignées de mélanome ainsi que dans des mélanomes primaires et métastatiques; nous avons aussi montré que PPAR/3 et 7 sont actifs sur le plan transcriptionnel dans les mélanocytes normaux et les cellules de mélanome. La rosiglitazone, un agoniste de PPAR7, a démontré des propriétés anti-mélanome essentiellement indépendantes de l'expression de PPAR7, qui semble très variable dans les lignées et les biopsies que nous avons évaluées; de plus, l'expression de PPAR7 n'est pas corrélée avec le stade de la lignée. En accord avec la vision communément admise de PPAR7 comme étant un gène suppresseur de tumeur, nous avons observé dans des échantillons humains que PPAR7 est moins exprimé dans les mélanomes que dans la peau normale. Une étude transcrip- tomique de cellules de mélanome métastatique a révélé que la modulation phar-macologique de PPAR7 est associée avec la transition épithélio-mésenchymateuse, même si la pertinence fonctionnelle de cette trouvaille reste à déterminer. Collec-tivement, ces résultats suggèrent que l'activité de PPAR/y dans le mélanome est hautement complexe et qu'une image claire du rôle de PPAR7 dans ce cancer est difficile à dessiner. Dans cette étude, nous avons également fourni de solides preuves que la thiore-doxin interacting protein (TXNIP) est, dans le mélanome, un gène cible bona fide de PPAR7 dont l'expression est réprimée par l'activation de PPAR7. Bien que TXNIP soit surtout connu comme un inhibiteur de la thiorédoxine -un anti-oxydant majeur - cette protéine a démontré une large gamme de fonctions biologiques et est généralement considérée comme un gène suppresseur de tumeur. En accord avec cette conception, nous avons trouvé que l'expression de TXNIP est associée avec l'arrêt de croissance des cellules de mélanome in vitro et que l'expression forcée de TXNIP freine considérablement la prolifération cellulaire. Nous avons aussi découvert que TXNIP favorise la migration des cellules de mélanome alors qu'elle diminue leur adhésion. Enfin, nous avons obtenu plusieurs preuves que TXNIP pourrait réguler ces processus tant au niveau transcriptionnel que par des interactions protéine-protéine au sein de la membrane plasmique. En conclusion, nos résultats suggèrent que la cible de PPAR7 TXNIP pourrait être une épée à double tranchant dans le mélanome, freinant la croissance tumorale mais favorisant l'invasion et la dissémination. Des expériences permettant d'évaluer l'effet biologique net de la modulation de TXNIP in vivo sont en cours.

7

Evidence of altered antioxidant systems and signs of elevated oxidative stress are reported in peripheral tissue and brain of schizophrenic patients, including low levels of glutathione (GSH), a major thiol antioxidant and redox buffer. Functional and genetic data indicate that an impaired regulation of GSH synthesis is a vulnerability factor for the disease. Impaired GSH synthesis from a genetic origin combined with environmental risk factors generating oxidative stress (e.g., malnutrition, exposure to toxins, maternai infection and diabetes, obstetrical complications, and psychological stress) could lead to redox dysregulation. This could subsequently perturb normal brain development and maturation with delayed functional consequences emerging in early adulthood. Depending on the nature and the time of occurrence of the environmental insults, the structural and functional delayed consequences could vary, giving rise to various endophenotypes. The use of animal models of GSH deficit represents a valuable approach to investigate how interactions between genetic and environmental factors lead to the emergence of pathologies found in the disease. Moreover, these models of GSH can be useful to investigate links between schizophrenia and comorbid somatic disorders, as dysregulation of the GSH system and elevated oxidative stress are also found in cardiovascular diseases and diabetes. This chapter reviews pharmacological and genetic rodent models of GSH synthesis dysregulation used to address some of the aforementioned issues. Up to date, these models revealed that GSH deficits lead to morphological, physiological, and behavioral alterations that are quite analogous to pathologies observed in patients. This includes hypofunction of NMDA receptors, alteration of dopamine neurotransmission, anomalies in parvalbumin-immunoreactive fast-spiking interneurons, and reduced myelination. In addition, a GSH deficit affects the brain in a region-specific manner, the anterior cingulate cortex and the ventral hippocampus being the most vulnerable regions investigated. Interestingly, a GSH deficit during a limited period of postnatal development is sufficient to have long-lasting consequences on the integrity of PV-IR interneurons in the anterior cingulate cortex and impairs cognitive functions in adulthood. Finally, these animal models of GSH deficit display behavioral impairments that could be related to schizophrenia. Altogether, current data strongly support a contributing role of a redox dysregulation on the development of pathologies associated with the illness and demonstrate the usefulness of these models to better understand the biological mechanisms leading to schizophrenia.

IFNalpha activates dormant haematopoietic stem cells in vivo.

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFNalpha), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFNalpha treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFNalpha target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFNalpha/beta receptor (IFNAR), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFNalpha stimulation, demonstrating that STAT1 and Sca-1 mediate IFNalpha-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil, HSCs pre-treated (primed) with IFNalpha and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFNalpha are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFNalpha pathway in HSCs impairs their function, acute IFNalpha treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFNalpha on leukaemic cells, and raise the possibility for new applications of type I interferons to target cancer stem cells.

Ubiquitination and the Ubiquitin-Proteasome System as regulators of transcription and transcription factorsin epithelial mesenchymal transition of cancer.

Epithelial to Mesenchymal Transition (EMT) in cancer is a process that allows cancer cells to detach from neighboring cells, become mobile and metastasize and shares many signaling pathways with development. Several molecular mechanisms which regulate oncogenic properties in neoplastic cells such as proliferation, resistance to apoptosis and angiogenesis through transcription factors or other mediators are also regulators of EMT. These pathways and downstream transcription factors are, in their turn, regulated by ubiquitination and the Ubiquitin-Proteasome System (UPS). Ubiquitination, the covalent link of the small 76-amino acid protein ubiquitin to target proteins, serves as a signal for protein degradation by the proteasome or for other outcomes such as endocytosis, degradation by the lysosome or directing these proteins to specific cellular compartments. This review discusses aspects of the regulation of EMT by ubiquitination and the UPS and underlines its complexity focusing on transcription and transcription factors regulating EMT and are being regulated by ubiquitination.

Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF.

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment.

Study of a sialyltransferase ST3GAL6, in adipogenesis and obesity

RésuméL'obésité et les maladies métaboliques qui lui sont associées tels que le diabète ou les maladies cardiovasculaires ont un impact épidémiologique croissant. Ainsi, les mécanismes moléculaires se produisant dans le tissu adipeux en expansion font l'objet de nombreuses investigations. Dans ce contexte, nous nous sommes particulièrement intéressés à l'adipogénèse, le procédé permettant la formation d'adipocytes matures et fonctionnels. Le gène St3gal6 code pour une enzyme appelée β-galactosidase a2,3-sialyltransferase 6 et participant à la voie de glycosylation. Cette protéine appartient à la famille des a2,3- sialyltransferases dont la fonction principale est de transférer un acide sialique à l'extrémité de chaînes glycosidiques présentes sur les glycoprotéines et les glycolipides. Dans une précédente étude de transcriptomique réalisée chez la souris, St3gal6 a été décrit comme un gène dont l'expression est augmentée dans le tissu adipeux blanc d'animaux en surpoids et dont l'expression est normalisée après une perte de poids. Afin d'étudier le rôle potentiel de St3gal6 dans le développement du tissu adipeux, nous nous sommes intéressés à la régulation de son expression en cas d'obésité ainsi qu'à ses effets sur l'adipogénèse. Nous avons d'abord montré que St3gal6 s'exprime aussi bien dans le tissu adipeux blanc que dans le tissu adipeux brun. Puis nous avons confirmé dans deux différents modèles animaux que l'expression de St3gal6 dans le tissu adipeux était augmentée en cas d'obésité. Nous avons aussi observé in vitro une induction de St3gal6 dans des adipocytes traités par des cytokines pro-inflammatoires sécrétées dans le tissu adipeux d'individus obèses. Enfin, parmi les six membres que compte la famille des a2,3-sialyltransferases, St3gal6 est celui dont l'expression est la plus significativement induite en situation d'obésité. En outre, au cours de la différenciation des adipocytes blancs et bruns, l'expression de St3gal6 est augmentée et son inhibition réduit le potentiel de maturation des adipocytes qui accumulent moins de lipides. A l'inverse, la surexpression de St3gal6 dans des préadipocytes blancs augmente leur taux de différenciation in vitro; la formation de gouttelettes lipidiques et l'expression de genes spécifiques de l'adipocyte mature sont accrues. Enfin, le traitement d'adipocytes blancs in vitro avec un inhibiteur pharmacologique des a2,3-sialyltransferases ou une sialidase clivant les résidus sialylés montre qu'un défaut de a2,3-sialylation affectant les adipocytes diminue leur potentiel adipogénique. Par conséquent, ces résultats suggèrent que St3gal6 est impliqué dans la voie de différenciation des adipocytes et que cette a2,3-sialylation joue un rôle dans le remodelage du tissu adipeux induit par l'obésité.AbstractIn order to better understand molecular events occurring in obesity and leading to its associated complications, we were interested in the biology of adipose tissue and particularly in the study of adipogenesis, the process by which new mature adipocytes develop and accumulate lipids.The β-galactosidase a2,3-sialyltransferase 6 (St3gal6) gene encodes for an enzyme involved in post-translational protein glycosylation. Thereby, St3gal6 enzyme belongs to the a2,3sialyltransferase family whose function is to add sialic acids at outer position on glycosidic chain of glycoproteins or glycolipids. Previously, in mouse, St3gal6 has been described as a gene whose expression in white adipose tissue is increased in overweighted animals and normalized after weight loss. Therefore, we have assumed that St3gal6 may play a role in adipose tissue development and in tissue remodelling triggered by obesity. First we show that St3gal6 is expressed in white but also in brown adipose tissue. St3gal6 upregulation upon weight gain was confirmed in two mouse models of obesity namely diet- induced and genetically-induced obesity. We also report that St3gal6 is induced by pro¬inflammatory cytokines known to be oversecreted in adipose tissue during obesity. Furthermore, St3gal6 is the a2,3-sialyltransferase whose expression is more markedly induced in adipose tissue. In addition, we demonstrate that St3gl6 expression is progressively increased in late stages of white and brown adipogenesis while St3gal6 knockdown inhibits adipocyte differentiation in vitro. Conversely, St3gal6 overexpression in a white preadipocyte cell line increases lipid accumulation during differentiation process and enhances gene expression of mature white adipocyte markers. Finally, using an a2-3 sialyltransferase inhibitor and a sialidase treatment on white adipocyte cell line, we observe that a decreased a2,3-sialylation impairs adipocyte differentiation in vitro. Altogether, these result suggest that St3gal6 plays a role in adipogenesis and in tissue remodelling associated with obesity likely through its enzymatic activity of a2,3-sialylation. Thus, a2,3-sialylation appears as a novel pathway of interest whose precise molecular mechanisms remain to be elucidated in the context of adipose tissue development and adipocyte functions.

SH3TC2, a protein mutant in Charcot-Marie-Tooth neuropathy, links peripheral nerve myelination to endosomal recycling

Patients with Charcot-Marie-Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90.

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.