Clathrin light chains CLCa and CLCb have non-redundant roles in epithelial lumen formation

To identify functional differences between vertebrate clathrin light chains (CLCa or CLCb), phenotypes of mice lacking genes encoding either isoform were characterised. Mice without CLCa displayed 50% neonatal mortality, reduced body weight, reduced fertility, and ∼40% of aged females developed uterine pyometra. Mice lacking CLCb displayed a less severe weight reduction phenotype compared with those lacking CLCa and had no survival or reproductive system defects. Analysis of female mice lacking CLCa that developed pyometra revealed ectopic expression of epithelial differentiation markers (FOXA2 and K14) and a reduced number of endometrial glands, indicating defects in the lumenal epithelium. Defects in lumen formation and polarity of epithelial cysts derived from uterine or gut cell lines were also observed when either CLCa or CLCb were depleted, with more severe effects from CLCa depletion. In cysts, the CLC isoforms had different distributions relative to each other, although they converge in tissue. Together, these findings suggest differential and cooperative roles for CLC isoforms in epithelial lumen formation, with a dominant function for CLCa

Activation of a-7 Nicotinic Acetylcholine Receptor Reduces Ischemic Stroke Injury through Reduction of Pro-Inflammatory Macrophages and Oxidative Stress

International audienceActivation of a-7 nicotinic acetylcholine receptor (a-7 nAchR) has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that a-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1) and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, a-7 nAchR agonist), methyllycaconitine (MLA, nAchR antagonist), or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO). Behavior test, lesion volume, CD68 + , M1 (CD11b + /Iba1 +) and M2 (CD206/Iba1 +) microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68 + and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA's effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in a-7 nAchR neuro-protective effect

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function

Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance. [Abstract copyright: Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin’s role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules

CHC22 clathrin mediates traffic from early secretory compartments for human GLUT4 pathway biogenesis

Post-prandial blood glucose is cleared by Glucose Transporter 4 (GLUT4) released from an intracellular GLUT4 storage compartment (GSC) to the surface of muscle and adipose tissue in response to insulin. Here we map the biosynthetic pathway for human GSC formation, which involves the clathrin isoform CHC22. We observe that GLUT4 transits more slowly through the early secretory pathway than the constitutively-secreted GLUT1 transporter, and show CHC22 colocalizes with p115 in the endoplasmic-reticulum-to-Golgi-intermediate compartment (ERGIC). We find CHC22 functions in membrane traffic from the early secretory pathway during formation of the replication vacuole of Legionella pneumophila, which also acquires components of the GLUT4 pathway. We show that p115 but not GM130 is required for GSC formation, indicating GSC biogenesis from the ERGIC bypasses the Golgi. This GSC biogenesis pathway is attenuated in mice, which lack CHC22, and rely mainly on recapture of surface GLUT4 to populate their GSC. GLUT4 traffic to the GSC is enhanced by CHC22 function at the human ERGIC, which has implications for pathways to insulin resistance

Innovative pathways of chemosensitization in pancreatic ductal adenocarcinoma : acoustic cavitation and NRF2 inhibition

L’adénocarcinome pancréatique (AP) connaît une forte augmentation d’incidence, qui en fait la quatrième cause de mortalité par cancer avec un pronostic extrêmement sombre, moins de 5% des patients étant en vie à 5 ans. De nombreuses avancées dans la compréhension de l’oncogénèse pancréatique notamment sur les aspects génétiques, immunitaires et sur les interactions cellulaires du stroma tumoral ont permis d’envisager le développement de nouvelles stratégies de traitement. Cependant malgré des résultats pré-cliniques très encourageants aucune de ces stratégies n’a encore permis l’émergence d’un traitement plus efficace que la chimiothérapie standard. Ce travail de thèse a abordé 2 approches thérapeutiques innovantes distinctes mais potentiellement complémentaires dans le traitement de l’adénocarcinome pancréatique en étudiant in vitro (cultures cellulaire 2D et 3D) et in vivo (modèles ectopiques et orthotopiques) les effets sur la croissance tumorale de l’inhibition d’un acteur important du stress oxydant (la voie Nrf2) d’une part, de la combinaison d’une chimiothérapie liposomale et d’un agent physique, la cavitation ultrasonore, d’autre part. La cavitation ultrasonore est un effet mécanique des ultrasons permettant d’augmenter l’internalisation de molécules ou de gènes dans les cellules. Dans cette thèse, la faisabilité et l'efficacité de la combinaison d’une chimiothérapie liposomale ciblée par la cavitation ultrasonore a été évaluée dans des modèles murins orthotopiques d’AP. Un système de délivrance d’ultrasons a été adapté afin d’appliquer une cavitation inertielle focalisée sur des xénogreffes d’AP créées après l'injection de doxorubicine liposomale (L-DOX) selon une étude pharmacocinétique préliminaire réalisée dans un modèle murin. La L-DOX, conçue à base de phospholipides non saturés de dioleoylphosphatidyléthanolamine, connue pour être stable dans la circulation sanguine, a été choisie afin de maximiser son accumulation et le relargage du principe actif lors de la délivrance des ultrasons. Nous montrons que l’association de la L-DOX à la cavitation inertielle permet de réduire in vivo le volume tumoral dans un modèle orthotopique d’AP chez la souris nude. La cavitation inertielle peut donc augmenter l'effet antitumoral des liposomes porteurs de chimiothérapie avec un effet mécanique minimal sur le tissu environnant la tumeur.Des études récentes suggèrent que Nrf2 est une cible de choix pour vaincre la chimiorésistance de l’AP. Des méthodes in vitro et in vivo ont été utilisées afin d’examiner l'effet du brusatol associé à des agents chimiothérapeutiques de référence sur la mort cellulaire et son impact sur le stress oxydant. Nous montrons que l’inhibition de la voie Nrf2 par le brusatol, un composé naturel issu de Fructus Bruceae, potentialise les effets de la chimiothérapie et permet l’inhibition de la croissance tumorale in vitro sur des lignées cellulaires d’AP cultivées en 2D et 3D. Cette inhibition s’accompagne d’une modulation du stress oxydant dont témoignent l’augmentation des espèces réactives de l’oxygène (ROS) et la diminution du glutathion (GSH). In vivo, la combinaison du brusatol et de l'oxaliplatine a réduit le volume tumoral dans deux modèles murins de xénogreffe d’AP. Ces résultats suggèrent l'efficacité du brusatol pour lutter contre la chimiorésistance et renforce l’hypothèse d’un rôle clinique potentiel de l’inhibition de Nrf2 comme adjuvant à la chimiothérapie dans l’AP. Un travail clinique a également été mené en parallèle sur une modalité de traitement physique innovante, la radiofréquence endobiliaire, dans la prise en charge endoscopique de l’ampullome vatérien, tumeur rare à la croisée entre le tube digestif et le système biliopancréatique. Les résultats de cette étude prospective multicentrique seront également présentés dans cette thèse.Pancreatic ductal adenocarcinoma (PDAC) has increased in incidence over the past decade, leading it to be the fourth lethal cause of cancer in the world with a very poor prognosis, since less than 5% of patients are alive at 5 years. Many advances in the understanding of pancreatic tumorigenesis, notably on the genetic, immune and cellular stroma interactions of the tumor, have led to the development of new treatment strategies in the last decade. However, despite very encouraging pre-clinical results, none of these strategies has yet led to the emergence of a truly effective treatment in comparison with standard chemotherapy. This thesis focused on two innovative therapeutic modalities in the treatment of PDAC at a preclinical stage by studying in vitro (2D and 3D cell cultures) and in vivo (ectopic, orthotopic xenografts) the effects on the tumor growth of an inhibitor of the Nrf2 pathway (involved in oxidative stress), on the first hand, and of a physical element, ultrasound cavitation associated with liposomal chemotherapy, on the second hand. Ultrasound cavitation is a mechanical effect of ultrasound to increase the uptake of molecules or genes in cells. The feasibility and effectiveness of the combination of liposomal chemotherapy targeted by ultrasonic cavitation was evaluated in murin orthotopic models of PDAC. An ultrasound delivery system has been adapted to apply focused inertial cavitation to PDAC xenografts created after the injection of liposomal doxorubicin (L-DOX) according to a preliminary pharmacokinetic study carried out in the murine model. L-DOX, designed on unsaturated phospholipids of dioleoylphosphatidylethanolamine, was known to be stable in the bloodstream and to maximize its accumulation and release of the active drug during ultrasound delivery. This thesis shows that this therapeutic combination (L DOX and inertial cavitation) makes it possible to reduce the tumor volume in vivo in a nude mouse orthotopic model of PDAC. Inertial cavitation may be generated to increase the therapeutic effect of chemotherapybearing liposomes accumulated in the tumor with minimal mechanical effect on the surrounding tissue. Recent studies strongly suggest that Nrf2 is an ideal target against chemoresistance of PDAC. In vitro and in vivo methods were combined to examine the effect of brusatol associated with chemotherapeutic agents on cell death in addition to its impact on oxidative stress (reactive oxygen species and gluthation levels). This thesis demonstrates that the inhibition of the Nrf2 pathway via brusatol, a natural compound derived from Fructus Bruceae, potentiates the effects of chemotherapy and allows the inhibition of tumor growth in vitro on PDAC cell lines. This inhibition is accompanied by a modulation of oxidative stress by brusatol, with increasing ROS and decreasing GSH. In vivo, the combination of brusatol and oxaliplatin reduced tumor volume in two mouse models of PDAC xenograft. These results suggest the efficacy of using brusatol to combat chemoresistance and reinforce the idea that brusatol could be developed as an adjuvant to chemotherapy in PA. Clinical work was also carried out in parallel on an innovative physical treatment modality, endobiliary radiofrequency, in the management of adenoma of the ampoule of Vater, a rare tumor located between the digestive and the bilio-pancreatic systems. The results of this work will also be presented in this thesis

Evolution à long terme des patients atteints de maladie de Crohn répondeurs à l azathioprine

PARIS7-Xavier Bichat (751182101) / SudocSudocFranceF