Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis

Claudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and -19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis

PATHOLOGIES GENETIQUES AFFECTANT LA MINERALISATION DE LA DENT : EXEMPLE DE L’AMELOGENESE IMPARFAITE ET DE LA DYSPLASIE DENTINAIRE

La dent est constituée par un ensemble de tissus dont l'association au sein de l'organe dentaire assure différentes fonctions : la pulpe, coeur vivant de la dent, et de trois tissus minéralisés, l’émail, la dentine, et le cément. L'organe dentaire est principalement impliqué, par son aspect morpho-fonctionnel, dans la fonction masticatrice, mais au-delà de la santé, fait la beauté du sourire, jouant un rôle important dans la construction et la stabilité de l’estime de soi. Les anomalies dentaires, qu’elles soient de nombre, forme, taille, structure ou d’éruption/résorption, font parties des manifestations phénotypiques de maladies rares ou syndromes fixant dans le temps les troubles issus du développement.Mon projet de recherche est centré sur les pathologies affectants les processus de la minéralisation de la dent. Notre étude est basée sur l’analyse de données cliniques, en parallèle à des données ex vivo de tissus humain et in vivo chez la souris, en particulier les modèles murins de maladies rares. J’ai étudié principalement deux pathologies affectant deux tissus dentaires minéralisés, l’émail et la dentine, dessinant ainsi mes deux axes de recherche avec pour objectif commun la mise en évidence et/ou compréhension des dysfonctionnements moléculaires associés aux pathologies étudiées.Le premier axe de recherche de ce projet a consisté à étudier les conséquences de la mutation du gène claudin-16 responsable d’une pathologie rénale sévère (l’hypomagnésémie hypercalciurie et néphrocalcinose familiale ou HHNF) sur la formation de l’émail.Nous avons démontré que la carence en CLDN16 conduit à une structure altérée des ameloblastes sécrétoires, à une réduction du pH extracellulaire dans la matrice de l’émail et à un traitement anormal de protéines dans cette matrice. Grâce à l’étude d’un autre modèle murin invalidé pour la claudin-3 nous avons pu confirmer ces résultats, soulignant l'importance des claudines pour la structure des jonctions serrées, dans la formation des dents.Pour le second axe de mon projet, je me suis intéressée à l’identification de la mutation d’un nouveau gène impliqué dans la dysplasie dentinaire de type 1, le gène Trip 10 (Thyroid hormone receptor interactor 10) codant pour la protéine CIP4 (CDC42-interacting protein 4), une protéine à domaine BAR qui serait notamment impliquée dans l’endocytose dépendante de la clathrine.Nous suggérons que l'absence de TRIP10 à la membrane pourrait induire un dérèglement de la voie β-caténine. Cette perturbation pourrait affecter à la fois la formation des racines et l'ancrage des dents au parodonte, ce qui explique la perte dentaire précoce subie par les patients.En étudiant les mécanismes impliqués dans les défauts de la minéralisation de l’émail et de la dentine, ce travail a permis d’identifier des nouveaux gènes et/ou leur implication dans des pathologies dentaires, d’apporter des éléments permettant une meilleure compréhension des mécanismes impliqués dans ces pathologies et un meilleur accompagnement du diagnostic associé à une meilleure prise en charge des patients

Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis

International audienceClaudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and-19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis

A New Wnt1-CRE Tomato Rosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity

International audienceNeural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 RosaTomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair

Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor.

International audienceThe mechanism by which hyaluronic acid (HA)-bearing lipoplexes target the A549 lung cancer cell line was evaluated. For this purpose, cationic liposomes targeting the CD44 receptor were designed thanks to the incorporation in their composition of a conjugate between high molecular weight HA and the lipid DOPE (HA-DOPE). Liposomes containing HA-DOPE were complexed at different lipids:DNA ratios with a reporter plasmid encoding the green fluorescent protein (GFP). Diameter, zeta potential, lipoplex stability and DNA protection from nucleases have been determined. Lipids:DNA ratios of 2, 4 and 6 provided a diameter around 250 nm with a zeta potential of -30 mV. The strength of lipids:DNA interaction and the fraction of DNA protected from enzymatic degradation increased with the lipids:DNA ratio. 2D-immunoelectrophoresis demonstrated the low capacity to activate the C3 fraction of the complement system of any of these three ratios, with and without HA-DOPE. Transfection efficiency in the presence of 0, 10 and 15% of HA-DOPE or unconjugated HA, was determined on the CD44-expressing A549 cells by flow cytometry. Lipoplexes at a lipids:DNA ratio of 2 containing 10% (w/w) of HA-DOPE were the most efficient for transfection. The maximal level of GFP expression was obtained after 6h of incubation demonstrating a slow transfection kinetics of lipoplexes. Finally, lipoplex cellular uptake, measured indirectly by the level of transfection using flow cytometry and validated by fluorescence microscopy, was shown to be mediated by the CD44 receptor and caveolae. These results demonstrate the strong specificity of DNA targeting through the CD44 receptor using HA of high molecular weight as a ligand

ABCG2 and ABCG4-Mediated Efflux of Amyloid-β Peptide 1-40 at the Mouse Blood-Brain Barrier.

International audienceThe accumulation of amyloid-β peptide (Aβ) in the brain is a critical hallmark of Alzheimer's disease. This high cerebral Aβ concentration may be partly caused by impaired clearance of Aβ across the blood-brain barrier (BBB). The low-density lipoprotein receptor-related protein-1 (LRP-1) and the ATP-binding cassette (ABC) protein ABCB1 (P-glycoprotein) are involved in the efflux of Aβ across the BBB. We hypothesized that other ABC proteins, such as members of the G subfamily, are also involved in the BBB clearance of Aβ. We therefore investigated the roles of ABCG2 (BCRP) and ABCG4 in the efflux of [3H] Aβ1-40 from HEK293 cells stably transfected with human ABCG2 or mouse abcg4. We showed that ABCG2 and Abcg4 mediate the cellular efflux of [3H] Aβ1-40. In addition, probucol fully inhibited the efflux of [3H] Aβ1-40 from HEK293-abcg4 cells. Using the in situ brain perfusion technique, we showed that GF120918 (dual inhibitor of Abcb1 and Abcg2) strongly enhanced the uptake (Clup, μl/g/s) of [3H] Aβ1-40 by the brains of Abcb1-deficient mice, but not by the brains of Abcb1/Abcg2-deficient mice, suggesting that Abcg2 is involved in the transport of Aβ at the mouse BBB. Perfusing the brains of Abcb1/Abcg2- and Abca1-deficient mice with [3H] Aβ1-40 plus probucol significantly increased the Clup of Aβ. This suggests that a probucol-sensitive transporter that is different from Abca1, Abcb1, and Abcg2 is involved in the brain efflux of Aβ. We suggest that this probucol-sensitive transporter is Abcg4. We conclude that Abcg4 acts in concert with Abcg2 to efflux Aβ from the brain across the BBB

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells

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EMMPRIN/CD147 deficiency disturbs ameloblast-odontoblast cross-talk and delays enamel mineralization

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