Modelisation of a laminopathy from pluripotent stem cells : phenotypic and genotypic study, search for new therapeutic targets

Les laminopathies regroupent des maladies rare dues à des mutations sur le gène Lmna, codant pour les lamines nucléaires A et C. Parmi des centaines de mutations identifiées jusqu’alors, la mutation Lmna+/H222P est responsable de la Dystrophie Musculaire d’Emery Dreifuss Autosomale Dominante (DMED-AD). Les patients atteints par DMED-AD souffrent d’une dystrophie et d’une cardiomyopathie. J’ai étudié tout particulièrement l’effet et la fonction de la mutation Lmna+/H222P au niveau du développement cardiaque en utilisant des cellules souches embryonnaires murines (CSEm) ainsi que des cellules humaines induites à la pluripotence (hiPS) sauvages et porteuses de la mutation Lmna+/H222P. Un défaut dans la cardiogenèse est retrouvé dans les corps embryoïdes (CE) dérivés des CSEm et dans les hiPS dont la différenciation est induite par le BMP2. En effet, dans les cellules différenciées porteuses de la mutation Lmna+/H222P, on observe une expression des gènes mésodermiques et cardiaques (ex : brachyury, MesP1, Nkx2.5, Mef2c, Isl1…) déficiente. Néanmoins, la formation du mésendoderme ne semble pas affectée dans ces cellules. De plus, des défauts de contraction dûs à une désorganisation de la structure sarcomérique est retrouvée dans les Ces dérivés des CSEm Lmna+/H222P. Mes travaux de thèse ont donc permis de mettre en place un modèle murin et humain de cellules souches pluripotentes pour laminopathies. Ces cellules pourront plus tard être utilisées afin de tester des médicaments permettant de trouver des traitements pour les personnes atteintes de la DMED-AD.Laminopathies are rare genetic disorders caused by mutations in Lmna which encodes nuclear lamins A/C. Among hundreds of mutations identified so far, Lmna+/H222P leads to an Autosomal Dominant Emery-Dreifuss Muscular Dystrophy (AD-EDMD). AD-EDMD patients suffer of both muscle dystrophy and cardiomyopathy. Herein, we investigated the effects of Lmna+/H222P in cardiac development and function using wild type and mutated mouse embryonic stem cells (mESC) and human induced pluripotent stem cells (hiPS). Lmna+/H222P impairs cardiogenesis of both mESC and hiPSC. Expression of mesodermal and cardiac genes (i.e., brachyury, MesP1, Nkx2.5, Mef2c, Isl1…) in mESC derived embryoid bodies (mEBs) and in BMP2-induced cardiac progenitors from hIPCs was deficient in mutated cells. Nevertheless, the formation of mesendoderm was not affected in cells carrying Lmna+/H222P mutation. Cell contractility was impaired in mutated mEBs which correlated with a poor sarcomeric network visualised by cell immunostaining. Thus, my thesis revealed that human and murine pluripotent stem cells can serve as cellular model for laminopahties. These cells could be used for drug screening in order to test pharmacological approach to relieve symptomns of AD-EDMD

Cardiomyopathie due à la lamine de type A mutée: Un traitement en vue grâce à la recherche sur le cancer ?

International audienc

HIV antiretroviral drugs, dolutegravir, maraviroc and ritonavir-boosted atazanavir use different pathways to affect inflammation, senescence and insulin sensitivity in human coronary endothelial cells

International audienceOBJECTIVES:Aging HIV-infected antiretroviral-treatment (ART)-controlled patients often present cardiovascular and metabolic comorbidities. Thus, it is mandatory that life-long used ART has no cardiometabolic toxicity. Protease inhibitors have been associated with cardiometabolic risk, integrase-strand-transfer-inhibitors (INSTI) with weight gain and the CCR5 inhibitor maraviroc with improved vascular function. We have previously reported that the INSTI dolutegravir and maraviroc improved, and ritonavir-boosted atazanavir(atazanavir/r) worsened, inflammation and senescence in human coronary artery endothelial cells (HCAEC)s from adult controls. Here, we analyzed the pathways involved in the drugs' effects on inflammation, senescence and also insulin resistance.METHODS:We analyzed the involvement of the anti-inflammatory SIRT-1 pathway in HCAECs. Then, we performed a transcriptomic analysis of the effect of dolutegravir, maraviroc and atazanavir/r and used siRNA-silencing to address ubiquitin-specific-peptidase-18 (USP18) involvement into ART effects.RESULTS:Dolutegravir reduced inflammation by decreasing NFκB activation and IL-6/IL-8/sICAM-1/sVCAM-1 secretion, as did maraviroc with a milder effect. However, when SIRT-1 was inhibited by splitomicin, the drugs anti-inflammatory effects were maintained, indicating that they were SIRT-1-independant. From the transcriptomic analysis we selected USP18, previously shown to decrease inflammation and insulin-resistance. USP18-silencing enhanced basal inflammation and senescence. Maraviroc still inhibited NFκB activation, cytokine/adhesion molecules secretion and senescence but the effects of dolutegravir and atazanavir/r were lost, suggesting that they involved USP18. Otherwise, in HCAECs, dolutegravir improved and atazanavir/r worsened insulin resistance while maraviroc had no effect. In USP18-silenced cells, basal insulin resistance was increased, but dolutegravir and atazanavir/r kept their effect on insulin sensitivity, indicating that USP18 was dispensable.CONCLUSION:USP18 reduced basal inflammation, senescence and insulin resistance in coronary endothelial cells. Dolutegravir and atazanavir/r, but not maraviroc, exerted opposite effects on inflammation and senescence that involved USP18. Otherwise, dolutegravir improved and atazanavir/r worsened insulin resistance independently of USP18. Thus, in endothelial cells, dolutegravir and atazanavir/r oppositely affected pathways leading to inflammation, senescence and insulin resistance

Brown and beige fat: From molecules to physiology and pathophysiology.

The adipose organ portrays adipocytes of diverse tones: white, brown and beige, each type with distinct functions. Adipocytes orchestrate their adaptation and expansion to provide storage to excess nutrients, the quick mobilisation of fuel to supply peripheral functional demands, insulation, and, in their thermogenic form, heat generation to maintain core body temperature. Thermogenic adipocytes could be targets for anti-obesity and anti-diabetic therapeutic approaches aiming to restore adipose tissue functionality and increase energy dissipation. However, for thermogenic adipose tissue to become therapeutically relevant, a better understanding of its development and origins, its progenitors and their characteristics and the composition of its niche, is essential. Also crucial is the identification of stimuli and molecules promoting its specific differentiation and activation. Here we highlight the structural/cellular differences between human and rodent brown adipose tissue and discuss how obesity and metabolic complication affects brown and beige cells as well as how they could be targeted to improve their activation and improve global metabolic homeostasis. Finally, we describe the limitations of current research models and the advantages of new emerging approaches

Lipodystrophic syndromes due to LMNA mutations: recent developments on biomolecular aspects, pathophysiological hypotheses and therapeutic perspectives

International audienceMutations in LMNA, encoding A-type lamins, are responsible for laminopathies including musculardystrophies, lipodystrophies, and premature ageing syndromes. LMNA mutations have been shownto alter nuclear structure and stiffness, binding to partners at the nuclear envelope or within thenucleoplasm, gene expression and/or prelamin A maturation. LMNA-associated lipodystrophicfeatures, combining generalized or partial fat atrophy and metabolic alterations associated withinsulin resistance, could result from altered adipocyte differentiation or from altered fat structure.Recent studies shed some light on how pathogenic A-type lamin variants could triggerlipodystrophy, metabolic complications, and precocious cardiovascular events. Alterations inadipose tissue extracellular matrix and TGF-beta signaling could initiate metabolic inflexibility.Premature senescence of vascular cells could contribute to cardiovascular complications. Inaffected families, metabolic alterations occur at an earlier age across generations, which couldresult from epigenetic deregulation induced by LMNA mutations. Novel cellular modelsrecapitulating adipogenic developmental pathways provide scalable tools for disease modelingand therapeutic screening

Functional Human Beige Adipocytes From Induced Pluripotent Stem Cells

International audienc