Etude des mécanismes moléculaires et cellulaires impliqués dans le développement de la dystrophie myotonique de type 1 à l'aide de cellules souches embryonnaires humaines porteuses de la mutation causale

Parmi leurs applications prometteuses, les cellules souches pluripotentes humaines présentent un potentiel inestimable pour améliorer la compréhension des mécanismes moléculaires et cellulaires impliqués dans le développement de maladies monogéniques. Cette application est dans un premier temps devenue possible grâce à l utilisation de lignées de cellules souches embryonnaires humaines (CSEh) porteuses de mutation causale de maladie monogénique, obtenues au cours d un diagnostique pré-implantatoire, puis dans un second temps par la reprogrammation des cellules somatiques en cellules souches pluripotentes (iPS). Dans le cadre de la validation de ce concept, nous avons démontré que des lignées de CSEh porteuses de la mutation causale de la dystrophie myotonique de type 1 (DM1), ainsi que leursprogénies neurales et mésodermiques, exprimaient des défauts moléculaires caractéristiques de la pathologie. Par l intermédiaire d une étude transcriptomique comparative, nous avons identifié une liste de biomarqueurs pouvant être considérés comme une nouvelle signature moléculaire de la DM1. Parmi ces derniers, nous avons montré que l anomalie d expression de certains gènes de la famille SLITRK était à l origine des défauts d arborisation neuritique mis en évidence dans des cellules motoneuronales dérivées des CSEh mutées, et que ces cellules peuvent interagir avec leur ciblemusculaire. Parallèlement, nous avons identifié un facteur de transcription à domaine Krab dont l expression est fortement altérée dans la DM1 et qui semble être impliqué dans les défauts de régénération musculaire associé à des fins thérapeutiques en définissant leur capacité à modéliser une maladie génétique de façon suffisamment précise pour permettre d élaborer des biothérapies spécifiquement liées aux mécanismes moléculaires mis en jeu.Human pluripotent stem cells present far reaching implication not only for their therapeutic potential but also for the understanding of the molecular and cellular mechanisms of monogenic diseases. This application became at first possible by using human embryonic stem cells lines (hES) carrying the causal mutation of the monogenic disease, obtained during pre-implantation genetic diagnosis, thereafter through the development of somatic cells reprogramming into pluripotent stem cells (iPS). In line with this concept, we provided evidence that hES lines carrying the causal mutation of myotonic dystrophy type 1 (DM1), as well as their neural and mesodermal progenitors, expressed characteristic molecular defects of the pathology. Through a comparative study of their transcriptome profile, we identified a list of biomarkers which can be considered as new molecular signature of DM1. Among these genes, we showed that abnormal expression of some genes of the SLITRK family was responsible for the defective neuritic outgrowth observed in motor neuron cells derived from mutated hES, but that these cells could nonetheless interact with their muscular target. In parallel, we identified a Krab domain transcription factor which expression is strongly altered in DM1 and which seems to be involved in muscular regeneration defects associated with DM1. In conclusion, the aim of this work was to extend the spectrum of hES cells use for therapeutic purposes by accurately defining their capacity to model a genetic disease, enabling the elaboration of biotherapies targeted to disease specific molecular mechanisms.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Etude des mécanismes moléculaires de la Dystrophie Myotonique de Type 1 à l'aide de cellules souches embryonnaires humaines porteuses de la mutation causale

Les cellules souches embryonnaires humaines (hESC) représentent un nouvel outilbiologique au potentiel prometteur pour l amélioration de la compréhension des mécanismes moléculaires et cellulaires impliqués dans le développement de maladies monogéniques. Cette application est dans un premier temps devenue possible grâce à l utilisation de lignées de cellules souches embryonnaires humaines porteuses de mutation causale de pathologie, obtenues au cours d un diagnostique pré-implantatoire. Mon travail de thèse s est inscrit dans la validation de ce nouveau concept en utilisant des lignées de cellules souches embryonnaires humaines porteuses de la mutation causale de la Dystrophie Myotonique de type 1 (DM1). Ces cellules, ainsi que leurs progenies neurales et mésenchymateuses représentent un modèle pertinent pour l étude des conséquences physiopathologiques de la mutation DM1 dans la mesure où elles reproduisent certaines caractéristiques moléculaires connues de la pathologie. Mon projet a eu pour objectif de caractériser d un point de vue moléculaire et physiopathologique deux nouvelles altérations géniques identifiées par transcriptome différentiel entre les cellules contrôles et DM1. Ainsi, ce travail nous a permis d identifier un nouveau marqueur, le facteur de transcription ZNF37A, dont l expression est diminuée en association avec la mutation DM1 et qui serait impliqué dans les défauts myogéniques caractérisant cette pathologie. Parallèlement nous avons identifié un nouveau défaut d épissage alternatif d un gène impliqué dans la guidance axonale, l EphA5 qui pourrait être impliqué dans les défauts cognitifs des patients DM1.Abstract not availableEVRY-Bib. électronique (912289901) / SudocSudocFranceF

A defective Krab-domain zinc-finger transcription factor contributes to altered myogenesis in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder caused by a non-coding CTG repeat expansion that, in particular, provokes functional alteration of CUG-binding proteins. As a consequence, several genes with misregulated alternative splicing have been linked to clinical symptoms. In our search for additional molecular mechanisms that would trigger functional defects in DM1, we took advantage of mutant gene-carrying human embryonic stem cell lines to identify differentially expressed genes. Among the different genes found to be misregulated by DM1 mutation, one strongly downregulated gene encodes a transcription factor, ZNF37A. In this paper, we show that this defect in expression, which derives from a loss of RNA stability, is controlled by the RNA-binding protein, CUGBP1, and is associated with impaired myogenesis—a functional defect reminiscent of that observed in DM1. Loss of the ZNF37A protein results in changes in the expression of the subunit α1 of the receptor for the interleukin 13. This suggests that the pathological molecular mechanisms linking ZNF37A and myogenesis may involve the signaling pathway that is known to promote myoblast recruitment during development and regeneratio

Etude des mécanismes impliqués dans l établissement de l'infection persistante du virus de Theiler dans le système nerveux central de la souris

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Unlocking the Complexity of Neuromuscular Diseases: Insights from Human Pluripotent Stem Cell-Derived Neuromuscular Junctions

Over the past 20 years, the use of pluripotent stem cells to mimic the complexities of the human neuromuscular junction has received much attention. Deciphering the key mechanisms underlying the establishment and maturation of this complex synapse has been driven by the dual goals of addressing developmental questions and gaining insight into neuromuscular disorders. This review aims to summarise the evolution and sophistication of in vitro neuromuscular junction models developed from the first differentiation of human embryonic stem cells into motor neurons to recent neuromuscular organoids. We also discuss the potential offered by these models to decipher different neuromuscular diseases characterised by defects in the presynaptic compartment, the neuromuscular junction, and the postsynaptic compartment. Finally, we discuss the emerging field that considers the use of these techniques in drug screening assay and the challenges they will face in the future

L’heure est venue d’un (re)positionnement thérapeutique des maladies ultra-rares

International audienceNo abstract availabl

Theiler's Virus Infection of Primary Cultures of Bone Marrow-Derived Monocytes/Macrophages

Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR(−/−) mice were fully permissive

Pluripotent Stem Cells in Disease Modeling and Drug Discovery for Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is a progressive multisystemic disease caused by the expansion of a CTG repeat tract within the 3′ untranslated region (3′ UTR) of the dystrophia myotonica protein kinase gene (DMPK). Although DM1 is considered to be the most frequent myopathy of genetic origin in adults, DM1 patients exhibit a vast diversity of symptoms, affecting many different organs. Up until now, different in vitro models from patients’ derived cells have largely contributed to the current understanding of DM1. Most of those studies have focused on muscle physiopathology. However, regarding the multisystemic aspect of DM1, there is still a crucial need for relevant cellular models to cover the whole complexity of the disease and open up options for new therapeutic approaches. This review discusses how human pluripotent stem cell–based models significantly contributed to DM1 mechanism decoding, and how they provided new therapeutic strategies that led to actual phase III clinical trials

Pluripotent Stem Cells in Disease Modeling and Drug Discovery for Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is a progressive multisystemic disease caused by the expansion of a CTG repeat tract within the 3′ untranslated region (3′ UTR) of the dystrophia myotonica protein kinase gene (DMPK). Although DM1 is considered to be the most frequent myopathy of genetic origin in adults, DM1 patients exhibit a vast diversity of symptoms, affecting many different organs. Up until now, different in vitro models from patients’ derived cells have largely contributed to the current understanding of DM1. Most of those studies have focused on muscle physiopathology. However, regarding the multisystemic aspect of DM1, there is still a crucial need for relevant cellular models to cover the whole complexity of the disease and open up options for new therapeutic approaches. This review discusses how human pluripotent stem cell–based models significantly contributed to DM1 mechanism decoding, and how they provided new therapeutic strategies that led to actual phase III clinical trials