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.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

Les lamines A et C sont deux composants majeurs de la lamina nucléaire, réseau de filaments intermédiaires situé sous la membrane nucléaire interne. Les mutations du gène LMNA, codant les lamines A/C, ont été associées à de nombreuses pathologies humaines, appelées laminopathies, et affectant un ou plusieurs tissus dont le muscle. Les mécanismes physiopathologiques sous-jacents ne sont encore que partiellement élucidés. Les lamines A/C jouant un rôle crucial dans l’architecture nucléaire et l’organisation de la chromatine, l’hypothèse d’une altération de l’expression de facteurs de transcription ou de gènes tissus-spécifiques a été formulée. De plus, au niveau musculaire, il a été décrit que les lamines A/C jouent un rôle majeur dans la mise en place d’une différenciation musculaire efficace.Afin d’identifier des altérations potentielles au sein des voies de signalisation régulant la différenciation musculaire, nous avons utilisés un modèle de myoblastes murins conditionnellement immortalisés et comparés le profil d’expression entre les myoblastes sauvages et inactivés pour le gène Lmna (Lmna-/-). Nous avons donc identifiés deux altérations majeures de la voie BMP (Bone Morphogenetic Pathway) : la diminution de l’expression du ligand Bmp4 et l’augmentation de celle de Smad6, un inhibiteur intracellulaire de la voie. Cette surexpression de Smad6 est responsable d’une séquestration cytoplasmique des Smads 1, 5 et 8 phosphorylées et d’une diminution de l’expression des gènes cibles, Id1 et Id2. Les myoblastes Lmna-/- montrent une différenciation myogénique prématurée, phénotype réversible par des expériences d’ARN interférent ciblant Smad6. Enfin, nous avons montré que ces défauts sont retrouvés dans des myoblastes humains porteurs hétérozygotes de la mutation LMNA R310X.Ces résultats apportent un nouveau mécanisme physiopathologique des laminopathies musculaires et identifient une nouvelle cible thérapeutique potentielle / LMNA gene encodes lamins A and C, two major components of the nuclear lamina, a network of intermediate filaments underlying the inner nuclear membrane. LMNA mutations have been associated with a wide spectrum of human diseases collectively called “laminopathies” affecting one or several tissues, such as muscles. The physiopathological mechanisms underlying laminopathies remain unclear. Given the crucial role of lamins A/C in nuclear architecture and chromatin organization, the “gene regulation” hypothesis have been proposed. It suggests that LMNA mutations could alter in a tissue-specific manner transcription factors and/or genes expression. Moreover, lamins A/C have been described as important regulators in muscle differentiation regulation.To identify potential alterations in signaling pathways regulating muscle differentiation in LMNA-mutated myoblasts, we used a previously described model of conditionally immortalized murine myoblasts and compared gene expression profiles in wild-type and Lmna-/- H-2K myoblasts. We identified two major alterations of the Bone Morphogenetic Protein (BMP) pathway in Lmna-/- myoblasts: Bmp4 downregulation and Smad6 overexpression. We demonstrated that Smad6 overexpression lead to Smad1/5/8 sequestration in the cytoplasm and to the downregulation of their target genes, Id1 and Id2. As a consequence, Lmna-null myoblasts displayed a premature differentiation which could be rescued by downregulating Smad6 expression. Finally, we showed that these defects are relevant for human laminopathies as they are also present in myoblasts from a human patient carrying a LMNA+/Q310X mutation.Taken together, these results provide a potential mechanism for the muscle stem cell exhaustion and muscle atrophy observed in muscle laminopathies and identify a new therapeutical target likely to reverse pathological phenotypes

Now that the functioning of microtubules and the actin cytoskeleton has been worked out in enormous detail, the next important task is defining the structure of intermediate filaments that are far behind the other two major skeletal networks due to their inherent resistance to most structural techniques. The evolution of novel structural approaches for flexible proteins is making this possible now. In my thesis I will aim to elucidate the structure and assembly principles of lamin A nuclear intermediate filament protein. To study lamin A, I principally employed chemical cross-linking that allows the capturing of full-length protein structures in solution. I combined this with mass spectrometry approaches to identify cross-linked residues at the various stages of lamin A assembly that were additionally tracked with SILAC labelling and rotary metal shadowing TEM. Unlike previous cross-linking studies on intermediate filaments I use a zero-length self-excluding cross-linking agent EDC that is better tailored for investigation of the polar interactions between multiple unstructured or otherwise flexible charged sequences of lamins. Using this composite approach I interrogated lamin A dimeric and tetrameric assemblies. I elucidated hinge-like properties of the L12 and found indications that L1 and the region containing coil 2A and L2 and the beginning of coil 2B possess properties of linker-like flexibility and of predicted linear α-helical bundle and could act as molecular springs or compression buffers for the nuclear intermediate filaments. Further I confirm the role of the N-terminal unstructured region in lamin A assembly and for the first time show similar role for the C-terminal unstructured region flanking the rod domain of lamin A. Collected data strongly supports the model where both positively charged unstructured regions participate in extensive interaction with acidic rod termini and act as molecular bridges between these in the head-to-tail interface, confirming the uniformity of this principle between cytoplasmic and nuclear intermediate filaments. Formation of these bridges requires conformational change likely happening due to proline residues in the mitotic phosphorylation sites. Finally I suggest a mechanism of regulation of the order of assembly unique to the nuclear intermediate filament where C-terminal unstructured region blocks lateral interactions until it is tethered to the head-to-tail interface. Collected data on the dynamic behaviour of the C-terminal unstructured region and its ability to tether lamin A Ig domain may have far reaching implications for filament assembly and regulation of binding of hundreds of lamin A partner proteins presenting an important step in our understanding of relationship between lamin A structure and function and how altering the former could lead to disease.

Dilated Cardiomyopathy (DCM) with conduction disease and Atrial Fibrillation (AF) are the two cardiac-specific diseases associated with lamin A/C gene (LMNA) mutations. Protein Kinase C Alpha, (PKCα) functions as a nodal integrator of cardiac contractility by “sensing” intracellular calcium and signal transduction. PKCα has been implicated in heart failure and cardiac hypertrophy. Moreover, abnormal PKCα function results in irregular atrial potassium channel activity associated with chronic AF PKCα is a lamin A/C binding partner. Thus, the deregulation of PKCα signaling can contribute to the development of DCM and AF. Our hypothesis is that the AF (Thr528Met), DCM-associated (Arg541Cys) and (Arg541Gly) and DCM/AF-associated (Tyr481Stop) LMNA variants will disrupt the cellular distribution of PKCα therefore resulting in impaired PKCα function. The first objective was to phenotypically characterise Arg541Cys LMNA variant in murine skeletal myoblasts cell line (C2C12) in comparison to cellular phenotypes induced by LMNA variants associated with AF, DCM and DCM with AF. Arg541Cys lamin A and C variants formed circular and sickle-shaped lamin A/C in the nucleus of C2C12 cells. The second objective was to determine the effect of these lamin variants on cellular distribution of PKCα in C2C12 cells. PKCα mislocalized into the nucleus of C2C12 cells transfected with AF and DCM-associated variants (Thr528Met and Arg541Cys). Colocalization analysis showed significant increase in PKCα in the nucleus of AF (Thr528Met) and DCM (Arg541Cys) variants when lamin A and C, were co-transfected compared to wild-type, DCM (Arg541Gly) and DCM/AF (Tyr481Stop) variants. Densitometry analysis showed statistically significant increase in phosphorylated PKCα, the active form of PKCα, in nuclear and cytoplasmic extracts of C2C12 cells expressing Arg541Cys variant. Densitometry analysis also showed statistically significant increase in non-phosphorylated PKCα in the nuclear extract of Thr528Met variant expressing cells. The third objective was to determine the effect of AF and DCM-associated variants on the activity of PKCα. PKCα activity is quantified by measuring the phosphorylation of a known phosphorylated PKCα substrate. Alpha-6-tubulin phospho (Ser165) is phosphorylated by PKCα. Hence, this was used to quantify PKCα activity. No statistical significance was observed in the level of phosphorylated alpha-6-tubulin at (Ser165) in the C2C12 cells that were transfected with lamin A and C variants compared to wild type. Furthermore, PKCα phosphorylation state is cyclic in nature and this could have had an impact on the phosphorylation state of the chosen substrate in this study. The functional consequence of nuclear translocation of PKCα with respect to laminopathies is unknown. Abnormal activation of the Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2) which are branches of the mitogen-activated protein kinase (MAPK) signalling cascade in hearts of mice, and humans prior to the onset of cardiomyopathy. These findings have been associated to cardiac disease-causing lamin A/C alteration to signal transduction pathways implicated in heart function and cardiomyopathy. Human LMNA cardiomyopathy, could lead to abnormal activation of MAPK signalling pathways via abnormal PKCα activation in cardiomyocytes.

No description available.

La mutation LMNA H222P est responsable de dystrophie musculaire d’Emery Dreifuss autosomale dominante (DMED-AD). Les patients atteints de DMED-AD souffrent d’une dystrophie musculaire et de cardiomyopathie dilatée. Les mécanismes moléculaires impliqués dans cette pathologie sont encore peu connus. Dans mes travaux de thèse, je me suis servie de cellules souches pluripotentes murines ainsi que de souris portant la mutation LMNA H222P afin d’étudier une approche thérapeutique potentielle. L'échocardiographie des souris LMNA H222P in utero révèle une dilatation des cœurs embryonnaires dès E13.5, ce qui indique une origine développementale de la maladie. La différenciation cardiaque des cellules souches pluripotentes murines est altérée dès le stade mésoderme. Aussi, les niveaux d’expression de Mesp1, snail1 et twist, gènes impliqués dans la transition épithélio-mésenchymateuse (TEM) sont diminués dans les cellules mutées en comparaison avec les cellules sauvages en cours de différenciation. L'immunoprécipitation de la chromatine dans les cellules différenciées révèle une diminution spécifique de la marque d'histone H3K4me1 sur des régions régulatrices de Mesp1 et Twist. L'inhibition de LSD1, une déméthylase spécifique de H3K4me1 rétablit le taux de la marque H3K4me1 sur les régions génomiques étudiées dans les cellules mutées. De plus, la baisse de LSD1 améliore la contraction des cardiomyocytes différenciés obtenus à partir des cellules souches embryonnaires portant la mutation LMNA H222P. L'inhibiteur de LSD1, utilisé dans les essais cliniques en cancérologie, pourrait être une molécule thérapeutique potentielle pour le traitement des laminopathies à phénotype cardiaque. / The LMNA H222P missense mutation in autosomal dominant Emery-Dreifuss muscular dystrophy patients is responsible for a muscular dystrophy and dilated cardiomyopathy. The molecular mechanisms underlying the origin and development of the pathology are still unknown. Herein, we used mouse pluripotent stem cells as well as a mutant mouse, all harboring the LMNA H222P mutation, to investigate potential therapeutic approaches. Echocardiography of LMNA H222P mice in utero revealed dilatation of heart as early as E13.5, pointing to a developmental origin of the disease. Cardiac differentiation of mouse pluripotent stem cells was impaired as early as the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene as well as snail1 and twist, involved in epithelial-mesenchymal transition (EMT) of epiblast cells, was decreased in mutated cells when compared to wild type in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. Chromatin immunoprecipitation assays of the H3K4me1 epigenetic mark in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of MesP1 and Twist. Downregulation or inhibition of LSD1, that specifically demethylates H3K4me1, rescued the epigenetic landscape in mutated cells. In turn downregulation of LSD1 rescued contraction in cardiomyocytes differentiated from LMNA H222P pluripotent stem cells. Our data point to LSD1 inhibitor, used in clinical trials in cancerology, as potential therapeutic molecule for laminopathies with a cardiac phenotype.