Three mammalian nuclear lamin proteins, lamin B1, lamin B2 and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility that farnesyl transferase inhibitors can represent effective treatment for HGPS. However, there are concerns about their use for this purpose given the potential for alternative prenylation pathways.

Radial chromosome positioning in interphase nuclei is non-random and can alter according to developmental, differentiation, proliferation or disease status. The aim of this thesis is to understand how chromosome re-positioning is elicited and to identify the nuclear structures that assist this re-localisation event. By positioning all human chromosomes in primary fibroblasts that have left the proliferative cell cycle, the study within this thesis has demonstrated that in cells made quiescent by reversible growth arrest, chromosome positioning is altered considerably. Upon removal of serum from the culture medium, chromosome re-positioning took less than 15 minutes, required energy and was inhibited by drugs affecting the polymerization of myosin and actin. The nuclear distribution of nuclear myosin 1β was dramatically different in quiescent cells as compared to proliferating cells. If the expression of nuclear myosin 1β was suppressed using interference RNA procedures the movement of chromosomes after 15 minutes in low serum was inhibited. When high serum was restored to the serum starved cultures chromosome repositioning was only evident after 24-36 hours that coincided with a return to a proliferating distribution of nuclear myosin 1β.

Multiple sclerosis (MS), a demyelinating disorder of the central nervous system (CNS), affects approximately 400,000 individuals in the United States, and 2.5 million people worldwide. It is a leading cause of disability in young adults. Current treatments for MS target the inflammatory aspects of the disease, but do not aid in remyelination. To address remyelination as a therapeutic strategy, it is imperative to identify mechanisms that regulate myelin formation, including epigenetic targets. In this study, we investigate the role of the LMNA, a gene encoding Lamins A and C, intermediate filaments of the nuclear lamina, in regulating oligodendrocyte development and myelination in the CNS. Using electron microscopic analyses, I examined levels of heterochromatin and its distribution in the oligodendrocyte nucleus as an indicator of gene expression, oligodendrocyte maturity, and myelin formation in the absence of A type lamins.. While overall levels of heterochromatin in oligodendrocytes were not altered in the absence of A type lamins, peripherally located heterochromatin was reduced and thinner myelin was observed in the spinal cord. My observations present novel findings for the role of LMNA in oligodendrocytes and myelination.

Many tissue-restricted diseases are linked to mutations in lamins and nuclear envelope transmembrane proteins (NETs). How these mutations in ubiquitously expressed proteins cause such defined diseases is still unknown. It is hypothesized that tissue restricted NETs that are partners of the nuclear lamins/existing linked proteins mediate tissue-specific disease pathologies. Proteomic studies have identified many tissue restricted NETs with effects on the cytoskeleton, gene positioning and regulation. This study investigates potential roles of candidate NETs in mediating tissue restricted disease pathology and their interactions with known factors such as emerin and lamins, mutations in which have been linked to a variety of tissue-specific dystrophies. This study looks into candidate tissue-specific NETs distribution in human tissues and in vitro using a solid phase binding assay to study candidate NETs interactions. I confirmed the tissue-specificity of the candidate NETs in human and mouse tissue sections but did not find clear reproducible distribution of these NETs in patient tissue biopsy. One postulate is that NETs bind WT lamin for localisation and/or function and disruption of this interaction leads to disease. Using a solid phase binding assay approach to study NETs/lamin interactions, we demonstrate that Tmem120a, an adipocyte-specific NET binds WT lamin but has a reduced Bmax when tested for binding against a lipodstrophy causing lamin mutant (R482Q and G465D). This is consistent with the hypothesis that tissue-specific NET partners might mediate tissue-specific disease pathology in lamin-linked nuclearenvelopathies.

The eukaryotic interphase nucleus is well organised and the genome positioned non-randomly. Nuclear structure is an important regulator of genome behaviour and function. Genome organisation and nuclear structure are compromised in diseases such as cancer and laminopathies. This study was to find out and to determine if there is any functional relationship between nuclear structure and genome mis-organisation in cancer cells. I have assessed the presence and distribution of specific nuclear structural proteins (A-type, B-type lamins and its receptor LBR, many of their binding proteins such as MAN1, LAP2α, LAP2, and Emerin and other nuclear proteins (PML, Nucleolin, and Ki67) using indirect immunofluorescence. From this study, it is found that the nuclear structure of breast cancer cells is often altered. The most severely affected proteins are the nuclear lamins B1 and B2 and they found as large foci within the nucleoplasm with little LBR expression to localise the lamin B. I also assessed the chromosome positioning (HSA 7, 10, 11, 14 and 17) and gene positioning (AKT1, CCND1, HSP90AA1, EGFR, ERRBB2/HER2 and PTEN) in breast cancer cell lines (T-47D, GI-101, Sk-Br-3 and BT-474) and in normal breast cell lines (MCF-10A) using 2D-FISH technique. I also assessed the position of the genes in nuclei and correlated with gene expression using qRT-PCR. Breast cell lines have treated with a drug named lovastatin and it was found that the cells have restored LBR expression and localisation of lamin B, leading to altered gene positioning and changed expression of breast cancer genes. Since the drug (lovastatin, 12 μM/48 hours) affects the prenylation as a post-translation modification process and lamins B biosythensis, it is found that B-type lamins and its receptor expression and distribution were improved and increased in expression by 2-fold in expression levels in the most affected cells (T-47D, and BT-474) compared to the normal cells (MCF-10A) and these cells also showed abnormal nuclei and dead cells. When analysing the nuclear positioning of the genes (AKT1, HSP90AA1 and ERRBB2/HER2), it is found that AKT1 was positioned periphery in BT-474 and T-47D cells and interiorly in the normal cells (MCF-10A) before treatment whereas the same gene was positioned periphery in T-47D and MCF-10A cells and interiorly in BT-474 after treatment with lovastatin. It is also found that HSP90AA1 was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 cells before and after treatment (no change). Moreover, ERRBB2/HER2 gene was positioned periphery in T-47D and BT-474 cells and interiorly in MCF-10A cells before treatment whereas the same gene was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 after treatment with the same drug. Regarding LMNB1, LMNB2, and LBR genes, the study focussed only on their expression levels and no work has done on their chromosome positioning as well as gene position before and after treatment. These three genes were over expressed when assessed by measuring the relative and fold changes in expression. Therefore, it is suggestive that 2D-FISH experiment to assess their localisation and their specific chromosome territories is required. The results shown in this thesis demonstrate the importance and roles of nuclear architecture specifically nuclear lamins and the integral nuclear membrane proteins (B-type lamins and LBR) in mediating correct genome organisation and function. The breast normal (immortalised cells) and cancerous cell lines showed different nuclear structures as lamin B affect the position of specific target chromosomes and genes. These results will strength the finding that the nuclear lamina is a significant nuclear structure which associates, organises, and regulates numerous vital nuclear processes and the stability of the genome.

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

Les patients lipodystrophiques porteurs de mutations du gène LMNA codant pour les lamines de type A, et les patients infectés par le VIH (Virus de l’Immunodéficience Humaine) traités par des antirétroviraux (ARV) sont à risque de développer une athérosclérose précoce avec calcifications et des comorbidités liées au vieillissement. Pour mieux comprendre la physiopathologie des atteintes vasculaires, j’ai étudié l’impact d’ARV ou de mutations LMNA sur des cellules musculaires lisses (CML) ou endothéliales d’artères coronaires humaines in vitro. Ce travail a révélé que certains ARV (les inhibiteurs de protéase lopinavir ou atazanavir associés au ritonavir) induisent une sénescence prématurée et des dysfonctions des cellules endothéliales, alors que d’autres ont peu ou pas de conséquences (maraviroc, dolutégravir, maraviroc/dolutégravir et darunavir/ritonavir). De plus, le lopinavir/ritonavir ou l’atazanavir/ritonavir, ou l’expression des mutations LMNA p.R482W, p.D47Y ou p.R133L induisent une sénescence prématurée, une transdifférenciation ostéogénique avec calcification, et un stress oxydant dans les CML. L’accumulation de prélamine A farnésylée (une lamine A immature) et la diminution de l’expression de ZMPSTE24, son enzyme de maturation, sont, au moins en partie, responsables de ces effets.Ainsi, les ARV étudiés ont des impacts différents et agissent par des mécanismes physiopathologiques pro-athérogéniques en partie communs avec certaines mutations LMNA associées aux lipodystrophies. Le rôle majeur de l’accumulation de prélamine A farnésylée liée à une diminution d’expression de ZMPSTE24 ouvrent de nouvelles perspectives thérapeutiques. / Patients with lipodystrophies dues to mutation in the LMNA gene encoding A-type lamins, or HIV-infected patients (Human Immunodeficiency Virus) receiving antiretroviral therapy (ARV) are prone to develop early atherosclerosis and vascular calcifications associated with comorbidities linked to premature aging. Our study focused on the impact of LMNA mutations or antiretroviral treatment in vitro on vascular smooth muscle cells (VSMC) or endothelial cells from human coronary arteries. The results obtained during my thesis showed that some ARV (the protease inhibitors lopinavir or atazanavir associated with ritonavir) induce a cellular premature senescence with associated dysfunctions in endothelial cells, whereas others have little or no consequences (maraviroc, dolutegravir, maraviroc/dolutegravir and darunavir/ritonavir). In addition, some ARV (lopinavir or atazanavir with ritonavir) or the expression of LMNA mutations p.R482W, p.D47Y or p.R133L induce premature senescence, osteogenic transdifferentiation with calcification and oxidative stress of VSMC. Our results reveal that the accumulation of farnesylated prelamin A (an immature lamin A) and the decreased expression of its processing enzyme ZMPSTE24 are, at least partly, responsible for these effects.This work shows the different effects of ARVs and highlights the existence of common pro-atherogenic pathophysiological mechanisms in HIV-infected patients receiving some protease inhibitors and in lipodystrophic patients with LMNA mutations, initiated by an accumulation of farnesylated prelamin A related to a decrease expression of ZMPSTE24. These abnormalities could give rise to new therapeutic perspectives.

Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LAΔ50/progerin). These modifications cause an abnormal association of LAΔ507 progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G 1 is impaired in cells expressing LAΔ50/ progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.

<div>As aging proceeds, the occurrence of cardiovascular diseases increases independent of other risk factors. At atherosclerotic sites, the rise in the senescent cell population was also observed. Patients with Hutchinson Gilford Progeria Syndrome (HGPS) also showed accelerated aging syndromes and extensive atherosclerosis progression, which was due to missense mutations on the LMNA gene that led to the production of progerin, an aberrant lamin A isoform instead of regular lamin A protein. Lamins act as structural and functional components in nuclear lamina, and recent findings suggested that the ectopic expression of mutant lamin A or lamin A precursor (prelamin A) not only caused defects in cell mechanics but also disturbed mechanotransduction pathways involving lamin A, both of which may contribute to vascular dysregulation. Moreover, the observation of the accumulation of prelamin A in normal aged vascular cells further suggests shared dysregulations involving lamin A in the vascular system between aged people and HGPS patients.</div><div>In the vascular system, endothelial cells were well regulated by hemodynamic forces in vivo to maintain vascular homeostasis. Endothelial dysfunction, including impaired vasodilation and increased permeability, was regarded as the initial marker of atherosclerosis. Despite recent advancements and discussions about the potential mechanisms of progerin-induced vascular disorders, how progerin triggers endothelial dysfunction in a mechanical environment as an early event during atherosclerotic lesion formation has not been studied intensively.</div><div>To help answer the gap question, we first set our goal to understand the effect of laminar flow at arterial levels on endothelial lamins as part of the aging process. Spatial and temporal changes in lamin A/C expression were observed as cell passage went up without flow present. As shear stress was applied, lamin A/C expressions were modulated on both transcriptional and translational levels, which were also dependent on PDL. To further examine how progerin was involved in EC functions with a particular focus on the flow effects, we next generated a stable endothelial cell line that expressed progerin as our EC aging model. Endothelial wound repair under laminar flow at different rates was characterized, and differential cell proliferation activities, as well as migration deficiencies in progerin-expressing ECs during the process, were also recognized. Furthermore, we also showed the overactivated mTORC2 pathway and unusual actin polymerization activities in these cells after flow application. Our results reported changes in cell migration by progerin with flow application for the first time and provided potential candidate pathways that were disturbed by progerin under arterial flow, which may help explain the high occurrence of atherosclerotic lesions in HGPS vasculature, even at straight portion. The reported progerin-induced wound recovery defects in endothelial cells in the presence of physiological flow may also suggest a mechanism of how progerin disturbs endothelial integrity and functions under mechanical stimuli in the development of vascular pathologies.</div><div>Further extended studies may help to understand the roles of progerin in initiating atherosclerosis, which will aid in the development of potential therapies for those suffering from prelamin A-associated accelerated aging syndromes.</div>

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.