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.

Cells regularly take on various types of force in the body. They have structures that are able to mediate, transfer and respond to the forces. A mutation in force regulating proteins such as lamin in the nucleus or the KASH domain which connects the nucleus to the cytoskeleton of the cell can cause catastrophic events to occur. The aims of this study were to better understand the response of the nucleus when structural proteins are mutated or are not present while under force. Progeria, a rare disease where an additional farnesyl group is attached to lamin was used in this study. Different types of forces were used to represent similar conditions in the body. Confinement of endothelial cell width showed an increase of surface defects. When restricting proteins such as actin was removed the nucleus appeared to rupture. This was shown to occur at a higher rate in the progeria groups. Endothelial cells under shear force showed high amount of nuclear rupture in progeria expressing group. prolonged exposure showed more rupture which eventually cased cell death and cells to come off the surface. Progeria expressing smooth muscle cells under cyclic stretch also showed similar results as endothelial cells. The amount and rate of deformation of the nucleus when the cytoskeleton is connected and not was looked at. When the connected the rate of deformation was higher. The high rate of nuclear defects and rupture while under force in progeria expressing cells shows that the nuclei have different structural properties and are weaker. It’s also been shown that the LINC complex contributes to nuclear deformation when stretching.

The genome of mammalian cells is under constant attack from DNA-damaging agents. To maintain genomic integrity, cells activate an array of pathways primarily consisting of DNA repair and DNA damage checkpoints. Human replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is essential for almost all DNA metabolic pathways. However, the role of RPA in nucleotide excision repair (NER), a DNA repair pathway for removing bulky DNA lesions, remains elusive. In this study, the binding of RPA to a battery of well-defined ssDNA substrates has been systematically examined using fluorescence spectroscopy. The results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA, and there was no direct contact between RPA residues and the lesion itself. These findings will help define the roles of RPA in DNA damage recognition in NER. In cells, RPA undergoes hyperphosphorylation in the N-terminus of RPA32 subunit after DNA damage. In this study, the hyperphosphorylation-induced conformational changes of RPA have been probed using mass spectrometry-based protein foot-printing, fluorescence spectroscopy and limited proteolysis. The data show that upon hyperphosphorylation RPA undergoes a subtle structural change involving its DNA-binding domain B (DBD-B), reducing its affinity for short ssDNA. These results suggest that hyperphosphorylation may modulate RPA functions by altering DBD-B-mediated RPA-DNA/protein interactions. Cellular accumulation of DNA damage has been widely implicated in premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is caused by defective maturation of lamin A and linked to accumulation of DNA double-strand breaks (DSBs). However, how lamin A dysfunction leads to genome instability and premature aging is not understood. Here evidence showed that in HGPS and RD fibroblasts DNA damage checkpoints are persistently activated and recruitment of repair factors to DSBs was impaired. Strikingly, xeroderma pigmentosum group A (XPA), a unique NER protein, formed foci and colocalized with the unrepairable DSBs in the patient cells. RNAi knockdown of XPA in HGPS cells significantly restored DSB repair. These results indicate that XPA dysfunction may play an important role in accumulating DSBs in HGPS, implicating a potential strategy for treatment of these premature aging diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Cellular structural mechanics play a key role in homeostasis by transducing mechanical signals to regulate gene expression and by providing adaptive structural stability for the cell. The alteration of nuclear mechanics in various laminopathies and in natural aging can damage these key functions. Arterial endothelial cells appear to be especially vulnerable due to the importance of shear force mechanotransduction to structure and gene regulation as is made evident by the prominent role of atherosclerosis in Hutchinson-Gilford progeria syndrome (HGPS) and in natural aging. Computational models of cellular mechanics may provide a useful tool for exploring the structural hypothesis of laminopathy at the intracellular level. This thesis explores this topic by introducing the biological background of cellular mechanics and lamin proteins in arterial endothelial cells, investigating disease states related to aberrant lamin proteins, and exploring computational models of the cell structure. It then presents a finite element model designed specifically for investigation of nuclear shear forces in arterial endothelial cells. Model results demonstrate that changes in nuclear material properties consistent with those observed in progerin-expressing cells may result in substantial increases in stress concentrations on the nuclear membrane. This supports the hypothesis that progerin disrupts homeostatic regulation of gene expression in response to hemodynamic shear by altering the mechanical properties of the nucleus.

<p>Cellular structural mechanics play a key role in homeostasis by transducing mechanical signals to regulate gene expression and by providing adaptive structural stability for the cell. The alteration of nuclear mechanics in various laminopathies and in natural aging can damage these key functions. Arterial endothelial cells appear to be especially vulnerable due to the importance of shear force mechanotransduction to structure and gene regulation as is made evident by the prominent role of atherosclerosis in Hutchinson-Gilford progeria syndrome (HGPS) and in natural aging. Computational models of cellular mechanics may provide a useful tool for exploring the structural hypothesis of laminopathy at the intracellular level. This thesis explores this topic by introducing the biological background of cellular mechanics and lamin proteins in arterial endothelial cells, investigating disease states related to aberrant lamin proteins, and exploring computational models of the cell structure. It then presents a finite element model designed specifically for investigation of nuclear shear forces in arterial endothelial cells. Model results demonstrate that changes in nuclear material properties consistent with those observed in progerin-expressing cells may result in substantial increases in stress concentrations on the nuclear membrane. This supports the hypothesis that progerin disrupts homeostatic regulation of gene expression in response to hemodynamic shear by altering the mechanical properties of the nucleus.</p>

Hallermann-Streiff syndrome (HSS) is a rare inherited disorder characterized by malformations of the cranium and facial bones, congenital cataracts, microphthalmia, skin atrophy, hypotrichosis, proportionate short stature, teeth abnormalities, and a typical facial appearance with prominent forehead, small pointed nose, and micrognathia. The genetic cause of this developmental disorder is presently unknown. Here we describe 8 new patients with a phenotype of HSS. Individuals with HSS present with clinical features overlapping with some progeroid syndromes that belong to the laminopathies, such as Hutchinson-Gilford progeria syndrome (HGPS) and mandibuloacral dysplasia (MAD). HGPS is caused by de novo point mutations in the LMNA gene, coding for the nuclear lamina proteins lamin A and C. MAD with type A and B lipodystrophy are recessive disorders resulting from mutations in LMNA and ZMPSTE24 , respectively. ZMPSTE24 in addition to ICMT encode proteins involved in posttranslational processing of lamin A. We hypothesized that HSS is an allelic disorder to HGPS and MAD. As the nuclear shape is often irregular in patients with LMNA mutations, we first analyzed the nuclear morphology in skin fibroblasts of patients with HSS, but could not identify any abnormality. Sequencing of the genes LMNA, ZMPSTE24 and ICMT in the 8 patients with HSS revealed the heterozygous missense mutation c.1930C>T (p.R644C) in LMNA in 1 female. Extreme phenotypic diversity and low penetrance have been associated with the p.R644C mutation. In ZMPSTE24 and ICMT , no pathogenic sequence change was detected in patients with HSS. Together, we found no evidence that HSS is another laminopathy.