The discovery of mammalian protein prenylation was originally motivated by an effort to identify a nonsterol isoprenoid which indirect evidence suggested was a coregulator of isoprenoid biosynthesis and played a critical role in cellular proliferation. The first prenylated proteins to be identified were the nuclear lamin proteins-B lamins and prelamin A-which were subsequently shown to be farnesylated at a carboxyl-terminal CAAX motif. In both types of lamin, the farnesylation and carboxymethylation play a role in targeting these proteins to the nuclear envelope. The nucleus can be demonstrated to be a CAAX processing compartment for the lamins. In the case of prelamin A, there is removal of a carboxyl-terminal polypeptide which is specifically catalyzed by the enzyme Zmpste24. This processing event is necessary for assembly of lamin A into the lamina and may play a role in cell cycle control. Because the nucleus contains only one target membrane, lamin farnesylation and carboxymethylation may be sufficient to allow association with this membrane. This stands in contrast to farnesylated proteins expressed in the cytoplasm.

Endothelial cells form the lining of the walls of blood vessels and are continuously subjected to mechanical stimuli from the blood flow. Microtubule-organizing center (MTOC),<br>also known as centrosome is a structure found in eukaryotic cells close to the nucleus. MTOC relocates relative to the nucleus when endothelial cells are exposed to shear stress which determines their polarization, thus it plays a critical role in cell migration and wound healing. The nuclear lamina, a mesh-like network that lies underneath the nuclear membrane, is composed of lamins, type V intermediate filament proteins. Mutations in LMNA gene that encodes A-type lamins cause the production of a mutant form of lamin A called progerin and leads to a rare premature aging disease known as Hutchinson-Gilford Progeria Syndrome<br><div>(HGPS). The goal of this study is to investigate how fluid flow affects the cytoskeleton of endothelial cells.</div><div><br></div>This thesis consists of two main sections; computational mechanical modeling and laboratory experimental work. The mechanical model was implemented using Ansys Workbench software as a tensegrity-based cellular model in order to simulate the state of an endothelial cell under the effects of induced shear stress from the blood fluid flow. This tensegrity-based cellular model - composed of a plasma membrane, cytoplasm, nucleus, microtubules, and<br><div>actin filaments - aims to understand the effects of the fluid flow on the mechanics of the cytoskeleton. In addition, the laboratory experiments conducted in this study examined the MTOC-nuclear orientation of endothelial cells under shear stress with the presence of wound healing. Wild-type lamin A and progerin-expressing BAECs were studied under static and sheared conditions.</div><div><br></div><div> Moreover, a custom MATLAB code was utilized to measure the MTOC-nuclear orientation</div>angle and classification. Results demonstrate that shear stress leads to different responses of the MTOC orientation between the wild-type and progerin-expressing cells around the vertical wound edge. Future directions for this study involve additional experimental work together with the improved simulation results to confirm the MTOC orientation<br>relative to the nucleus under shear stress.

Cellular accumulation of DNA damage has been widely implicated in cellular senescence, aging, and premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is linked to accumulation of DNA double-strand breaks (DSBs), which results in genome instability. However, how DSBs accumulate in cells despite the presence of intact DNA repair proteins remains unknown. Here we report that the recruitment of DSB repair factors Rad50 and Rad51 to the DSB sites, as marked by γ-H2AX, was impaired in human HGPS and Zmpste24-deficient cells. Consistently, the progeria-associated DSBs appeared to be unrepairable although DSBs induced by camptothecin were efficiently removed in the progeroid cells. We also found that these progeroid cells exhibited nuclear foci of xeroderma pigmentosum group A (XPA), a unique nucleotide excision repair protein. Strikingly, these XPA foci colocalized with the DSB sites in the progeroid cells. This XPA-DSB association was further confirmed and found to be mediated by DNA, using a modified chromatin immunoprecipitation assay and coimmunoprecipitation. RNA interference (RNAi) knockdown of XPA in HGPS cells partially restored DSB repair as evidenced by Western blot analysis, immunofluorescence and comet assays. We propose that the uncharacteristic localization of XPA to or near DSBs inhibits DSB repair, thereby contributing to the premature aging phenotypes observed in progeria arising from genetic defects in prelamin A maturation.

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.

Le vieillissement est un processus complexe qui peut être influencé par des facteurs environnementaux et génétiques. Le syndrome progéroïde de Hutchinson-Gilford (HGPS ou progéria) fourni une preuve irréfutable de l'implication de l'épissage dans le processus de vieillissement. La progéria est une maladie due à une mutation hétérozygote silencieuse qui renforce l'utilisation d'un site 5' d'épissage interne dans l'exon 11 de l'ARN pré-messager LMNA, ce qui entraîne la production d'une protéine tronquée appelée «progérine». Le défaut d'épissage du gène LMNA a aussi lieu dans les cellules de personnes âgées, et la correction de ce défaut permet un sauvetage partiel des anomalies qu'il provoque. Ceci fait de l'ARN pré-messager LMNA une cible très attractive pour des thérapies ayant pour but de corriger l'épissage. Mes travaux de thèse ont montré que cette mutation silencieuse active un site d'épissage 5' dans l'exon 11 en changeant la structure de l'ARN. Ce changement de structure facilite l'interaction de la snRNP U1 avec le site d'épissage et permet ainsi sa modulation par les protéines SR SRSF1 et SRSF6. J'ai aussi participé à la caractérisation d'un nouveau modèle murin qui reproduit l'altération d'épissage des patients HGPS au niveau du gène Lmna souris. De façon surprenante, ce modèle récapitule tous les phénotypes du syndrome HGPS. Les souris homozygotes, dans lesquelles la plupart de la lamine A est convertie en progérine, ne vivent pas plus de 5 mois, alors que les souris hétérozygotes vivent autour d'un an et que les contrôles sauvages vivent deux ans. Étonnamment, des souris qui n'expriment ni la lamine A ni la progérine, mais uniquement de la lamine C, vivent plus longtemps que les souris contrôle, suggérant que la lamine A et la progérine, qui sont produites à partir du même transcrit, participent à la régulation de la durée de la vie. De plus, la caractérisation initiale des souris HGPS indique que l'expression de la progérine est délétère pour le tissu adipeux, établissant ainsi un lien inattendu entre l'épuisement du tissu adipeux et le vieillissement accéléré. Ce nouveau modèle murin est actuellement en train d'être utilisé pour des approches de modulation de l'épissage aberrant du gène LMNA avec des oligonucléotides antisense et des petites molécules chimiques. / Aging is a complex cellular and organismal process that can be influenced by environmental as well as genetic factors. A striking proof-of-concept that splicing regulation plays an important role in the aging process is provided by Hutchinson-Gilford progeria syndrome (HGPS), a disease caused by a heterozygous silent mutation that enhances the use of an internal 5' splice site in exon 11 of LMNA pre-mRNA and leads to the production of a truncated protein called “progerin”. The LMNA splicing defect also occurs with increased frequency in cells from healthy aged individuals and correction of this defect leads to partial reversal of age-related dysfunction. This makes LMNA pre-mRNA an attractive target for splicing-correction therapies. During my PhD thesis I have characterized the splicing mechanism responsible for progerin production and demonstrated that this process is conserved from mouse to human. I have found that HGPS mutation changes the accessibility of the exon 11 internal 5' splice site, allowing its modulation by U1 snRNP and a subset of SR proteins, namely SRSF6 and SRSF1. I have also participated to the characterization of a new mouse model reproducing human HGPS splicing alteration in the mouse Lmna gene. Strikingly, this model recapitulates all phenotypic manifestations of HGPS. The homozygous mice, where most lamin A is converted to progerin, lived no longer than 5 months, whereas heterozygous mice lived in average one year and wild type littermates up to two years. Unexpectedly, mice expressing neither lamin A nor progerin, but only lamin C, lived longer than wild type littermates mice, suggesting that lamin A and progerin which are produced from the same transcript, control critical steps of lifespan. Furthermore, initial characterization of HGPS mouse model indicated that progerin expression is deleterious for adipose tissue, establishing an unexpected link between adipose tissue depletion and accelerated aging. The new mouse model is currently being used for pharmacological modulation of LMNA aberrant splicing by antisense oligonucleotides and small molecules.

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial cells form the lining of the walls of blood vessels and are continuously subjected to mechanical stimuli from the blood flow. Microtubule-organizing center (MTOC), also known as centrosome is a structure found in eukaryotic cells close to the nucleus. MTOC relocates relative to the nucleus when endothelial cells are exposed to shear stress which determines their polarization, thus it plays a critical role in cell migration and wound healing. The nuclear lamina, a mesh-like network that lies underneath the nuclear membrane, is composed of lamins, type V intermediate filament proteins. Mutations in LMNA gene that encodes A-type lamins cause the production of a mutant form of lamin A called progerin and leads to a rare premature aging disease known as Hutchinson-Gilford Progeria Syndrome (HGPS). The goal of this study is to investigate how fluid flow affects the cytoskeleton of endothelial cells. This thesis consists of two main sections; computational mechanical modeling and laboratory experimental work. The mechanical model was implemented using Ansys Workbench software as a tensegrity-based cellular model in order to simulate the state of an endothelial cell under the effects of induced shear stress from the blood fluid flow. This tensegrity-based cellular model - composed of a plasma membrane, cytoplasm, nucleus, microtubules, and actin filaments - aims to understand the effects of the fluid flow on the mechanics of the cytoskeleton. In addition, the laboratory experiments conducted in this study examined the MTOC-nuclear orientation of endothelial cells under shear stress with the presence of wound healing. Wild-type lamin A and progerin-expressing BAECs were studied under static and sheared conditions. Moreover, a custom MATLAB code was utilized to measure the MTOC-nuclear orientation angle and classification. Results demonstrate that shear stress leads to different responses of the MTOC orientation between the wild-type and progerin-expressing cells around the vertical wound edge. Future directions for this study involve additional experimental work together with the improved simulation results to confirm the MTOC orientation relative to the nucleus under shear stress.

<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>

Le syndrome de Hutchinson-Gilford, ou progéria, est une pathologie génétique rare qui se caractérise par des symptômes assimilés à un vieillissement prématuré. Les mutations à l'origine de la progéria affectent le gène LMNA, codant la lamine A, qui joue un rôle majeur dans la formation, la maintenance et la résistance du noyau. Ces mutations activent l'utilisation de sites 5' alternatif ou cryptique d'épissage présents dans l'exon 11 du pré-ARNm LMNA en amont du site normalement utilisé. Nous avons révélé un effet des mutations sur la structure secondaire de l'ARN aux alentours des mutations, qui permet l'augmentation de l'utilisation des sites d'épissage mutants. De plus, nous avons montré l'implication de plusieurs protéines SR (SRSF1, SRSF5 et SRSF6) dans la régulation de l'utilisation des différents sites d'épissage. D'autre part, il a déjà été observé que les noyaux des cellules des patients atteints de progéria contiennent des granules de stress, les nSB, situés dans les régions péricentromériques des chromosomes et contenant des ARN dits satellite III et des facteurs d'épissage. Des nSB similaires sont formés dans les cellules saines suite à divers stress, comme le stress thermique. Il est possible que ces nSB séquestrent ces facteurs d'épissage afin de réguler le profil d'épissage alternatif des cellules pendant la régénération après un stress. Nous avons purifié les protéines associées aux ARN satellite III in vitro afin de trouver de nouveaux composants des nSB et analysé, par emploi de puces jonction-exon, le transcriptome de cellules soumises à un choc thermique, pour mieux comprendre à terme comment la formation des nSB peut affecter l'épissage alternatif / The Hutchinson-Gilford syndrome, also called progeria, is a rare genetic disease, characterized by symptoms that can be assimilated to accelerated natural ageing. Mutations that cause progeria affect the LMNA gene, which codes the lamin A that plays a major role in the shaping, maintenance and resistance of the nucleus. These mutations lead to the activation of alternative or cryptic 5' splice sites located within the exon 11 of LMNA pre-mRNA upstream from the normal 5' splice site. Our work revealed an effect of the mutations on the 2D RNA structure of the splice sites, which contributes to the increased use of the mutant sites. On top of it, we showed the impact of several SR proteins, (SRSF1, SRSF5 and SRSF6) on the regulation of the use of the exon 11 5' splice sites. On the other hand, it was previously observed that cells from progeria patients contain nuclear stress bodies (nSB), located in chromosomal pericentromeric regions and containing satellite III RNAs and several splicing regulatory proteins. Similar bodies are formed in healthy cells submitted to various stresses such as heat shock. A work hypothesis is that those nSBs sequester splicing factors in order to regulate the global alternative splicing profile in cells during the recovery period after stress. We purified proteins associated with satellite III RNAs in vitro, to find new components of the nSBs, and analyzed the transcriptome of cells subjected to heat shock using exon junction microarrays, in order to eventually understand how nSB formation can affect alternative splicing

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.