La scoliose est une déformation de la colonne vertébrale dans les trois plans de l’espace (frontal, sagittal et transverse), et la forme la plus fréquente est la scoliose idiopathique (SI) dont la cause est encore très mal comprise. La scoliose idiopathique de l’adolescent (SIA) est une forme de SI qui se développe au cours de la puberté, plus souvent chez les filles, et présente une importante hétérogénéité phénotypique. Les très nombreuses recherches afin de comprendre les mécanismes qui entrent en jeu dans le développement de cette maladie et afin de déterminer l’étiologie ont permis de mettre en évidence un très grand nombre de théories regroupant des facteurs endocriniens et métaboliques, des facteurs biomécaniques et musculosquelettiques, des facteurs neurologiques et vestibulaires ainsi que des facteurs génétiques et épigénétiques. L’identification du gène candidat POC5 dans la SI par notre laboratoire, un gène crucial dans la formation du cil primaire, a été un tournant majeur apportant la première pièce d’un puzzle plaçant le cil primaire au centre des hypothèses concernant la mise en place de la maladie. Le cil primaire est une organelle non-motile retrouvée à la surface de la totalité des cellules du corps humain à l’exception des cellules sanguines. Grâce à son axonème formé de tubuline capable de subir des modifications post-traductionnelles appelées « code tubuline » et sa membrane ciliaire ultra spécialisée, le cil primaire joue un rôle mécanosenseur important tout au long de la vie, dès le développement embryonnaire. Il prend part à de nombreux mécanismes dont la formation et l’homéostasie osseuse et a déjà été mis en cause dans les maladies osseuses. En effet, lorsque le cil primaire est défectueux, il est responsable de syndromes complexes regroupés sous le nom de ciliopathie dont un des symptômes est la scoliose. L’hypothèse générale de la présente thèse est que des variants rares dans des gènes ciliaires, particulièrement le gène POC5 et le gène TTLL11, agissent sur les mécanismes cellulaires responsables de la mise en place de la SIA faisant d’elle une maladie appartenant à la grande famille des ciliopathies. L’objectif de cette thèse a été de documenter l’implication et la pathogénicité des gènes POC5 et TTLL11 dans la SIA. Nous avons d’abord déterminé la prévalence des variants du gène POC5 dans une cohorte de patients d’origine franco-canadienne ou britannique atteints de SIA (Manuscrit 1). Ensuite, nous avons créé trois modèles de souris Knock-in pour l’équivalent des variants du gène POC5 humain afin d’analyser la pathogénicité et le phénotype éventuel de ciliopathie (Manuscrit 2). Finalement, la pathogénicité du gène TTLL11 muté a été analysée dans des cellules issues de patients atteints de SIA et dans un modèle animal, le poisson zèbre (Manuscrit 3). Les travaux réalisés au cours de cette thèse ont montré que les deux gènes ciliaire POC5 et TTLL11 sont des gènes candidats probablement impliqués dans les mécanismes cellulaires fondamentaux induisant la mise en place de la SIA et ont permis d’identifier un mécanisme, la polyglutamylation du cil primaire, comme étant relié à la SIA. / Scoliosis is a 3D spinal curvature, in the frontal, sagittal and transverse plans, and the most common form is the idiopathic scoliosis (IS) with a cause that remains very poorly understood. Adolescent Idiopathic Scoliosis (AIS) is a subtype of IS that develops during the puberty, most commonly in girls, and has a significant phenotypic heterogeneity. To date, a large amount of research on scoliosis etiology highlighted various hypotheses based on endocrine and metabolic factors, biomechanical and musculoskeletal factors, neurological and vestibular factors, as well as genetic and epigenetic factors. The identification of the IS candidate gene POC5 by our laboratory, a crucial gene in the formation of the primary cilium, was a major turning point bringing the first piece of a puzzle where the primary cilium is a central hypothesis concerning the onset of the disease. The primary cilium is a non-motile organelle found at the cell surface of all the human body cells, except for blood cells. The primary cilium plays an important mechano-sensor role throughout life, from embryonic development through its axoneme composed by tubulin that hosted post-translational modifications called “tubulin code”, and its ultra-specialized ciliary membrane. It is involved in many mechanisms including bone formation and homeostasis and has already been implicated in bone diseases. Indeed, the defective primary cilium is responsible for human ciliopathy syndromes that are associated with scoliosis. The main hypothesis of this thesis is that rare variants in ciliary genes, especially the POC5 gene and the TTLL11 gene, participate on cellular mechanisms responsible for the onset of AIS supporting the idea that scoliosis is a form of ciliopathy. The specific objective of this thesis was to document the involvement and pathogenicity of the POC5 and TTLL11 genes in AIS. We first determined the prevalence of POC5 gene variants in a cohort of French-Canadian and British AIS patients (Manuscript 1). Then, we created three knock-in mouse models carrying the equivalent of the human POC5 gene variants to analyze the pathogenicity and the possible phenotype of ciliopathy (Manuscript 2). Finally, the pathogenicity of the mutated TTLL11 gene was analyzed in cells extracted from AIS patients and in an animal model, the zebrafish (Manuscript 3). The work presented in this thesis showed that the two ciliary genes POC5 and TTLL11 are candidate genes probably involved in the fundamental cellular mechanisms inducing the onset of AIS and allowed us to identify the primary cilium polyglutamylation, as a mechanism being related to AIS.

Primary cilia are nonmotile, hair-shaped organelles that extend from the basal body in the centrosome. The present study is the first investigation of this organelle in the oligodendrocyte lineage in vivo. I used immunohistochemical approaches in normal and cilia-deficient mutant mice to study cilia in relation to oligodendrogenesis and myelination. Primary cilia immunoreactive for Arl13b and ACIII were commonly present in NG2+ oligodendrocyte progenitor cells (OPCs), in which cilia-associated pathways control proliferation, differentiation, and migration. The loss of primary cilia is generally associated with enhanced Wnt/β-catenin signaling, and Wnt/β-catenin signaling has been shown to promote myelin gene expression. I examined whether the lack of cilia in the oligodendrocyte lineage is associated with elevated Wnt/β-catenin activity. I found that absence of a primary cilium was associated with with higher levels of TCF3, and with β-galactosidase in Axin2-lacZ Wnt reporter mice. This evidence supports the proposal that cilia loss in oligodendrocytes leads to enhanced Wnt/β-catenin activity, which promotes myelination. Cilia are dependent on the centrosome, which assembles microtubules for the cilium, the cytoskeleton, and the mitotic spindle. Centrosomes are the organizing center for microtubule assembly in OPCs, but this function is decentralized in oligodendrocytes. I found that the intensity of centrosomal pericentrin was reduced in oligodendrocytes relative to OPCs, and γ-tubulin was evident in centrosomes of OPCs but not in mature oligodendrocytes. These decreases in centrosomal proteins might contribute to functional differences between OPCs and oligodendrocytes. The importance of cilia in the oligodendrocyte lineage was examined in Tg737orpk mice, which have a hypomorphic IFT88 mutation resulting in decreased cilia numbers and lengths. These mice showed marked, differential decreases in numbers of oligodendrocytes and myelin, yet little or no change in OPC populations. It appears that sufficient cells were available for maturation, but lineage progression was stalled. There were no evident effects of the mutation on Wnt/β-catenin. Factors that might contribute to the abnormalities in the oligodendrocyte lineage of Tg737orpk mice include decreased cilia-dependent Shh mitogenic signaling and dysregulation in cilia-associated pathways such as Notch and Wnt/β-catenin.

No description available.

The primary cilium is an immotile, microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Critically, the transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, this work employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. Using SPEED microscopy and single molecule tracking, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins. While the 3D transport pathways in this work are always replicated multiple times with a high degree of consistency, several experimental parameters directly affect the 3D transport routes’ error, such as single molecule localization precision and the number of single molecule localizations. In fact, if these experimental parameters do not meet a minimum threshold, the resultant 3D transport pathways may not have significant resolution to determine any biological details. To estimate the 3D transport routes’ error, this work will explain in detail the component of SPEED microscopy that estimates 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric bio-structures, such as the primary cilium. This component is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions based on prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the sub-structural localization of a particular protein is not. This work will demonstrate how the 2D-to-3D component of SPEED microscopy can be successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as the primary cilium and microtubules. Furthermore, this work will provide comprehensive analyses of this method by using computational simulations which investigate the role of various experimental parameters on the 3D transport pathway error. Lastly, this work will demonstrate that this method can distinguish different types of 3D transport pathway distributions in addition to their locations. / Biology

The objective of this study was to investigate primary cilia and their mechanotransduction role in lung adenocarcinoma tumor progression. The main focus investigated the effect of primary cilia on cell cycle progression, survival, adhesion and migration analysis of these cells and the role of sonic hedgehog signaling pathway in mechanotransduction. Human Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma biopsies contain more primary cilia than non-tumor lung sections. To observe the effects of primary cilia presence in lung cancer cells in-vitro, formation of primary cilia is inhibited using small interfering RNA. A549 cells with intact primary cilia observe less cell cycle progression than cells deficient in primary cilia under static and cyclic stretch conditions. Primary cilia cause higher cell survival and adhesion. Increase in cell adhesion also increases the migration and wound closure rates in control samples compared to samples treated with inhibition of IFT88, thereby increasing the metastasis of these cells. Several downstream regulatory genes in sonic hedgehog signaling pathway observe significantly decreased gene expressions in primary cilia deficient cells, thus indicating inefficient mechanotransduction. Therefore, cancer cells need primary cilia to survive, adhere and migrate and continue tumor progression.

The objective of this study is to investigate the role of microtubule based organelle, the primary cilia in lung adenocarcinoma by i) Quantifying the presence of primary cilia in several Non Small Cell Lung Cancer (NSCLC) cell lines in response to mechanical stimuli, ii) Attempting to determine the role of primary cilia in cell migration, iii) Investigating the effects of Paclitaxel(Taxol) resistance in lung cancer cells, iv) Analyzing the response of lung cancer cells to Smoothened Inhibitors and v) Determining the effects of Transforming Growth Factor Beta-1(TGF-β1) induced Epithelial to Mesenchymal Transition(EMT) in lung cancer cells. To ascertain the effects of primary cilia in the hall marks of tumor progression, several experiments involved prohibition of primary cilia formation by silencing IFT88, the gene responsible using small interfering RNA. Three out of the five cell lines tested, showed increased expression of primary cilia under mechanical stretch. IFT88 inhibition of H460 cells decreased their migration rate to the injury site under stretch conditions. Smoothened (SMO) Inhibitors decreased proliferation and migration rates in human lung adenocarcinoma cell lines (A549luc) similar to the effects observed in IFT88 silenced cells. IFT88 silenced A549luc cells showed a partial reversal of TGF-beta1 induced up-regulation of a mesenchymal marker. These results indicate that primary cilia play a role in the progression and metastasis of lung cancer by aiding the adhesion, proliferation, migration and EMT of lung cancer cells.

La glycogénose de type Ia (GSDIa) est une maladie métabolique rare causée par une déficience en glucose-6-phosphatase (G6Pase), due à des mutations de la sous-unité catalytique (G6PC). Cette enzyme confère au foie, aux reins et à l’intestin la capacité de produire du glucose. Les patients atteints de GSDIa sont donc incapables de produire du glucose et souffrent d’hypoglycémies sévères lors de jeûnes courts. De plus, la déficience en G6Pase provoque une accumulation de glucose-6 phosphate dans le foie et les reins, conduisant à l’accumulation de glycogène et de lipides. A long terme, la plupart des patients souffre d’une maladie chronique rénale (MCR), qui peut évoluer en insuffisance rénale, nécessitant une mise sous dialyse ou une transplantation rénale. Cette MCR se caractérise par une fibrose, ainsi que par le développement de kystes dans les stades tardifs. Au niveau du foie, les patients développent une hépatomégalie et une stéatose hépatique qui peut évoluer vers le développement d’adénomes ou carcinomes hépatocellulaires. Le but de mes travaux de thèse a été d’identifier les mécanismes moléculaires impliqués dans l’établissement de la pathologie rénale et la formation des kystes, à l’aide de modèles murins invalidés pour le gène G6pc spécifiquement dans les reins (souris K.G6pc-/-). Alors que la GSDIa est une maladie caractérisée par l’accumulation hépatique et rénale de glycogène, nous avons d’abord montré que le développement de la fibrose, à l’origine de la perte de la fonction rénale, était induit par l’accumulation de lipides, indépendamment du contenu en glycogène. De plus, l’utilisation d’un agoniste de PPARα, le fénofibrate, en diminuant le contenu lipidique rénal, a ralenti l’installation de la fibrose et l’évolution de la MCR. Le mécanisme moléculaire impliqué est l’activation du système rénine angiotensine par les dérivés lipidiques, qui induit l’expression du facteur profibrotique TGFβ1. De même, le fénofibrate en limitant l’accumulation de lipides hépatiques a prévenu le développement d’atteintes hépatiques caractéristiques de la GSDI. Ainsi, l’activation du catabolisme des lipides par des agonistes de PPARα semble une stratégie thérapeutique intéressante pour réduire la progression des maladies rénales et hépatique de la GSDI. La deuxième partie de mes résultats suggèrent que le développement de kystes rénaux chez les patients atteints de la GSDI pourrait être causé par une altération du cil primaire, organelle jouant un rôle clé dans le maintien d’une structure et fonction normale des reins. En effet, une augmentation de la longueur du cil primaire a pu être observée dans les reins des souris K.G6pc-/- associée à une dérégulation de différentes protéines impliquées dans sa structure et sa fonction, par rapport aux souris contrôles. Nous avons également mis en évidence une reprogrammation métabolique de type Warburg, caractérisée par une activation accrue de la glycolyse aérobie, une inhibition de l’oxydation mitochondriale du pyruvate et une production de lipides. Ainsi, l’ensemble de ces perturbations va favoriser la prolifération cellulaire et le développement de kystes, et pourrait mener au développement de tumeur rénale comme observée chez une souris K.G6pc-/-. En conclusion nous avons démontré que, dans le cadre de la GSDI, l’accumulation de lipides dans les reins et le foie, secondaire à la déficience en G6Pase, joue un rôle clé dans le développement des complications hépatiques et rénales à long terme. Également, la reprogrammation métabolique rénale de type Warburg, prenant place dans le cadre de la GSDI, associée à un défaut du cil primaire pourrait être à l’origine de la formation des kystes et de tumeurs rénales. Ces études, en permettant une meilleure compréhension de la physiopathologie des complications à long terme de la GSDIa, offrent de nouvelles perspectives concernant les stratégies thérapeutiques à développer pour une meilleure prise en charge des patients atteints de GSDIa / Glycogen storage disease type Ia (GSDIa) is a rare metabolic disease caused by glucose-6-phosphatase (G6Pase) deficiency, due to mutations on the gene encoding G6Pase catalytic subunit (G6PC). This enzyme confers to the liver, kidneys and intestine the ability to produce glucose. Thus, patients with GSDIa are unable to ensure endogenous glucose production and suffer from severe hypoglycemia during fasting in the absence of nutritional control. In addition, G6Pase deficiency causes intracellular accumulation of glucose-6 phosphate in the liver and kidneys, leading to metabolic defects and the accumulation of glycogen and lipids. Over time, most adult patients suffer from chronic kidney disease (CKD), which can progress to kidney failure, requiring dialysis or kidney transplantation. This nephropathy is characterized in particular by tubulo-interstitial fibrosis and glomerulosclerosis, as well as by the development of cysts in the late stages. Moreover, patients develop hepatomegaly and hepatic steatosis that may progress to the development of hepatocellular adenomas or carcinomas. The aim of my thesis was to identify the molecular mechanisms involved in the establishment of renal pathology and cyst formation in GSDIa, by using mouse models where G6pc gene is specifically deleted in the kidneys (K.G6pc-/- mice). While GSDIa is a disease characterized by glycogen accumulation in the liver and kidneys, we first showed that the development of fibrosis, which causes progressive loss of kidney function, was induced by intracellular accumulation of lipids, regardless of glycogen content. The molecular mechanism probably involved is the activation of the renin angiotensin system by lipid derivatives such as diacylglycerol, which induced the expression of the profibrotic factor TGFβ1 and an epithelial-mesenchymal transition. In addition, the use of a PPARα agonist, i.e. fenofibrate, by decreasing renal lipid content, reduced the development of fibrosis and CKD evolution. Similarly, fenofibrate treatment prevented the accumulation of lipids in the liver and the development of liver damages that cause tumor development. Thus, the activation of lipid catabolism by PPARα agonists such as fenofibrate seems to be an interesting therapeutic strategy to reduce the progression of renal and hepatic diseases of GSDIa. The second part of my results suggest that the development of renal cysts in GSDI patients may be caused by an alteration of the primary cilia, a non-motile organelle that plays a key role in maintaining normal kidney structure and function. Indeed, defects in the primary cilia are involved in many polycystic kidney diseases. In summary, an increase in the length of the primary cilia was observed in the kidneys of K.G6pc-/- mice, which could be explained by a deregulation of the expression of different proteins involved in cilia structure and function, compared to control mice. We also demonstrated a metabolic reprogramming leading to a Warburg metabolism, characterized by the increased activation of aerobic glycolysis and the inhibition of mitochondrial pyruvate oxidation and lipid production in K.G6pc-/- mice. Thus, all these disorders would promote cell proliferation and cyst development, and could lead to the development of renal tumor, as recently observed in one K.G6pc-/- mouse (out of 36 studied mice). In conclusion, we have shown that, in GSDI, the accumulation of lipids in the kidneys and liver that occurs secondary to G6Pase deficiency plays a key role in the development of hepatic and renal long-term complications. In addition, the Warburg like metabolic reprogramming taking place in the GSDIa kidneys, associated with a defect in the primary cilia, could be at the origin of cysts formation and renal tumors. These new studies, by providing a better understanding of the pathophysiology of long-term complications of GSDIa, offer new perspectives on therapeutic strategies to be developed for better management of patients

Ttc39c has been identified as a novel gene in skeletal muscle that is upregulated in response to neurogenic atrophy in mice. Quantitative PCR and Western blot analysis confirmed that Ttc39c is expressed in both proliferating and differentiated muscle cells. Furthermore, comparison of Ttc39c expression in undifferentiated and differentiated C2C12 cells demonstrated that Ttc39c levels peak in early differentiation, but decreases as cells become fully differentiated myotubes. The transcriptional regulation of Ttc39c was examined by cloning promoter fragments of the gene and fusing it with the SEAP reporter gene. The Ttc39c reporter gene constructs were transfected into muscle cells and confirmed to have significant transcriptional activity in cultured muscle cells and were also found to be transcriptionally repressed in response to ectopic expression of myogenic regulatory factors (MRF). Furthermore, conserved E-box elements in the proximal promoter region were identified, mutated, and analyzed for their role in the transcriptional regulation of Ttc39c expression. Mutation of the conserved E-box sequences reduced the activity of the Ttc39c reporter gene, suggesting that these elements are potentially necessary for full Ttc39c expression. To determine the sub-cellular location of Ttc39c in muscle cells, the Ttc39c cDNA was fused with the green fluorescent protein (GFP), expressed in muscle cells, and visualized by confocal microscopy revealing that Tct39c is localized to the cytoplasm of proliferating myoblasts and differentiating myotubes. Furthermore, Ttc39c appears to localize to the microtubule network and differentiating muscle cells developed elongated primary cilia in response to Ttc39c ectopic expression. Additionally, Ttc39c overexpression resulted in impaired muscle cell differentiation, attenuated Hedgehog and MAP Kinase signaling, and increased expression of IFT144, a component of the intraflagellar transport complex A involved in retrograde movement in primary cilia. Interestingly, Ttc39c knockdown also resulted in abrogated muscle cell differentiation and impaired Hedgehog and MAP Kinase signaling, but did not affect IFT144 expression levels. These results suggest that muscle cell differentiation is sensitive to aberrant Ttc39c expression, that Ttc39c is necessary for proper muscle cell differentiation, and that Ttc39c may participate in retrograde transport of the primary cilia of developing muscle cells.

<p>  </p> <p>Cilia are cell appendages that sense our environment and are critical in cell-to-cell communication. Dysfunction of cilia can result in several disease states including obesity. While cilia in the brain are known to be important for feeding behavior, it is unclear how they regulate energy homeostasis. Classically, cilia coordinate signaling through surface receptors called G-protein coupled receptors (GPCRs). For example, cilia mediated GPCR signaling is critical for both our senses of vision and smell. How cilia regulate the signaling of GPCRs in other areas of the body including the brain is only now emerging. To answer cell biology questions around cilia mediated GPCR signaling in neurons, we developed a system for primary neuronal cultures. We discovered that the cilia mediated hedgehog pathway influences the ability of neurons to respond to GPCR ligands. For the first time, this result highlights the role of the hedgehog pathway in neurons. We continue to explore how cilia integrate the hedgehog pathway and GPCR signaling in the central nervous system, and the potential connections to energy homeostasis. We discovered that hedgehog pathway activity in feeding centers of the brain changes based upon feeding conditions like fasting. We also learned that activating the hedgehog pathway in these brain regions is sufficient to cause obesity in mice. These novel results highlight an unrecognized role for the hedgehog pathway in the regulation of feeding behavior. Overall, this work provides a better understanding of ciliopathy associated obesity and may reveal more common mechanisms of obesity in the general population. In addition, this work implicates the hedgehog pathway in regulating behaviors and new modes of cell-cell communication within the central nervous system.</p>