<p>Lowe Syndrome (LS) is a lethal developmental disease characterized by mental retardation, cataracts at birth and kidney dysfunction. LS children unfortunately die by adolescence from renal failure. The gene responsible for the disease (<i>OCRL1</i>) encodes an inositol 5’ phosphatase Ocrl1. In addition to its 5’ phosphatase domain, this protein has other domains that allow protein-protein interactions, facilitating diverse sub-cellular distribution and functions. LS patient cells lacking Ocrl1 display defects in cell spreading, ciliogenesis and vesicle trafficking. Currently the mechanisms underlying these cellular defects are not known, and hence no LS-specific therapies exist. </p> <p>We have uncovered the mechanisms underlying two LS-specific cellular phenotypes- namely cell spreading and ciliogenesis and identified 2 FDA-approved candidates- statins and rapamycin that could revert these abnormalities. We found that Ocrl1-deficient cells exhibit hyperactivation in mTOR signaling, resulting in ciliogenesis as well as autophagy defects, which were rescued by administering rapamycin. We also identified a novel RhoGTPase signaling-dependent cell adhesion defect in LS patient cells which resulted in focal adhesion abnormalities and sensitivity to fluid shear stress (critical for kidney function). Both RhoGTPase signaling dependent cell spreading and adhesion defects were corrected by treatment with statins. </p> <p>Importantly, over 200 unique mutations in <i>OCRL1</i> cause LS and patients demonstrate heterogeneity in symptoms. However, the correlation between genotype and cellular phenotypes is unknown. We have determined that different <i>OCRL1</i> patient mutations have a differential impact on the two cellular phenotypes described above. Mutants exhibit behavior, sub-cellular distribution and cellular phenotypes unique to the domain and relevant to LS pathogenesis. We also propose that a subset of non-catalytic phosphatase domain mutations are conformationally affecting the protein, suggesting that LS has a conformational disease component. Importantly, we tested an FDA-approved drug, 4-phenyl butyric acid (4-PBA), used as a therapeutic in conformational diseases and found that it could revert phenotypes and restore the catalytic activity of these mutants. These findings collectively contribute to provide the cellular basis for LS patient heterogeneity as well as to propose a conformational disease component for LS (allowing the use of chemical chaperones as a therapeutic strategy for a subset of LS patients). Together, we hope that these studies will help lay the foundation of better prognosis and tailoring personalized therapeutic strategies for LS patients.</p>

Mutations of the gene encoding the inositol polyphosphate 5-phosphatase OCRL1 are responsible for causing two disorders in humans: Lowe syndrome and type 2 Dent's disease (Dent-2). Lowe syndrome (oculocerebrorenal syndrome of Lowe) is an X-linked genetic disorder that causes multisystem defects affecting predominantly the eyes, brain and kidneys. Dent-2 disease is very similar to Lowe syndrome, but it affects primarily the kidneys with little or no symptoms in the brain and eyes. The enzymatic activity, structure and binding partners of the OCRL1 protein have been described and progress on the cellular functions of OCRL1 has been made. However the studies to date have not provided the necessary insight to explain the tissue-specific defects observed in Lowe syndrome and Dent-2 patients. In order to investigate the role of OCRL1 and the consequences of its deficiency in a physiological context an animal model is required. We have chosen the zebrafish for this study due to its suitability for investigating vertebrate early development and the abundance of research techniques available for this model organism. We have studied the expression of OCRL1 in zebrafish and its role in the early embryonic development. We have also investigated its role in the endocytic function of the zebrafish larval pronephric kidney. Finally we have investigated its role in ciliogenesis and function of pronephric cilia. Our studies show that OCRL1 depletion does not cause gross developmental defects, nor affects the development of pronephros, but impairs their endocytic activity. We have also shown, that efficient pronephric uptake requires OCRL1 interactions with clathrin, Rab GTPase family proteins, APPL1 and IPIP27A/B. Our studies link the reduced uptake with lowered levels of megalin receptor, which is responsible for the bulk of protein reabsorption in the kidney. Together our results strongly suggest that defects in this process are responsible for low molecular weight proteinuria present in Lowe syndrome and Dent-2 patients and zebrafish is a suitable model to study the renal aspect of these diseases.

The complete role of inositol signaling in plants and humans is still elusive. The plant Arabidopsis thaliana contains fifteen predicted inositol polyphosphate 5- phosphatases (5PTases, E.C. 3.1.3.36) that have the potential to remove a 5-phosphate from various inositol second messenger substrates. To examine the substrate specificity of one of these Arabidopsis thaliana 5PTases (At5PTases), recombinant At5PTase1 was obtained from a Drosophila melanogaster expression system and analyzed biochemically. This analysis revealed that At5PTase1 has the ability to catalyze the hydrolysis of four potential inositol second messenger substrates. To determine whether At5PTase1 can be used to alter the signal transduction pathway of the major drought-sensing hormone abscisic acid (ABA), plants ectopically expressing At5PTase1 under the control of a constitutive promoter were characterized. This characterization revealed that plants ectopically expressing At5PTase1 had an altered response to ABA. These plants have stomata that are insensitive to ABA, and have lower basal and ABA-induced inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] levels. In addition, At5PTase1 mRNA and protein levels are transiently regulated by ABA. These data strongly suggest that At5PTase1 can act as a signal terminator of ABA signal transduction. Like the Arabidopsis At5PTase1, a human 5PTase, Ocrl, has the ability to catalyze the hydrolysis of a 5-phosphate from several inositol-containing substrates. The loss of functional Ocrl protein results in a rare genetic disorder known as Lowe oculocerebrorenal syndrome. To gather information concerning the specificity determinants of the Ocrl protein, a structure-function analysis of Ocrl was conducted using a vibrational technique, difference Fourier transform infrared (FT-IR) spectroscopy. Upon the introduction of Ins(1,4,5)P₃ substrate, structural changes in carboxylic acid and histidine residues were observed. The net result of changes in these residues indicates that upon Ins(1,4,5)P₃ introduction, a carboxylic acid-containing residue is protonated, and a histidine residue is deprotonated. This interpretation supports the idea that the deprotonation of the histidine residue is concomitant with the coordination of a divalent cation upon Ins(1,4,5)P₃ introduction. This work allows for the proposal of a new model for the role of the active site histidine of OCRL. / Ph. D.

No description available.

Après la transcription, le pré ARNm subit des étapes de maturation avant de sortir du noyau pour être traduit. Une des étapes de maturation est l'épissage. Il permet de souder les séquences codantes de l'ARNm entre elles (les exons) et d'exclure les régions non codantes (les introns).Des mutations génétiques sont à l'origine de défaut d'épissage. Elles peuvent conduire à des rétentions d'intron, des sauts d'exon et des inclusions de séquences introniques appelées pseudo exons.Ma thèse a porté sur l'utilisation du saut d'exon pour corriger ces inclusions. Je me suis intéressé à deux pathologies : la myopathie à cores et le syndrome de LowePour le premier cas, je me suis intéressé à une mutation dans l'intron 101 du gène RyR1. Cette mutation est à l'origine de la création d'un site donneur d'épissage qui active un site accepteur provoquant l'inclusion d'un pseudo exon de 99 nucléotides. Cette inclusion induit une baisse de la quantité du canal calcique RyR1 dans les cellules du patient. Ce canal permet le couplage entre l'excitation et la contraction musculaire. Ses défauts conduisent à diverses myopathies dont la myopathie à cores. Le patient présentait une hypotonie néonatale, une scoliose et des défauts respiratoires, et n'a jamais acquis la marche. J'ai dessiné des oligonucléotides, je les ai transfectés dans les cellules du patient en culture et ainsi montré par RT PCR que le saut du pseudo exon était possible. Afin d'optimiser l'efficacité pour pouvoir évaluer la restauration au niveau protéique et fonctionnel, j'ai développé un lentivirus exprimant une cassette U7 SmOPT avec les AON choisis. Après transduction des cellules, j'ai pu montrer que le saut du pseudo exon permettait le retour de la protéine et de sa fonctionnalité, cette approche pourrait donc permettre une correction chez le patient.Pour le deuxième cas, j'ai tenté de corriger une mutation du gène OCRL. Cette mutation crée un site donneur d'épissage dans l'intron 4 du gène OCRL et active un site accepteur d'épissage 66 nucléotides en amont. L'inclusion du pseudo exon induit la chute du taux de transcrit OCRL par un mécanisme de "Non sense mediated mRNA Decay". OCRL est une phosphatidyl inositol 5 Phosphatase permettant de réguler la quantité de Ptd Ins(4,5)P2 dans la cellule. Les défauts dans OCRL sont responsables d'une pathologie multisystémique, le syndrome de Lowe. J'ai pu obtenir des fibroblastes cutanés du patient. J'ai transfecté ces cellules avec des AONs choisis pour permettre un saut de l'exon pathogène. J'ai ensuite intégré la séquence des AONs efficaces dans un lentivirus U7. J'ai transduit les cellules du patient en culture et observé un retour de la protéine et un retour de l'activité enzymatique, cette approche pourrait donc théoriquement permettre une correction chez le patient.Ces deux travaux sont les premières preuves de principes de thérapie par modulation de l'épissage pour les myopathies congénitales et pour le syndrome de Lowe. Ils ouvrent la voie à des perspectives de traitement pour ces maladies génétiques. / After transcription, the pre mRNA will undergo different maturation step before getting out of the nucleus for translation. One of these step of maturation is the splicing. It allows to concatenate the coding sequences of the mRNA (the exons) and induces the exclusion of the non coding sequences (the introns).Genetics mutations can lead to splicing defects. These defects could be intron retention, exon skipping and exonisation of intronic sequences called pseudo exons.My thesis work was to evaluate the exon skipping therapy to correct these exonisation. I focuses on two diseases: core myopathy and Lowe syndrome.For the first one, my interest was on a mutation in the 101 th intron of RYR1 gene. This mutation creates a splicing donor site wich unveils a cryptic acceptor site. This leads to the inclusion of a 99 nucleotides pseudo exon. This inclusion induces a decrease of the quantity of the calcium channel RyR1 in the patient cells. This channel allows the excitation-contraction coupling, and therefore the muscular contraction. Defects in this channel lead to different myopathies (eg. core myopathy). The patient present at birth a major neonatal hypotonia, scoliosis and respiratory defects. He has never walked. I designed oligonucleotides (AON), transfected them in the cultured patient cells and showed by RT PCR that exon skipping was possible. In order to optimise the efficiency and to evaluate the rescue at a protein level and at a fonctionnal level, I devellopped a lentivirus which express a U7 Sm OPT cassette with the choosen AONs. After cell transduction, I have shown that exon skipping allowed the rescue of the protein and of its functionnality. This approach could permit a genetic correction for the patientFor the second case, I have tried to correct an OCRL mutation. This upstream creates a splicing donor site and unveils an acceptor site 66 nucleotides before. This leads to the inclusion of a pseudo exon which induces a severe decrease of OCRL transcripts level due to a "non sense mediated mRNA Decay". OCRL is a phosphatidyl inositol 5 Phosphatase, which regulates the Ptd Ins(4,5)P2 pool in the cell. OCRL defects induces a multi systemic disease the Lowe syndrome. I obtained patient cutaneous fibroblasts. I transfected these cells with choosen AONs to correct the splicing defect. I integrated the AONs sequence into a U7 lentiviral cassette. I transduced the cultured patient cells and observed a rescue of the protein with a rescue of its activity. This approach could, theoritically permit a correction in the patient.These two studies are the first proof of concept of splicing modulation therapy for congenital myopathy and for Lowe syndrome. This work offers a lot of perspective for the tratment of these genetic illness

Le Phosphatidylinositol(4,5)P2 (PtdIns(4,5)P2) est essentiel pour réguler divers processus cellulaires, y compris la signalisation cellulaire, le trafic intracellulaire et la cytocinèse. Le contrôle strict de son homéostasie est donc crucial et la dérégulation des kinases, des phosphatases et des phospholipases qui la contrôlent conduit à de multiples pathologies. Parmi elles, le syndrome de Lowe est une maladie rare et incurable causée par des mutations du gène OCRL qui code pour la PtdIns(4,5)P2 phosphatase OCRL1. La déplétion de dOCRL, l’orthologue d’OCRL1 chez la drosophile altère l’homéostasie du PtdIns(4,5)P2 avec (i) une accumulation anormale de PtdIns(4,5)P2 sur les endomembranes conduisant (ii) à des défauts de cytocinèse et à de la multinucléation. L’objectif de cette thèse était de comprendre comment le PtdIns(4,5)P2 est régulé sur les endomembranes. Dans les cellules de drosophile, nous avons découvert une fonction nouvelle et inattendue pour le suppresseur de tumeur PTEN, indépendante de son activité phosphatase. En effet, nous avons constaté que PTEN réduit les niveaux de PtdIns(4,5)P2 sur les endosomes grâce à l’action enzymatique de dPLCXD, une phospholipase C (PLC) atypique. Ainsi la voie de signalisation PTEN/dPLCXD peut compenser pour les défauts de cytocinèse dus à la perte de dOCRL. Enfin, nous avons identifié un activateur chimique des PLC qui restaure la perte fonctionnelle d’OCRL dans trois modèles de syndrome de Lowe distincts. Par la suite, nous avons étudié le rôle de la PTEN/PLCXD pendant l’autophagie, mécanisme d’autodigestion du matériel cellulaire. En effet, l’homéostasie du PtdIns(4,5)P2 lysosomale est essentielle pour l’étape autophagique de fusion des autophagosomes avec lysosomes. Nous avons observé que la déplétion de PLCXD et la surexpression d’un mutant catalytiquement inactif de PTEN altèrent l’autophagie chez les cellules de drosophile et de mammifère. Ces données suggèrent que la voie PTEN/PLCXD nouvellement identifiée régule le flux autophagique. Dans cette thèse, nous avons mis en lumière une nouvelle voie de signalisation PTEN/dPLCXD qui contrôle les niveaux de PtdIns(4,5)P2 sur les endolysosomes. Cette voie peut réguler l’autophagie et compenser la perte de dOCRL. Il s’agit d’une nouvelle fonction de PTEN indépendante de son activité phosphatase et c’est une première fonction biologique connue pour PLCXD. Cette découverte a conduit à l’identification d’une stratégie thérapeutique potentielle pour traiter les patients atteints du syndrome de Lowe. / Phosphatidylinositol(4,5)P2 (PtdIns(4,5)P2) is essential for various cellular processes, including cell signaling, intracellular traffic and cytokinesis. Therefore, strict control of its homeostasis is crucial. Indeed, the deregulation of the kinases, phosphatases and phospholipases which controls PtdIns(4,5)P2 leads to multiple pathologies. Among them, the Lowe syndrome is a rare and incurable disease caused by mutations in the OCRL gene which codes for PtdIns(4,5)P2 phosphatase OCRL1. Depletion of dOCRL, the orthologue of OCRL1 in drosophila, alters the homeostasis of PtdIns(4,5)P2 with (i) an abnormal accumulation of PtdIns(4,5)P2 on the endomembranes leading (ii) to cytokinesis defects and multinucleation. The objective of this thesis was to understand how PtdIns(4,5)P2 is regulated on endomembranes. In drosophila cells, we have discovered a new and unexpected function for the tumor suppressor PTEN independent of its phosphatase activity. Indeed, we have found that PTEN reduces the levels of PtdIns(4,5)P2 on endolysosomes thanks to the enzymatic action of dPLCXD, an atypical phospholipase C (PLC). Thus, the PTEN/dPLCXD signaling pathway can compensate for cytokinesis defects due to the loss of dOCRL. Finally, we identified a chemical activator of PLC that restores the functional loss of OCRL in three distinct Lowe syndrome models. Next, we studied the role of this newly identified PTEN/PLCXD pathway during autophagy, a self-digestion mechanism. Indeed, the homeostasis of lysosomal PtdIns(4,5)P2 is essential for the fusion of autophagosomes with lysosomes during autophagy. We have observed that depletion of PLCXD and overexpression of a catalytically inactive mutant of PTEN both alter autophagy in Drosophila and mammalian cells. These data suggest that this newly identified PTEN/PLCXD pathway regulates the autophagic flux. In this thesis, we have highlighted a new PTEN/dPLCXD signaling pathway which controls the levels of PtdIns(4,5)P2 on endolysosomes. This new PTEN function is independent of its phosphatase activity and the first biological function for PLCXD can regulate autophagy and compensate for the loss of dOCRL. This discovery led to the identification of a potential therapeutic strategy for treating patients with Lowe’s syndrome.

No description available.

L’hémostase primaire est un processus permettant la formation d’un clou plaquettaire qui sera stabilisé par un réseau de fibrine. Ce caillot est également consolidé grâce à des phases tardives de l’hémostase primaire résultant des fonctions plaquettaires ; il s’agit principalement de la rétraction qui diminue la taille du caillot afin de le stabiliser. Cette phase est déclenchée par une signalisation « outside-in », consécutive à l’activation de l’intégrine αIIbβ3 et à l’agrégation plaquettaire, et est dépendante d’une réorganisation du cytosquelette. Le premier objectif de ce travail a été d’étudier la signalisation impliquée dans la rétraction, et en particulier l’implication des protéines ROCK, MLCK, Rac-1 et de l’actine dans l’activité de la chaine légère de la myosine (MLC) . MLC est en effet une protéine clé de la réorganisation du cytosquelette. Nous avons mis en évidence une phosphorylation biphasique de MLC dont le deuxième pic, corrélé à la rétraction, est dépendant de Rac1 et de la polymérisation de l’actine. Cette étude a été appliquée à une pathologie, le syndrome de Lowe. Il s’agit d’une maladie génétique rare, également appelée OCRL (Oculo cérébro rénal de Lowe) en référence aux organes majoritairement touchés. Suite à l’observation d’événements hémorragiques per et postopératoires suggérant une instabilité du caillot et l’observation dans une étude précédente d’un temps d’occlusion allongé au PFA100®, nous avons mis en place une étude sur 15 patients et 15 témoins pour lesquels nous avons étudié les différentes phases de l’hémostase primaire. Outre une anomalie et un retard de maturation des mégacaryocytes, nous avons mis en évidence pour la première fois chez ces patients un défaut de la voie « outside-in » responsable d’une anomalie de l’étalement plaquettaire et de la rétraction du caillot. Ce défaut de rétraction, dû à un défaut d’activation de MLC, pourrait être en partie responsable des événements hémorragiques observés chez ces patients. / Primary hemostasis is a mechanism allowing platelet clot formation that is thereafter stabilized by a fibrin network. Fibrin clot is also consolidated following post occupancy events, mainly clot retraction that decrease clot size and thus strengthen it. This phase is triggered by « outside-in » signaling. It is consecutive to αIIbβ3 integrin activation and platelet aggregation, dependent on cytoskeleton organization. Our first objective was to investigate signaling events underlying retraction, and particularly the involvement of ROCK, MLCK, Rac-1, and actin in MLC (Myosin Light Chain) phosphorylation. Indeed, MLC, involved in cytoskeleton rearrangement, is a key protein of this mechanism. We described a MLC biphasic phosphorylation profile, which second peak was dependent of Rac1 and actin polymerization. In a second part, we studied clot retraction signaling in patients with the Lowe syndrome. It is a rare genetic disease, caused by absence of OCRL (oculo cerebro renal of Lowe) protein in reference to the majority of affected organs. The rationale of this study was a previous observation of hemorrhagic events during and after surgeries, suggesting clot instability. A thrombopathy was suggested by a closure time lengthening in the PFA-100 system. The study enrolled 15 patients and 15 controls. Besides a defect of megakaryocyte maturation, we described a defect of « outside-in » signaling responsible for spreading and clot retraction abnormality. This retraction defect, caused by a MLC activity defect, could be partly responsible for hemorrhagic events reported in these patients.