Thèse de doctorat : Sciences du vivant : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 227-261.

Thèse doctorat : Sciences du Vivant : Strasbourg 1 : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 8 p.

Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder affecting approximately 1 every 40,000 individuals in Western Europe, is characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive weakness of central origin. Additional features particularly include an hypertrophic cardiomyopathy that can cause premature death. A large GAA repeat expansion in the first intron of the FXN gene is the most common mutation underlying FRDA. Patients show severely reduced levels of the FXN-encoded mitochondrial protein frataxin.<p>Frataxin function is not yet completely elucidated. In frataxin deficiency conditions abnormalities of iron metabolism occur: decreased activities of iron-sulfur cluster (ISC) containing proteins, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake, and, in some models, reduced heme synthesis. <p>Evidence of oxidative stress has also been found in most though not all models of frataxin deficiency. Accordingly, yfh1-deficient yeast and cells from FRDA patients are highly sensitive to oxidants. Respiratory chain dysfunction further aggravate oxidative stress by increasing leakage of electrons and the formation of superoxide. Frataxin deficient cells not only generate more free radicals, but, they also show a reduced ability to mobilize antioxidant defenses, in particular to induce superoxide dismutase 2 (SOD2).<p>Peroxisome proliferator-activated receptor (PPAR) isoform-gamma play a key role in numerous cellular functions and is a key regulator of mitochondrial biogenesis and of the ROS metabolism. Recruitment of the PPAR coactivator-1a (PGC-1a) mediates many effects of the PPAR-γ activation.<p>In a first work we assessed the potential beneficial effects of a potent PPAR-gamma agonist on frataxin expression in primary fibroblasts from healthy controls and FRDA patients, and Neuroblastoma cells. We used the APAF molecule (1-0-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocoline; C33H66NO9P). Our results show that this compound is able to increase frataxin amount both at transcriptional and post-transcriptional level. At a dose of 20µM frataxin mRNA significantly increases in both controls (p=0.03) and FRDA patients (p=0.002) fibroblasts (1). The finding was confirmed in Neuroblastoma cells (p=0.042). According to previous publications APAF, as others PPAR-gamma agonists is able to up-regulate PGC-1a transcription.<p>In a second part of the study we investigate the role of the PPAR-gamma/PGC-1a pathway in the pathogenesis of FRDA. We performed a microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency and we found molecular evidence of increased lipogenesis in skeletal muscle and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the PPAR-gamma pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of Pgc1a and the transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Particularly, PGC-1a was found significantly reduced (2) in primary fibroblasts and lymphocytes from FRDA patients (p<0.05). Furthermore, PGC-1a mRNA levels strongly correlate with frataxin relative mRNA levels (r2=0.9, p<0.001). According to this observation, in C2C12 myoblasts, PGC-1a and a reporter gene under the control of the PGC-1a promoter are rapidly down-regulated (p<0.05) when frataxin expression is inhibited by an shRNA in vitro. To further investigate this relation, we then generate PGC-1a deficient fibroblasts cells using a specific siRNA; at 72 hours of transfection frataxin was found down-regulate (p<0.05) in control cells. <p>Taken together those data indicate that some mechanism directly links an early effect of frataxin deficiency with reduced PGC-1a transcription in this cell type, and presumably in other cells that also down-regulate PGC-1α when frataxin levels are low.<p>Finally, since PGC-1a has also emerged as a key factor in the induction of many antioxidant programs in response to oxidative stress, both in vivo and in vitro, in particular in neurons, we tested whether the PGC-1a down-regulation occurring in FRDA cells could be in part responsible for the blunted antioxidant response observed in frataxin deficiency.<p>Using primary fibroblasts from FRDA patients we found reduced SOD2 levels (p<0.05), according to PGC1& / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

La dysfonction ventriculaire gauche reste sous-diagnostiquée en pratique clinique actuelle car les paramètres conventionnels d’échographie ne sont pas suffisamment sensibles pour détecter des modifications fines de la fonction cardiaque. L’introduction récente de l’échocardiographie de suivi des marqueurs acoustiques (speckle tracking echocardiography) a permis par ses capacités descriptives de la mécanique cardiaque, de revisiter la contraction cardiaque et, de ce fait, de proposer une nouvelle approche échographique de l’évaluation de la fonction ventriculaire gauche.<p><p>A travers trois études, nous avons montré que l’analyse des indices de déformation permet d’objectiver des modifications de fonction ventriculaire gauche indétectables en échocardiographie conventionnelle dans des situations complexes à fraction d’éjection conservée, en dépit de l’augmentation modérée de la fréquence cardiaque qui leur est associée. Les situations que nous avons étudiées sont les suivantes :l’adaptation physiologique à l’hypoxie au niveau de la mer et en altitude, et les situations pathologiques que sont le syndrome de mal d’altitude chronique, et la cardiomyopathie associée à l’ataxie de Friedreich.<p><p>L’intégration de nos résultats et des informations disponibles dans la littérature permet de suggérer que l’utilisation de toutes les ressources offertes par l’échocardiographie de suivi des marqueurs acoustiques permet d’améliorer l’évaluation de la fonction cardiaque au-delà de la fraction d’éjection, en offrant une meilleure identification de situations pathologiques mais également une meilleure compréhension de situations physiologiques et pathologiques. <p><p>L’utilisation généralisée des indices de déformation pour l’évaluation de la fonction ventriculaire gauche en pratique clinique connaît néanmoins d’importantes limitations que nous abordons dans la discussion de ce travail. Au terme d’investigations complémentaires et de standardisation de la technique, l’incorporation d’un algorithme d’évaluation échographique de la fonction cardiaque à FEVG conservée combinant les paramètres échographiques conventionnels et les indices de déformation pourra être évalué de façon prospective pour sa translation en pratique clinique, avec pour finalité la proposition d’une définition mieux adaptée de l’insuffisance cardiaque à FEVG conservée.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Friedreich’s ataxia (FRDA) is an inherited recessive disorder characterized by progressive neurological disability and heart disease. It is caused by a pathological intronic hyperexpansion of a GAA repeat in the FXN gene, encoding the essential mitochondrial protein frataxin. At the homozygous state, the GAA expansion induces a heterochromatin state with decreased histone acetylation and increased methylation, resulting in a partial deficiency of frataxin expression. This was established in cells from FRDA patients. We showed that the same chromatin changes exist in a GAA based mouse model, KIKI, generated in our laboratory. Furthermore, treatment of KIKI mice with a novel Histone Deacetylase Inhibitor (HDACi), 106, a pimelic diphenylamide that increases frataxin levels in FRDA cell culture, restored frataxin levels in the nervous system and heart of KIKI mice and induced histone hyperacetylation near the GAA repeat. As shown by microarrays, most of the differentially expressed genes in KIKI were corrected towards wild type. In an effort to improve the pharmacological profile of compound 106, we synthesized more compounds based on its structure and specificity. We characterized two of these compounds in FRDA patients’ peripheral blood lymphocytes and in the KIKI mouse model. We observed a sustained frataxin upregulation in both systems, and, by following the time course of the events, we concluded that the effects of these compounds last longer than the time of direct exposure to HDACi. Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACis for FRDA. Laboratory tools to follow disease progression and assess drug efficacy are needed in a slowly progressive neurodegenerative disease such as FRDA. We used microarrays to characterize the gene expression profile in peripheral lymphocytes from FRDA patients, carriers and controls. We identified gene expression changes in heterozygous, clinically unaffected GAA expansion carriers, suggesting that they present a biochemical phenotype, consistent with data from animal models of frataxin deficiency. We identified a subset of genes changing in patients as a result of pathological frataxin deficiency establishing robust gene expression changes in peripheral lymphocytes. These changes can be used as a biomarker to monitor disease progression and potentially assess drug efficacy. To this end, we used he same methodology to characterize the gene expression profiles in peripheral lymphocytes after treatment with pimelic diphenylamide HDACi. This treatment had relevant effects on gene expression on peripheral patients’ blood lymphocytes. It increased frataxin levels in a dose-dependent manner, and partially rescued the gene expression phenotype associated with frataxin deficiency in the tested cell model, thus providing the first application of a biomarker gene set in FRDA. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished