Limitations in a frataxin knockdown cell model for Friedreich ataxia in a high-throughput drug screen

<p>Abstract</p> <p>Background</p> <p>Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.</p> <p>Methods</p> <p>Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.</p> <p>Results</p> <p>The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.</p> <p>Conclusion</p> <p>Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.</p

Analyse par microfaisceau d'ions. Application à l'étude de la fonction barrière cutanée et à la nanotoxicologie in vitro

Depuis plusieurs années, les nanosciences promettent des progrès remarquables dans de nombreux domaines, mais soulèvent aussi de nombreuses inquiétudes en regard de leur impact sur la santé humaine (expositions environnementales, industrielles, médicales). Il se pose, entre autres, la question de la détection, de la quantification et du devenir d oxydes métalliques et de nanoparticules manufacturées au sein de tissus, et plus particulièrement de la peau. Il est donc essentiel de préciser les mécanismes impliqués dans ces processus de fonction barrière cutanée et de les caractériser dans des modèles biologiques in vitro/in vivo. Ainsi, au cours de ma thèse, il m a été donné l occasion de mettre en œuvre des méthodes d analyses quantitatives en association avec des techniques d imagerie très résolutives (microscopies confocale et électronique et analyse par microfaisceau d ions) afin de caractériser : (i) la fonction barrière d un modèle de peau d oreille de porc maintenu en survie en définissant le comportement de l homéostasie ionique en réponse à différents stress chimiques ou physiques (Collaboration Hélène Duplan, Institut de Recherche Pierre Fabre); (ii) l impact sur la viabilité, l accumulation et la distribution intracellulaire de nanoparticules (oxydes de titane) natives ou fonctionnalisées à l aide d agents fluorescents (FITC, Rhodamine) (Collaboration M.H. Delville, Institut de Chimie de la Matière Condensée de Bordeaux). Les résultats obtenus montrent la possibilité de définir (i) le rôle des ions dans la fonction barrière cutanée d un modèle biologique maintenu en survie et exposé à différents stress, (ii) la toxicologie des nanoparticules manufacturées in vivo; (iii) leur devenir au sein de modèle biologique d intérêt (kératinocytes).Since many years, nanosciences are of great interest for researchers and industrialists, with numerous applications in various domains, however, their potential effects on human health have also attracted attention (environmental, industrial and medical exposures). Up to now, it has been very difficult to detect and track metallic oxides and manufactured nanoparticles in biological tissues, most particularly in skin. Thus, it is essential to precise the mechanisms involved in skin barrier function processes face to these exogenous agents and to characterize them in biological models in vitro/in vivo. During my PhD, I had the opportunity to combine quantitative methods of analysis with high resolution imagery techniques (confocal microscopy, transmission electron microscopy and ion beam analysis) in order to characterize: (i) the skin barrier function of an ex vivo pig ear skin model understanding the ion homeostasis behavior face to different stresses, chemical or physical (Collaboration with Hélène Duplan, Pierre Fabre Research Institute), (ii) the impact on viability, accumulation and intracellular distribution of nanoparticles (Titanium Oxides) naked or functionalized with fluorescent dyes (FITC, Rhodamine) (Collaboration with M.H. Delville, Bordeaux Institute of Condense Matter Chemistry). Results show the possibility to define (i) the role of ions in skin barrier function of a biological ex vivo model in native conditions and after exposure to different stresses (ii) in vivo toxicology of manufactured nanoparticles (iii) their future in a biological model of interest (keratinocytes)BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Nanoparticules et santé (des applications aux risques potentiels)

Les nanotechnologies constituent un champ de recherche et de développement important depuis le début du XXIème siècle. Elles reposent sur la capacité à manipuler la matière à l échelle du nanomètre (10-9 m) voire de l atome. L utilisation des nanoparticules au niveau mondial ne cesse de s accroître notamment sur le plan médical, environnemental et industriel. Elles permettent la création de nouvelles classes de matériaux aux propriétés innovantes. Ainsi, ces nanoparticules produites intentionnellement sont utilisées dans des produits cosmétiques et d hygiène, dentifrices, additifs alimentaires, articles de sport, pneus... De plus, leurs propriétés particulières en font des outils extrêmement intéressants dans plusieurs domaines de la recherche fondamentale et clinique, tels que l oncologie, l imagerie médicale, le diagnostic, l administration de médicaments, la fabrication de biomatéri! aux, ou encore la vaccination. Il est indispensable de clarifier l impact de ces nanoparticules sur la santé en raison de leur multiplicité et des différentes voies d exposition possibles. Il est à ce jour difficile de se prononcer quant à l innocuité de ces éléments, les résultats étant parfois contradictoires, à l instar des données actuelles relatives à la pénétration de la barrière cutanée par les nanoparticules du dioxyde de titane (TiO2) contenues dans les dermo-cosmétiques. L étude du devenir des nanoparticules dans notre organisme (toxicocinétique) et de leurs effets cellulaires (stress oxydatif, cytotoxicité, génotoxicité) reste indispensable afin de garantir leur sécurité d utilisation. Ce manuscrit propose une introduction générale aux nanotechnologies et à leurs applications par une approche du nanomonde et l étude particulière de l éventuelle toxicité du TiO2 contenu dans les topiques cosmétiques.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Mise en évidence de cassures double brin de l'ADN induites par irradiation de kératinocytes humains en microfaisceau alpha

Comprendre les modes d'interaction des rayonements ionisants avec la matière vivante, notamment lors de l'exposition à de faibles doses telles que celles que l'on peut trouver dans un environnement industriel ou dans la nature, reste un enjeu majeur pour l'évaluation du risque associé. Il s'agit d'un problème de société qui n'a pu malheureusement trouver de réponse dans les études épidémiologiques classiques dans la mesure où les seules données fiables concernent des expositions accidentelles à des doses beaucoup plus élevées. L'exposition naturelle représente pourtant la première source dans la vie courante juste devant les sources d'origines médicales (radiologie, radiothérapie). De plus, ce type d'exposition est très difficile à reproduire en laboratoire sur des lignées cellulaires. La méthode principalement utilisée, basée sur l'irradiation aléatoire de populations cellulaires, consiste à calculer le nombre moyen de particules ayant interagi par cellule et repose ainsi sur des lois de distributions statistique (loi de Poisson). En plus des inévitables impacts multiples, la variété des cibles intracellulaires touchées (noyau, cytoplasme), les effets indirects induits par des impacts sur les cellules voisines ou simplement extracellulaires sont autant de phénomènes qui compliquent sérieusement l'interprétation des données. Un microfaisceau de particules alpha a été developpé au CENBG pour réaliser une irradiation ciblée à l'échelle sub-cellulaire avec une précision de quelques micromètres. Il est ainsi possible de contrôler le nombre exact de particules délivrées par cellule (jusqu'à la dose ultime d'un ion par cellule), de prédéterminer avec précision le point d'impact et d'irradier certaines cellules tout en vérifiant la réponse de cellules voisines. La valisation de ce dispositif a été rééalisée au cours de ce travail de thèse, sur des keratinocytes humains exprimant une protéine recombinante nucléaire fluorescente (histone H2B-GFP) en mettant en évidence des dommages nucléaires radio-induits spécifiques et dose-dépendant. La combinaison des techniques en microfaisceau d'ions, de microscopie confocale et d'analyse quantitative numérique a permis de mesurer, in situ et à l'échelle de la cellule unique, la cinétique de phosphorylation de la protéine histone H2A.X et d'aborder l'étude des processus de réparation de l'ADN et d'induction de l'apoptose. Les résultats expérimentaux valident la méthodologie développée en démontrant la reproductibilité du tir et le contrôle de dose à la mise en évidence d'une relation dose-effet qui a été également étudiée en fonction du temps.BORDEAUX1-BU Sciences-Talence (335222101) / SudocSTRASBOURG-Bib.Central Recherche (674822133) / SudocSudocFranceF

2003 International Friedreich's Ataxia Research Conference, 14-16 February 2003, Bethesda, MD, USA

No abstract availabl

Titanium dioxide nanoparticles induced intracellular calcium homeostasis modification in primary human keratinocytes. Towards an in vitro explanation of titanium dioxide nanoparticles toxicity.

Abstract Deciphering the molecular basis of toxicology mechanism induced by nanoparticles (NPs) remains an essential challenge. Ion Beam Analysis (IBA) was applied in combination with Transmission Electron Microscopy and Confocal Microscopy to analyze human keratinocytes exposed to TiO(2)-NPs. Investigating chemical elemental distributions using IBA gives rise to a fine quantification of the TiO(2)-NPs uptake within a cell and to the determination of the intracellular chemical modifications after TiO(2)-NPs internalization. In addition, fluorescent dye-modified TiO(2)-NPs have been synthesized to allow their detection, precise quantification and tracking in vitro. The internalization of these TiO(2)-NPs altered the calcium homeostasis and induced a decrease in cell proliferation associated with an early keratinocyte differentiation, without any indication of cell death. Additionally, the relation between the surface chemistry of the TiO(2)-NPs and their in vitro toxicity is clearly established and emphasizes the importance of the calcium homeostasis alteration in response to the presence of TiO(2)-NPs

Proton Microbeam Targeted Irradiation of the Gonad Primordium Region Induces Developmental Alterations Associated with Heat Shock Responses and Cuticle Defense in Caenorhabditis elegans

International audienceWe describe a methodology to manipulate Caenorhabditis elegans (C. elegans) and irradiate the stem progenitor gonad region using three MeV protons at a specific developmental stage (L1). The consequences of the targeted irradiation were first investigated by considering the organogenesis of the vulva and gonad, two well-defined and characterized developmental systems in C. elegans. In addition, we adapted high-throughput analysis protocols, using cell-sorting assays (COPAS) and whole transcriptome analysis, to the limited number of worms (>300) imposed by the selective irradiation approach. Here, the presented status report validated protocols to (i) deliver a controlled dose in specific regions of the worms; (ii) immobilize synchronized worm populations (>300); (iii) specifically target dedicated cells; (iv) study the radiation-induced developmental alterations and gene induction involved in cellular stress (heat shock protein) and cuticle injury responses that were found

Recruitment Kinetics of XRCC1 and RNF8 Following MeV Proton and α-Particle Micro-Irradiation

International audienceTime-lapse fluorescence imaging coupled to micro-irradiation devices provides information on the kinetics of DNA repair protein accumulation, from a few seconds to several minutes after irradiation. Charged-particle microbeams are valuable tools for such studies since they provide a way to selectively irradiate micrometric areas within a cell nucleus, control the dose and the micro-dosimetric quantities by means of advanced detection systems and Monte Carlo simulations and monitor the early cell response by means of beamline microscopy. We used the charged-particle microbeam installed at the AIFIRA facility to perform micro-irradiation experiments and measure the recruitment kinetics of two proteins involved in DNA signaling and repair pathways following exposure to protons and α-particles. We developed and validated image acquisition and processing methods to enable a systematic study of the recruitment kinetics of GFP-XRCC1 and GFP-RNF8. We show that XRCC1 is recruited to DNA damage sites a few seconds after irradiation as a function of the total deposited energy and quite independently of the particle LET. RNF8 is recruited to DNA damage sites a few minutes after irradiation and its recruitment kinetics depends on the particle LET

Accuracy of three-dimensional proton imaging of an inertial confinement fusion target assessed by Geant4 simulation

International audience•Proton micro-tomography experiments have been simulated using Geant4.•Geant4 gives access to information that is not possible to get from experiments.•A phantom of inertial confinement fusion target was constructed.•Accuracy of mass density images reconstructed by different methods was assessed.•Accuracy of the corrections implemented for X-ray attenuation was evaluated. The Geant4 toolkit was used to perform benchmark Monte Carlo simulations of proton micro-tomography imaging. A phantom of an inertial confinement fusion (ICF) target was designed, based on experimental data. Simulations of STIM (Scanning Transmission Ion Microscopy) and PIXE (Particle Induced X-ray Emission) tomography were performed. Quantitative images were obtained from the TomoRebuild and JPIXET software packages, chosen for their ability to handle large solid angles of X-ray detection. The tomographic images were compared with the original phantom, used as a reference. For STIM-T, the accuracy of the calculated mass density was ≤ 2 % for TomoRebuild and ≤ 14 % for JPIXET. Corrections of X-ray production cross section and X-ray absorption were tested for the quantification of Ge, used as a dopant in the ICF target. The accuracy on the obtained Ge density was ≤ 2.9 % for TomoRebuild and ≤ 6.4 % for JPIXET, whereas the error was about 40 % without correction

Idebenone delays the onset of cardiac functional alteration without correction of Fe-S enzymes deficit in a mouse model for Friedreich ataxia

Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associated with cardiomyopathy, is caused by severely reduced frataxin, a mitochondrial protein involved in Fe-S cluster assembly. We have recently generated mouse models that reproduce important progressive pathological and biochemical features of the human disease. Our frataxin-deficient mouse models initially demonstrate time-dependent intramitochondrial iron accumulation, which occurs after onset of the pathology and after inactivation of the Fe-S dependent enzymes. Here, we report a more detailed pathophysiological characterization of our mouse model with isolated cardiac disease by echocardiographic, biochemical and histological studies and its use for placebo-controlled therapeutic trial with Idebenone. The Fe-S enzyme deficiency occurs at 4 weeks of age, prior to cardiac dilatation and concomitant development of left ventricular hypertrophy, while the mitochondrial iron accumulation occurs at a terminal stage. From 7 weeks onward, Fe-S enzyme activities are strongly decreased and are associated with lower levels of oxidative stress markers, as a consequence of reduced respiratory chain activity. Furthermore, we demonstrate that the antioxidant Idebenone delays the cardiac disease onset, progression and death of frataxin deficient animals by 1 week, but does not correct the Fe-S enzyme deficiency. Our results support the view that frataxin is a necessary, albeit non-essential, component of the Fe-S cluster biogenesis, and indicate that Idebenone acts downstream of the primary Fe-S enzyme deficit. Furthermore, our results demonstrate that Idebenone is cardioprotective even in the context of a complete lack of frataxin, which further supports its utilization for the treatment of FRDA