Friedreich's ataxia: the vicious circle hypothesis revisited

Friedreich's ataxia, the most frequent progressive autosomal recessive disorder involving the central and peripheral nervous systems, is mostly associated with unstable expansion of GAA trinucleotide repeats in the first intron of the FXN gene, which encodes the mitochondrial frataxin protein. Since FXN was shown to be involved in Friedreich's ataxia in the late 1990s, the consequence of frataxin loss of function has generated vigorous debate. Very early on we suggested a unifying hypothesis according to which frataxin deficiency leads to a vicious circle of faulty iron handling, impaired iron-sulphur cluster synthesis and increased oxygen radical production. However, data from cell and animal models now indicate that iron accumulation is an inconsistent and late event and that frataxin deficiency does not always impair the activity of iron-sulphur cluster-containing proteins. In contrast, frataxin deficiency appears to be consistently associated with increased sensitivity to reactive oxygen species as opposed to increased oxygen radical production. By compiling the findings of fundamental research and clinical observations we defend here the opinion that the very first consequence of frataxin depletion is indeed an abnormal oxidative status which initiates the pathogenic mechanism underlying Friedreich's ataxia

Friedreich's Ataxia, Frataxin, PIP5K1B: Echo of a Distant Fracas

“Frataxin fracas” were the words used when referring to the frataxin-encoding gene (FXN) burst in as a motive to disqualify an alternative candidate gene, PIP5K1B, as an actor in Friedreich's ataxia (FRDA) (Campuzano et al., 1996; Cossee et al., 1997; Carvajal et al., 1996). The instrumental role in the disease of large triplet expansions in the first intron of FXN has been thereafter fully confirmed, and this no longer suffers any dispute (Koeppen, 2011). On the other hand, a recent study suggests that the consequences of these large expansions in FXN are wider than previously thought and that the expression of surrounding genes, including PIP5K1B, could be concurrently modulated by these large expansions (Bayot et al., 2013). This recent observation raises a number of important and yet unanswered questions for scientists and clinicians working on FRDA; these questions are the substratum of this paper

Rôle des protéases Pim1/Lon et ClpP dans la mitochondrie ( implication en conditions de stress et dans le vieillissement)

Un dysfonctionnement mitochondrial a été mis en cause dans le processus de vieillissement, en lien avec une augmentation du niveau de stress oxydant et une accumulation de protéines oxydées dans les cellules. Les systèmes protéolytiques occupent une place importante en éliminant les protéines altérées produites en condition de stress. Dans le cytosol, le protéasome constitue le principal système de dégradation des protéines oxydées. Dans les mitochondries, les protéases Pim1/Lon et ClpP sont localisées dans la matrice et participent au renouvellement des protéines endommagées. Plusieurs études soulignent leur importance dans la réponse au stress dans la mitochondrie. La protéase Pim1/Lon est également impliquée dans le maintien des fonctions mitochondriales. Mon objectif pendant cette thèse a été de préciser le rôle de ces deux protéases dans la mitochondrie, notamment en conditions de stress. L implication de la protéase Pim1 dans le vieillissement a aussi été analysée. Nous avons identifié les substrats endogènes de la protéase Pim1 (S. cerevisiae) en utilisant un modèle de levure 0 (dépourvu d ADNmt). Des protéines partenaires de Pim1 ont également été obtenues. Ce travail a permis de mettre en évidence le rôle prépondérant de la protéase Pim1 en condition de stress spécifique de la mitochondrie. Des précisions quant à ses fonctions dans l organite ont aussi été apportées. Dans un deuxième temps, une diminution de la longévité a été observée chez une souche pim1. Etonnamment, ce vieillissement prématuré met en jeu un dysfonctionnement cytosolique. Nous avons ensuite réussi à reverser ce phénotype de vieillissement par surexpression d Hsp104, validant de cette manière les mécanismes mis à jour. D autre part, nous avons établi des lignées de cellules humaines permettant d éteindre l une ou l autre des protéases Lon ou ClpP par une approche shRNA inductible. Ces cellules ont été caractérisées en conditions basales, en portant une attention particulière aux fonctions mitochondriales, en lien avec la prolifération et la mort cellulaire. A l exception d une baisse de prolifération, aucun phénotype n a été observé dans les cellules déficientes en ClpP. En revanche, l extinction de la protéase Lon révèle une altération du réseau mitochondrial et semble indiquer une accumulation de protéines non dégradées dans les mitochondries. A terme, ces modèles cellulaires présentent un intérêt pour identifier les substrats de ces deux protéases et pour évaluer leur rôle en conditions de stressPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress.

International audienceFriedreich's ataxia is generally associated with defects in [Fe-S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich's ataxia to study the physiological consequences of modulating the expression of the frataxin gene (YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2muYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe-S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2muYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2muYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2muYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe-S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different

Mitochondrial dysfunction caused by novel ATAD3A mutations

International audienceMitochondrial respiratory chain integrity depends on a number of proteins encoded by nuclear and mitochondrial genomes. Mutations of such factors can result in isolated or combined respiratory chain deficits, some of which can induce abnormal morphology of the mitochondrial network or accumulation of intermediary metabolites. Consequently, affected patients are clinically heterogeneous, presenting with central nervous system, muscular, or neurodegenerative disorders. ATAD3A is a nuclear-encoded ATPase protein of the AAA+ family and has been localized to the inner mitochondrial membrane. Recently reported mutations or large deletions in the ATDA3A gene in patients have been shown to induce altered mitochondrial structure and function and abnormal cholesterol metabolism in a recessive or dominant manner. Here, we report two siblings presenting axonal sensory-motor neuropathy associated with neonatal cataract. Genetic analyses identified two novel mutations in ATAD3A; a point mutation and an intronic 15 bp deletion affecting splicing and leading to exon skipping. Biochemical analysis in patient cells and tissues showed abnormal function of the mitochondrial respiratory chain in muscle and abnormal mitochondrial cristae structure. These new cases underline the large spectrum of biochemical and clinical presentations of ATAD3A deficiency and the different modes of inheritance, making it an atypical mitochondrial disorder

Induced pluripotent stem cells-derived neurons from patients with Friedreich ataxia exhibit differential sensitivity to resveratrol and nicotinamide

International audienceAbstract Translation of pharmacological results from in vitro cell testing to clinical trials is challenging. One of the causes that may underlie these discrepant results is the lack of the phenotypic or species-specific relevance of the tested cells; today, this lack of relevance may be reduced by relying on cells differentiated from human pluripotent stem cells. To analyse the benefits provided by this approach, we chose to focus on Friedreich ataxia, a neurodegenerative condition for which the recent clinical testing of two compounds was not successful. These compounds, namely, resveratrol and nicotinamide, were selected because they had been shown to stimulate the expression of frataxin in fibroblasts and lymphoblastoid cells. Our results indicated that these compounds failed to do so in iPSC-derived neurons generated from two patients with Friedreich ataxia. By comparing the effects of both molecules on different cell types that may be considered to be non-relevant for the disease, such as fibroblasts, or more relevant to the disease, such as neurons differentiated from iPSCs, a differential response was observed; this response suggests the importance of developing more predictive in vitro systems for drug discovery. Our results demonstrate the value of utilizing human iPSCs early in drug discovery to improve translational predictability

Effect of Lon protease knockdown on mitochondrial function in HeLa cells.

International audience: ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI-38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type

Dymeclin deficiency causes postnatal microcephaly, hypomyelination and reticulum-to-Golgi trafficking defects in mice and humans

International audienceDymeclin is a Golgi-associated protein whose deficiency causes Dyggve–Melchior–Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation