Des mécanismes moléculaires pathologiques aux stratégies de correction génomique in vitro de la Dystrophie Facio-Scapulo-Humérale

Facioscapulohumeral dystrophy (FSHD) is one of the most common genetic myopathies characterized by a progressive and asymmetric weakening of a specific group of skeletal muscles, typically facial, shoulder girdle and upper arms muscles. FSHD is a multifactorial disease that results from the combination of genetic and epigenetic events mapped at the 4q35 locus. These genetic and epigenetic alterations lead to chromatin relaxation and the subsequent overexpression of the majority of 4q35 genes, notably DUX4, the major actor in FSHD pathology. These genomic alterations lead to molecular and cellular defects observed in vitro. Cultured-FSHD myoblasts show a distinct transcription profile, they exhibit morphological differentiation defects and are sensitive to oxidative stress. Several aspects of the disease remain poorly understood, and the elaboration of an appropriate therapeutic strategy is limited by the complexity of this myopathy. However, the discovery of genome editing tools and their successful therapeutic applications in vitro and in animal models of several human diseases, including myopathies, open doors to potential therapeutic strategies for FSHD.This work highlighted the involvement of DNA damage and oxidative stress in the pathophysiology of FSHD, by revealing their constitutive presence in FSHD myoblasts, their link to DUX4 expression and their participation in morphological defects of FSHD myotubes observed in vitro. The second part of this work was aimed at developing genome- and epigenome-editing tools capable of specifically targeting one of the genetic events causing FSHD, a pathogenic variant 4qA161 that contains an insulator and a nuclear matrix attachment site (FR-MAR). These engineered tools will be then used to develop in vitro therapeutic strategies, with the intention of restoring the insulator activity of FR-MAR and the chromatin organization of 4q35 locus.La dystrophie Facio-Scapulo-Humérale (FSHD) fait partie des maladies musculaires génétiques les plus fréquentes. Elle se caractérise par une dégénérescence progressive et asymétrique d’un groupe spécifique de muscles striés squelettiques, dont principalement les muscles faciaux, scapulaires et huméraux. D’un point de vue génétique, la FSHD est une maladie multifactorielle qui résulte d’évènements génétiques situés sur la région sub-télomérique du chromosome 4, ainsi que d’évènements épigénétiques altérant l’organisation chromatinienne du locus 4q35. Ces anomalies provoquent une relaxation chromatinienne et une surexpression de la majorité des gènes du locus 4q35, dont DUX4, gène majeur impliqué dans la FSHD. Les répercussions de l’ensemble de ces altérations se traduisent notamment par une dérégulation de la signature transcriptionnelle des myoblastes primaires issus des patients FSHD, et par des anomalies de leur différenciation myogénique in vitro et leur hypersensibilité au stress oxydant. Plusieurs aspects de la maladie demeurent incompris, et la complexité de cette myopathie rend difficile le choix d’une stratégie thérapeutique optimale. Cependant, la découverte des outils de l’édition du génome et la multiplication de leurs applications à visée thérapeutique dans le cadre de maladies humaines, notamment les myopathies, ouvre de nouvelles perspectives pour la FSHD qui reste, jusque-là, incurable.Le travail de thèse a concerné, dans un premier temps, l’implication des dommages de l’ADN et du stress oxydant dans la pathophysiologie de la FSHD. Nous avons mis en évidence l’omniprésence de ces caractéristiques cellulaires dans les myoblastes FSHD, leur lien à l’expression aberrante de DUX4 et leur participation à la morphologie défectueuse des myotubes FSHD in vitro. Dans un second temps, le travail de thèse a consisté à concevoir et à développer des outils de l’édition génomique et épigénomique, capables de cibler spécifiquement un des évènements génétiques causal de la FSHD, le variant pathogénique 4qA161 touchant un site d’attachement à la matrice nucléaire, FR-MAR. A partir de ces outils développés, deux stratégies de corrections génomique et épigénomique à visée thérapeutique peuvent être alors envisagées in vitro, ayant pour but ultime de rétablir la fonction d’insulation de FR-MAR et la conformation chromatinienne de la région 4q35

Molecular mechanisms and in vitro genome correction strategies of Facioscapulohumeral dystrophy

La dystrophie Facio-Scapulo-Humérale (FSHD) fait partie des maladies musculaires génétiques les plus fréquentes. Elle se caractérise par une dégénérescence progressive et asymétrique d’un groupe spécifique de muscles striés squelettiques, dont principalement les muscles faciaux, scapulaires et huméraux. D’un point de vue génétique, la FSHD est une maladie multifactorielle qui résulte d’évènements génétiques situés sur la région sub-télomérique du chromosome 4, ainsi que d’évènements épigénétiques altérant l’organisation chromatinienne du locus 4q35. Ces anomalies provoquent une relaxation chromatinienne et une surexpression de la majorité des gènes du locus 4q35, dont DUX4, gène majeur impliqué dans la FSHD. Les répercussions de l’ensemble de ces altérations se traduisent notamment par une dérégulation de la signature transcriptionnelle des myoblastes primaires issus des patients FSHD, et par des anomalies de leur différenciation myogénique in vitro et leur hypersensibilité au stress oxydant. Plusieurs aspects de la maladie demeurent incompris, et la complexité de cette myopathie rend difficile le choix d’une stratégie thérapeutique optimale. Cependant, la découverte des outils de l’édition du génome et la multiplication de leurs applications à visée thérapeutique dans le cadre de maladies humaines, notamment les myopathies, ouvre de nouvelles perspectives pour la FSHD qui reste, jusque-là, incurable.Le travail de thèse a concerné, dans un premier temps, l’implication des dommages de l’ADN et du stress oxydant dans la pathophysiologie de la FSHD. Nous avons mis en évidence l’omniprésence de ces caractéristiques cellulaires dans les myoblastes FSHD, leur lien à l’expression aberrante de DUX4 et leur participation à la morphologie défectueuse des myotubes FSHD in vitro. Dans un second temps, le travail de thèse a consisté à concevoir et à développer des outils de l’édition génomique et épigénomique, capables de cibler spécifiquement un des évènements génétiques causal de la FSHD, le variant pathogénique 4qA161 touchant un site d’attachement à la matrice nucléaire, FR-MAR. A partir de ces outils développés, deux stratégies de corrections génomique et épigénomique à visée thérapeutique peuvent être alors envisagées in vitro, ayant pour but ultime de rétablir la fonction d’insulation de FR-MAR et la conformation chromatinienne de la région 4q35.Facioscapulohumeral dystrophy (FSHD) is one of the most common genetic myopathies characterized by a progressive and asymmetric weakening of a specific group of skeletal muscles, typically facial, shoulder girdle and upper arms muscles. FSHD is a multifactorial disease that results from the combination of genetic and epigenetic events mapped at the 4q35 locus. These genetic and epigenetic alterations lead to chromatin relaxation and the subsequent overexpression of the majority of 4q35 genes, notably DUX4, the major actor in FSHD pathology. These genomic alterations lead to molecular and cellular defects observed in vitro. Cultured-FSHD myoblasts show a distinct transcription profile, they exhibit morphological differentiation defects and are sensitive to oxidative stress. Several aspects of the disease remain poorly understood, and the elaboration of an appropriate therapeutic strategy is limited by the complexity of this myopathy. However, the discovery of genome editing tools and their successful therapeutic applications in vitro and in animal models of several human diseases, including myopathies, open doors to potential therapeutic strategies for FSHD.This work highlighted the involvement of DNA damage and oxidative stress in the pathophysiology of FSHD, by revealing their constitutive presence in FSHD myoblasts, their link to DUX4 expression and their participation in morphological defects of FSHD myotubes observed in vitro. The second part of this work was aimed at developing genome- and epigenome-editing tools capable of specifically targeting one of the genetic events causing FSHD, a pathogenic variant 4qA161 that contains an insulator and a nuclear matrix attachment site (FR-MAR). These engineered tools will be then used to develop in vitro therapeutic strategies, with the intention of restoring the insulator activity of FR-MAR and the chromatin organization of 4q35 locus

Effects of cellular senescence on metabolic pathways in non-immune and immune cells

•Senescent cells are metabolically active.•Cellular senescence induces metabolic changes in non-immune and immune cells.•Senescent cells are flexible in the use of different metabolic pathways.•Metabolic reprogramming in senescent cells leads to increased survival and supports function. Many cellular stresses induce cellular senescence and the irreversible arrest of cell proliferation in different cell types. Although blocked in their capacity to divide, senescent cells are metabolically active and are characterized by a different metabolic phenotype as compared to non-senescent cells. Changes observed in senescent cells depend from the cell type and lead to an adaptative flexibility in the type of metabolism. This metabolic reprogramming is needed to cope with survival and with the energetic demands of the senescent program that include the increased secretion of senescence-associated secretory phenotype factors

Control of DNA integrity in skeletal muscle under physiological and pathological conditions

International audienceSkeletal muscle is a highly oxygen-consuming tissue that ensures body support and movement, as well as nutrient and temperature regulation. DNA damage induced by reactive oxygen species is present in muscles and tends to accumulate with age. Here, we present a summary of data obtained on DNA damage and its implication in muscle homeostasis, myogenic differentiation and neuromuscular disorders. Controlled and transient DNA damage appears to be essential for muscular homeostasis and differentiation while uncontrolled and chronic DNA damage negatively affects muscle health

Facioscapulohumeral dystrophy myoblasts efficiently repair moderate levels of oxidative DNA damage.

International audienceFacioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24 h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants

A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of Genomic DNA-Editing Tools

International audienceDespite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools

Protective effect of Rhus coriaria fruit extracts against hydrogen peroxide-induced oxidative stress in muscle progenitors and zebrafish embryos

Background and Purpose Oxidative stress is involved in normal and pathological functioning of skeletal muscle. Protection of myoblasts from oxidative stress may improve muscle contraction and delay aging. Here we studied the effect of R. coriaria sumac fruit extract on human myoblasts and zebrafish embryos in conditions of hydrogen peroxide-induced oxidative stress. Study Design and Methods Crude ethanolic 70% extract (CE) and its fractions was obtained from sumac fruits. The composition of sumac ethyl acetate EtOAc fraction was studied by 1H NMR. The viability of human myoblasts treated with CE and the EtOAc fraction was determined by trypan blue exclusion test. Oxidative stress, cell cycle and adhesion were analyzed by flow cytometry and microscopy. Gene expression was analyzed by qPCR. Results The EtOAc fraction (IC50 2.57 µg/mL) had the highest antioxidant activity and exhibited the best protective effect against hydrogen peroxide-induced oxidative stress. It also restored cell adhesion. This effect was mediated by superoxide dismutase 2 and catalase. Pre-treatment of zebrafish embryos with low concentrations of the EtOAc fraction protected them from hydrogen peroxide-induced death in vivo. 1H NMR analysis revealed the presence of gallic acid in this fraction. Conclusion Rhus coriaria extracts inhibited or slowed down the progress of skeletal muscle atrophy by decreasing oxidative stress via superoxide dismutase 2 and catalase-dependent mechanisms

DUX4-induced constitutive DNA damage and oxidative stress contribute to aberrant differentiation of myoblasts from FSHD patients

International audienceFacioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts

A homoeostatic switch causing glycerol-3-phosphate and phosphoethanolamine accumulation triggers senescence by rewiring lipid metabolism

International audienceCellular senescence affects many physiological and pathological processes and is characterized by durable cell cycle arrest, an inflammatory secretory phenotype and metabolic reprogramming. Here, by using dynamic transcriptome and metabolome profiling in human fibroblasts with different subtypes of senescence, we show that a homoeostatic switch that results in glycerol-3-phosphate (G3P) and phosphoethanolamine (pEtN) accumulation links lipid metabolism to the senescence gene expression programme. Mechanistically, p53-dependent glycerol kinase activation and post-translational inactivation of phosphate cytidylyltransferase 2, ethanolamine regulate this metabolic switch, which promotes triglyceride accumulation in lipid droplets and induces the senescence gene expression programme. Conversely, G3P phosphatase and ethanolamine-phosphate phospho-lyase-based scavenging of G3P and pEtN acts in a senomorphic way by reducing G3P and pEtN accumulation. Collectively, our study ties G3P and pEtN accumulation to controlling lipid droplet biogenesis and phospholipid flux in senescent cells, providing a potential therapeutic avenue for targeting senescence and related pathophysiology