Oxidative Stress in Disease and Aging: Mechanisms and Therapies 2016

International audienceOxidative stress (OS) is an imbalance between the formation of reactive oxygen species (ROS) and antioxidant defense mechanisms. This phenomenon increases with age and affects the normal functioning of several tissues. Furthermore, numerous chronic diseases associated with older age, such as diabetes and cardiovascular, renal, pulmonary, and skeletal muscle disorders, are also directly related to OS. Considering this relationship, the aim of many ongoing studies is to elucidate the underlying mechanisms and role of OS in disease onset and development. In particular, there is considerable emphasis on finding new therapeutic strategies for decreasing OS

Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology.</p> <p>Results</p> <p>Using RNA interference (RNAi) to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (<it>Pvalb </it>and <it>Casq1</it>), or cardiomyopathy (<it>Obscurin</it>, <it>Tcap</it>). In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels <it>in vivo </it>and <it>in vitro</it>.</p> <p>Conclusion</p> <p>Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage. Therefore, it becomes apparent that any gene therapy approaches for DMD should target early stages in muscle development to attain a maximum clinical benefit. With a clear and specific definition of the transcriptome of dystrophin deficiency, manipulation of identified dysregulated molecules downstream of dystrophin may lead to novel ameliorative approaches for DMD.</p

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knock-down and replacement of mutant expanded PABPN1

International audienc

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy

International audienc

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

<p>Abstract</p> <p>Background</p> <p>Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.</p> <p>Methods</p> <p>Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.</p> <p>Results</p> <p>The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both <it>in vitro </it>and <it>in vivo </it>after transplantation into regenerating muscle of immunodeficient mice.</p> <p>Conclusions</p> <p>Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess <it>in vivo </it>the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.</p

Transfert de gène in vivo:<br />étude, régulation et application de l'électrotransfert

We have been dealing with different projects using a physical method of in vivo gene transfer,called electrotransfer, widely used in mouse muscle and skin with high gene expression.By in vivo optical imaging, we have been working on optimization of electrotransfer. We havedeveloped an artificial regulation system to in vivo control an exogenous gene expression based onthe antisense technology using muscular electrotransfer in mouse. We have worked on molecularoptimization of DNA electrotransfer for genetic immunization for botulinum antigen screening.This kind of DNA immunization has been used to induce antibodies production in mouse andrabbit. We have been studying the methylation status and the reasons for persistence of plasmidDNA in muscle cells after electrotransfer since intramuscularly injected and electrotransferredplasmid DNA leads to a persisting expression for more than a year.L'électrotransfert est une technique de transfert de gène in vivo, permettant une expressionélevée du transgène dans un grand nombre de tissus. Dans ce contexte, nous avons utilisé l'imagerieoptique pour optimiser différents protocoles d'électrotransfert dans le muscle et la peau.Nous avons développé un système de régulation de l'expression des gènes combinant le systèmede régulation répressible à la tétracycline Tet-Off et une stratégie antisens. Nous avons examinéle potentiel de l'électrotransfert d'ADN plasmidique pour l'obtention d'anticorps neutralisants àhaut titre contre les toxines botuliques A, B et E. Les animaux immunisés avec des plasmidescodant les fragments immunogènes des toxines expriment de façon endogène ces fragments detoxines, qui servent d'antigènes et déclenchent une réponse immune. Nous avons finalement étudiél'état de méthylation de l'ADN plasmidique après injection et électrotransfert dans le musclesquelettique et sa possible intégration dans le génome

Transfert de gène in vivo (étude, régulation et application de l' électrotransfert)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF