Oxidative stress and calcium handling in mdx muscle, a model for Duchenne Muscular Dystrophy: Involvement of mitochondria and NADPH oxidase

La dystrophie musculaire de Duchenne (DMD) est une maladie génétique due à une mutation du gène codant la dystrophine situé sur le chromosome X. Elle touche 1 garçon sur 3500. Les premiers signes de faiblesse musculaire apparaissent vers l'âge de 2-3 ans puis la maladie évolue progressivement vers la mort. A l'heure actuelle, les mécanismes moléculaires conduisant à la dégénération du muscle dystrophique sont mal connus mais un rôle crucial est accordé dans la maladie à la perte de l'intégrité du cytosquelette et de la membrane de la fibre musculaire, à la dérégulation de l'homéostasie du calcium et à l'augmentation du stress oxydatif. Cette thèse met en évidence le rôle crucial de la mitochondrie et de la NADPH oxidase (NOX) dans la signalisation des radicaux libres et dans l'homéostasie du calcium dans les cellules musculaires dystrophiques. Tout d'abord, nous montrons que la mélatonine protège la fonction de la mitochondrie contre le stress oxydatif et empêche la mort par apoptose des cellules musculaires. Ensuite, comparées au souris non traitées, les souris dystrophiques traitées avec la mélatonine sont plus fortes, contractent et se relâchent plus vite et présentent une meilleure stabilité de leur membrane. Enfin, il apparaît que l'expression et l'activité de la NOX sont augmentées dans les cellules musculaires dystrophiques et que les radicaux libres générés par la NOX contribuent aux réponses calciques anormales observées dans ces cellules

Concise Review: Methods and Cell Types Used to Generate Down Syndrome Induced Pluripotent Stem Cells

Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Several models have been used to investigate the mechanisms by which the extra copy of chromosome 21 leads to the DS phenotype. In the last five years, several laboratories have been successful in reprogramming patient cells carrying the trisomy 21 anomaly into induced pluripotent stem cells, i.e., T21-iPSCs. In this review, we summarize the different T21-iPSCs that have been generated with a particular interest in the technical procedures and the somatic cell types used for the reprogramming

DYRK1A Protein, A Promising Therapeutic Target to Improve Cognitive Deficits in Down Syndrome

Down syndrome (DS) caused by a trisomy of chromosome 21 (HSA21), is the most common genetic developmental disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment, early onset of Alzheimer’s disease, congenital heart disease, hypotonia, muscle weakness and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Among the numerous protein coding genes of HSA21, dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) encodes a proline-directed serine/threonine and tyrosine kinase that plays pleiotropic roles in neurodevelopment in both physiological and pathological conditions. Numerous studies point to a crucial role of DYRK1A protein for brain defects in patients with DS. Thus, DYRK1A inhibition has shown benefits in several mouse models of DS, including improvement of cognitive behaviour. Lastly, a recent clinical trial has shown that epigallocatechine gallate (EGCG), a DYRK1A inhibitor, given to young patients with DS improved visual recognition memory, working memory performance and adaptive behaviour

Melatonin improves muscle function of the dystrophic mdx(5Cv) mouse, a model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine(®) implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD

Data in brief: Transcriptome analysis of induced pluripotent stem cells from monozygotic twins discordant for trisomy 21

Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. In this “Data in Brief” paper, we sum up the whole genome analysis by mRNA sequencing of normal and DS induced pluripotent stem cells that was recently published by Hibaoui et al. in EMBO molecular medicine

Modeling Klinefelter Syndrome Using Induced Pluripotent Stem Cells Reveals Impaired Germ Cell Differentiation

Klinefelter syndrome (KS), with an incidence between 1/600 and 1/1,000, is the main genetic cause of male infertility. Due to the lack of an accurate study model, the detailed pathogenic mechanisms by which this X chromosome aneuploidy leads to KS features remain unknown. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from a patient with KS: 47XXY-iPSCs. In order to compare the potentials of both 47XXY-iPSCs and 46XY-iPSCs to differentiate into the germ cell lineage, we developed a directed differentiation protocol by testing different combinations of factors including bone morphogenetic protein 4 (BMP4), glial-derived neurotrophic factor (GDNF), retinoic acid (RA) and stem cell factor (SCF) for 42 days. Importantly, we found a reduced ability of 47XXY-iPSCs to differentiate into germ cells when compared to 46XY-iPSCs. In particular, upon germ cell differentiation of 47XXY-iPSCs, we found a reduced proportion of cells positive for BOLL, a protein required for germ cell development and spermatogenesis, as well as a reduced proportion of cells positive for MAGEA4, a spermatogonia marker. This reduced ability to generate germ cells was not associated with a decrease of proliferation of 47XXY-iPSC-derived cells but rather with an increase of cell death upon germ cell differentiation as revealed by an increase of LDH release and of capase-3 expression in 47XXY-iPSC-derived cells. Our study supports the idea that 47XXY-iPSCs provides an excellent in vitro model to unravel the pathophysiology and to design potential treatments for KS patients

Optimization of thymidine kinase-based safety switch for neural cell therapy

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.</p

Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21

Down syndrome (trisomy 21) is the most common viable chromosomal disorder with intellectual impairment and several other developmental abnormalities. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background. The alterations observed by genetic analysis at the iPSC level and at first approximation in early development illustrate the developmental disease transcriptional signature of Down syndrome. Moreover, we observed an abnormal neural differentiation of Down syndrome iPSCs in vivo when formed teratoma in NOD-SCID mice, and in vitro when differentiated into neuroprogenitors and neurons. These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation. Furthermore, we provide novel evidence that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) on chromosome 21 likely contributes to these defects. Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects