Immortalized Skin Fibroblasts Expressing Conditional MyoD as a Renewable and Reliable Source of Converted Human Muscle Cells to Assess Therapeutic Strategies for Muscular Dystrophies: Validation of an Exon-Skipping Approach to Restore Dystrophin in Duchenne Muscular Dystrophy Cells

Chaouch, Soraya; Mouly, Vincent; Goyenvalle, Aurélie; Vulin, Adeline; Mamchaoui, Kamel; Négroni, Elisa; Santo, James P. Di; Butler-Browne, Gillian; Torrente, Yvan; Garcı́a, Luis; Furling, Denis

doi:10.1089/hum.2008.163

Cited by 62 publications

(59 citation statements)

References 17 publications

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“…Myoblast Splicing Assay-Two human fibroblast cell lines with a doxycycline-inducible MyoD construct for differentiation into myoblasts were kindly provided by Denis Furling (Université Pierre et Marie Curie-Paris, Paris, France) (31). The WT control cell line was derived from a 19-year-old healthy individual with a normal CTG repeat length in the DMPK locus, whereas the DM1 cell line was derived from an 11-yearold individual with a CTG 1300 repeat length in the same locus.…”

Section: Methodsmentioning

confidence: 99%

“…Identified Alkaloids Improve Splicing in Human DM1 Myoblasts-The alkaloids identified in our in vitro screening assay were tested for their ability to ameliorate splicing in a human DM1 myoblast cell line containing a CTG 1300 repeat in the 3Ј UTR of the DMPK gene (31). The WT control cell line contained a normal repeat length in the same locus.…”

Section: Cug 78 -Mbnl1 Complex Inhibitors Of Naturalmentioning

confidence: 99%

“…We identified several alkaloids as CUG 78 -MBNL1 complex inhibitors. Testing their bioactivity in a human myoblast model of DM1 (31) and in the human skeletal ␣-actin long repeat (HSA LR ) mouse model of DM1 (4) showed that the alkaloids ameliorate certain aspects of the DM1 pathology.…”

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confidence: 99%

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Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Herrendorff

Faleschini

Stiefvater

et al. 2016

Journal of Biological Chemistry

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Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3 UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG) n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG) n -MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG) n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ␤-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases. Myotonic dystrophy type I (DM1)4 is the most common muscular dystrophy in the adult population, with a relatively high prevalence of about 1:8000 (1). This autosomal dominantly inherited disease affects multiple organs, most prominently the skeletal muscle, with wasting, weakness, and an inability to relax (myotonia) (1). Currently, there is no effective treatment for this disabling disease. The pathomechanism of DM1 is linked to a CTG n expansion in the 3Ј UTR of the dystrophia myotonica protein kinase (DMPK) gene (2, 3), leading to a toxic gain-of-function RNA (4, 5). The mutant DMPK transcript is entrapped within nuclei of affected cells, where it forms aggregates (foci) with splicing factors such as muscleblind-like 1 (MBNL1) (6, 7). Bound to mutant DMPK (CUG) n RNA, MBNL1 is no longer available for correct splicing of its target pre-mRNAs (8, 9). Thus, the splicing of a multitude of pre-mRNAs is misregulated, including the skeletal muscle chloride channel (CLCN1), the insulin receptor (INSR), sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATPase 1 (SERCA1), and cardiac troponin T type 2 (TNNT2) pre-mRNA (10 -16). Interestingly, the missplicing of some pre-mRNAs can be linked directly to a certain disease symptom, e.g. myotonia in the case of the CLCN1 pre-mRNA. MBNL1 sequestration by (CUG) n RNA causes inclusion of alternative exon 7a, leading to a shift in the open reading frame and to premature termination of translation (12, 1...

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Section: Methodsmentioning

confidence: 99%

Section: Cug 78 -Mbnl1 Complex Inhibitors Of Naturalmentioning

confidence: 99%

See 1 more Smart Citation

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Herrendorff

Faleschini

Stiefvater

et al. 2016

Journal of Biological Chemistry

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“…G. Kollberg and E. Holme (Sahlgrenska University Hospital, Goteborg, Sweden) provided the original frozen cells (second passage) and suitable age-matched normal controls. Before use, both cell lines were immortalized with plasmid CMVhTERT/PGK-Pura, as described previously (36). The cells for the experiments were seeded at 0.35 × 10 6 cells/10-cm dish and grown in MEM/10% FCS.…”

Section: Methodsmentioning

confidence: 99%

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Pontarin

Ferraro

Bee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

114

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In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage.DNA precursors | dNTP de novo synthesis | cell cycle | mitochondrial disease D NA replication and repair require the continued synthesis of the four dNTPs. They are synthesized by evolutionary-related ribonucleotide reductases operating with slightly different mechanisms in aerobic and anaerobic organisms (1). Each ribonucleotide reductase provides the required amounts of all four dNTPs. A similar allosteric mechanism, maintained throughout evolution, regulates both the enzyme's activity and its substrate specificity. Cells contain small dNTP pools of similar sizes, approximately 10-fold larger during DNA replication than during quiescence. Regulation of pool sizes by ribonucleotide reductases is of great importance for correct DNA replication, and changes in the actual sizes or in their balance lead to increased mutation rates (2). For mammalian cells, the induction of mutations by pool imbalances has been described in detail, along with possible mechanisms (3). In yeast, a recent elegant study (4) linked specific amino acid substitutions in the catalytic subunit of ribonucleotide reductase to defined pool imbalances, which result in increased mutation rates.In mammalian cells, the canonical ribonucleotide reductase is a complex between two proteins: the large catalytic protein R1 that contains the allosteric sites and the smaller protein R2 that contributes a stable tyrosyl free radical during the reaction (1). Both proteins are transcriptionally activated during early S-phase (5) a...

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“…MyoD-transduced fibroblasts from control individuals and patients were cultured in growth medium (DMEM, 10% FBS, 50 μg/ml gentamicin) at 37°C under 5% CO 2 . At confluence, transduction into myoblasts was induced by differentiation medium (DMEM, 50 μg/ml gentamicin, 3 μg/ml doxycycline hyclate, 10 μg/ml human recombinant insulin) (67). Myotubes obtained after 10 days were incubated for 3 hours in growth medium (refed), PBS (starved), or PBS supplemented with chloroquine (100 μM).…”

Section: Author Contributionsmentioning

confidence: 99%

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

Brockhoff¹,

Rion²,

Chojnowska³

et al. 2017

Journal of Clinical Investigation

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Immortalized Skin Fibroblasts Expressing Conditional MyoD as a Renewable and Reliable Source of Converted Human Muscle Cells to Assess Therapeutic Strategies for Muscular Dystrophies: Validation of an Exon-Skipping Approach to Restore Dystrophin in Duchenne Muscular Dystrophy Cells

Cited by 62 publications

References 17 publications

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

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