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      Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1

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          Summary

          There is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases.

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          Highlights

          • •

            Myotonic dystrophy type 1 hPSCs were adapted for high content screening

          • •

            FDA-approved cardiac glycosides normalize in vitro and in vivo DM1 biological markers

          • •

            Cardiac glycosides synergize with the ERK pathway to normalize DM1 biomarkers

          • •

            This study emphasizes the value of human pluripotent stem cells for drug discovery

          Abstract

          Physiology; Molecular Biology; Cell Biology

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          Most cited references44

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          Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy.

          R S Savkur, A. V. Philips, T A Cooper … (2001)
          Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.
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            Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2.

            A Mankodi, G C Urbinati, M Krym … (2001)
            The phenotypes in myotonic dystrophy types 1 and 2 (DM1 and DM2) are similar, suggesting a shared pathophysiologic mechanism. DM1 is caused by expansion of a CTG repeat in the DMPK gene. Pathogenic effects of this mutation are likely to be mediated, at least in part, by the expanded CUG repeat in mutant mRNA. The mutant transcripts are retained in the nucleus in multiple discrete foci. We investigated the possibility that DM2 is also caused by expansion of a CTG repeat or related sequence. Analysis of DNA by repeat expansion detection methods, and RNA by ribonuclease protection, did not show an expanded CTG or CUG repeat in DM2. However, hybridization of muscle sections with fluorescence-labeled CAG-repeat oligonucleotides showed nuclear foci in DM2 similar to those seen in DM1. Nuclear foci were present in all patients with symptomatic DM1 (n = 9) or DM2 (n = 9) but not in any disease controls or healthy subjects (n = 23). The foci were not seen with CUG- or GUC-repeat probes. Foci in DM2 were distinguished from DM1 by lower stability of the probe-target duplex, suggesting that a sequence related to the DM1 CUG expansion accumulates in the DM2 nucleus. Muscleblind proteins, which interact with expanded CUG repeats in vitro, localized to the nuclear foci in both DM1 and DM2. These results support the idea that nuclear accumulation of mutant RNA is pathogenic in DM1, suggest that a similar disease process occurs in DM2, and point to a role for muscleblind in the pathogenesis of both disorders.
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              Splicing biomarkers of disease severity in myotonic dystrophy.

              Masayuki Nakamori, Krzysztof Sobczak, Araya Puwanant … (2013)
              To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2). In a discovery cohort, we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects, we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides to reduce levels of toxic RNA. Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue. Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy. © 2013 American Neurological Association.
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                Author and article information

                Contributors
                Cécile Martinat
                Journal
                Journal ID (nlm-ta): iScience
                Journal ID (iso-abbrev): iScience
                Title: iScience
                Publisher: Elsevier
                ISSN (Electronic): 2589-0042
                Publication date PMC-release: 27 December 2018
                Publication date Collection: 25 January 2019
                Publication date (Electronic): 27 December 2018
                Volume: 11
                Pages: 258-271
                Affiliations
                [1 ]CECS, I-STEM, AFM, 91100 Corbeil-Essonnes, France
                [2 ]INSERM, UMR 861, UEVE, ISTEM, AFM, 91100 Corbeil-Essonnes, France
                [3 ]LBMC, 69007 Lyon, France
                [4 ]Sorbonne Universités UPMC Univ Paris 06, INSERM, Centre de Recherche en Myologie - UMRS974, Institut de Myologie, 75013 Paris, France
                Author notes
                [∗ ]Corresponding author cmartinat@ 123456istem.fr
                [5]

                Lead Contact

                Article
                Publisher ID: S2589-0042(18)30249-9
                DOI: 10.1016/j.isci.2018.12.019
                PMC ID: 6327858
                PubMed ID: 30639849
                SO-VID: cd3714a9-af0b-48f4-af8a-ea66af8f39c4
                Copyright © © 2018 The Author(s)
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 9 July 2018
                Date revision received : 14 December 2018
                Date accepted : 20 December 2018
                Categories
                Subject: Article

                Keywords: physiology,molecular biology,cell biology
                Data availability:
                Keywords: physiology, molecular biology, cell biology

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