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Background: Myotonic dystrophy type 1 (DM1) is a multiorgan disorder with significant cardiac involvement. ECG abnormalities, including arrhythmias, occur in 80 % of DM1 patients and are the second-most common cause of death after respiratory complications; however, the mechanisms underlying the arrhythmogenesis remain unclear. The objective of this study was to investigate the basis of the electrophysiological abnormalities in DM1 using the DMSXL mouse model., Methods: ECG parameters were evaluated at baseline and post flecainide challenge. Calcium transient and action potential parameters were evaluated in Langendorff-perfused hearts using fluorescence optical mapping. Calcium transient/sparks were evaluated in ventricular myocytes via confocal microscopy. Protein and mRNA levels for calcium handling proteins were evaluated using western blot and RT-qPCR, respectively., Results: DMSXL mice showed arrhythmic events on ECG including premature ventricular contractions and sinus block. DMSXL mice showed increased calcium transient time to peak without any change to voltage parameters. Calcium alternans and both sustained and non-sustained ventricular tachyarrhythmias were readily inducible in DMSXL mice. The confocal experiments also showed calcium transient alternans and increased frequency of calcium sparks in DMSXL cardiomyocytes. These calcium abnormalities were correlated with increased RyR2 phosphorylation without changes to the other calcium handling proteins., Conclusions: The DMSXL mouse model of DM1 exhibited enhanced arrhythmogenicity associated with abnormal intracellular calcium handling due to hyperphosphorylation of RyR2, pointing to RyR2 as a potential new therapeutic target in DM1 treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the increased number of CTG repeats in 3' UTR of Dystrophia Myotonia Protein Kinase (DMPK) gene. DM1 patients experience conduction abnormalities as well as atrial and ventricular arrhythmias with increased susceptibility to sudden cardiac death. The ionic basis of these electrical abnormalities is poorly understood. Methods: We evaluated the surface electrocardiogram (ECG) and key ion currents underlying the action potential (AP) in a mouse model of DM1, DMSXL, which express over 1000 CTG repeats. Sodium current (I Na ), L-type calcium current (I CaL ), transient outward potassium current (I to ), and APs were recorded using the patch-clamp technique. Results: Arrhythmic events on the ECG including sinus bradycardia, conduction defects, and premature ventricular and atrial arrhythmias were observed in DMSXL homozygous mice but not in WT mice. PR interval shortening was observed in homozygous mice while ECG parameters such as QRS duration, and QTc did not change. Further, flecainide prolonged PR, QRS, and QTc visually in DMSXL homozygous mice. At the single ventricular myocyte level, we observed a reduced current density for I to and I CaL with a positive shift in steady state activation of L-type calcium channels carrying I CaL in DMSXL homozygous mice compared with WT mice. I Na densities and action potential duration did not change between DMSXL and WT mice. Conclusion: The reduced current densities of I to , and I CaL and alterations in gating properties in L-type calcium channels may contribute to the ECG abnormalities in the DMSXL mouse model of DM1. These findings open new avenues for novel targeted therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2023 Ginjupalli, Cupelli, Reisqs, Sleiman, El-Sherif, Gourdon, Puymirat, Chahine and Boutjdir.)

Myotonic Dystrophy type 1 (DM1) is a neuromuscular disease associated with toxic RNA containing expanded CUG repeats. The developing therapeutic approaches to DM1 target mutant RNA or correct early toxic events downstream of the mutant RNA. We have previously described the benefits of the correction of the GSK3β-CUGBP1 pathway in DM1 mice ( HSA LR model) expressing 250 CUG repeats using the GSK3 inhibitor tideglusib (TG). Here, we show that TG treatments corrected the expression of ~17% of genes misregulated in DM1 mice, including genes involved in cell transport, development and differentiation. The expression of chloride channel 1 ( Clcn1 ), the key trigger of myotonia in DM1, was also corrected by TG. We found that correction of the GSK3β-CUGBP1 pathway in mice expressing long CUG repeats (DMSXL model) is beneficial not only at the prenatal and postnatal stages, but also during adulthood. Using a mouse model with dysregulated CUGBP1, which mimics alterations in DM1, we showed that the dysregulated CUGBP1 contributes to the toxicity of expanded CUG repeats by changing gene expression and causing CNS abnormalities. These data show the critical role of the GSK3β-CUGBP1 pathway in DM1 muscle and in CNS pathologies, suggesting the benefits of GSK3 inhibitors in patients with different forms of DM1.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by the abnormal expansion of CTG-repeats in the 3'-untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene, characterized by multisystemic symptoms including muscle weakness, myotonia, cardio-respiratory problems, hypersomnia, cognitive dysfunction and behavioral abnormalities. Sleep-related disturbances are among the most reported symptoms that negatively affect the quality of life of patients and that are present in early and adult-onset forms of the disease. DMSXL mice carry a mutated human DMPK transgene containing >1,000 CTGrepeats, modeling an early onset, severe form of DM1. They exhibit a pathologic neuromuscular phenotype and also synaptic dysfunction resulting in neurological and behavioral deficits similar to those observed in patients. Additionally, they are underweight with a very high mortality within the first month after birth presenting several welfare issues. To specifically explore sleep/rest-related behaviors of this frail DM1 mouse model we used an automated home cage-based system that allows 24/7 monitoring of their activity non-invasively. We tested male and female DMSXL mice and their wild-type (WT) littermates in Digital Ventilated Cages (DVCR) assessing activity and rest parameters on day and night for 5 weeks. We demonstrated that DMSXL mice show reduced activity and regularity disruption index (RDI), higher percentage of zero activity per each hour and longer periods of rest during the active phase compared to WT. This novel rest-related phenotype in DMSXL mice, assessed unobtrusively, could be valuable to further explore mechanisms and potential therapeutic interventions to alleviate the very common symptom of excessive daytime sleepiness in DM1 patients., Competing Interests: MRi was employed by Tecniplast S.p.A., which provided support in the form of salaries for the author MRi. Tecniplast S.p.A. did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Golini, Rigamonti, Raspa, Scavizzi, Falcone, Gourdon and Mandillo.)

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder that affects many organs. It is caused by the expansion of a cytosine-thymine-guanine triplet repeat in the 3' untranslated region of the human dystrophia myotonica protein kinase (h DMPK ) gene, which results in a toxic gain of function of mutant h DMPK RNA transcripts. Antisense oligonucleotides (ASOs) have emerged in recent years as a potential gene therapy to treat DM1. However, the clinical efficacy of the systemic administration of ASOs is limited by a combination of insufficient potency and poor tissue distribution. In the present study, we assessed the potential of a new ligand-conjugated ASO (IONIS-877864; C16-HA-ASO) to target mutant h DMPK mRNA transcripts in the DMSXL mouse model of DM1 carrying over 1000 CTG pathogenic repeats. DMSXL mice were treated subcutaneously for 9 weeks with either IONIS-877864 (12.5 or 25 mg/kg) or IONIS-486178 (12.5 or 25 mg/kg), an unconjugated ASO with the same sequence. At 25 mg/kg, IONIS-877864 significantly enhanced ASO delivery into the striated muscles of DMSXL mice following systemic administration compared with the unconjugated control. IONIS-877864 was also more efficacious than IONIS-486178, reducing mutant h DMPK transcripts by up to 92% in the skeletal muscles and 78% in the hearts of DMSXL mice. The decrease in mutant h DMPK transcripts in skeletal muscles caused by IONIS-877864 was associated with a significant improvement in muscle strength. IONIS-877864 was nontoxic in the DMSXL mouse model. The present study showed that the C16-HA-conjugated ASO is a powerful tool for the development of gene therapy for DM1.

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function., (© 2022. The Author(s).)

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.

CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3' untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo ., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disease caused by an abnormal CTG repeat expansion in the 3'UTR region of the DMPK gene. In patients, the CTG repeat size varies from fifty to thousands CTG and usually increases across generations (intergenerational instability) and over time in tissues (somatic instability). Larger expansions are associated with more severe symptoms and a decreasing age of onset. Thus, the larger expansions are often associated with the most severe clinical form of DM1 (congenital form). Our PhD project is to identify new genetic and chemical factors reducing the number of repeats and to better understand the mechanisms underlying instability. To this end, genetic and pharmacological screenings are carried out in a HEK293 cell model allowing the rapid detection of expansions (increase in CTG repeat number) and contractions (decrease in CTG repeat number). The effects of different genes and chemical factors, selected during the screening, on the dynamics of the CTG repeat instability will be studied in a DM1 cell model. The results of our work will provide a better understanding of the mechanisms behind contractions. In addition, the identification of new pharmacological compounds promoting CTG contractions and thus reducing or even reversing the progression of disease will offer new therapeutic prospects for DM1 but also for other triplet repeat diseases., (© 2021 médecine/sciences – Inserm.)

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, primarily characterized by muscle wasting and weakness. Many biomarkers already exist in the rapidly developing biomarker research field that aim to improve patients' care. Limited work, however, has been performed on rare diseases, including DM1. We have previously shown that specific microRNAs (miRNAs) can be used as potential biomarkers for DM1 progression. In this report, we aimed to identify novel serum-based biomarkers for DM1 through high-throughput next-generation sequencing. A number of miRNAs were identified that are able to distinguish DM1 patients from healthy individuals. Two miRNAs were selected, and their association with the disease was validated in a larger panel of patients. Further investigation of miR-223-3p, miR-24-3p, and the four previously identified miRNAs, miR-1-3p, miR-133a-3p, miR-133b-3p, and miR-206-3p, showed elevated levels in a DM1 mouse model for all six miRNAs circulating in the serum compared to healthy controls. Importantly, the levels of miR-223-3p, but not the other five miRNAs, were found to be significantly downregulated in five skeletal muscles and heart tissues of DM1 mice compared to controls. This result provides significant evidence for its involvement in disease manifestation., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease., Competing Interests: AG-B was a former employee of BioMarin Pharmaceutical Inc., and holds shares of the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 González-Barriga, Lallemant, Dincã, Braz, Polvèche, Magneron, Pionneau, Huguet-Lachon, Claude, Chhuon, Guerrera, Bourgeois, Auboeuf, Gourdon and Gomes-Pereira.)

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease caused by an unstable cardiotocography (CTG) repeat expansion in the DMPK gene. This disease is characterized by high clinical and genetic variability, leading to some difficulties in the diagnosis and prognosis of DM1. Better understanding the origin of this variability is important for developing new challenging therapies and, in particular, for progressing on the path of personalized treatments. Here, we reviewed CTG triplet repeat instability and its modifiers as an important source of phenotypic variability in patients with DM1., Competing Interests: The authors no conflict of interest.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1., Competing Interests: EA, MN, YR, AH, CC, CD, LM, MT, GG, GJ, TJ, MZ, KJ No competing interests declared, (© 2019, Auxerre-Plantié et al.)

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Myotonic dystrophy type 1 (DM1) is a multisystem neuromuscular disease without cure. One of the possible therapeutic approaches for DM1 is correction of the RNA-binding proteins CUGBP1 and MBNL1, misregulated in DM1. CUGBP1 activity is controlled by glycogen synthase kinase 3β (GSK3β), which is elevated in skeletal muscle of patients with DM1, and inhibitors of GSK3 were suggested as therapeutic molecules to correct CUGBP1 activity in DM1. Here, we describe that correction of GSK3β with a small-molecule inhibitor of GSK3, tideglusib (TG), not only normalizes the GSK3β-CUGBP1 pathway but also reduces the mutant DMPK mRNA in myoblasts from patients with adult DM1 and congenital DM1 (CDM1). Correction of GSK3β in a mouse model of DM1 ( HSA LR mice) with TG also reduces the levels of CUG-containing RNA, normalizing a number of CUGBP1- and MBNL1-regulated mRNA targets. We also found that the GSK3β-CUGBP1 pathway is abnormal in skeletal muscle and brain of DMSXL mice, expressing more than 1,000 CUG repeats, and that the correction of this pathway with TG increases postnatal survival and improves growth and neuromotor activity of DMSXL mice. These findings show that the inhibitors of GSK3, such as TG, may correct pathology in DM1 and CDM1 via several pathways., (Copyright © 2019 American Society for Microbiology.)

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Intensive effort has been directed toward the modeling of myotonic dystrophy (DM) in mice, in order to reproduce human disease and to provide useful tools to investigate molecular and cellular pathogenesis and test efficient therapies. Mouse models have contributed to dissect the multifaceted impact of the DM mutation in various tissues, cell types and in a pleiotropy of pathways, through the expression of toxic RNA transcripts. Changes in alternative splicing, transcription, translation, intracellular RNA localization, polyadenylation, miRNA metabolism and phosphorylation of disease intermediates have been described in different tissues. Some of these events have been directly associated with specific disease symptoms in the skeletal muscle and heart of mice, offering the molecular explanation for individual disease phenotypes. In the central nervous system (CNS), however, the situation is more complex. We still do not know how the molecular abnormalities described translate into CNS dysfunction, nor do we know if the correction of individual molecular events will provide significant therapeutic benefits. The variability in model design and phenotypes described so far requires a thorough and critical analysis. In this review we discuss the recent contributions of mouse models to the understanding of neuromuscular aspects of disease, therapy development, and we provide a reflective assessment of our current limitations and pressing questions that remain unanswered.

Numerous human diseases are associated with abnormal expansion of unstable trinucleotide repeats (TNRs). TNR instability mechanisms are complex, and remain only partially understood. Small-pool-PCR (SP-PCR) is the reference method to assess TNR instability. SP-PCR amplifies a low number of DNA molecules and is followed by Southern blot. However, SP-PCR remains expensive and time consuming. Here, we describe an optimized SP-PCR that can be done in a day, which reduces cost and experimental biases: the flash-small-pool PCR (FSP-PCR). This method consists of a fluorescent PCR on a few DNA molecules, followed by an alkaline gel electrophoresis revealed with a near infra-red detector system. With reduced experimental steps, cost, and time consumption, microsatellite analysis will become more accessible due to FSP-PCR.

Brain function is compromised in myotonic dystrophy type 1 (DM1), but the underlying mechanisms are not fully understood. To gain insight into the cellular and molecular pathways primarily affected, we studied a mouse model of DM1 and brains of adult patients. We found pronounced RNA toxicity in the Bergmann glia of the cerebellum, in association with abnormal Purkinje cell firing and fine motor incoordination in DM1 mice. A global proteomics approach revealed downregulation of the GLT1 glutamate transporter in DM1 mice and human patients, which we found to be the result of MBNL1 inactivation. GLT1 downregulation in DM1 astrocytes increases glutamate neurotoxicity and is detrimental to neurons. Finally, we demonstrated that the upregulation of GLT1 corrected Purkinje cell firing and motor incoordination in DM1 mice. Our findings show that glial defects are critical in DM1 brain pathophysiology and open promising therapeutic perspectives through the modulation of glutamate levels., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG) n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUG exp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUG exp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Myotonic dystrophies are multisystemic diseases characterized not only by muscle and heart dysfunction but also by CNS alteration. They are now recognized as brain diseases affecting newborns and children for myotonic dystrophy type 1 and adults for both myotonic dystrophy type 1 and type 2. In the past two decades, much progress has been made in understanding the mechanisms underlying the DM symptoms allowing development of new molecular therapeutic tools with the ultimate aim of curing the disease. This review describes the state of the art for the characterization of CNS related symptoms, the development of molecular strategies to target the CNS as well as the available tools for screening and testing new possible treatments.

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG) n -repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Myotonic dystrophy type 1 (DM1) is caused by an unstable expanded CTG repeat located within the DMPK gene 3'UTR. The nature, severity and age at onset of DM1 symptoms are very variable in patients. Different forms of the disease are described, among which the congenital form (CDM) is the most severe. Molecular mechanisms of DM1 are well characterized for the adult form and involve accumulation of mutant DMPK RNA forming foci in the nucleus. These RNA foci sequester proteins from the MBNL family and deregulate CELF proteins. These proteins are involved in many cellular mechanisms such as alternative splicing, transcriptional, translational and post-translational regulation miRNA regulation as well as mRNA polyadenylation and localization. All these mechanisms can be impaired in DM1 because of the deregulation of CELF and MBNL functions. The mechanisms involved in CDM are not clearly described. In order to get insight into the mechanisms underlying CDM, we investigated if expanded RNA nuclear foci, one of the molecular hallmarks of DM1, could be detected in human DM1 fetal tissues, as well as in embryonic and neonatal tissues from transgenic mice carrying the human DMPK gene with an expanded CTG repeat. We observed very abundant RNA foci formed by sense DMPK RNA and, to a lesser extent, antisense DMPK RNA foci. Sense DMPK RNA foci clearly co-localized with MBNL1 and MBNL2 proteins. In addition, we studied DMPK sense and antisense expression during development in the transgenic mice. We found that DMPK sense and antisense transcripts are expressed from embryonic and fetal stages in heart, muscle and brain and are regulated during development. These results suggest that mechanisms underlying DM1 and CDM involved common players including toxic expanded RNA forming numerous nuclear foci at early stages during development.

With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

The toxicity of expanded transcripts in myotonic dystrophy type 1 (DM1) is mainly mediated by the disruption of alternative splicing. However, the detailed disease mechanisms in the central nervous system (CNS) have not been fully elucidated. In our recent study, we demonstrated that the accumulation of mutant transcripts in the CNS of a mouse model of DM1 disturbs splicing in a region-specific manner. We now discuss that the spatial- and temporal-regulated expression of splicing factors may contribute to the region-specific spliceopathy in DM1 brains. In the search for disease mechanisms operating in the CNS, we found that the expression of expanded CUG-containing RNA affects the expression and phosphorylation of synaptic vesicle proteins, possibly contributing to DM1 neurological phenotypes. Although mediated by splicing regulators with a described role in DM1, the misregulation of synaptic proteins was not associated with missplicing of their coding transcripts, supporting the view that DM1 mechanisms in the CNS have also far-reaching implications beyond the disruption of a splicing program.

Acute and chronic respiratory failure is one of the major and potentially life-threatening features in individuals with myotonic dystrophy type 1 (DM1). Despite several clinical demonstrations showing respiratory problems in DM1 patients, the mechanisms are still not completely understood. This study was designed to investigate whether the DMSXL transgenic mouse model for DM1 exhibits respiratory disorders and, if so, to identify the pathological changes underlying these respiratory problems. Using pressure plethysmography, we assessed the breathing function in control mice and DMSXL mice generated after large expansions of the CTG repeat in successive generations of DM1 transgenic mice. Statistical analysis of breathing function measurements revealed a significant decrease in the most relevant respiratory parameters in DMSXL mice, indicating impaired respiratory function. Histological and morphometric analysis showed pathological changes in diaphragmatic muscle of DMSXL mice, characterized by an increase in the percentage of type I muscle fibers, the presence of central nuclei, partial denervation of end-plates (EPs) and a significant reduction in their size, shape complexity and density of acetylcholine receptors, all of which reflect a possible breakdown in communication between the diaphragmatic muscles fibers and the nerve terminals. Diaphragm muscle abnormalities were accompanied by an accumulation of mutant DMPK RNA foci in muscle fiber nuclei. Moreover, in DMSXL mice, the unmyelinated phrenic afferents are significantly lower. Also in these mice, significant neuronopathy was not detected in either cervical phrenic motor neurons or brainstem respiratory neurons. Because EPs are involved in the transmission of action potentials and the unmyelinated phrenic afferents exert a modulating influence on the respiratory drive, the pathological alterations affecting these structures might underlie the respiratory impairment detected in DMSXL mice. Understanding mechanisms of respiratory deficiency should guide pharmaceutical and clinical research towards better therapy for the respiratory deficits associated with DM1.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

An expanded CTG-repeat in the 3' UTR of the DMPK gene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreased DMPK sense and SIX5 transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability.

Sharka is a devastating viral disease caused by the Plum pox virus (PPV) in stone fruit trees and few sources of resistance are known in its natural hosts. Since any knowledge gained from Arabidopsis on plant virus susceptibility factors is likely to be transferable to crop species, Arabidopsis's natural variation was searched for host factors essential for PPV infection. To locate regions of the genome associated with susceptibility to PPV, linkage analysis was performed on six biparental populations as well as on multiparental lines. To refine quantitative trait locus (QTL) mapping, a genome-wide association analysis was carried out using 147 Arabidopsis accessions. Evidence was found for linkage on chromosomes 1, 3 and 5 with restriction of PPV long-distance movement. The most relevant signals occurred within a region at the bottom of chromosome 3, which comprises seven RTM3-like TRAF domain-containing genes. Since the resistance mechanism analyzed here is recessive and the rtm3 knockout mutant is susceptible to PPV infection, it suggests that other gene(s) present in the small identified region encompassing RTM3 are necessary for PPV long-distance movement. In consequence, we report here the occurrence of host factor(s) that are indispensable for virus long-distance movement., (© 2012 INRA. New Phytologist © 2012 New Phytologist Trust.)