Understanding the pathogenesis of myotonic dystrophy type 1

To identify the full range of targets and the pathogenic consequences, we sought to mimic the pathogenesis of myotonic dystrophy type 1 with temporal and spatial control: temporal to reproduce the developmental pathogenesis of the congenital form, and spatial to isolate tissue specific pathology. To do this, we attempted to use the Cre-lox system for the conditional expression of an EGFP reporter-linked expanded CUG repeat RNA in the mouse. Expression of the transgene was controlled by Cre excision of a transcriptional stop, placed upstream of the EGFP-expanded repeat open reading frame. The transgenes were constructed and tested successfully, and a normal length repeat transgenic line was established. Unfortunately generation of the expanded repeat line was not successful. The constructs were used to generate cell-culture models of DM1, in both human and murine cells, which mimicked the nuclear foci formation and MBNL1 co-localisation seen in patient cells. Expression of exogenous MBNL1/GFP fusion protein in this model resulted in an increase in the size of foci, indicating that MBNL1 protein is limiting within the cell, and may possibly play a protective role. The murine DM1 cell-culture model was used to investigate the effects of expanded CUG repeat expression on splicing within the transcriptome. The differential effect between 5 and 250 repeat RNA expression using Affymetrix whole transcript and exon arrays was compared. Using whole genome arrays, 6 genes were down-regulated and 128 upregulated. With exon arrays, 58 genes showed alternative exon usage. Six genes were selected for further bioinformatics analysis: MtmR4, which has possible neuromuscular involvement; Kcnk4, Narg1, Ttyh1 and Bptf, potentially related to brain development; and Cacna1c, a promising candidate for heart conductance defects and sudden death

Molecular Effects of the CTG Repeats in Mutant Dystrophia Myotonica Protein Kinase Gene

Myotonic Dystrophy type 1 (DM1) is a multi-system disorder characterized by muscle wasting, myotonia, cardiac conduction defects, cataracts, and neuropsychological dysfunction. DM1 is caused by expansion of a CTG repeat in the 3´untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. A body of work demonstrates that DMPK mRNAs containing abnormally expanded CUG repeats are toxic to several cell types. A core mechanism underlying symptoms of DM1 is that mutant DMPK RNA interferes with the developmentally regulated alternative splicing of defined pre-mRNAs. Expanded CUG repeats fold into ds(CUG) hairpins that sequester nuclear proteins including human Muscleblind-like (MBNL) and hnRNP H alternative splicing factors. DM1 cells activate CELF family member CUG-BP1 protein through hyperphosphorylation and stabilization in the cell nucleus. CUG-BP1 and MBNL1 proteins act antagonistically in exon selection in several pre-mRNA transcripts, thus MBNL1 sequestration and increase in nuclear activity of CUG-BP1 both act synergistically to missplice defined transcripts. Mutant DMPK-mediated effect on subcellular localization, and defective phosphorylation of cytoplasmic CUG-BP1, have additionally been linked to defective translation of p21 and MEF2A in DM1, possibly explaining delayed differentiation of DM1 muscle cells. Mutant DMPK transcripts bind and sequester transcription factors such as Specificity protein 1 leading to reduced transcription of selected genes. Recently, transcripts containing long hairpin structures of CUG repeats have been shown to be a Dicer ribonuclease target and Dicer-induced downregulation of the mutant DMPK transcripts triggers silencing effects on RNAs containing long complementary repeats. In summary, mutant DMPK transcripts alter gene transcription, alternative splicing, and translation of specific gene transcripts, and have the ability to trigger gene-specific silencing effects in DM1 cells. Therapies aimed at reversing these gene expression alterations should prove effective ways to treat DM1

Conséquences pathologiques des expansions CTG sur le système nerveux central d'un modèle murin de la dystrophie myotonique de Steinert (approches moléculaires, protéomiques et cellulaires)

La dystrophie myotonique de type I (DM1) constitue la plus fréquente des pathologies musculaires héréditaires chez l adulte. Bien qu initialement considérée comme une maladie musculaire, la DM1 présente une atteinte neurologique très handicapante. Cette maladie autosomique dominante résulte de l expansion anormale d un triplet CTG dans la partie 3 UTR du gène DMPK. Un effet trans du transcrit DMPK muté entraine une dérégulation de l épissage alternatif dans de nombreux tissus. Cependant, les mécanismes pathologiques de la DM1 dans le cerveau restent encore peu compris. Afin de disséquer ce mécanisme, notre laboratoire a créé des souris transgéniques exprimant le transcrit DMPK avec de larges expansions CUG dans de nombreux tissus. Ces souris nommées DMSXL, recréent d importants aspects pathologiques de la DM1, comme des anomalies du comportement et électrophysiologiques du cerveau. Elles représentent donc un excellent outil pour explorer l effet pathologique de la mutation dans le SNC. En m appuyant sur ce modèle, j ai exploré dans un premier temps l effet trans des ARNs toxiques et l ampleur de la splicéopathie dans le SNC. De façon intéressante, certains défauts d épissage sont régions spécifiques, et ne montrent pas d aggravation avec l âge des souris DMSXL. Mes résultats démontrent que les ARNs mutés sont capables de déréguler l épissage alternatif dans l ensemble du SNC. La région du cervelet a aussi montré des anomalies de l épissage dans les souris DMSXL, qui, en plus, présentent des perturbations cognitives dépendantes de cette région cérébrale. Le cervelet des souris DMSXL présente aussi des déficits électrophysiologiques suggérant une dysfonction cérébelleuse et plus précisément une dysfonction des cellules de Purkinje. Dans la recherche des populations cellulaires les plus affectées dans le cervelet, j ai démontré la présence de signes de la toxicité de l ARN plus marqués dans la glie de Bergman, entourant les cellules de Purkinje. Pour trouver les voies moléculaires perturbées dans le cervelet, et disséquer le mécanisme derrière les anomalies observées, j ai réalisé une approche protéomique globale et trouvé une sévère baisse de l expression du transporteur glial de glutamate GLT1/EAAT2, suggérant une dysfonction du cervelet, en conséquence d un possible métabolisme anormal du glutamate. L analyse protéomique globale du cerveau des souris DM1 a aussi identifié des différences d expression et des modifications post-traductionnelles de protéines impliquées dans la signalisation du calcium. L étude du métabolisme des ARNm dans la DM1 a mis en évidence la dérégulation de l épissage de gènes impliqués dans le métabolisme du calcium, soutenant l hypothèse d une dysfonction calcique dans le SNC. Pour étudier les conséquences de la mutation sur les variations calciques cellulaires, j ai caractérisé un modèle cellulaire astrocytaire de la DM1. Ce modèle m a permis de démontrer une localisation anormale du récepteur GRIN1/NMDAR1, ainsi qu une réponse calcique anormale dans les astrocytes primaires porteurs des amplifications CTG. Malgré les avancés thérapeutiques dans le muscle, on ne sait pas à quel point les stratégies en cours de développement sont efficaces dans le SNC. Pour étudier ce problème, j ai utilisé le modèle astrocytaire de la DM1 afin de valider in cellulo une stratégie thérapeutique qui vise à rétablir une activité normale du facteur d épissage MBNL1 endogène. Mes travaux de thèse ont permis d avancer dans la compréhension de la neuropathologie de la DM1. Ils ont mis en évidence pour la première fois une dysfonction du cervelet, ainsi que la possible dérégulation de la voix calcique dans le SNC. Mes résultats ont donc contribué à mieux comprendre le mécanisme de la DM1 dans le SNC, pour, à long terme, développer des approches thérapeutiques ciblant des évènements moléculaires précis.Myotonic dystrophy type 1 (DM1) is the most frequent inherited muscular disorder in adults. Although traditionally regarded as a muscle disease, DM1 presents debilitating neurological manifestations. DM1 is an autosomic dominant disease caused by the abnormal expansion of a CTG triplet within the 3 UTR of the DMPK gene. Many molecular aspects of the DM1 are mediated by a trans effect of the expanded DMPK transcripts, whose accumulation leads to splicing deregulation in many tissues. Despite recent progress in the understanding of DM1 pathogenesis in muscle and central nervous system (CNS), the detailed molecular disease mechanism operating in the brain is still poorly understood. In order to investigate the pathophysiology, our laboratory has generated DMSXL transgenic mice expressing DMPK transcripts containing large CUG expansions in many tissues. DMSXL mice mimic important features of the DM1, notably in the CNS, showing behaviour as well as electrophysiological abnormalities. Therefore, this mouse line represents an excellent tool to investigate the toxic effects of the mutation in the CNS. Taking advantages of this transgenic model, I have first explored the trans effect of the toxic RNA and the extent of DM1-associated spliceopathy in the CNS. Interestingly, some splicing defects were region-specific, and their severity did not increase with the age of the DMSXL mice. My data demonstrate that CUG-containing RNAs have a wide deleterious effect and deregulate alternative splicing in many areas of the CNS. In addition to splicing abnormalities in cerebellum, DMSXL mice also displayed deficits in cerebellum-dependant motor coordination. Plus, DMSXL cerebellum showed electrophysiological abnormalities, suggesting cerebellar dysfunction and more precisely Purkinje cell dysfunction. In the search for the cellular populations showing the greatest susceptibility to RNA toxicity in the cerebellum, I have found extensive foci accumulation as well as pronounced splicing defects in the Bergman glia, surrounding Purkinje cells, in DMSXL and DM1 patients cerebellum. In order to identify molecular pathways and mechanisms behind the behaviour and electrophysiological abnormalities detected, I have performed a global proteomics approach and found a severe decrease in the expression of a glial glutamate transporter GLT1/EAAT2, suggesting that DM1 causes cerebellum dysfunction, through abnormal glutamate metabolism. Global proteomic analysis of DMSXL cerebellum also identified expression and post-translational changes of several proteins involved in calcium signalling. Missplicing of different transcripts involved in calcium metabolism reinforces the idea of calcium dysfunction in the neuropathogenesis of the DM1. To study the effects of toxic RNA on calcium homeostasis and flux, I have established and characterised a brain cell model of DM1. DMSXL primary astrocyte cultures allowed me to show the mislocalisation of the glutamate receptor GRIN1/NMDAR1, as well as abnormal calcium responses to stimulation. Despite recent therapeutic advances in muscle, we do not know the CNS efficiency of the therapeutic strategies currently being developed. To address this problem, I have used the DM1 astrocyte cell model to validate in cellulo a therapeutic strategy aiming to restore the activity of the endogenous splicing factor MBNL1. My thesis work provided a significant step in the understanding of the DM1 pathology in the CNS. My results revealed for the first time signs of cerebellum dysfunction in DM1, as well as signs of calcium homeostasis deregulation in the SNC. My work contributed to better understand the pathological mechanisms of DM1, the brain pathways and cell types most susceptible to toxic RNA. In the long term, my data will contribute to the rational development of therapeutic strategies targeting precise and deleterious molecular events.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Análisis de la función molecular de las proteínas Muscleblind de Drosophila.

RESUMEN El gen muscleblind (mbl) es necesario para el adecuado desarrollo del sistema nervioso periférico embrionario y la diferenciación terminal de los fotorreceptores y los músculos en Drosophila. A partir del único gen muscleblind de Drosophila se generan cuatro transcritos por procesado alternativo que codifican para cuatro proteínas caracterizadas por la presencia de dedos de zinc del tipo Cys3H. Los homólogos de muscleblind en vertebrados son los genes Muscleblind-like1, 2 y 3 (MBNL1-3). Las proteínas humanas MBNL1, 2 y 3 tienen la capacidad de modificar el procesado alternativo de diversos transcritos y tienen un papel importante en la patogénesis de la distrofia miotónica (DM). La DM es una enfermedad autonómica dominante generada por la expansión del trinucleótido CTG en una región no codificante del gen DMPK. La presente tesis recoge experimentos realizados en diferentes sistemas para desvelar la función molecular de las proteínas Muscleblind de Drosophila. Este trabajo demuestra la conservación de la actividad como factores de splicing alternativo y la ruta patogénica de la DM en moscas. Se describen dos nuevas dianas moleculares de las proteínas Muscleblind, los transcritos de la alpha-actinina y la troponinaT, cuyo patrón de splicing está alterado en mutantes mbl y en moscas que expresan repeticiones CUG. Además, mediante experimentos en cultivo celular se muestra la capacidad de estas proteínas de inducir muerte celular al ser sobre-expresadas en células S2 de Drosophila. Las isoformas de Muscleblind mostraron diferente capacidad en los distintos ensayos funcionales realizados. Mediante experimentos de sobre-expresión en células de vertebrado y mutagénesis dirigida mostramos la implicación de los extremos carboxilo en la diversificación funcional de las isoformas de Muscleblind. Las isoformas se localizan en distintos compartimentos sub-celulares y la eliminación de un sitio putativo de sumolización (FKRP) conservado altera la capacidad de inducir muerte celular de MuscleblindC. __________________________________________________________________________________________________The human Muscleblind-like proteins MBNL1-3 bind RNAs through pairs of zinc fingers of the Cys3His type. They have the ability to modify alternative splicing and sub-cellular localisation of defined transcripts and their function is impaired in myotonic dystrophies type 1 and 2 (DM1 and DM2). DMs are autosomal dominant neuromuscular diseases characterized by myotonia, muscle weakness, and iridescent cataracts, among other symptoms. At a molecular level, DM patients show disruption of alternative splicing regulation of specific transcripts. The genetic cause of these diseases is the expansion of either a CTG or a CCTG repeat in non-coding regions of the DMPK (DM1) and ZNF9 (DM2) genes. Upon transcription, expanded RNAs form stable hairpins, which sequester nuclear factors depleting them from their normal function. Among those factors are the MBNL proteins. The relevance of MBNL sequestration in DM pathogenesis is supported by muscleblind like 1 knock-out mice (Mbnl1DE3/DE3), which reproduce the main features of DM patients including myotonia, cataracts, and RNA splicing defects in transcripts such as troponinT 2 and 3. Furthermore, expression of Mbnl1 protein reverts the DM-like alterations of mice expressing expanded CUG containing RNA

Approches globales afin d’élucider les mécanismes pathogéniques de la dystrophie myotonique de type 1

La dystrophie myotonique de type 1 (DM1) est une maladie dégénérative impliquant des symptômes d’atrophie musculaire et de myotonie. Au niveau moléculaire, elle est caractérisée par une expansion aberrante de CUG dans la région 3’UTR de l’ARNm de DMPK (Dystrophia Myotonica protein kinase). Ces répétitions CUG forment des agrégats toxiques (appelés foci) majoritairement nucléaires dans les cellules de patients DM1 et causent la séquestration anormale de ribonucléoprotéines (RBP), tel que le facteur «Muscleblind-like 1» (MBNL1), qui lieraient normalement les motifs CUG d’autres ARN. Les fonctions normales de ces RBPs seraient alors perturbées. En plus de leur rôle dans l’épissage alternatif, MBNL a récemment été caractérisé pour son implication dans la localisation intracellulaire de ses ARN cibles. Ceci suggèrerait que la pathogénèse de la DM1 pourrait résulter de l’effet perturbateur des répétitions CUG sur la localisation d’ARN précis et de protéines RBPs. À cet effet, un criblage basé sur de la microscopie fluorescente de 322 RBPs dans des myoblastes de patients DM1 a permis d’identifier des nouveaux facteurs qui colocaliseraient avec les expansions pathogéniques CUG. De plus, ces myoblastes DM1 ont été fractionnés et un séquençage-ARN a par la suite permis l’identification de transcrits délocalisés. Les deux banques de données ainsi générées, tant par le criblage que par le fractionnement/séquençage-ARN, pourraient ouvrir des nouvelles avenues de recherches dans la compréhension des anomalies moléculaires associées à la DM1, et potentiellement d’autres maladies à expansions microsatellites.Myotonic dystrophy of type 1 (DM1) is a degenerative disorder implicating symptoms of muscular atrophy and myotony. In a molecular level, it is caused by the aberrant expansion of CUG repeats in the 3’-UTR region of the DMPK mRNA (Dystrophia Myotonica protein kinase). Excessive CUG repeats then form toxic aggregates (foci) enriched within the nucleus of DM1 patient cells. These RNA foci cause the abnormal sequestration of RNA Binding Proteins (RBP), in particular members of the Muscleblind-like protein 1 (MBNL), that normally bind the CUG motif of other target RNAs, and will hence alter their normal functions. In addition to their role in alternative splicing, MBNL1 has recently been implicated in the intracellular localisation of its RNA targets. It remains elusive whether the pathogenesis of DM1 could result from the deregulating effect of CUG repeats on the localisation of specific RNAs and RBP proteins. In this thesis, a fluorescent imaging-based screening of 322 RBPs in DM1 patient’s myoblasts has been conducted and this had led to the identification of new factors that may colocalize with pathogenic CUG expansions. Moreover, these DM1 myoblasts have been fractionated and subsequent RNA-sequencing has permitted the identification of transcripts that are delocalised between subcellular compartments. From the two large datasets generated from the RBP imaging-based screening and fractionation/RNA-sequencing, new avenues of research can be initiated to further understand not only DM1, but perhaps also other disorders that implicate microsatellite expansions

Can human pluripotent stem cell-derived cardiomyocytes advance understanding of muscular dystrophies?

Muscular dystrophies (MDs) are clinically and molecularly a highly heterogeneous group of single-gene disorders that primarily affect striated muscles. Cardiac disease is present in several MDs where it is an important contributor to morbidity and mortality. Careful monitoring of cardiac issues is necessary but current management of cardiac involvement does not effectively protect from disease progression and cardiac failure. There is a critical need to gain new knowledge on the diverse molecular underpinnings of cardiac disease in MDs in order to guide cardiac treatment development and assist in reaching a clearer consensus on cardiac disease management in the clinic. Animal models are available for the majority of MDs and have been invaluable tools in probing disease mechanisms and in pre-clinical screens. However, there are recognized genetic, physiological, and structural differences between human and animal hearts that impact disease progression, manifestation, and response to pharmacological interventions. Therefore, there is a need to develop parallel human systems to model cardiac disease in MDs. This review discusses the current status of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC) to model cardiac disease, with a focus on Duchenne muscular dystrophy (DMD) and myotonic dystrophy (DM1). We seek to provide a balanced view of opportunities and limitations offered by this system in elucidating disease mechanisms pertinent to human cardiac physiology and as a platform for treatment development or refinement

Protein Kinase C inhibitor Molecule Effect Partial Reversal of Spliceopathy in Myotonic Dystrophy Type 2 Cell Line

Myotonic dystrophy is a neuromuscular disease which manifest as two forms namely type 1 (DM1) and type 2 (DM2). One of the major molecular events associated with the disease condition is misregulation of splicing resulting in spliceopathy. This study was performed to assess the effect of protein kinase C (PKC) inhibitors- Ro 31-8220 and hypericin- on the splicing of insulin receptor (IR) and muscleblind-like (MBNL1) in myotonic dystrophy type 2 (DM2) cell line. The results of this study demonstrated that only Ro 31-8820 was able to effect partial rescue of missplicing of insulin receptor and muscleblind-like 1. It indicates that it could be a possible therapeutic agent for treatment of DM2. Keywords: Myotonic dystrophy, Protein Kinase C inhibitor, spliceopathy, therapeutics DOI: 10.7176/JNSR/11-14-03 Publication date:July 31st 202

Evaluating the effects of CELF1 deficiency in a mouse model of RNA toxicity.

International audienceMyotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by an expanded (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The toxic RNA transcripts produced from the mutant allele alter the function of RNA-binding proteins leading to the functional depletion of muscleblind-like (MBNL) proteins and an increase in steady state levels of CUG-BP1 (CUGBP-ETR-3 like factor 1, CELF1). The role of increased CELF1 in DM1 pathogenesis is well studied using genetically engineered mouse models. Also, as a potential therapeutic strategy, the benefits of increasing MBNL1 expression have recently been reported. However, the effect of reduction of CELF1 is not yet clear. In this study, we generated CELF1 knockout mice, which also carry an inducible toxic RNA transgene to test the effects of CELF1 reduction in RNA toxicity. We found that the absence of CELF1 did not correct splicing defects. It did however mitigate the increase in translational targets of CELF1 (MEF2A and C/EBPβ). Notably, we found that loss of CELF1 prevented deterioration of muscle function by the toxic RNA, and resulted in better muscle histopathology. These data suggest that while reduction of CELF1 may be of limited benefit with respect to DM1-associated spliceopathy, it may be beneficial to the muscular dystrophy associated with RNA toxicity

Development of assays for therapeutic screening in myotonic dystrophy

Myotonic dystrophy (DM) is an autosomal dominant inherited multisystemic neuromuscular disease. The molecular mechanism for DM is mediated by toxic RNAs containing expanded repeat units. DMI is caused by a CTG repeat expansion in 3'-UTR of the DMPK gene while DM2 is caused by CCTG repeat in intronl of the ZNF9 gene. The molecular features of DM are formation of RNA foci, co-localisation of MBNL proteins with ribonuclear foci, splicing defects of a subset of pre-mRNAs with elevation ofCUGBPI in DMl. In order to develop therapy for DM, assays were designed based on the molecular characteristics of the disease to screen compounds. Two primary assays were based on disruption of nuclear foci and on correction of misregulated splicing involving intron2 CLCNI. The first part of this report deals with the development of a nuclear foci assay and splicing construct assay. Both assays were optimised in HTS and utilized in screens for molecules that clear nuclear foci from DM cells and correct misregulated splicing in intron2 of CLCNI respectively. High throughput screens of kinase and phosphatase inhibitor libraries using the nuclear foci assay and CLCNI splicing construct assay yielded two positive hits: protein kinase C inhibitors designated in the compound library as D8 (hypericin) and D9 (Ro-31-8220). The second part of this thesis deals with the confirmation of compound hits obtained from the primary screen. I examined two aspects: mutant DMPKtranscript entrapment in nucleus and splicing defects associated with the disease. A BpmJ restriction assay was used to test the effect of compounds on mutant DMPK transcripts showed that both D8 and D9 were unable to release the mutant transcript into the cytoplasm. D8 demonstrated efficacy in reversing spliceopathy in alternative splicing assays of JR, SERCAI MBNLI and MBNL2 while D9 did not have this effect except on MBNLI which showed a very minor efficacy