DM1 Mouse Model Research Articles

Ultrasound-enhanced delivery of Morpholino with Bubble liposomes ameliorates the myotonia of myotonic dystrophy model mice

Phosphorodiamidate morpholino oligonucleotide (PMO)-mediated control of the alternative splicing of the chloride channel 1 (CLCN1) gene is a promising treatment for myotonic dystrophy type 1 (DM1) because the abnormal splicing of this gene causes myotonia in patients with DM1. In this study, we optimised a PMO sequence to correct Clcn1 alternative splicing and successfully remedied the myotonic phenotype of a DM1 mouse model, the HSALR mouse. To enhance the efficiency of delivery of PMO into HSALR mouse muscles, Bubble liposomes, which have been used as a gene delivery tool, were applied with ultrasound exposure. Effective delivery of PMO led to increased expression of Clcn1 protein in skeletal muscle and the amelioration of myotonia. Thus, PMO-mediated control of the alternative splicing of the Clcn1 gene must be important target of antisense therapy of DM1.

Manumycin A corrects aberrant splicing of Clcn1 in myotonic dystrophy type 1 (DM1) mice

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.

Systemic Delivery of a Peptide-Linked Morpholino Oligonucleotide Neutralizes Mutant RNA Toxicity in a Mouse Model of Myotonic Dystrophy

Expansions of CUG trinucleotide sequences in RNA transcripts provide the basis for toxic RNA gain-of-function that leads to detrimental changes in RNA metabolism. A CTG repeat element normally resides in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, but when expanded it is the genetic lesion of myotonic dystrophy type 1 (DM1), a hereditary neuromuscular disease. The pathogenic DMPK transcript containing the CUG expansion is retained in ribonuclear foci as part of a complex with RNA-binding proteins such as muscleblind-like 1 (MBNL1), resulting in aberrant splicing of numerous RNA transcripts and consequent physiological abnormalities including myotonia. Herein, we demonstrate molecular and physiological amelioration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic administration of peptide-linked morpholino (PPMO) antisense oligonucleotides bearing a CAG repeat sequence. Intravenous administration of PPMO conjugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnormal RNA splicing. Additionally, myotonia was completely eliminated in PPMO-treated HSA(LR) mice. These studies provide proof of concept that neutralization of RNA toxicity by systemic delivery of antisense oligonucleotides that target the CUG repeat is an effective therapeutic approach for treating the skeletal muscle aspects of DM1 pathology.

GSK3β is a new therapeutic target for myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1), an incurable, neuromuscular disease, is caused by the expansion of CTG repeats within the 3′ UTR of DMPK on chromosome 19q. In DM1 patients, mutant DMPK transcripts deregulate RNA metabolism by altering CUG RNA-binding proteins. Several approaches have been proposed for DM1 therapy focused on specific degradation of the mutant CUG repeats or on correction of RNA-binding proteins, affected by CUG repeats. One such protein is CUG RNA-binding protein (CUGBP1). The ability of CUGBP1 to increase or inhibit translation depends on phosphorylation at Ser302, which is mediated by cyclin D3-CDK4. The mutant CUG repeats increase the levels of CUGBP1 protein and inhibit Ser302 phosphorylation, leading to the accumulation of CUGBP1 isoforms that repress translation (i.e., CUGBP1REP). Elevation of CUGBP1REP in DM1 is caused by increased GSK3β kinase, which reduces the cyclin D3-CDK4 pathway and subsequent phosphorylation of CUGBP1 at Ser302. In this review, we discuss our recent discovery showing that correction of GSK3β activity in the DM1 mouse model (i.e., HSALR mice) reduces DM1 muscle pathology. These findings demonstrate that GSK3β is a novel therapeutic target for treating DM1.

Synaptic protein dysregulation in myotonic dystrophy type 1: Disease neuropathogenesis beyond missplicing.

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.

GSK3β mediates muscle pathology in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease characterized by skeletal muscle wasting, weakness, and myotonia. DM1 is caused by the accumulation of CUG repeats, which alter the biological activities of RNA-binding proteins, including CUG-binding protein 1 (CUGBP1). CUGBP1 is an important skeletal muscle translational regulator that is activated by cyclin D3-dependent kinase 4 (CDK4). Here we show that mutant CUG repeats suppress Cdk4 signaling by increasing the stability and activity of glycogen synthase kinase 3β (GSK3β). Using a mouse model of DM1 (HSA(LR)), we found that CUG repeats in the 3' untranslated region (UTR) of human skeletal actin increase active GSK3β in skeletal muscle of mice, prior to the development of skeletal muscle weakness. Inhibition of GSK3β in both DM1 cell culture and mouse models corrected cyclin D3 levels and reduced muscle weakness and myotonia in DM1 mice. Our data predict that compounds normalizing GSK3β activity might be beneficial for improvement of muscle function in patients with DM1.

Rational Design of Bioactive, Modularly Assembled Aminoglycosides Targeting the RNA that Causes Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)(exp)) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6'-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)(exp). However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a d-Arg(9) molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r(CUG)(exp); pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA.

Targeting nuclear RNA for in vivo correction of myotonic dystrophy

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat1–3 and are retained in the nucleus4, 5. The mutant RNA exerts a toxic gain-of-function6, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target mRNAs7, 8. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are extraordinarily sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUGexp RNA in skeletal muscle, correcting the physiological, histopathologic, and transcriptomic features of the disease. The effect was sustained for up to one year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat-1, a long noncoding RNA (lncRNA) that is retained in the nucleus9. These results provide a general strategy to correct RNA gain-of-function and modulate the expression of expanded repeats, lncRNAs, and other transcripts with prolonged nuclear residence.

From dynamic combinatorial ‘hit’ to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy

The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG–CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG–RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.

Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching.

Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5'CUG/3'GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, sarco(endo)plasmic reticulum Ca(2+) ATPase 1, and cardiac troponin T. Based on these observations, the development of small-molecule ligands that target specifically expanded DM1 repeats could be of use as therapeutics. In the present study, chemical similarity searching was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of in vitro inhibitors of the RNA-protein complex were identified with low micromolar IC(50)'s, which are >20-fold more potent than the query compounds. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with chemical similarity searching.

Repression of nuclear CELF activity can rescue CELF-regulated alternative splicing defects in skeletal muscle models of myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CUG repeats in the 3’ UTR of DMPK transcripts. DM1 pathogenesis has been attributed in part to alternative splicing dysregulation via elevation of CUG-BP, Elav-like family member 1 (CELF1). Several therapeutic approaches have been tested in cells and mice, but no previous studies had specifically targeted CELF1. Here, we show that repressing CELF activity rescues CELF-dependent alternative splicing in cell culture and transgenic mouse models of DM1. CELF-independent splicing, however, remained dysregulated. These data highlight both the potential and limitations of targeting CELF1 for the treatment of DM1.

Abstract P104: Reactivation of Embryonic Gene Program due to CUG Repeat RNA Expression in Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. Cardiac involvement, which is characterized by conduction defects and arrhythmias, is the second leading cause of death in DM1 patients. The causative mutation is a CTG expansion in the 3' untranslated region of DMPK gene resulting in aberrant expression of CUG repeat RNA that accumulates into nuclear foci and causes misregulation in alternative splicing. Here we show that heart-specific and inducible expression of CUG repeat RNA in a DM1 mouse model results in global reactivation of embryonic gene expression program in adult heart that is distinct from a general hypertrophic stress response. Using q-PCR TaqMan arrays, we identified 54 miRNAs that were differentially expressed in DM1 mouse hearts one week following induction of CUG repeat RNA. Interestingly, 83% (45/54) of them exhibited a developmental shift in expression towards the embryonic pattern. Because over 90% (41/45) of them were down regulated within 72 hr after induction of repeat RNA and only 2/22 examined decreased in two unrelated mouse models of heart disease, we conclude their reduced expression is specific to DM1 and not simply a general response to cardiac injury. Microarray studies revealed a developmental switch not only in the miRNA expression patterns but also a pervasive shift in mRNA steady state levels of a number of genes to embryonic stage. Intriguingly, we found that loss of MBNL1 or gain of CELF1 activity, two major RNA binding proteins disrupted in DM1, are not driving the miRNA misregulation since their expression is indistinguishable between wild type, MBNL1 knock out and CELF1 over expressing mice. Moreover, comparable decrease in ten out of ten primary miRNA transcripts examined suggests loss of expression is not due to a processing defect. Instead, we discovered that adult-to-embryonic shift in expression of select micro- and messenger RNAs in DM1 heart occurs due to specific inactivation of a Mef2 transcriptional program. We are currently determining causal contributions of this Mef2-miRNA circuitry in the developmental reprogramming of gene expression in DM1 as well as its direct role in cardiac manifestations of this disease.

O.6 Antisense approach for myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by a dominant-negative pathogenic effects of RNA containing CUG expansions cause DM1. The knockdown of the CUG repeat RNA could be a major approach for the development of a gene therapy for DM1. The proof of principle regarding this approach was originally demonstrated in hDM1 myoblasts. We have shown that antisense RNAs are more efficient than shRNAs to target mutant DMPK RNAs and to restore a normal behavior to hDM1 myoblasts. In a DM1 mouse model, we showed that antisense RNAs can decreased up to 55% the levels of mutant transcripts, one month following a single i.m.

Myotonic dystrophy type 1-associated CTG repeats disturb the expression and subcellular distribution of microtubule-associated proteins MAP1A, MAP2, and MAP6/STOP in PC12 cells

To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3'-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.

Non-ATG–initiated translation directed by microsatellite expansions

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1

The neuromuscular disease myotonic dystrophy type I (DM1) affects multiple organ systems with the major symptoms being severe muscle weakness, progressive muscle wasting and myotonia. The causative mutation in DM1 is a CTG repeat expansion in the 3'-untranslated region of the DM protein kinase (DMPK) gene. RNA transcribed from the expanded allele contains the expanded CUG repeats and leads to the nuclear depletion of Muscleblind-like 1 (MBNL1) and to the increased steady-state levels of CUG-binding protein 1 (CUGBP1). The pathogenic effects of MBNL1 depletion have previously been tested by the generation of MBNL1 knockout mice, but the consequence of CUGBP1 overexpression in adult muscle is not known. In a DM1 mouse model expressing RNA containing 960 CUG repeats in skeletal muscle, CUGBP1 up-regulation is temporally correlated with severe muscle wasting. In this study, we generated transgenic mice with doxycycline-inducible and skeletal muscle-specific expression of CUGBP1. Adult mouse skeletal muscle overexpressing CUGBP1 reproduces molecular and physiological defects of DM1 tissue. The results from this study strongly suggest that CUGBP1 has a major role in DM1 skeletal muscle pathogenesis.

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for approximately 100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUG(exp) RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion within the 3'-untranslated region of the DMPK gene. The predominant mechanism of pathogenesis is a toxic gain of function of CUG repeat containing RNA transcribed from the expanded allele. The molecular mechanisms by which the RNA containing expanded repeats produce pathogenic effects include: sequestration of muscleblind-like 1 (MBNL1) protein and up-regulation of CUG binding protein 1 (CUGBP1). MBNL1 and CUGBP1 are RNA binding proteins that regulate alternative splicing transitions during development. Altered functions of these proteins in DM1 lead to misregulated splicing of their target genes, resulting in several features of the disease. The role of MBNL1 depletion in DM1 is well established through a mouse knock-out model that reproduces many disease features. Here we directly test the hypothesis that CUGBP1 up-regulation also contributes to manifestations of DM1. Using tetracycline-inducible CUGBP1 and heart-specific reverse tetracycline trans-activator transgenes, we expressed human CUGBP1 in adult mouse heart. Our results demonstrate that up-regulation of CUGBP1 is sufficient to reproduce molecular, histopathological and functional changes observed in a previously described DM1 mouse model that expresses expanded CUG RNA repeats as well as in individuals with DM1. These results strongly support a role for CUGBP1 up-regulation in DM1 pathogenesis.

PKC inhibition ameliorates the cardiac phenotype in a mouse model of myotonic dystrophy type 1

Cardiac complications are a common cause of death in individuals with the inherited multisystemic disease myotonic dystrophy type 1 (DM1). A characteristic molecular feature of DM1 is misregulated alternative splicing due to disrupted functioning of the splicing regulators muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CUGBP1 is upregulated in DM1 due to PKC pathway activation and subsequent CUGBP1 protein hyperphosphorylation and stabilization. Here, we blocked PKC activity in a heart-specific DM1 mouse model to determine its pathogenic role in DM1. Animals given PKC inhibitors exhibited substantially increased survival that correlated with reduced phosphorylation and decreased steady-state levels of CUGBP1. Functional studies demonstrated that PKC inhibition ameliorated the cardiac conduction defects and contraction abnormalities found in this mouse model. The inhibitor also reduced misregulation of splicing events regulated by CUGBP1 but not those regulated by MBNL1, suggesting distinct roles for these proteins in DM1 cardiac pathogenesis. The PKC inhibitor did not reduce mortality in transgenic mice with heart-specific CUGBP1 upregulation, indicating that PKC inhibition did not have a general protective effect on PKC-independent CUGBP1 increase. Our results suggest that pharmacological blockade of PKC activity mitigates the DM1 cardiac phenotype and provide strong evidence for a role for the PKC pathway in DM1 pathogenesis.