Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1) disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor) or muscleblind, or by RNA interference (RNAi)-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of noncoding CTG repeats in the dystrophia myotonica-protein kinase gene. Mutant transcripts form CUG hairpins that sequester RNAbinding factors into nuclear foci, including Muscleblind-like-1 protein (MBNL1), which regulate alternative splicing and gene expression. To identify molecules that target toxic CUG transcripts in vivo, we performed a positional scanning combinatorial peptide library screen using a Drosophila model of DM1. The screen identified a D-amino acid hexapeptide (ABP1) that reduced CUG foci formation and suppressed CUG-induced lethality and muscle degeneration when administered orally. Transgenic expression of natural, L-amino acid ABP1 analogues reduced CUG-induced toxicity in fly eyes and muscles. Furthermore, ABP1 reversed muscle histopathology and splicing misregulation of MBNL1 targets in DM1 model mice. In vitro, ABP1 bound to CUG hairpins and induced a switch to a single-stranded conformation. Our findings demonstrate that ABP1 shows antimyotonic dystrophy activity by targeting the core of CUG toxicity.disease model | drug discovery | non-coding RNA disease | medicinal chemistry | RNA secondary structure M yotonic dystrophy type 1 (DM1, OMIM #160900) is an autosomal dominant disease caused by the expansion of a CTG trinucleotide repeat in the 3′ untranslated region (UTR) of the dystrophia myotonica-protein kinase (DMPK) gene. Characteristic symptoms include myotonia, progressive muscle wasting, and cardiac conduction defects, among other systemic manifestations. The molecular mechanisms underlying DM1 pathogenesis are complex and affect a large number of cellular processes (1). However, most data suggest that the main triggering event is a toxic gain-of-function of the expanded CUG RNA. CUG-repeat expansions form double-stranded hairpins that are retained as inclusions within the nucleus. These hairpins recruit a number of transcription and splicing factors, including Muscleblind-like 1 (MBNL1) (2-6). Sequestration of MBNL1 originates a loss of function, which plays a key role in the development of DM1 symptoms. Mbnl1 knockout mice reproduced typical features of DM1, and overexpression of Mbnl1 in a mouse model that expressed CTG repeats reversed these phenotypes (7,8). CTGrepeat expression in mice caused misregulation of at least 156 alternative splicing events. Of these, 128 also occurred in Mbnl1 knockout animals (9-12). The splicing factor CUG-binding protein 1 (CUGBP1) is another key component in the development of DM1 phenotypes. CUGBP1 antagonizes MBNL1 activity in the regulated use of alternative exons in a number of transcripts and is abnormally upregulated in patients with DM1, further contributing to splicing misregulation (13-15).Mahadevan et al. provided the first in vivo proof-of-principle for a therapeutic strategy based on ablating toxic RNA molecules in DM1 (16). They demonstrated that expanded CTG-induced effects could be reverted if CTG-repeat transgene expression was interrupted in a DM1 mouse model. Several ...

Functional depletion of the alternative splicing factors Muscleblind-like (MBNL 1 and 2) is at the basis of the neuromuscular disease myotonic dystrophy type 1 (DM1). We previously showed the efficacy of miRNA downregulation in Drosophila DM1 model. Here, we screen for miRNAs that regulate MBNL1 and MBNL2 in HeLa cells. We thus identify miR-23b and miR-218, and confirm that they downregulate MBNL proteins in this cell line. Antagonists of miR-23b and miR-218 miRNAs enhance MBNL protein levels and rescue pathogenic missplicing events in DM1 myoblasts. Systemic delivery of these “antagomiRs” similarly boost MBNL expression and improve DM1-like phenotypes, including splicing alterations, histopathology, and myotonia in the HSALR DM1 model mice. These mammalian data provide evidence for therapeutic blocking of the miRNAs that control Muscleblind-like protein expression in myotonic dystrophy.

Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.