Modeling of Myotonic Dystrophy Cardiac Phenotypes in Drosophila

After respiratory distress, cardiac dysfunction is the second most common cause of fatality associated with the myotonic dystrophy (DM) disease. Despite the prevalance of heart failure in DM, physiopathological studies on heart symptoms have been relatively scarce because few murine models faithfully reproduce the cardiac disease. Consequently, only a small number of candidate compounds have been evaluated in this specific phenotype. To help cover this gap Drosophila combines the amenability of its invertebrate genetics with the possibility of quickly acquiring physiological parameters suitable for meaningful comparisons with vertebrate animal models and humans. Here we review available descriptions of cardiac disease in DM type 1 and type 2, and three recent papers reporting the cardiac toxicity of non-coding CUG (DM1) and CCUG (DM2) repeat RNA in flies. Notably, flies expressing CUG or CCUG RNA in their hearts developed strong arrhythmias and had reduced fractional shortening, which correlates with similar phenotypes in DM patients. Overexpression of Muscleblind, which is abnormally sequestered by CUG and CCUG repeat RNA, managed to strongly suppress arrhythmias and fractional shortening, thus demonstrating that Muscleblind depletion causes cardiac phenotypes in flies. Importantly, small molecules pentamidine and daunorubicin were able to rescue cardiac phenotypes by releasing Muscleblind from sequestration. Taken together, fly heart models have the potential to make important contributions to the understanding of the molecular causes of cardiac dysfunction in DM and in the quick assessment of candidate therapeutics

rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

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.Peer reviewe

Two enhancers control transcription of Drosophila muscleblind in the embryonic somatic musculature and in the central nervous system.

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene

Sequence and melting temperature (Tm) of PCR primers used for cloning genomic DNA.

<p>Restriction sites are highlighted in bold.</p

Genomic organization of the human MBNL1 gene.

<p>(A) Scale representation of 198 kb of the human MBNL1 locus. Green boxes correspond to exons and black lines to introns. Tested promoter regions are indicated as P1 and P2; a yellow circle denotes a predicted CpG island. (B) Schematic representation of H3K27Ac marks, typical of promoter regions, on seven human cell lines. (C) 5′-Ends of ESTs mapping to the MBNL1 locus support two potential transcription start sites for the gene. Data according to the UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093125#pone.0093125-Meyer1" target="_blank">[58]</a>. (E,F,H,I) Direct visualization of the eGFP reporter under the control of the ME enhancer. (D–F) Enhancer-less Hsa-P1 construct is a negative control. (G–I) Flies carrying the ME enhancer upstream of the human MBNL1 promoter (Hsa-P1) reproduce Muscleblind expression in the somatic musculature. (E,H) Lateral and (F,I) ventral views of late embryos. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up, unless otherwise stated.</p

ME reproduces <i>muscleblind</i> expression in the embryonic somatic musculature.

<p>(A) Localization of the putative <i>cis</i>-regulatory modules M1, M2, ML, H and M3, indicated as orange boxes, in the context of the <i>muscleblind</i> genomic locus. Fluorescence confocal images of lateral (B,E,H,) and ventral (C,F,I,) views of late <i>Drosophila</i> embryos. (D,G) Schematic representation of the reporter constructs used to transform the germline of <i>Drosophila</i>. In control <i>yw</i> flies (B,C) an anti-Mbl antibody detects robust expression in the somatic musculature and in the CNS (green). Direct visualization of the GFP reporter under the control of the ME enhancer in the pH-Stinger vector (E,F,H,I). Promoter-less ME constructs (D–F) do not activate GFP expression and serve as negative controls. Flies carrying the ME enhancer upstream of <i>Hsp70</i> (G–I) reproduce Muscleblind expression in the somatic musculature but not in the CNS. All micrographs were taken at 200× magnification. Anterior is to the left and dorsal up unless otherwise stated.</p

NE reproduces <i>muscleblind</i> expression in the central nervous system.

<p>(A) Schematic representation of the reporter construct used to transform the <i>Drosophila</i> germline. Fluorescence confocal images of lateral (B–D) and ventral (E–J) views of late <i>Drosophila</i> embryos expressing construct (A) co-stained with anti-GFP (green) and anti-Mbl antibodies (red). NE drives expression in the CNS (arrows; C,I) overlapping Muscleblind expression (B,E,H; D,G,J shows the merge in yellow). No signal of the reporter was observed in tissues other than CNS. Endogenous Muscleblind expression in the muscles is in focus in (E,G,H,J). Micrographs were taken at 200× (B–G) and 400× magnification (H–J). Anterior is to the left and dorsal up unless otherwise stated.</p

Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model

SUMMARY Myotonic dystrophy type 1 (DM1) is a genetic disease caused by the pathological expansion of a CTG trinucleotide repeat in the 3′ UTR of the DMPK gene. In the DMPK transcripts, the CUG expansions sequester RNA-binding proteins into nuclear foci, including transcription factors and alternative splicing regulators such as MBNL1. MBNL1 sequestration has been associated with key features of DM1. However, the basis behind a number of molecular and histological alterations in DM1 remain unclear. To help identify new pathogenic components of the disease, we carried out a genetic screen using a Drosophila model of DM1 that expresses 480 interrupted CTG repeats, i(CTG)480, and a collection of 1215 transgenic RNA interference (RNAi) fly lines. Of the 34 modifiers identified, two RNA-binding proteins, TBPH (homolog of human TAR DNA-binding protein 43 or TDP-43) and BSF (Bicoid stability factor; homolog of human LRPPRC), were of particular interest. These factors modified i(CTG)480 phenotypes in the fly eye and wing, and TBPH silencing also suppressed CTG-induced defects in the flight muscles. In Drosophila flight muscle, TBPH, BSF and the fly ortholog of MBNL1, Muscleblind (Mbl), were detected in sarcomeric bands. Expression of i(CTG)480 resulted in changes in the sarcomeric patterns of these proteins, which could be restored by coexpression with human MBNL1. Epistasis studies showed that Mbl silencing was sufficient to induce a subcellular redistribution of TBPH and BSF proteins in the muscle, which mimicked the effect of i(CTG)480 expression. These results provide the first description of TBPH and BSF as targets of Mbl-mediated CTG toxicity, and they suggest an important role of these proteins in DM1 muscle pathology

RbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

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