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

Abstract

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.

Figure 1. Construction of the Clcn1-L reporter minigene and effect of triplet repeat expansion on the reporter vector Clcn1-L.

(a) Schematic structure of the Clcn1-L minigene reporter. A genomic segment of mouse Clcn1 containing exons 6 to 7 (including the intron) was sub-cloned downstream of EGFP in the pEGFP-C1 plasmid. Firefly luciferase was inserted in-frame with a correct Clcn1 splicing pattern. (b) Schematic of how Clcn1-L functions. Exon 7A exclusion results in luciferase expression to detect correct Clcn1 splicing. (c) Inclusion of Clcn1 exon 7A increased upon expression of the expanded CUG repeat. (d) Bar charts show the quantified percentages of exon 7A inclusion (mean + SEM, n = 4). (e) Luciferase analysis showed that relative luciferase activity decreased upon expression of the expanded CUG repeat (mean + SEM, n = 3). The gel image was cropped around the region of interest and the samples (n = 4) were resolved in the same gel. Statistical significances were determined using t-tests (*p < 0.05, ***p < 0.001).

Figure 2. Identification of small-molecule compounds that correct aberrant splicing of Clcn1.

(a) A luciferase reporter assay showed that manumycin A corrected aberrant splicing in the presence of the expanded CUG repeat (mean + SEM, n = 3). (b) Cellular splicing analysis showed that manumycin A corrects aberrant splicing of Clcn1. (c) Quantification of the results shown in (b) (mean + SEM, n = 3). (d) Structure of manumycin A. The gel image was cropped around the region of interest and the samples (n = 3) were resolved in the same gel. Statistical significance was determined using t-tests (*p < 0.05, **p < 0.01).

Figure 3. Manumycin A corrects aberrant splicing of Clcn1 in HSA^LR DM1 model mice.

(a) RT-PCR analysis showed increased inclusion of Clcn1 exon 7A in HSA^LR mouse. (b) Quantification of the results shown in (a) (mean + SEM, n = 3). (c) RT-PCR analysis showed reduced inclusion of Clcn1 exon 7A 5 days after injection of manumycin A into TA muscles. (d) Quantification of the results shown in (a) (Mean + SEM, n = 3). The gel image was cropped around the region of interest and the samples (n = 3) were resolved in the same gel. Statistical significance was determined using t-tests (*p < 0.05, **p < 0.01).

Figure 4. The knockdown of H-Ras corrects aberrant splicing of Clcn1 in the presence of the expanded CUG repeat.

(a) Representative result of Western blot analysis of H-Ras in C2C12 cells. (b) Results of cellular splicing assays using Clcn1-L minigene, DM480 and siRNA. (c) Quantification of the results shown in (b) (mean + SEM, n = 3). The gel and blot image were cropped around the region of interest and the samples (n = 3) were resolved in the same gel or blot. Statistical significance was determined using t-tests (**p < 0.01).

Figure 5. Model of how manumycin A affects Clcn1 splicing.

In DM1, it is known that expanded CUG repeat RNA transcripts trap MBNL1 and up-regulate CUGBP1, resulting in aberrant regulation of alternative splicing. However, manumycin A does not affect the expression of these proteins. Manumycin A might act as a Ras farnesyltransferase inhibitor, thereby altering H-Ras signaling. This could in turn result in alteration of a trans-acting factor involved in alternative splicing other than MBNL1 and CUGBP1.