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, 19 (18), 3614-22

CUGBP1 Overexpression in Mouse Skeletal Muscle Reproduces Features of Myotonic Dystrophy Type 1

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CUGBP1 Overexpression in Mouse Skeletal Muscle Reproduces Features of Myotonic Dystrophy Type 1

Amanda J Ward et al. Hum Mol Genet.

Abstract

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.

Figures

Figure 1.
Figure 1.
MDAFrtTA/TRECUGBP1 mice display CUGBP1 up-regulation in skeletal muscle upon dox administration. (A) Western blot for CUGBP1 following serial dilution of gastrocnemius muscle protein lysates from induced MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) mice show an 8-fold elevation in CUGBP1 expression compared with uninduced control mice. (B) Western blot using anti-Flag antibody to detect exogenous CUGBP1 shows that the expression is specific to the skeletal muscle of MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) mice. Ponceau staining of the blot was used as the loading control. (C) CUGBP1 is highly expressed in all MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) skeletal muscles tested including the gastrocnemius, quadriceps, triceps and soleus muscles. Western blots for CUGBP2 and MBNL1 and the regeneration marker eMHC are also shown. Both GAPDH and Ponceau stain were used as loading controls. (D) CUGBP1 western blot over the 8-week time course on dox diet shows short-term induction in MDAFrtTA/TRECUGBP1 mice. CUGBP2 and MBNL1 protein expression are also shown.
Figure 2.
Figure 2.
Induced MDAFrtTA/TRECUGBP1 mice exhibit reduced muscle weight and muscle function. (A) Total mouse body weight at 1, 2, 4 and 8 weeks following CUGBP1 induction relative to the pre-induction weight. Induced mice (open circles, n ≥ 5) have a significant reduction in body weight when compared with uninduced control mice (closed circles, n ≥ 5), **P < 0.001. (B) Induced mice (open bars, n = 8 muscles) have a significant reduction in gastrocnemius muscle weight when compared with uninduced control mice (closed bars, n = 8 muscles), **P < 0.001. Only male mice were used for this assay and muscle weights were normalized to the length of the tibia. (C) Mice were subject to an acute treadmill exercise regime at each time point and the time to drop-off is represented in a box plot. The top and bottom of each box denote the 75th and 25th percentiles, respectively, and the median is shown by the bold middle line. Induced mice (red boxes, n ≥ 4) have a significant reduction in muscle function at 1–4 weeks on dox diet compared with uninduced control mice (black boxes, n ≥ 4), P < 0.001.
Figure 3.
Figure 3.
Induced MDAFrtTA/TRECUGBP1 mice exhibit DM1-like histological features. Light microscope analysis of gastrocnemius muscle stained with hematoxylin and eosin. (A) Uninduced control mice have normal myofiber size and peripherally located nuclei. Scale bar: 20 µm. (B and C) Induced MDAFrtTA/TRECUGBP1 mice have an abundance of central nuclei (arrows) and evidence of degeneration (small fibers with inflammatory infiltrate, double asterisks) by 2 and 4 weeks on dox diet. (D) Skeletal muscles from EpA960/HSA-Cre-ERT2 mice 4 weeks after the induction of DMPK-CUG960 RNA show similar changes as CUGBP1-overexpressing muscle, including hypotrophic myofibers with central nuclei (arrows) and degenerative changes (double asterisks). All panels are 40× magnification.
Figure 4.
Figure 4.
Long-term expression of CUGBP1 in MDAFrtTA/TRECUGBP1 mice induced with a low dosage dox diet (0.05 g dox/kg food). (A) Western blot of protein extracted from gastrocnemius muscle shows a 2-fold up-regulation of CUGBP1 in MDAFrtTA/TRECUGBP1 mice (two mice per time point) fed a low dosage dox diet compared with uninduced controls (one mouse per time point). The induction is stable over the 6-month time course. There is no detectable expression of the regeneration marker, eMHC. (B) Hematoxylin and eosin staining of gastrocnemius cross-sections from low dosage dox-induced MDAFrtTA/TRECUGBP1 mice shows an increase in myofibers containing centrally located nuclei (arrows) (20× magnification).
Figure 5.
Figure 5.
Induced MDAFrtTA/TRECUGBP1 mice reproduce the characteristic alternative splicing misregulation observed in DM1 patients. (A) MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) mice have increased expression of the embryonic splice variants in adult gastrocnemius muscle for the indicated alternative splicing events, *P < 0.01 and **P < 0.001. t-Test for statistical significance was performed between adult uninduced and induced mice. (B) Alternative splicing events that are not significantly misregulated in induced MDAFrtTA/TRECUGBP1 gastrocnemius muscle. Newborn limb (n = 1, pooled limbs from 12 mice), adult uninduced (n = 4, individual) and adult induced (n = 4, individual).

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