Myotonic Dystrophy in Transgenic Mice Expressing an Expanded CUG Repeat

Mankodi, Ami; Logigian, Eric L.; Callahan, Linda M.; McClain, Carolyn; White, Robert J.; Henderson, Don; Krym, Matt; Thornton, Charles A.

doi:10.1126/science.289.5485.1769

Cited by 662 publications

(730 citation statements)

References 27 publications

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“…Standard mouse anesthetics were cardiotoxic, especially in mice with preexisting conduction defects, and after extensive experimentation we settled on the valium and ketamine combination. We performed and scored EMGs as previously reported 7 . We performed three lead ECGs using a BioAmp/Powerlab from ADInstruments and collected data on a computer for later analysis.…”

Section: Emgs and Ecgsmentioning

confidence: 99%

“…2 ). Myoblast cell culture models 5,6 and subsequently a transgenic mouse model 7 have provided strong evidence for the involvement of RNA containing expanded CUG repeat tracts in aspects of DM1 skeletal muscle pathology. However, there is no clear model of RNA toxicity in the heart, and instead it has been suggested that DM1 cardiac pathology may be due to misexpression of DMPK 8,9 .…”

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confidence: 99%

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Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

et al. 2006

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Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG) n tract in the 3′ UTR of the gene encoding myotonic dystrophy protein kinase (DMPK) 1 , which results in nuclear entrapment of the 'toxic' mutant RNA and interacting RNAbinding proteins (such as MBNL1) in ribonuclear inclusions 2 . It is unclear if therapy aimed at eliminating the toxin would be beneficial. To address this, we generated transgenic mice expressing the DMPK 3′ UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP). We were surprised to find that mice overexpressing a normal DMPK 3′ UTR mRNA reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. However, we observed increased levels of CUG-binding protein (CUG-BP1) in skeletal muscle, as seen in individuals with DM1. Notably, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represent the first in vivo proof of principle for a therapeutic strategy for treatment of myotonic dystrophy by ablating or silencing expression of the toxic RNA molecules.Common features of adult-onset DM1 include myotonia, progressive skeletal muscle loss, cardiac conduction defects, smooth muscle dysfunction, cataracts and insulin resistance 2 . The normal number of CTG repeats (n = 5 to ~30) is higher (n = 50 to >3,000) in individuals with DM1 (ref. 1 ). Unlike the wild-type transcript, mutant DMPK mRNA forms nuclear aggregates 3,4 and is thought to trigger dominant effects by aberrant interactions with or altered activity of RNA splicing factors, principally members of the muscleblind-like (MBNL) family (such as MBNL1) and the CUG-BP and ETR3-like factor (CELF) family (such as CUG-BP1), leading to abnormal splicing of specific RNAs such as chloride channel (Clcn1), insulin Correspondence should be addressed to M.S.M. (mahadevan@virginia.edu). 4 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSM.S.M., R.S.Y., Q.Y., C.D.F.-M., T.D.B. and L.H.P. performed experimental work and data analysis. S.B. generated the transgene constructs. M.S.M. was responsible for conceptual design and execution. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. One potential therapeutic approach in DM1 is to get rid of the toxic RNA from cells. However, it is unclear if this will alleviate the effects of the disease. We used the tetracycline (Tet) inducible system with the reverse tetracycline transactivator (rtTA) to generate double transgenic mice harboring (i) a Tet-responsive, DMPK promoter 10,11 -driven transgene (named GFP-DMPK 3′ UTR) expressing the DMPK 3′ UTR mRNA as part of a GFP transcript, and (ii) a constitutively expressed rtTA transgene (Fig. 1a) Fig. 1). Notably, RNA blots of skeletal muscle RNA showed two major species due to alternative use of polyadenylation signal...

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Section: Emgs and Ecgsmentioning

confidence: 99%

mentioning

confidence: 99%

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

et al. 2006

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“…In this transgenic mouse model, the expanded repeat is inserted in a genomic fragment containing the entire human skeletal actin gene, including the 5' regulatory sequences, and the pattern of transgene expression is similar to endogenous skeletal actin [20,29]. To our knowledge, the paucity of ribonuclear foci at the NMJ in HSA LR transgenic mice is the first evidence that genes encoding myofibrillar proteins are downregulated in subsynaptic nuclei.…”

Section: Discussionmentioning

confidence: 99%

“…Spinal cord tissue was obtained at autopsy and stored at -70°C. HSA LR transgenic mice that express CUG exp RNA in skeletal muscle were described previously [20]. CUG exp expression in these transgenic mice is controlled by regulatory elements from the human skeletal actin gene, which results in ribonuclear foci, myotonia, myopathy, and a spliceopathy that is similar to human DM1 [6].…”

Section: Tissue Samplesmentioning

confidence: 99%

Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1

Wheeler

Krym

Thornton

2007

Neuromuscular Disorders

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In myotonic dystrophy type 1 (DM1) the muscle fibers express RNA containing an expanded CUG repeat (CUG exp ). The CUG exp RNA is retained in the nucleus, forming ribonuclear foci. Splicing factors in the muscleblind (MBNL) family are sequestered in ribonuclear foci, resulting in abnormal regulation of alternative splicing. In extrajunctional nuclei, these effects on splicing regulation lead to reduced chloride conductance and altered insulin receptor signaling. Here we show that CUG exp RNA is also expressed in subsynaptic nuclei of muscle fibers and in motor neurons in DM1, causing sequestration of MBNL1 protein in both locations. In a transgenic mouse model, expression of CUG exp RNA at high levels in extrajunctional nuclei replicates many features of DM1, but the toxic RNA is poorly expressed in subsynaptic nuclei and the mice fail to develop denervation-like features of DM1 myopathology. Our findings indicate that subsynaptic nuclei and motor neurons are at risk for DM1-induced spliceopathy, which may affect function or stability of the neuromuscular junction.

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“…1,2 Although it has been suggested that haploinsufficiency and position effect variegation may contribute to DM1 phenotype, there is increasing evidence that the accumulation of mutant DMPK mRNA with expanded CUG repeats [3][4][5][6][7][8] has a central function in the pathogenetic mechanism. 9,10 Induced expression of expanded CUG repeats in a transgenic mouse model resulted in a DM1 phenotype, which reverted to normal when the induction was switched off. 11 This provided strong evidence that blockade of mutant DMPK transcript accumulation can restore cellular functions.…”

Section: Introductionmentioning

confidence: 99%

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

et al. 2008

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Dystrophia myotonica type 1 (DM1), the most common muscular dystrophy in adults, results from expansion of a CTG repeat in the 3 0 -untranslated region of the dystrophia myotonica protein kinase gene (DMPK). Correction of the mutant DMPK transcript is a potential therapeutic strategy in DM1. We investigated the efficacy of artificial trans-splicing molecules (ATMs) to target and correct DMPK transcripts. ATMs designed to target intron 14 of DMPK pre-mRNA transcripts were tested for their ability to trans-splice the transcripts of a DMPK mini-gene construct and the endogenous DMPK transcripts of human myosarcoma cells (CCL-136). On agarose gel electrophoresis analysis, six of eight ATMs showed trans-splicing efficacy when applied to DMPK mini-gene construct transcripts, of which three were able to trans-splice endogenous DMPK pre-mRNA transcripts in myosarcoma cells, with trans-splicing efficiency ranging from 1.81 to 7.41%. These findings confirm that artificial trans-splicing can repair DMPK pre-mRNA and provide proof-of-principle evidence for this approach as potential therapeutic strategy for DM1.

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Myotonic Dystrophy in Transgenic Mice Expressing an Expanded CUG Repeat

Cited by 662 publications

References 27 publications

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

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