A Muscleblind Knockout Model for Myotonic Dystrophy

Kanadia, Rahul N.; Johnstone, Karen A.; Mankodi, Ami; Lungu, Codrin; Thornton, Charles A.; Esson, Douglas W.; Timmers, Adrian M.; Hauswirth, William W.; Swanson, Maurice S.

doi:10.1126/science.1088583

Cited by 662 publications

(752 citation statements)

References 21 publications

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“…In (CTG) 5 mice in which expression of GFP-DMPK 3′ UTR is induced, the elevated CUG-BP1 levels in the absence of MBNL1 changes still results in an imbalance, albeit one that affects only one side of the equation. This is analogous to results from the CUG-BP1 transgenic mouse 28 and the Mbnl1ΔE3 knockout mouse 16 , in which perturbations of only one (CUG-BP1) or the other (MBNL-1) protein results in DM1-like splicing defects and DM1 pathology. Therapeutic strategies aimed at addressing only one side of the balance (that is, correction of MBNL1 sequestration or decreasing CUG-BP1) may not be sufficient, as both these proteins may have other functions in addition to their roles as splicing factors 17 .…”

supporting

confidence: 74%

“…Furthermore, we performed RT-PCR for Clcn1 and Tnnt3 in our mice and uncovered splicing abnormalities (Fig. 2c) similar to those in transgenic mice overexpressing CUG repeats 15 , in the Mbnl1ΔE3 knockout mouse 16 and in individuals with myotonic dystrophy [14][15][16] . Skeletal muscle histology also showed induction of central nuclei, nuclear clumping and fiber size variation as seen in individuals with DM1 (Fig.…”

mentioning

confidence: 62%

“…We performed RT-PCR for Clcn1 and Tnnt3 as described 16 . All RT-PCR products were cloned and their identity confirmed by DNA sequencing.…”

Section: Rna Analysesmentioning

confidence: 99%

“…We used protein blots of 50 μg of total protein to detect CUG-BP1 using monoclonal antibody 3B1 (Upstate, cat.# 05-621) at a 1:4,000 dilution. We detected MBNL1 as previously described 16 . Glyceraldehyde phosphate dehydrogenase (GAPDH) levels were detected using a GAPDH antiserum (Ambion) and were used as a loading control.…”

Section: Protein Blottingmentioning

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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supporting

confidence: 74%

mentioning

confidence: 62%

“…We performed RT-PCR for Clcn1 and Tnnt3 as described 16 . All RT-PCR products were cloned and their identity confirmed by DNA sequencing.…”

Section: Rna Analysesmentioning

confidence: 99%

Section: Protein Blottingmentioning

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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“…A multi-step model for DM1 pathogenesis has been proposed [reviewed in reference 2]: (1) the mutant gene is transcribed, giving rise to transcripts that contain an expanded CUG repeat (CUG exp ) [3]; (2) the CUG exp transcripts accumulate in RNA nuclear (ribonuclear) foci [4]; (3) RNA binding proteins, including muscleblind 1 (MBNL1), are sequestered in the ribonuclear foci [5,6]; (4) altered activity of splicing factors, such as MBNL1 and CUG binding protein 1, leads to abnormal alternative splicing for a sub-group of pre-mRNAs [7,8]; and (5) expression of inappropriate splice products leads to symptoms of DM1. For example, CUG exp RNA triggers abnormal alternative splicing of the ClC-1 chloride channel, and the predominant ClC-1 splice products expressed in DM1 muscle are devoid of ion channel activity [9][10][11].…”

Section: Introductionmentioning

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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Cancer Risk Among Patients With Myotonic Muscular Dystrophy

Gadalla¹,

Lund²,

Pfeiffer³

et al. 2011

JAMA

103

108

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Context Myotonic muscular dystrophy (MMD) is an autosomal dominant multisystem neuromuscular disorder characterized by unstable nucleotide repeat expansions. Case reports have suggested that MMD patients may be at increased risk of malignancy, putative risks which have never been quantified. Objective To quantitatively evaluate cancer risk in patients with MMD, overall, and by sex and age. Design, Setting, and Participants We identified 1,658 patients with an MMD discharge diagnosis in the Swedish Inpatient Hospital or Danish Patient Discharge Registries between 1977 and 2008. We linked these patients to their corresponding cancer registry. Patients were followed from date of first MMD-related inpatient or outpatient contact, to first cancer diagnosis, death, emigration, or completion of cancer registration. Main Outcome Measures Risks of all cancers combined, and by anatomic site, stratified by sex and age. Results 104 patients with an inpatient or outpatient discharge diagnosis of MMD developed cancer during post-discharge follow-up. This corresponds to an observed cancer rate of 73.4/10,000 person-years in MMD versus an expected rate of 36.9/10,000 in the general Swedish and Danish populations combined (SIR =2.0, 95% CI =1.6–2.4). Specifically, we observed significant excess risks of cancers of the endometrium (observed rate=16.1/10,000 person-years: SIR=7.6, 95%CI=4.0–13.2), brain (observed rate=4.9/10,000 person-years: SIR=5.3, 95%CI=2.3–10.4), ovary (observed rate=10.3/10,000 person-years: SIR=5.2, 95% CI=2.3–10.2), and colon (observed rate=7.1/10,000 person-years: SIR=2.9, 95%CI=1.5–5.1). Cancer risks were similar in females and males after excluding genital organ tumors (SIR=1.9, 95% CI=1.4–2.5 vs. 1.8, 95% CI=1.3–2.5, respectively, p-heterogeneity=0.81; observed rates=64.5 and 47.7/10,000 person-years in women and men, respectively), The same pattern of cancer excess was observed first in the Swedish, and then in the Danish cohorts, which were studied sequentially and initially analyzed independently. Conclusions MMD patients identified from the Swedish and Danish patient registries were at increased risk of cancer both overall and for selected anatomic sites.

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A Muscleblind Knockout Model for Myotonic Dystrophy

Cited by 662 publications

References 21 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

Cancer Risk Among Patients With Myotonic Muscular Dystrophy

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