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A New Protein Is Implicated in Myotonic Dystrophy, Pointing the Way to a Therapeutic Target

Robinson, Richard

doi: 10.1097/01.NT.0000420994.78613.b7
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TOXIC RNAs (red dots) are shown here accumulating in the nucleus (blue) of a myotonic dystrophy induced pluripotent stem cell; the neuronal marker is in green

TOXIC RNAs (red dots) are shown here accumulating in the nucleus (blue) of a myotonic dystrophy induced pluripotent stem cell; the neuronal marker is in green

Investigators report that sequestration of muscleblind-like 2 protein in the CNS likely accounts for the neurologic manifestations of myotonic dystrophy. Combined with new proof-of-principle experiments showing the potential of antisense therapy to improve myotonia, these results also suggest that comprehensive treatment of this multisystem disorder may not be out of the question.

Myotonic dystrophy (DM) may get its name from the disease's effects on muscle, but from the patient's perspective, it's usually the effects on the brain that matter most. Recent progress in understanding DM pathogenesis has drawn attention to a muscle-specific transcription factor, muscleblind-like protein 1 (MBNL1), which becomes sequestered in expanded RNAs within muscle.

Now, an Aug. 9 paper in Neuron shows that sequestration of a related protein in the CNS likely accounts for the neurologic manifestations of the disease. Combined with new proof-of-principle experiments showing the potential of antisense therapy to improve myotonia, these results also suggest that comprehensive treatment of this multisystem disorder may not be out of the question.

DM is caused by an expansion of a trinucleotide CTG (for DM type 1) — an expanded CTG repeat occurs in the untranslated portion of the DMPK gene, which leads to a CUG repeat in the transcribed mRNA; tetranucleotide CCTG is expanded in DM type 2. When transcribed, each leads to the accumulation of long and tangled strands of RNA, which, recent studies have shown, bind to and prevent the activity of muscleblind-like protein 1 (MBNL1). MBNL1 is a peripheral splicing factor, responsible for directing the RNA splicing machinery to targets on pre-mRNA, causing some exons to be included, and others excluded, in the final mRNA. Splicing factors such as MBNL1 act on the majority of genes to allow the relatively small human genome to create a wide array of proteins.

A related gene, muscleblind-like protein 2 (MBNL2), is expressed in the CNS, and in DM, also becomes sequestered in accumulating RNA, in this case in neurons. But the effects of that sequestration have not been clear, according to Maurice Swanson, PhD, professor of molecular genetics and microbiology at the University of Florida in Gainesville.

In mice lacking MBNL1, Dr. Swanson said, “the skeletal muscle effects were strikingly similar to what we see in myotonic dystrophy,” but the CNS aspects of the disease were lacking. To many in the field, that didn't seem like a critical flaw in the model, “because we were very focused on muscle symptoms.” But that changed at an international myotonic dystrophy meeting several years back, when a group of patients addressed the assembled researchers. “They emphatically told us that one of the major concerns they had were the problems with cognitive function in the brain. That really caused a shift in the attention everybody in the field was giving to what was abnormal in the myotonic dystrophy brain,” and it laid the foundation for the current study, said Dr. Swanson.



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To determine the effect of depleting MBNL2 in the CNS, Dr. Swanson created a mouse model lacking the gene. As expected, the mice had normal skeletal muscle structure and function, displaying none of the pathology associated with lack of MBNL1. They did, however, develop features reminiscent of DM's effect on the central nervous system. The mice, like DM patients, had sleep abnormalities, most strikingly an increase in the number of REM sleep episodes and the total amount of REM sleep. The mice also had learning and memory deficits, which appeared to be due to decreased activity of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus.

Dr. Swanson examined which genes were affected by MBNL2 in the brain, and found over 800 targets. Many of them had one thing in common: the encoded protein could be made in both fetal and adult forms, depending on the exons used, and MBNL2 appeared to regulate which version was made.

It is quite common to have fetal and adult isoforms of the same protein, Dr. Swanson explained. “As you mature, each developmental window produces a new set of proteins. All those proteins have to work together on the fly, producing a structure that evolves as the organism becomes more adult-like. Other gene-protein families are involved as well, but muscleblind appears to be one of the major ones responsible for this fetal-to-adult conversion.”

In general, MBNL2 promotes the adult version of its target proteins, so in its absence, fetal versions predominate. But other splicing regulators are unaffected by DM, and the clash between adult and fetal proteins means that evolving structure begins to malfunction.

One significant MBNL2 target may be casein kinase 1 delta (CSNK1D), a key component of the mammalian circadian clock. “We feel this is an excellent candidate” for explaining sleep aberrations in DM, Dr. Swanson said, especially since mutations in this gene have also been linked to another sleep disorder.

Whether there is a single gene that corresponds to each of the disease's many symptoms is unknown. “That is the ultimate question we would like to address,” he said. “We believe the field has explained myotonia, which is due to the missplicing of the major chloride channel in skeletal muscle.” The hope is to do the same for each of the neurologic symptoms in DM.



Even if the picture isn't that simple, it may be possible to identify, and target, a small number of the most important genes involved, and have a major effect on the disorder. “If you asked the average myotonic dystrophy patients, 'If we could erase the problems you have with sleep regulation, would this be important to you?' they would uniformly say yes.” And there is some hope that therapy may be on the way, as recently shown for myotonia in the mouse model. (See “Antisense Therapy Successful Against Myotonia in Mouse Model.”)

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According to Stephen Tapscott, MD, PhD, professor of neurology at the University of Washington in Seattle, one of the major outstanding questions in myotonic dystrophy has been whether the neurologic manifestations are mediated through the same molecular mechanism as the muscle disease. “I think the major advance in this paper is showing that the dominant mechanism is probably the same as the dominant mechanism in skeletal muscle.” The implication, he said, is that therapies developed to treat skeletal muscle should be equally applicable to the neurologic symptoms.

“The exciting part of the paper,” he said, “is that it raises the possibility that if the sequestration of MBNL2 can be overcome, either by degrading the mutant RNA or by replacing the protein, you should be able to reverse the neurologic symptoms. This paper gives a road map for how to develop treatments.”

The study also identifies downstream mechanisms that may contribute to components of the clinical manifestations. Modulating these may provide symptomatic relief even in the absence of curative therapy.

And finally, Dr. Tapscott said, the development of the mouse model used in this study “should be a huge step forward in transitioning the development of therapy from just targeting skeletal muscle, to now targeting the neurologic symptoms.”

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An Aug. 2 paper in Nature has shown that antisense therapy may hold promise for treatment of the peripheral symptoms of myotonic dystrophy. Researchers administered antisense molecules targeting the expanded RNA in a mouse that expresses the expanded CUG RNA repeat. Unlike previous, unsuccessful attempts, in this case the antisense molecule was tailored to be retained in the cell nucleus, providing maximum exposure to the mutant RNA. Binding of the antisense molecule to the RNA triggered degradation of the RNA by the cellular defense machinery, and led to improvements in myotonia and muscle pathology, as well as normalization of the pathologic transcription changes caused by the absence of muscleblind 1. Remarkably, the beneficial effects of short-term treatment were sustained for up to one year.

“These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats,” Charles A. Thornton, MD, professor of neurology at the University of Rochester Medical Center, and colleagues reported.

—Richard Robinson

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• Charizanis K, Lee KY, Swanson MS, et al. Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy. Neuron 2012;75(3):437–450.
    • Wheeler TM, Leger AJ, Thornton CA, et al. Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature 2012;488(7409):111–115.
      © 2012 American Academy of Neurology