A Reversal of Misfortune for Myotonic Dystrophy?
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《新英格兰医药杂志》
Myotonic dystrophy, the most common cause of adult-onset muscular dystrophy, is a dominantly inherited disorder in which death is usually caused by the wasting of skeletal muscle and defects in cardiac conduction. Mutations in two unrelated genes cause strikingly similar disease phenotypes. The more common form, myotonic dystrophy 1, is caused by an expanded CTG repeat (with expansions ranging from 50 to 2000 repeats) within the noncoding 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The less common form of the disease, myotonic dystrophy 2, is caused by an expanded CCTG repeat (with expansions ranging from 80 to 11,000 repeats) in the first intron of the zinc finger protein 9 (ZNF9) gene.1
The disease mechanism remained a mystery while investigations focused on how dysfunction of DMPK and ZNF9 might cause disease — and how expansions in noncoding regions of the genes might disrupt protein expression. The major pathogenic mechanism is now clear, and it has nothing to do with DMPK and ZNF9 or their expression. The expanded allele is transcribed into RNA, which contains unusually long tracts of CUG or CCUG repeats. These RNA repeats fold into an unusual hairpin structure, and the contorted, mutant RNAs accumulate in nuclear foci and disrupt the regulation of alternative splicing of messenger RNA (mRNA).
The majority of human genes undergo alternative splicing so that pre-mRNAs from individual genes are processed to produce multiple mRNAs encoding different but related proteins that usually have long stretches of sequence in common. Splicing is often regulated according to cell type or developmental stage. In myotonic dystrophy, a subgroup of developmentally regulated splicing events fails to switch from an embryonic to an adult splicing pattern, resulting in aberrant expression of embryonic isoforms that are unable to support the functional requirements of adult tissues. For example, the characteristic clinical features of myotonia (muscle hyperexcitability) and insulin resistance correlate with the retention of embryonic splicing patterns of the muscle-specific chloride channel (ClC-1) and insulin receptor, respectively, in skeletal muscle of patients with myotonic dystrophy.2
Kanadia et al.3 have recently described a study that builds on this knowledge to reverse some aspects of the disease. Splicing misregulation in myotonic dystrophy results from altered functions of two RNA binding proteins — CUG-binding protein 1 (CUG-BP1) and muscleblind-like 1 (MBNL1) — which were identified because they bind CUG repeats in RNA. CUG-BP1 and MBNL1 are direct and antagonistic regulators of alternative splicing events that are normally regulated during development and misregulated in myotonic dystrophy. Several lines of evidence support models in which increased activity of CUG-BP1 and decreased activity of MBNL1 induce "embryonic pattern" splicing. The fact that MBNL1 colocalizes with nuclear RNA foci in the cells of patients with myotonic dystrophy suggests that MBNL1 is sequestered by mutant RNAs (Figure 1).4
Figure 1. Reversal of Splicing Abnormalities in a Mouse Model of Myotonic Dystrophy.
Myotonic dystrophy is caused by CTG repeats within a noncoding region of the Dmpk gene. It is also caused by the expansion of a 4-base motif CCTG in a noncoding region of the Znf9 gene (not shown). Transcribed RNA repeats fold into a hairpin, and the RNA is retained in the nucleus, where it alters the ratio of CUG RNA-binding proteins, such as CUG-BP1 and MBNL1. These proteins are mutually antagonistic mediators of a subgroup of alternative splicing events that are disrupted in myotonic dystrophy, in which "embryonic" forms of some proteins — that is, isoforms typically expressed in the developing embryo and fetus — predominate. A recent study by Kanadia et al.3 showed that increasing the expression of MBNL1 in a mouse model of myotonic dystrophy restored the adult splicing pattern of the ClC-1 protein and reversed the myotonia associated with ClC-1 misregulated splicing.
It follows that splicing abnormalities and associated symptoms due to MBNL sequestration should be reversed by increased expression of MBNL. Kanadia et al. tested this hypothesis by inducing the expression of exogenous MBNL1 in skeletal muscle in a mouse model of myotonic dystrophy 1 called HSALR. These mice, in which a human skeletal -actin transgene is expressed that contains 250 CTG repeats in its 3' untranslated region, have myotonia, histologic abnormalities, and embryonic alternative splicing patterns that are characteristic of myotonic dystrophy. Exogenous MBNL1 not only restored ClC-1 and other splicing abnormalities to the adult patterns but also reversed the myotonia.
The next step is to address the potential downsides of overexpressing a potent splicing regulator. For example, to avoid the potential for large-scale misregulated splicing resulting from MBNL1 overexpression, versions of MBNL1 could be developed that retain RNA-binding activity but do not have domains required for splicing activity. A "binding only" version could release endogenous MBNL1 from expanded repeat RNA without introducing additional MBNL1 splicing activity. Alternatively, a high-throughput screen could be used to identify small molecules that prevent sequestration of MBNL1 on CUG or CCUG repeat RNA.
Muscle degeneration is a major clinical feature of myotonic dystrophy. Kanadia et al. made the important observation that MBNL1 overexpression does not reverse histologic abnormalities in HSALR skeletal muscle. One explanation is that the level of MBNL1 expression was insufficient to reverse all aspects of pathogenesis caused by loss of function of MBNL1. However, it is also possible that muscle degeneration is due not to the loss of MBNL1 but rather to altered activities of other proteins.2 Addressing this issue may contribute to a full understanding of the pathogenic mechanism and to the development of a broad approach for treatment.
The results of the study by Kanadia et al. are relevant to other "expansion" disorders, such as the fragile X-associated tremor–ataxia syndrome, in which a pathogenic mechanism that parallels that of myotonic dystrophy has been suggested.2 Overall, these new findings provide additional support for a primary role of MBNL1 depletion in the pathogenesis of the disease and represent a viable therapeutic approach.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Departments of Pathology and Molecular and Cellular Biology, Baylor College of Medicine, Houston.
References
Harper PS. Myotonic dystrophy. 3rd ed. London: W.B. Saunders, 2001.
Ranum LP, Cooper TA. RNA-mediated neuromuscular disorders. Annu Rev Neurosci 2006;29:259-277.
Kanadia RN, Shin J, Yuan Y, et al. Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl Acad Sci U S A 2006;103:11748-11753.
Lin X, Miller JW, Mankodi A, et al. Failure of MBNL1-dependent postnatal splicing transitions in myotonic dystrophy. Hum Mol Genet 2006;15:2087-2097.(Thomas A. Cooper, M.D.)
The disease mechanism remained a mystery while investigations focused on how dysfunction of DMPK and ZNF9 might cause disease — and how expansions in noncoding regions of the genes might disrupt protein expression. The major pathogenic mechanism is now clear, and it has nothing to do with DMPK and ZNF9 or their expression. The expanded allele is transcribed into RNA, which contains unusually long tracts of CUG or CCUG repeats. These RNA repeats fold into an unusual hairpin structure, and the contorted, mutant RNAs accumulate in nuclear foci and disrupt the regulation of alternative splicing of messenger RNA (mRNA).
The majority of human genes undergo alternative splicing so that pre-mRNAs from individual genes are processed to produce multiple mRNAs encoding different but related proteins that usually have long stretches of sequence in common. Splicing is often regulated according to cell type or developmental stage. In myotonic dystrophy, a subgroup of developmentally regulated splicing events fails to switch from an embryonic to an adult splicing pattern, resulting in aberrant expression of embryonic isoforms that are unable to support the functional requirements of adult tissues. For example, the characteristic clinical features of myotonia (muscle hyperexcitability) and insulin resistance correlate with the retention of embryonic splicing patterns of the muscle-specific chloride channel (ClC-1) and insulin receptor, respectively, in skeletal muscle of patients with myotonic dystrophy.2
Kanadia et al.3 have recently described a study that builds on this knowledge to reverse some aspects of the disease. Splicing misregulation in myotonic dystrophy results from altered functions of two RNA binding proteins — CUG-binding protein 1 (CUG-BP1) and muscleblind-like 1 (MBNL1) — which were identified because they bind CUG repeats in RNA. CUG-BP1 and MBNL1 are direct and antagonistic regulators of alternative splicing events that are normally regulated during development and misregulated in myotonic dystrophy. Several lines of evidence support models in which increased activity of CUG-BP1 and decreased activity of MBNL1 induce "embryonic pattern" splicing. The fact that MBNL1 colocalizes with nuclear RNA foci in the cells of patients with myotonic dystrophy suggests that MBNL1 is sequestered by mutant RNAs (Figure 1).4
Figure 1. Reversal of Splicing Abnormalities in a Mouse Model of Myotonic Dystrophy.
Myotonic dystrophy is caused by CTG repeats within a noncoding region of the Dmpk gene. It is also caused by the expansion of a 4-base motif CCTG in a noncoding region of the Znf9 gene (not shown). Transcribed RNA repeats fold into a hairpin, and the RNA is retained in the nucleus, where it alters the ratio of CUG RNA-binding proteins, such as CUG-BP1 and MBNL1. These proteins are mutually antagonistic mediators of a subgroup of alternative splicing events that are disrupted in myotonic dystrophy, in which "embryonic" forms of some proteins — that is, isoforms typically expressed in the developing embryo and fetus — predominate. A recent study by Kanadia et al.3 showed that increasing the expression of MBNL1 in a mouse model of myotonic dystrophy restored the adult splicing pattern of the ClC-1 protein and reversed the myotonia associated with ClC-1 misregulated splicing.
It follows that splicing abnormalities and associated symptoms due to MBNL sequestration should be reversed by increased expression of MBNL. Kanadia et al. tested this hypothesis by inducing the expression of exogenous MBNL1 in skeletal muscle in a mouse model of myotonic dystrophy 1 called HSALR. These mice, in which a human skeletal -actin transgene is expressed that contains 250 CTG repeats in its 3' untranslated region, have myotonia, histologic abnormalities, and embryonic alternative splicing patterns that are characteristic of myotonic dystrophy. Exogenous MBNL1 not only restored ClC-1 and other splicing abnormalities to the adult patterns but also reversed the myotonia.
The next step is to address the potential downsides of overexpressing a potent splicing regulator. For example, to avoid the potential for large-scale misregulated splicing resulting from MBNL1 overexpression, versions of MBNL1 could be developed that retain RNA-binding activity but do not have domains required for splicing activity. A "binding only" version could release endogenous MBNL1 from expanded repeat RNA without introducing additional MBNL1 splicing activity. Alternatively, a high-throughput screen could be used to identify small molecules that prevent sequestration of MBNL1 on CUG or CCUG repeat RNA.
Muscle degeneration is a major clinical feature of myotonic dystrophy. Kanadia et al. made the important observation that MBNL1 overexpression does not reverse histologic abnormalities in HSALR skeletal muscle. One explanation is that the level of MBNL1 expression was insufficient to reverse all aspects of pathogenesis caused by loss of function of MBNL1. However, it is also possible that muscle degeneration is due not to the loss of MBNL1 but rather to altered activities of other proteins.2 Addressing this issue may contribute to a full understanding of the pathogenic mechanism and to the development of a broad approach for treatment.
The results of the study by Kanadia et al. are relevant to other "expansion" disorders, such as the fragile X-associated tremor–ataxia syndrome, in which a pathogenic mechanism that parallels that of myotonic dystrophy has been suggested.2 Overall, these new findings provide additional support for a primary role of MBNL1 depletion in the pathogenesis of the disease and represent a viable therapeutic approach.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Departments of Pathology and Molecular and Cellular Biology, Baylor College of Medicine, Houston.
References
Harper PS. Myotonic dystrophy. 3rd ed. London: W.B. Saunders, 2001.
Ranum LP, Cooper TA. RNA-mediated neuromuscular disorders. Annu Rev Neurosci 2006;29:259-277.
Kanadia RN, Shin J, Yuan Y, et al. Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl Acad Sci U S A 2006;103:11748-11753.
Lin X, Miller JW, Mankodi A, et al. Failure of MBNL1-dependent postnatal splicing transitions in myotonic dystrophy. Hum Mol Genet 2006;15:2087-2097.(Thomas A. Cooper, M.D.)