Charles A. Thornton

Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy

Myotonic dystrophy (DM1) is an autosomal dominant neuromuscular disorder associated with a (CTG)n expansion in the 3′‐untranslated region of the DM1 protein kinase (DMPK) gene. To explain disease pathogenesis, the RNA dominance model proposes that the DM1 mutation produces a gain‐of‐function at the RNA level in which CUG repeats form RNA hairpins that sequester nuclear factors required for proper muscle development and maintenance. Here, we identify the triplet repeat expansion (EXP) RNA‐binding proteins as candidate sequestered factors. As predicted by the RNA dominance model, binding of the EXP proteins is specific for dsCUG RNAs and proportional to the size of the triplet repeat expansion. Remarkably, the EXP proteins are homologous to the Drosophila muscleblind proteins required for terminal differentiation of muscle and photoreceptor cells. EXP expression is also activated during mammalian myoblast differentiation, but the EXP proteins accumulate in nuclear foci in DM1 cells. We propose that DM1 disease is caused by aberrant recruitment of the EXP proteins to the DMPK transcript (CUG)n expansion.

Expanded CUG Repeats Trigger Aberrant Splicing of ClC-1 Chloride Channel Pre-mRNA and Hyperexcitability of Skeletal Muscle in Myotonic Dystrophy

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.

Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA.

Resisting Repeats A set of diseases, including myotonic dystrophy, are caused by the expansion of a simple repeat in genomic DNA, which, when transcribed into RNA, can be toxic to other cellular processes. Ameliorating the effects of this toxic, repeat-laden RNA may also relieve the symptoms of the disease. Wheeler et al. (p. 336; see the Perspective by Cooper) developed an antisense morpholino oligonucleotide complementary to the expanded repeats found in the myotonic dystrophy protein kinase messenger RNA (mRNA). The morpholino bound the repeats in vitro and displaced the inappropriately bound and sequestered RNA splicing factor, Muscleblind-like 1. In an in vivo mouse model for myotonic dystrophy, local injection of the morpholino corrected a number of cellular defects in muscle, including the alternative mRNA splicing of several genes, among them the muscle-specific chloride channel, CIC1, leading to a marked reduction in the myotonia. An antisense oligonucleotide ameliorates the symptoms of myotonic dystrophy in transgenic mice. Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.

Targeting nuclear RNA for in vivo correction of myotonic dystrophy

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUGexp RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

Randomized controlled trial of IVIg in untreated chronic inflammatory demyelinating polyradiculoneuropathy

Objective: To determine the efficacy of IV immunoglobulin (IVIg) given patients with untreated chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Methods: A randomized, double-blind, multicenter, investigator-initiated study compared IVIg (Aventis Behring LLC, King of Prussia, PA) with placebo (5% albumin). On days 1, 2, and 21, IVIg (1 g/kg) or placebo was given. The primary outcome measure was the change in muscle strength from baseline to day 42, using the average muscle score (AMS). Secondary outcome measures included change from baseline AMS at days 10 and 21, the Hughes’ functional disability scale, forced vital capacity (FVC), and nerve conduction studies (NCS) of four motor nerves (median, ulnar, peroneal, and tibial). Results: The patients (n = 33) were randomized. Of these, 30 (14 women, 16 men, aged 54 ± 20 years, range 13 to 82) received IVIg and 23 were given placebo (12 women, 11 men, aged 50 ± 18 years, range 23 to 73). Baseline AMS values of the groups were similar (IVIg 7.06 ± 1.31 versus placebo 7.28 ± 1.18, p = 0.53). There were two dropouts in placebo group and one in the IVIg group. Mean AMS improved at day 42 comparing IVIg with placebo (0.63 versus −0.1, p = 0.006). Improved strength was seen by day 10. The placebo group lost strength over this same interval. In the IVIg, 11 subjects improved by the functional disability scale; none worsened. This differed (p = 0.019) from those in the placebo-treated group (two improved, two got worse, remainder unchanged). Forced vital capacity did not improve with IVIg treatment. IVIg improved ulnar motor distal latency (p = 0.005), tibial distal compound muscle amplitude (p = 0.003), and peroneal nerve conduction velocity (p = 0.03). Conclusions: IVIg improves strength in patients with untreated CIDP by day 10 with continued benefit through day 42; more than one third improve by at least a functional grade on a disability scale. This study provides data supporting IVIg as the initial treatment for CIDP.

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTGexp transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for ∼100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.

Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast−myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2′-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

Subcutaneous IGF-1 is not beneficial in 2-year ALS trial

Background: Previous human clinical trials of insulin-like growth factor type I (IGF-1) in amyotrophic lateral sclerosis (ALS) have been inconsistent. This phase III, randomized, double-blind, placebo-controlled study was undertaken to address whether IGF-1 benefited patients with ALS. Methods: A total of 330 patients from 20 medical centers were randomized to receive 0.05 mg/kg body weight of human recombinant IGF-1 given subcutaneously twice daily or placebo for 2 years. The primary outcome measure was change in their manual muscle testing score. Secondary outcome measures included tracheostomy-free survival and rate of change in the revised ALS functional rating scale. Intention to treat analysis was used. Results: There was no difference between treatment groups in the primary or secondary outcome measures after the 2-year treatment period. Conclusions: Insulin-like growth factor type I does not provide benefit for patients with amyotrophic lateral sclerosis. GLOSSARY: ALS = amyotrophic lateral sclerosis; ALSFRS-r = revised ALS functional rating scale; AUC = area under the curve; DVT = deep venous thromboses; IGF-1 = insulin-like growth factor type I; MMT = manual muscle testing; PE = pulmonary embolisms.

Pentamidine reverses the splicing defects associated with myotonic dystrophy

Myotonic dystrophy (DM) is a genetic disorder caused by the expression (as RNA) of expanded CTG or CCTG repeats. The alternative splicing factor MBNL1 is sequestered to the expanded RNA repeats, resulting in missplicing of a subset of pre-mRNAs linked to symptoms found in DM patients. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. We identified the small molecules pentamidine and neomycin B as compounds that disrupt MBNL1 binding to CUG repeats in vitro. We show in cell culture that pentamidine was able to reverse the missplicing of 2 pre-mRNAs affected in DM, whereas neomycin B had no effect. Pentamidine also significantly reduced the formation of ribonuclear foci in tissue culture cells, releasing MBNL1 from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of 2 pre-mRNAs in mice expressing expanded CUG repeats.

Quantitative assessment of motor fatigue and strength in MS

Objective: To determine the test–retest reliability of strength and fatigue measurements in patients with MS and in healthy control subjects, and to examine associations among motor fatigue, strength, and ambulatory impairment in MS patients. Background: Motor fatigue, defined as the loss of the maximal capacity to generate force during exercise, and weakness are common in patients with MS. Method:— Twenty ambulatory MS patients and 20 age- and sex-matched healthy control subjects participated in the study. Test–retest reliability was assessed in two identical testing sessions, separated by 3 to 5 days. Maximal voluntary isometric strength was determined by fixed myometry of seven muscle groups on each side. Motor fatigue was assessed using three exercise protocols: sustained maximal contractions (static fatigue), repetitive maximal contractions, and walking as far as 500 m. Four analysis models for static fatigue were examined for their test–retest reliability and their ability to discriminate between normal fatigue and pathologic fatigue from MS. Results: Test–retest reliability in MS patients was excellent for isometric strength and very good for static fatigue. Test–retest reliability was lower for exercise protocols that involved repetitive contractions or ambulation. Compared with healthy control subjects, MS patients were weak in lower extremity muscles, but upper extremity strength was relatively preserved. Fatigue was greater in MS patients, even in muscles that were not clearly weak. There were no significant associations between strength and fatigue in any of the muscles tested. A fatigue analysis model based on the area under the force-versus-time curve gave the best combination of reliability and sensitivity to detect differences between MS patients and healthy control subjects. Conclusions: Strength and motor fatigue can be measured reliably in patients with MS. MS patients experience more fatigue than healthy control subjects during sustained contractions, repetitive contractions, and ambulation. Motor fatigue appears to be distinct from weakness because the degree of fatigue was not associated with the degree of weakness in individual muscles. Quantitative assessment of strength and fatigue may be useful to monitor changes in motor function over time in MS patients.