The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.
Muscle degeneration and myotonia are clinical hallmarks of myotonic dystrophy type 1 (DM1), a multisystemic disorder caused by a CTG repeat expansion in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Transgenic mice engineered to express mRNA with expanded (CUG)250 repeats (HSALR mice) exhibit prominent myotonia and altered splicing of muscle chloride channel gene (Clcn1) transcripts. We used whole-cell patch clamp recordings and nonstationary noise analysis to compare and biophysically characterize the magnitude, kinetics, voltage dependence, and single channel properties of the skeletal muscle chloride channel (ClC-1) in individual flexor digitorum brevis (FDB) muscle fibers isolated from 1–3-wk-old wild-type and HSALR mice. The results indicate that peak ClC-1 current density at −140 mV is reduced >70% (−48.5 ± 3.6 and −14.0 ± 1.6 pA/pF, respectively) and the kinetics of channel deactivation increased in FDB fibers obtained from 18–20- d-old HSALR mice. Nonstationary noise analysis revealed that the reduction in ClC-1 current density in HSALR FDB fibers results from a large reduction in ClC-1 channel density (170 ± 21 and 58 ± 11 channels/pF in control and HSALR fibers, respectively) and a modest decrease in maximal channel open probability(0.91 ± 0.01 and 0.75 ± 0.03, respectively). Qualitatively similar results were observed for ClC-1 channel activity in knockout mice for muscleblind-like 1 (Mbnl1ΔE3/ΔE3), a second murine model of DM1 that exhibits prominent myotonia and altered Clcn1 splicing (Kanadia et al., 2003). These results support a molecular mechanism for myotonia in DM1 in which a reduction in both the number of functional sarcolemmal ClC-1 and maximal channel open probability, as well as an acceleration in the kinetics of channel deactivation, results from CUG repeat–containing mRNA molecules sequestering Mbnl1 proteins required for proper CLCN1 pre-mRNA splicing and chloride channel function.
Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUGexp RNA, when compared with Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding pre-mRNAs. These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.
Phosphorodiamidate morpholino oligonucleotide (PMO)-mediated control of the alternative splicing of the chloride channel 1 (CLCN1) gene is a promising treatment for myotonic dystrophy type 1 (DM1) because the abnormal splicing of this gene causes myotonia in patients with DM1. In this study, we optimised a PMO sequence to correct Clcn1 alternative splicing and successfully remedied the myotonic phenotype of a DM1 mouse model, the HSALR mouse. To enhance the efficiency of delivery of PMO into HSALR mouse muscles, Bubble liposomes, which have been used as a gene delivery tool, were applied with ultrasound exposure. Effective delivery of PMO led to increased expression of Clcn1 protein in skeletal muscle and the amelioration of myotonia. Thus, PMO-mediated control of the alternative splicing of the Clcn1 gene must be important target of antisense therapy of DM1.
Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3’UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG5) and DM1 patient (DMPK CTG1000) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.
Alternative splicing; HTS; myotonic dystrophy; DM1; cell-based assay.
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.
In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain. Here we show that a morpholino antisense oligonucleotide (AON) targeting the 3′ splice site of ClC-1 exon 7a reversed the defect of ClC-1 alternative splicing in 2 mouse models of DM. By repressing the inclusion of this exon, the AON restored the full-length reading frame in ClC-1 mRNA, upregulated the level of ClC-1 mRNA, increased the expression of ClC-1 protein in the surface membrane, normalized muscle ClC-1 current density and deactivation kinetics, and eliminated myotonic discharges. These observations indicate that the myotonia and chloride channelopathy observed in DM both result from abnormal alternative splicing of ClC-1 and that antisense-induced exon skipping offers a powerful method for correcting alternative splicing defects in DM.
The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL.
Two full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL.
For the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds.
The absence of the chloride channel CLC-3 in Clcn3−/− mice results in hippocampal degeneration with a distinct temporal-spatial sequence reminiscent of neuronal loss in temporal lobe epilepsy. We examined how the loss of CLC-3 might impact GABAergic synaptic transmission in the hippocampus. An electrophysiological study of synaptic function in Clcn3+/+ and Clcn3-−/− mice in hippocampal slices before the onset of neurodegeneration, revealed a significant decrease in the amplitude and frequency of mIPSCs. We found that CLC-3 colocalizes with the vesicular GABA transporter VGAT in the CA1 region of the hippocampus. Cl−-induced acidification of inhibitory synaptic vesicles showed a significant dependence on CLC-3 expression. The decrement in inhibitory transmission in the Clcn3−/− animals suggests a decrease in neurotransmitter loading of synaptic vesicles which we attributed to defective vesicular acidification. Our observations extend the role of Cl− in inhibitory transmission from that of a postsynaptic permeant species to a presynaptic regulatory element.
Myotonia congenita (MC) is a form of nondystrophic myotonia caused by a mutation of CLCN1, which encodes human skeletal muscle chloride channel (CLC-1). We performed sequence analysis of all coding regions of CLCN1 in patients clinically diagnosed with MC, and identified 10 unrelated Korean patients harboring mutations. Detailed clinical analysis was performed in these patients to identify their clinical characteristics in relation to their genotypes. The CLCN1 mutational analyses revealed nine different point mutations. Of these, six (p.M128I, p.S189C, p.M373L, p.P480S, p.G523D, and p.M609K) were novel and could be unique among Koreans. While some features including predominant lower extremity involvement and normal to slightly elevated creatine kinase levels were consistently observed, general clinical features were highly variable in terms of age of onset, clinical severity, aggravating factors, and response to treatment. Our study is the first systematic study of MC in Korea, and shows its expanding clinical and genetic spectrums.
Myotonia Congenita; CLCN1; Clinical Features
Myotonic dystrophy type 1 and type 2 (DM1 and DM2) are genetic diseases in which mutant transcripts containing expanded CUG or CCUG repeats cause cellular dysfunction by altering the processing or metabolism of specific mRNAs and miRNAs. The toxic effects of mutant RNA are mediated partly through effects on proteins that regulate alternative splicing. Here we show that alternative splicing of exon 29 (E29) of CaV1.1, a calcium channel that controls skeletal muscle excitation–contraction coupling, is markedly repressed in DM1 and DM2. The extent of E29 skipping correlated with severity of weakness in tibialis anterior muscle of DM1 patients. Two splicing factors previously implicated in DM1, MBNL1 and CUGBP1, participated in the regulation of E29 splicing. In muscle fibers of wild-type mice, the CaV1.1 channel conductance and voltage sensitivity were increased by splice-shifting oligonucleotides that induce E29 skipping. In contrast to human DM1, expression of CUG-expanded RNA caused only a modest increase in E29 skipping in mice. However, forced skipping of E29 in these mice, to levels approaching those observed in human DM1, aggravated the muscle pathology as evidenced by increased central nucleation. Together, these results indicate that DM-associated splicing defects alter CaV1.1 function, with potential for exacerbation of myopathy.
Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3−/−) mice and their age-matched heterozygous (Clcn3+/−) and wild-type (Clcn3+/+) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3−/− mice had significant slower heart rate (648±12 bpm) than Clcn3+/+ littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3−/− mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3+/+ (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3+/− mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3−/− littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3−/− myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.
Ischemia; Myocardial infarction; Hemodynamics; Ion channels
Generalised myotonia Becker (GM) is an autosomal recessively inherited muscle disorder. Affected subjects exhibit myotonic muscle stiffness in all skeletal muscles with marked hypertrophy in the legs. A transient muscle weakness is particularly pronounced in the arms and hands and is a typical symptom of the disorder. Recently, we showed complete linkage of the disorder GM to the gene (CLCN1) coding for the skeletal muscle chloride channel CLC-1 and the TCRB gene on chromosome 7 in German families. In the study presented here we performed linkage analysis on 14 new GM families. The GM locus was again completely linked to both the CLCN1 and the TCRB gene in all families with a combined lod score of Z = 9.26 at a recombination fraction of theta = 0.00. This confirms our previous data and supports the hypothesis that GM is a genetically homogeneous disorder. The previously detected T to G missense mutation is found on 15% of the 66 GM chromosomes counted so far.
The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.
An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.
This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing.
We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.
Myotonia congenita is a genetic muscle disorder characterized by clinical and electrical myotonia, muscle hypertrophy, and stiffness. It is inherited as either autosomal-dominant or –recessive, known as Thomsen and Becker diseases, respectively. These diseases are distinguished by the severity of their symptoms and their patterns of inheritance. Becker disease usually appears later in childhood than Thomsen disease and causes more severe muscle stiffness and pain. Mutations in the muscular voltage-dependent chloride channel gene (CLCN1), located at 7q35, have been found in both types. We report here the case of a Moroccan consanguineous family with a myotonic autosomal-recessive condition in two children. The molecular studies showed that the patients reported here are homozygous for mutation p.Gly482Arg in the CLCN1 gene. The parents were heterozygote carriers for mutation p.Gly482Arg. This diagnosis allowed us to provide an appropriate management to the patients and to make a genetic counselling to their family.
Autosomal recessive; CLCN1gene; myotonia congenital
Mutations of the human CLCN5 gene, which encodes the CLC-5 Cl−/H+ exchanger, lead to Dent's disease. Mutations result in functional defects that range from moderate reductions to complete loss of whole cell currents, although the severity of the functional defect rarely correlates with the severity of the disease. To further elucidate the basis of CLC-5 mutations causing Dent's disease, we examined the functional and cell biological consequences of seven previously reported missense mutants, utilizing electrophysiological and cell biological techniques. This revealed three classes of Dent's disease-causing CLC-5 mutations. Class 1 mutations lead to endoplasmic reticulum retention and degradation of CLC-5. Class 2 mutations appear to have little effect on subcellular distribution of CLC-5 but cause defective function resulting in severe defects in endosomal acidification. Class 3 mutations lead to alterations in the endosomal distribution of CLC-5 but are otherwise able to support endosomal acidification. Molecular modeling demonstrates a structural basis that may underlie the nature of the defect resulting from each mutation with each class occupying discrete regions of the protein quaternary structure. Thus these results demonstrate that the cell biological consequences of CLC-5 mutations are heterogeneous and can be classified into three major groups and that a correlation between the nature of the defect and the location of the mutation in the structure may be drawn. This model may prove to be useful as a tool to aid in the diagnosis and future therapeutic intervention of the disease.
chloride channel; endocytosis; endosomal acidification; proteinuria
Mutations in the chloride channel 7 gene (CLCN7) cause osteopetrosis, and polymorphisms of CLCN7 in the non-disease allele are associated with penetrance of the autosomal dominant osteopetrosis (ADO) phenotype. Studies have also shown an association between CLCN7 polymorphisms and bone mineral density (BMD) in women. However, there is no study to date that has examined whether CLCN7 polymorphisms underlie normal variation of peak BMD in healthy premenopausal white women and in white men.
Six single nucleotide polymorphisms (SNPs) and one variable number tandem repeat (VNTR) polymorphism in the CLCN7 gene were genotyped. Association was tested between CLCN7 gene polymorphisms and both lumbar spine and femoral neck BMD. Healthy premenopausal white sisters (age 33.1 ± 7.2, n=1692) and healthy white brothers (age 33.6 ± 10.9, n=715) were studied.
No significant association between CLCN7 gene polymorphisms and BMD at the lumbar spine or femoral neck was found in white women or white men.
Genetic variation in the CLCN7 gene is not a major contributor to the variability in peak BMD at the femoral neck and lumber spine in healthy premenopausal white women and in white men.
bone mineral density (BMD); association study; chloride channel 7 gene (CLCN7); single nucleotide polymorphism (SNP); variable number tandem repeat (VNTR)
Myotonic dystrophy (DM; also known as dystrophia myotonica) is an autosomal dominant disorder that affects the heart, eyes, brain and endocrine system, but the predominant symptoms are neuromuscular, with progressive muscle weakness and wasting. DM presents in two forms, DM1 and DM2, both of which are caused by nucleotide repeat expansions: CTG in the DMPK gene for DM1 and CCTG in ZNF9 (CNBP) for DM2. Previous studies have shown that the mutant mRNAs containing the transcribed CUG or CCUG repeats are retained within the nuclei of cells from individuals with DM, where they bind and sequester the muscleblind-like proteins MBNL1, MBNL2 and MBNL3. It has been proposed that the sequestration of these proteins plays a key role in determining the classic features of DM. However, the functions of each of the three MBNL genes are not completely understood. We have generated a zebrafish knockdown model in which we demonstrate that a lack of mbnl2 function causes morphological abnormalities at the eye, heart, brain and muscle levels, supporting an essential role for mbnl2 during embryonic development. Major features of DM are replicated in our model, including muscle defects and splicing abnormalities. We found that the absence of mbnl2 causes disruption to the organization of myofibrils in skeletal and heart muscle of zebrafish embryos, and a reduction in the amount of both slow and fast muscle fibres. Notably, our findings included altered splicing patterns of two transcripts whose expression is also altered in DM patients: clcn1 and tnnt2. The studies described herein provide broader insight into the functions of MBNL2. They also lend support to the hypothesis that the sequestration of this protein is an important determinant in DM pathophysiology, and imply a direct role of MBNL2 in splicing regulation of specific transcripts, which, when altered, contributes to the DM phenotype.
Introduction: Myotonia Congenita is an inherited myotonia that is
due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations
lead to reduced sarcolemmal chloride conductance, causing delayed muscle
relaxation that is evident as clinical and electrical myotonia.
Methods: We report the clinical presentations of two individuals
with Myotonia Congenita (MC).
Results: Patient 1 has been diagnosed with the recessive form of MC,
known as the Becker variant, and Patient 2 has been diagnosed with the dominant
form of MC, known as the Thomsen variant. In both patients, the diagnosis was
made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient
1 also had a muscle biopsy.
Conclusions: Genetic testing in both patients reveals previously
unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We
report the salient clinical features of each patient and discuss the effects and
common types of CLCN1 mutations and review the literature.
Myotonia Congenita; Becker variant; Thomsen variant; CLCN1 mutation
Muscleblind-like proteins, Muscleblind (Mbl) in Drosophila and MBNL1-3 in vertebrates, are regulators of alternative splicing. Human MBNL1 is a key factor in the etiology of myotonic dystrophy (DM), a muscle wasting disease caused by the occurrence of toxic RNA molecules containing CUG/CCUG repeats. MBNL1 binds to these RNAs and is sequestered in nuclear foci preventing it from exerting its normal function, which ultimately leads to mis-spliced mRNAs, a major cause of the disease. Muscleblind-proteins bind to RNAs via N-terminal zinc fingers of the Cys3-His type. These zinc fingers are arranged in one (invertebrates) or two (vertebrates) tandem zinc finger (TZF) motifs with both fingers targeting GC steps in the RNA molecule. Here I show that mbl genes in Drosophila and in other insects also encode proteins with two TZF motifs, highly similar to vertebrate MBNL proteins. In Drosophila the different protein isoforms have overlapping but possibly divergent functions in vivo, evident by their unequal capacities to rescue the splicing defects observed in mbl mutant embryos. In addition, using whole transcriptome analysis, I identified several new splicing targets for Mbl in Drosophila embryos. Two of these novel targets, kkv (krotzkopf-verkehrt, coding for Chitin Synthase 1) and cora (coracle, coding for the Drosophila homolog of Protein 4.1), are not muscle-specific but expressed mainly in epidermal cells, indicating a function for mbl not only in muscles and the nervous system.
Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, Sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1), and cardiac troponin T (cTNT). Based on these observations, the development of small molecule ligands that target specifically expanded DM1 repeats could serve as therapeutics. In the present study, computational screening was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of inhibitors of the RNA-protein complex with low micromolar IC50’s, which are >20-fold more potent than the query compounds, were identified. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with virtual screening.
In the cell nucleus, the gene primary transcripts undergo molecular processing to generate mature RNAs, which are finally exported to the cytoplasm. These mRNA maturation events are chronologically and spatially ordered, and mostly occur on distinct ribonucleoprotein (RNP)-containing structures. Defects in the mRNA maturation pathways have been demonstrated in myotonic dystrophy type 1 (DM1) and type 2 (DM2) whose characteristic multisystemic features are caused by the expansion of two distinct nucleotide sequences: (CTG)n in the DMPK gene on chromosome 19q13 in DM1, and (CCTG)n in the ZNF9 gene on chromosome 3q21 in DM2. By combining biomolecular and cytochemical techniques, it has been shown that the basic mechanisms of DMs reside in the accumulation of CUG- or CCUG-containing transcripts in intranuclear foci where several RNA-binding proteins necessary for the physiological processing of pre-mRNA are sequestered. Moreover, a nucleoplasmic accumulation of splicing and cleavage factors has been found in DMs. This suggests that the dystrophic phenotype could depend on a general alteration of the pre-mRNA post-transcriptional pathway. Interestingly, the accumulation of pre-mRNA processing factors in the myonuclei of DM1 and DM2 patients is reminiscent of the nuclear alterations typical of sarcopenia, i.e., the loss of muscle mass and function which physiologically occurs during ageing. Consistently, in an in vitro study, we observed that satellite-cell-derived DM2 myoblasts show cell senescence alterations and impairment of the pre-mRNA maturation pathways earlier than the myoblasts from healthy patient. These results suggest possible common cellular mechanisms responsible for skeletal muscle wasting in sarcopenia and in myotonic dystrophy.
cell nucleus; myotonic dystrophy; sarcopenia.
Myotonic dystrophy type 2 (DM2) is a dominantly inherited autosomal disease with multi-systemic clinical features and it is caused by expansion of a CCTG tetranucleotide repeat in the first intron of the zinc finger protein 9 (ZNF9) gene in 3q21.The expanded-CCUG-containing transcripts are retained in the cell nucleus and accumulate in the form of focal aggregates which specifically sequester the muscleblind-like 1 (MBNL1) protein, a RNA binding factor involved in the regulation of alternative splicing. The structural organization and composition of the foci are still incompletely known. In this study, the nuclear foci occurring in cultured myoblasts from DM2 patients were characterised at fluorescence and transmission electron microscopy by using a panel of antibodies recognizing transcription and processing factors of pre-mRNAs. MBNL1 proved to co-locate in the nuclear foci with snRNPs and hnRNPs, whereas no co-location was observed with RNA polymerase II, the non-RNP splicing factor SC35, the cleavage factor CStF and the PML protein. At electron microscopy the MBNL1-containing nuclear foci appeared as roundish domains showing a rather homogeneous structure and proved to contain snRNPs and hnRNPs. The sequestration of splicing factors involved in early phases of pre-mRNA processing supports the hypothesis of a general alteration in the maturation of several mRNAs, which could lead to the multiple pathological dysfunctions observed in dystrophic patients.
myotonic dystrophy type 2 – DM2; myoblast; immunocytochemistry; fluorescence microscopy; electron microscopy; RNA processing.
Kennedy disease, a degenerative disorder characterized by androgen-dependent neuromuscular weakness, is caused by a CAG/glutamine tract expansion in the androgen receptor (Ar) gene. We developed a mouse model of Kennedy disease, using gene targeting to convert mouse androgen receptor (AR) to human sequence while introducing 113 glutamines. AR113Q mice developed hormone and glutamine length–dependent neuromuscular weakness characterized by the early occurrence of myopathic and neurogenic skeletal muscle pathology and by the late development of neuronal intranuclear inclusions in spinal neurons. AR113Q males unexpectedly died at 2–4 months. We show that this androgen-dependent death reflects decreased expression of skeletal muscle chloride channel 1 (CLCN1) and the skeletal muscle sodium channel α-subunit, resulting in myotonic discharges in skeletal muscle of the lower urinary tract. AR113Q limb muscles show similar myopathic features and express decreased levels of mRNAs encoding neurotrophin-4 and glial cell line–derived neurotrophic factor. These data define an important myopathic contribution to the Kennedy disease phenotype and suggest a role for muscle in non–cell autonomous toxicity of lower motor neurons.
Misregulation of alternative splicing has become a molecular hallmark of myotonic dystrophy type 1 (DM1) in which neonatal splice variants are expressed in adult skeletal muscle. Splicing misregulation is induced by RNA containing expanded CUG repeats expressed from the expanded mutant allele by sequestration of Muscleblind-like 1 (MBNL1) protein within nuclear RNA foci and increased CUGBP, Elav-like family member 1 (CELF1) protein levels. Misregulated splicing has also been identified in other neuromuscular disorders suggesting either that diseases with different molecular causes share a common pathogenic mechanism or that misregulated splicing can also be a common secondary consequence of muscle degeneration and regeneration.
In this study we examined regulation of alternative splicing in four different mouse models of muscular dystrophy including DM1, limb-girdle muscular dystrophy, congenital merosin-deficient muscular dystrophy, Duchenne muscular dystrophy, and two myotoxin (cardiotoxin and notexin) muscle injury models.
We show that DM1-like alternative splicing misregulation and altered expression of MBNL1 and CELF1 occurs in non-DM1 mouse models of muscular dystrophy and muscle injury, most likely due to recapitulation of neonatal splicing patterns in regenerating fibers. In contrast, CELF1 was elevated in nuclei of mature myofibers of the DM1 model consistent with a primary effect of pathogenic RNA expression.
Splicing misregulation in DM1 is a primary effect of RNA containing expanded CUG repeats. However, we conclude that splicing changes can also be observed secondary to muscle regeneration and this possibility must be taken into account when evaluating cause-effect relationships between misregulated splicing and disease processes.