Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.     

The hallmarks of myotonic dystrophy type 1 muscle dysfunction

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3′ untranslated region (3′UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin–proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.     

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.     

孤独症谱系障碍小鼠肠道屏障功能研究

研究孤独症谱系障碍（ASD）模型小鼠肠道屏障功能及肠道动力的改变情况,为微生物−肠−脑轴在ASD发病中的作用提供理论基础。

C57bl/6J雌鼠和雄鼠交配,丙戊酸钠（VPA）诱导的ASD模型及对照组小鼠分别于孕12.5天皮下注射VPA或生理盐水。BTBR模型组为BTBR T⁺Itpr3^tf/J小鼠交配所产幼雄鼠。各组小鼠出生3周后,每窝随机选择2只雄鼠,各组5窝,共计10只,纳入相应组别。各组小鼠6周开始按如下顺序进行行为学检测：旷场实验、埋珠实验、三室社交实验、发声检测和自我梳理实验,各检测间隔5 d。评估小鼠肠道动力（粪便含水量及小肠推进率）和小鼠肠道屏障功能：FITC-葡聚糖灌胃后光谱仪检测小鼠血清中FITC-葡聚糖水平;Western blot检测各组小鼠结肠TREK1,CLDN1,CLDN3,OCLN及ZO1蛋白的表达;qPCR检测各组小鼠结肠Il6,Tnf-α和Ifn-γ mRNA的表达情况。

行为学检测结果显示：与对照组相比,BTBR组和VPA组小鼠穿越旷场中间区次数和停留时间显著降低（P＜0.05）;于目标小鼠侧室的停留时间率显著降低（P＜0.05）;埋珠率和自我梳理时间均显著升高（P＜0.05）;在（20～50）kHz和（50～100）kHz频率范围的发声次数及持续时间均显著降低（P＜0.05）。与对照组相比,BTBR组小鼠粪便含水量显著降低（P＜0.05）,而VPA组小鼠的差异无统计学意义（P＞0.05）。BTBR组和VPA组小鼠小肠推进率与对照组相比无差异（P＞0.05）。此外,与对照组相比,BTBR组和VPA组小鼠血清FITC−葡聚糖水平均升高,结肠TREK1,CLDN1,CLDN3,OCLD蛋白的表达水平降低,结肠Il6,Tnf-α和Ifn-γ mRNA的表达显著升高,差异均具有统计学意义（P＜0.05）,而ZO1的表达差异无统计学意义（P＞0.05）。

鉴于BTBR及VPA模型小鼠均表现出与ASD患者类似的广泛行为障碍,上述模型可作为ASD研究的可靠参考。与此同时,两种ASD小鼠模型均出现肠道紧密连接蛋白表达下调,肠道屏障通透性和促炎细胞因子水平的增高,表明微生物−肠−脑轴在ASD发病中扮演重要角色,且对治疗ASD症状具有潜在临床价值。

     

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.     

Myogenic defects in myotonic dystrophy

Myogenesis is the developmental program that generates and regenerates skeletal muscle. This process is impaired in patients afflicted with myotonic dystrophy type 1 (DM1). Muscle development is disrupted in infants born with congenital DM1, and recent evidence suggests that defective regeneration may contribute to muscle weakness and wasting in affected adults. DM1 represents the first example of a human disease that is caused, at least in part, by pathogenic mRNA. Cell culture models have been used to demonstrate that mutant DM1 mRNA takes on a gain-of-function and inhibits myoblast differentiation. Although the molecular mechanism(s) by which this mutant mRNA disrupts myogenesis is not fully understood, recent findings suggest that anomalous RNA-protein interactions have downstream consequences that compromise key myogenic factors. In this review, we revisit morphological studies that revealed the nature of myogenic abnormalities seen in patients, describe cell culture systems that have been used to investigate this phenotype and discuss recent discoveries that for the first time have identified myogenic events that are disrupted in DM1.     

Expanding complexity in myotonic dystrophy

Myotonic dystrophy (DM) is a highly variable multisystemic disease belonging to the rather special class of trinucleotide expansion disorders. DM results from dynamic expansion of a perfect (CTG)_n repeat situated in a gene-dense region on chromosome 19q. Based on findings in patient materials or cellular and animal models, many mechanisms for the causes and consequences of repeat expansion have been proposed; however, none of them has enjoyed prolonged support. There is now circumstantial evidence that long (CTG)_n repeats may affect the expression of any of at least three genes, myotonic dystrophy protein kinase (DMPK), DMR-N9 (gene 59), and a DM-associated homeodomain protein (DMAHP). Furthermore, the new findings suggest that DM is not a simple gene-dosage or gain-or-loss-of-function disorder but that entirely new pathological pathways at the DNA, RNA, or protein level may play a role in its manifestation. BioEssays 20: 901–912, 1998. © 1998 John Wiley & Sons, Inc.     

Insulin resistance in myotonic dystrophy.

The aim of the present study was to obtain a comprehensive picture of the rate of insulin secretion and of tissue sensitivity to the endogenous hormone in myotonic dystrophy patients (MyD). The minimal model approach was utilized for the analysis of frequently sampled intravenous glucose tolerance test data (FSIGT). This method provided the characteristic parameters: SI, insulin sensitivity index; SG fractional glucose disappearance independent of dynamic insulin; n, fractional insulin clearance; phi 1 and phi 2 first and second phase insulin delivery sensitivities to glucose stimulation. In MyD patients SI was reduced (p less than 0.01) by 71% to 1.4 +/- 0.3 x 10(-4) min-1/(microU/ml), whereas in controls it was 4.85 +/- 0.77; SG was within the normal range: 0.044 +/- 0.012 min-1 in MyD patients and 0.036 +/- 0.017 min-1 in controls; phi 1 increased in MyD patients (7.4 +/- 1.3 min (microU/ml)/(mg/dl) versus 4.1 +/- 1.2 in controls); phi 2 increased in MyD patients (126 +/- 47 x 10(4) min-2/(microU/ml)/(mg/dl) versus 17 +/- 6 in controls; p less than 0.05). MyD patients showed a normal tolerance with the glucose disappearance constant, KG within the normal range: 2.75 versus 2.62% min-1 in controls. In MyD patients insulin resistance was associated with a higher than normal insulin delivery for both secretory phases, although the second phase was responsible for releasing a greater amount of hormone. In conclusion MyD patients try to compensate for overall insulin resistance by a more marked pancreatic response.     

Transcriptome alterations in myotonic dystrophy frontal cortex

1. Download : Download high-res image (241KB)
2. Download : Download full-size image

     

Deregulated microRNAs in myotonic dystrophy type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.     

Oxidative stress in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults. The genetic basis of DM1 consists of a mutational expansion of a repetitive trinucleotide sequence (CTG). The number of triplets expansion divides patients in four categories related to the molecular changes (E1, E2, E3, E4). The pathogenic mechanisms of multi-systemic involvement of DM1 are still unclear. DM1 has been suspected to be due to premature aging, that is known to be sustained by increased free radicals levels and/or decreased antioxidants activities in neurodegenerative disorders. Recently, the gain-of-function at RNA level hypothesis has gained great attention, but oxidative stress might act in the disease progression. We have investigated 36 DM1 patients belonging to 22 unrelated families, 10 patients with other myotonic disorders (OMD) and 22 age-matched healthy controls from the clinical, biochemical and molecular point of view. Biochemical analysis detected blood levels of superoxide dismutase (SOD), malonilaldehyde (MDA), vitamin E (Vit E), hydroxyl radicals (OH) and total antioxidant system (TAS). Results revealed that DM1 patients showed significantly higher levels of SOD (+40%; MAL (+57%; RAD 2 (+106%; and TAS (+20%; than normal controls. Our data support the hypothesis of a pathogenic role of oxidative stress in DM1 and therefore confirm the detrimental role played by free radicals in this pathology and suggest the opportunity to undertake clinical trials with antioxidants in this disorder.     

Oxidative stress in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults. The genetic basis of DM1 consists of a mutational expansion of a repetitive trinucleotide sequence (CTG). The number of triplets expansion divides patients in four categories related to the molecular changes (E1, E2, E3, E4). The pathogenic mechanisms of multi-systemic involvement of DM1 are still unclear. DM1 has been suspected to be due to premature aging, that is known to be sustained by increased free radicals levels and/or decreased antioxidants activities in neurodegenerative disorders. Recently, the gain-of-function at RNA level hypothesis has gained great attention, but oxidative stress might act in the disease progression. We have investigated 36 DM1 patients belonging to 22 unrelated families, 10 patients with other myotonic disorders (OMD) and 22 age-matched healthy controls from the clinical, biochemical and molecular point of view. Biochemical analysis detected blood levels of superoxide dismutase (SOD), malonilaldehyde (MDA), vitamin E (Vit E), hydroxyl radicals (OH) and total antioxidant system (TAS). Results revealed that DM1 patients showed significantly higher levels of SOD (+40%; MAL (+57%; RAD 2 (+106%; and TAS (+20%; than normal controls. Our data support the hypothesis of a pathogenic role of oxidative stress in DM1 and therefore confirm the detrimental role played by free radicals in this pathology and suggest the opportunity to undertake clinical trials with antioxidants in this disorder.     

Glycomic analyses of mouse models of congenital muscular dystrophy

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1(-/-)). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with β1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical β1,2-elongation and β1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.     

Myotonic dystrophy mouse models: towards rational therapy development

DNA repeat expansions can result in the production of toxic RNA. RNA toxicity has been best characterised in the context of myotonic dystrophy. Nearly 20 mouse models have contributed significant and complementary insights into specific aspects of this novel disease mechanism. These models provide a unique resource to test pharmacological, anti-sense, and gene-therapy therapeutic strategies that target specific events of the pathobiological cascade. Further proof-of-principle concept studies and preclinical experiments require critical and thorough analysis of the multiple myotonic dystrophy transgenic lines available. This review provides in-depth assessment of the molecular and phenotypic features of these models and their contribution towards the dissection of disease mechanisms, and compares them with the human condition. More importantly, it provides critical assessment of their suitability and limitations for preclinical testing of emerging therapeutic strategies.     

Electrocardiographic abnormalities in patients with myotonic dystrophy.

In examining the incidence and progression of electrocardiographic abnormalities in 45 patients with myotonic dystrophy, 26 (58%) of whom at entry had at least 1 electrocardiographic abnormality, we found conduction abnormalities in 17 (38%). In 21 patients (47%), new abnormalities developed during follow-up (mean, 4.6 years). The overall incidence of electrocardiographic abnormalities increased to 78%, and the incidence of conduction defects increased to 62%. Second-degree or complete atrioventricular block did not develop in any of the patients. Pseudoinfarction patterns were common at entry and during follow-up and were not correlated with evidence of clinical coronary artery disease. There was no correlation between the presence of electrocardiographic abnormalities and apparent disease severity.