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.     

Imbalanced oxidant and antioxidant ratio in myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults and is due to trinucleotide sequence (CTG) in the 3′ UTR region of DMPK gene located at 19q13.3 chromosome. The pathogenic mechanisms of multisystemic involvement of DM1 are still unclear. The increased levels of reactive oxygen species/free radicals and lipid peroxides and decreased antioxidant levels play an important role in the pathogenesis of DM1. Present study includes 20 DM1 patients and 40 age- and sex-matched controls. Malonilaldehyde (MDA), superoxide dismutase (SOD), glutathione peroxidise (GPX), glutathione-S-transferase (GST), reduced glutathione (GSH), and TAS levels were measured and its association with clinical phenotype were evaluated. Results revealed significantly higher levels of MDA (p = 0.002), SOD (p = 0.006), and TAS p = 0.004) and lower level of GPX (p = 0.003), GST (P < 0.001) and GSH (P = 0.016) in DM1 patients. A significant negative correlation of MDA level with dyspepsia and CK-MB and GST level with serum SCK, CK-MB, and diabetes were observed. However, a significant positive correlation of SOD level with serum CK-MB, CK-MM, and diabetes and negative correlation with facial weakness were noted. Though, GSH level had significant positive correlation with learning and writing disability, speech, and languages disability yet found negative correlation with duration of disease. The GPX and TAS showed no correlation with any clinical findings. Our data further support the pathogenic role of oxidative stress in DM1 of Indian origin and support the opportunity to undertake clinical trials with antioxidants in this disorder.     

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.     

Redox imbalance in peripheral blood of type 1 myotonic dystrophy patients

Objectives: The aim of our study was to determine if redox imbalance caused by the activities of antioxidant enzymes existed in erythrocytes of type 1 myotonic dystrophy (DM1) patients.

Methods: The activities of erythrocyte superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were measured in 30 DM1 patients and 15 healthy controls (HCs). The obtained values were correlated with the Muscular Impairment Rating Scale (MIRS) score and creatine kinase (CK).

Results: Superoxide dismutase and catalase activities were lower in DM1 patients compared to HCs. A positive correlation was found between disease duration and MIRS score as well as with glutathione reductase activity. In DM1 patients, there were positive correlations between catalase, glutathione peroxidase, and glutathione reductase activities. After sub-dividing DM1 patients according to CK levels, superoxide dismutase activity was still statistically different from HCs. However, catalase activity was significantly lower only in DM1 patients with increased CK.

Discussion: Undesirable alterations in antioxidant enzyme activities during DM1 disease progression may result in conditions favoring oxidative stress and changes in metabolism which together could contribute to muscle wasting.     

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.     

Myotonic dystrophy and myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic reticulum. The functional role of DMPK is not fully understood, however, it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc.     

Decreased DMPK transcript levels in myotonic dystrophy 1 type IIA muscle fibers

Concentrations of free polyamines were investigated in Trypanosoma granulosum cultured in a semidefined medium containing traces of polyamines. Spermidine content peaked in early logarithmic growth while putrescine was not detectable. Unlike African trypanosomes and Leishmania, spermine was measured at equivalent amounts to spermidine in mid to late logarithmic stage cells. Addition of d,l-alpha-difluoromethylornithine to cultures did not decrease polyamine content nor was ornithine decarboxylase activity detected. In contrast, incubation of parasites with tritiated putrescine showed rapid uptake and subsequent conversion to spermidine and spermine. At late logarithmic growth, parasites contained glutathione (77% of total sulphydryl groups) and ovothiol A as major low molecular mass thiols with glutathionylpolyamine conjugates undetectable. However, the addition of exogenous putrescine elevated trypanothione and glutathionylspermidine content to 48% of total sulphydryl groups. Correspondingly, the addition of exogenous cadaverine increased homotrypanothione content. This first report of polyamines and low molecular mass thiols in Trypanosoma granulosum indicates intriguing similarities with the metabolism of the human pathogen Trypanosoma cruzi.     

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.     

Non-B DNA conformations formed by long repeating tracts of myotonic dystrophy type 1, myotonic dystrophy type 2, and Friedreich's ataxia genes, not the sequences per se, promote mutagenesis in flanking regions

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.     

Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.     

Brain-specific change in alternative splicing of Tau exon 6 in myotonic dystrophy type 1

Alternative splicing is altered in myotonic dystrophy of type 1 (DM1), a syndrome caused by an increase of CTG triplet repeats in the 3' untranslated region of the myotonic dystrophy protein kinase gene. Previously, we reported the preferential skipping of Tau exon 2 in DM1 brains. In this study, we analyze the alternative splicing of Tau exon 6 which can be inserted in three different forms (c, p and d) depending on the 3' splice site used. In fact, inclusion of exon 6c decreases in DM1 brains compared to control brains whereas inclusion of 6d increases. Alteration of exon 6 splicing was not observed in DM1 muscle although this exon was inserted in RNAs from normal muscle and DM1 splicing alterations were first described in this organ. In contrast, alteration of exon 2 of Tau mRNA was observed in both muscle and brain. However, co-transfections of a minigene containing exon 6 with CELF or MBNL1 cDNAs, two splicing factor families suspected to be involved in DM1, showed that they influence exon 6 splicing. Altogether, these results show the importance of determining all the exons and organs targeted by mis-splicing to determine the dysregulation mechanisms of mis-splicing in DM1.