Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function.

Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. Here, we show that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. We demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, we show a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, we provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. We suggest that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.

Optical Mapping Approaches on Muscleblind-Like Compound Knockout Mice for Understanding Mechanistic Insights Into Ventricular Arrhythmias in Myotonic Dystrophy.

BACKGROUND: Cardiac arrhythmias are common causes of death in patients with myotonic dystrophy (dystrophia myotonica [DM]). Evidence shows that atrial tachyarrhythmia is an independent risk factor for sudden death; however, the relationship is unclear. METHODS AND RESULTS: Control wild-type (Mbnl1+/+; Mbnl2+/+ ) and DM mutant (Mbnl1-/-; Mbnl2+/- ) mice were generated by crossing double heterozygous knockout (Mbnl1+/-; Mbnl2+/- ) mice. In vivo electrophysiological study and optical mapping technique were performed to investigate mechanisms of ventricular tachyarrhythmias. Transmission electron microscopy scanning was performed for myocardium ultrastructural analysis. DM mutant mice were more vulnerable to anesthesia medications and program electrical pacing: 2 of 12 mice had sudden apnea and cardiac arrest during premedication of general anesthesia; 9 of the remaining 10 had atrial tachycardia and/or atrioventricular block, but none of the wild-type mice had spontaneous arrhythmias; and 9 of 10 mice had pacing-induced ventricular tachyarrhythmias, but only 1 of 14 of the wild-type mice. Optical mapping studies revealed prolonged action potential duration, slower conduction velocity, and steeper conduction velocity restitution curves in the DM mutant mice than in the wild-type group. Spatially discordant alternans was more easily inducible in DM mutant than wild-type mice. Transmission electron microscopy showed disarranged myofibrils with enlarged vacuole-occupying mitochondria in the DM mutant group. CONCLUSIONS: This DM mutant mouse model presented with clinical myofibril ultrastructural abnormality and cardiac arrhythmias, including atrial tachyarrhythmias, atrioventricular block, and ventricular tachyarrhythmias. Optical mapping studies revealed prolonged action potential duration and slow conduction velocity in the DM mice, leading to vulnerability of spatially discordant alternans and ventricular arrhythmia induction to pacing.

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I.

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3' UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG)n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG)n-MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG)n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ß-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases.

Altered expression and splicing of Ca(2+) metabolism genes in myotonic dystrophies DM1 and DM2.

AIMS: Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, has been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca(2+) plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca(2+) signalling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca(2+) metabolism in DM patients, including Ca(2+) channels and Ca(2+) binding proteins. METHODS: We used patient muscle biopsies to analyse mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting. RESULTS: Most of the genes studied showed mRNA up-regulation in expression profiling. When analysed by immunohistochemistry the Ca(2+) release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca(2+) storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1. CONCLUSIONS: We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca(2+) metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.

Skeletal muscle metabolism in myotonic dystrophy A 31P magnetic resonance spectroscopy study.

We have used 31P magnetic resonance spectroscopy to investigate skeletal muscle bioenergetics in a total of 31 patients with myotonic dystrophy. Results from resting flexor digitorum superficialis and calf muscle showed a significant elevation in the concentration ratio of inorganic phosphate to ATP and a significant reduction in the phosphorylation potential. In addition, in resting calf muscle the concentration ratio of phosphocreatine to ATP was reduced, and the resting intracellular pH and calculated free cytosolic ADP concentration were elevated. In general, the abnormalities observed were more marked in those patients who were more severely affected as judged by their ability to exercise. During aerobic exercise in both calf muscle and flexor digitorum superficialis, phosphocreatine was depleted more rapidly in patients than in control subjects but the muscle acidified less and ADP concentrations were higher. Calculated ATP turnover was significantly elevated. Analysis of the recovery kinetics for phosphocreatine following exercise provides evidence for a small but significant reduction in mitochondrial function. Analysis of the response of flexor digitorum superficialis to ischaemic exercise provides evidence of a reduction in the relative utilization of glycogen to produce ATP which may account, in part, for the reduced acidification seen in exercising muscle in myotonic dystrophy. There was no definite evidence of an alteration in proton handling capacity in this condition.

Immunohistochemical study of intracytoplasmic inclusion bodies of the thalamus in myotonic dystrophy.

Intracytoplasmic inclusion bodies of the thalamus in eight patients with myotonic dystrophy (MyD) were studied immunohistochemically. The intracytoplasmic inclusion bodies of the thalamus (thalamic inclusions, TIs) were strongly immunostained with anti-ubiquitin antibody (Ab) and some of them were mildly stained with anti-microtubule associated protein 1 (MAP 1) and anti-MAP 2 antibodies. However, TIs did not react with any of the following: anti-neurofilament protein Ab, anti-tau Ab, anti-paired helical filament Ab, anti-tubulin Abs (alpha and beta), anti-neuron-specific enolase Ab, anti-glial fibrillary acidic protein Ab, anti-synaptophysin Ab, anti-myelin basic protein Ab, anti-actin Ab and anti-phosphorylated epitope of neurofilaments Ab. Thus, our study demonstrates the unique immunohistochemistry of TIs in MyD which differentiates them from other intracytoplasmic inclusions in various neurodegenerative disorders.

[Abnormalities of the muscular bioenergetics in Steinert's disease]. / Anomalies de la bioénergétique musculaire dans une maladie de Steinert.

The thenar muscles and gastrocnemius of a patient with myotonic dystrophy were investigated, at rest, by phosphorus nuclear magnetic resonance spectroscopy. A decrease in phosphocreatine level and an increase in inorganic phosphate and phosphodiester levels were found in the gastrocnemius, which was clinically spared, whilst the thenar muscles, which were wasted and affected by myotonia, exhibited only an increased inorganic phosphate level and an elevated pH. These findings were comparable with those found in other muscular disorders, such as Duchenne's and Becker's dystrophies, as well as in limb girdle dystrophy. They suggested that the abnormalities observed were unrelated to myotonia or wasting, and the possibility of a secondary mitochondrial disorder in myotonic dystrophy, is to be considered.

[Endocrine aspects of Steinert's disease]. / Aspects endocriniens de la maladie de Steinert

The authors report a case of Steinert's disease in a woman and discuss the endocrine profile of this disease after giving an account of the criteria of diagnosis. Disorders of gonad function are mild in women, primary testicular atrophy is very frequent in man with reduction in 17-ketosteroids and testosterone. Thyroid function was normal but, in a few cases, a low fixation curve was found (our case) corrected by TSH stimulation. The frequency of cataract emphasizes the interest of this sign for detection. Diabetes, associated with hyperinsulinism, seemed more frequent than in a population without Steinert's disease. The pathogenesis of these endocrine disorders appears secondary and is ill explained if one considers it as a single disease. Better knowledge, no doubt linked to progress in biochemistry of normal and myopathic muscle, will help to explain the pathogenesis.