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Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1) disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor) or muscleblind, or by RNA interference (RNAi)-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis.

Unstable CTG expansions in the 3' UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy-and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1.

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3′ UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSALR) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSALR mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.

SUMMARYMyotonic dystrophy type 1 (DM1) is a genetic disease caused by the pathological expansion of a CTG trinucleotide repeat in the 3′ UTR of the DMPK gene. In the DMPK transcripts, the CUG expansions sequester RNA-binding proteins into nuclear foci, including transcription factors and alternative splicing regulators such as MBNL1. MBNL1 sequestration has been associated with key features of DM1. However, the basis behind a number of molecular and histological alterations in DM1 remain unclear. To help identify new pathogenic components of the disease, we carried out a genetic screen using a Drosophila model of DM1 that expresses 480 interrupted CTG repeats, i(CTG)480, and a collection of 1215 transgenic RNA interference (RNAi) fly lines. Of the 34 modifiers identified, two RNA-binding proteins, TBPH (homolog of human TAR DNA-binding protein 43 or TDP-43) and BSF (Bicoid stability factor; homolog of human LRPPRC), were of particular interest. These factors modified i(CTG)480 phenotypes in the fly eye and wing, and TBPH silencing also suppressed CTG-induced defects in the flight muscles. In Drosophila flight muscle, TBPH, BSF and the fly ortholog of MBNL1, Muscleblind (Mbl), were detected in sarcomeric bands. Expression of i(CTG)480 resulted in changes in the sarcomeric patterns of these proteins, which could be restored by coexpression with human MBNL1. Epistasis studies showed that Mbl silencing was sufficient to induce a subcellular redistribution of TBPH and BSF proteins in the muscle, which mimicked the effect of i(CTG)480 expression. These results provide the first description of TBPH and BSF as targets of Mbl-mediated CTG toxicity, and they suggest an important role of these proteins in DM1 muscle pathology.

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.

Influence of morphine self-administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self-administer morphine, each having two 'yoked' control animals, receiving passive injections of either morphine or saline. After 40 sessions of self-administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip Ò arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real-time PCR showed significant changes in abundance of five transcripts: c protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self-administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, c 1 subunit of GABA A receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC c, c 1 subunit of GABA A and hsp70 genes. Keywords: addiction, amygdala, DNA arrays, morphine selfadministration.Addiction, a persistent state in which compulsive drug use escapes control, is the most serious consequence of repetitive drug taking. Although the number of known drugs that produce dependence is increasing, little is known regarding cellular and molecular mechanisms of addiction. It is believed that the development of addiction is associated with plastic changes and long-term potentiation in discrete parts of the central nervous system (Nestler 2001). Nevertheless, molecular changes in gene expression underlying these phenomena are still poorly understood.Brain regions involved in reinforcing drug effects, development of addiction and drug craving comprise the mesocorticostriatal areas and the so-called extended amygdala (Alheid and Heimer 1988;Kreek and Koob 1998). The best known targets for drugs of abuse are the dopaminergic neurones that project from the ventral tegmental area of the midbrain to the nucleus accumbens, as well as to other forebrain sites, including amygdala. The amygdala has been characterized as a relay station between the input of emotionally significant stimuli and the output of behavioural responses (Dent et al. 2001). It processes an information about the emotional and motivational significance of environmental stimuli (Everitt et al. 1991;McDonald 1991;Hatfield et al. 1996). Animal models of drug craving and relapse are based largely on conditioned reinforcement. The neural substrates for such conditioned positive reinforcement appear to involve parts of the extended amygdala and afferent pathways from the basolateral amygdala (Everitt et al. 1991). Thus, the amygdala is implicated in conditioned reinforcement and may play a crucial role in conditioned and emotional aspects of addiction. Received December 8, 2003; revised manuscri...

Skeletal muscle symptoms strongly contribute to mortality of myotonic dystrophy type 1 (DM1) patients. DM1 is a neuromuscular genetic disease caused by CTG repeat expansions that, upon transcription, sequester the Muscleblind-like family of proteins and dysregulate alternative splicing of hundreds of genes. However, mis-splicing does not satisfactorily explain muscle atrophy and wasting, and several other contributing factors have been suggested, including hyperactivated autophagy leading to excessive catabolism. MicroRNA ( miR ) -7 has been demonstrated to be necessary and sufficient to repress the autophagy pathway in cell models of the disease, but the origin of its low levels in DM1 was unknown. We have found that the RNA-binding protein Musashi-2 (MSI2) is upregulated in patient-derived myoblasts and biopsy samples. Because it has been previously reported that MSI2 controls miR-7 biogenesis, we tested the hypothesis that excessive MSI2 was repressing miR-7 maturation. Using gene-silencing strategies (small interfering RNAs [siRNAs] and gapmers) and the small molecule MSI2-inhibitor Ro 08-2750, we demonstrate that reducing MSI2 levels or activity boosts miR-7 expression, represses excessive autophagy, and downregulates atrophy-related genes of the UPS system. We also detect a significant upregulation of MBNL1 upon MSI2 silencing. Taken together, we propose MSI2 as a new therapeutic target to treat muscle dysfunction in DM1.