The InteractiveFly: Drosophila as a Model for Human Diseases


Muscular dystrophy: a group of diseases that cause progressive weakness and loss of muscle mass
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Drosophila genes associated with
Muscular dystrophy
Dystroglycan
Dystrophin
lamin C
muscleblind
Pabp2
Sarcoglycan δ
wunen
Related terms


Muscle
Overview of the disease

Muscular dystrophy as a triplet expansion disease Myotonic dystrophy type 1 (DM1) is a common neuromuscular disorder affecting 1 in 8000 people worldwide. DM1 patients display myotonia, muscle weakness and degeneration, together with insulin resistance, cataracts, cardiac conduction defects and hypogonadism. This autosomal-dominant disease is caused by an unstable CTG triplet expansion in the 3′ untranslated region of the DMPK gene. The severity and age of onset of DM1 are correlated with the number of repeats. Briefly, normal individuals have 5–37 CTG repeats, patients with the adult or childhood form display from 50 to 1000 CTG repeats, and congenital DM1 patients can have several thousand triplet repeats (Picchio, 2013 and references therein).

Despite the complexity of DM1 pathogenesis, it is now well established that non-coding CUG repeat transcripts play a toxic gain-of-function role. Abnormal DMPK transcripts form secondary structures, which aggregate into foci within muscle nuclei and which sequester RNA-binding proteins such as Muscleblind-like 1 (MBNL1). Also, by a still undetermined mechanism activating protein kinase C, CUG-binding protein 1 (CUGBP1) is stabilized by phosphorylation. MBNL1 and CUGBP1 are both splicing factors, but play antagonistic roles. Thus, in DM1 patients, several transcripts are mis-spliced due to an inverse ratio of MBNL1/CUGBP1. Among mis-spliced transcripts, insulin receptor (IR), chloride ion channel-1 (ClC-1), Bin1 and troponin T (cTNT) are involved in insulin resistance, myotonia, muscle weakness and reduced myocardial function observed in patients. However, recent reports indicate that other molecular aberrations such as altered maturation of miRNAs or CUG repeat-dependent transcription factors leaching can also contribute to the pathogenesis of DM1. To characterize molecular defects underlying this pathology, several animal models have been generated. The first to be developed were mice models, but it was found that Drosophila also represents an accurate model system to study DM1. Accordingly, fruit flies expressing CUG repeats in adult muscles develop DM1 symptoms and can be used to screen modifiers of transcript toxicity. Recently, applying a Drosophila model has revealed the role of the anti-sense DMPK transcript in DM1 pathogenesis. It has also been shown that Muscleblind (Mbl), the Drosophila MBNL1 ortholog, as in humans, is involved in muscle phenotypes observed in DM1 flies (Picchio, 2013 and references therein).

Myotonic dystrophy type 1 (DM1, OMIM no. 160900) is a neuromuscular disorder linked to a major misregulation of alternative splicing and is considered to be the first described spliceopathy. DM1 is caused by the expansion of a CTG trinucleotide repeat tract located in the 3′ untranslated region (UTR) of the dystrophia myotonica-protein kinase (DMPK) gene. The main pathogenic effect in DM1 is a deleterious gain-of-function of the mutant expanded CUG-containing mRNA (CUG-RNA), which triggers the biochemical and clinical features of DM1. The current model of disease progression derives from the strong interaction of expanded CUG-RNA with splicing regulators such as the muscleblind-like proteins (MBNL1–MBNL3) and CUG-BP Elav-like family member 1 (CELF1), key proteins involved in DM1 pathophysiology. Importantly, MBNL1 is sequestered by the expanded CUG-RNA in anomalous ribonuclear aggregates (foci), which causes the deregulation of alternative splicing in a large group of pre-mRNAs. The muscleblind gene (mbl) is not only conserved in the Drosophila genome, but it also plays a role in alternative splicing in this organism, suggesting the conservation of key disease pathways in Drosophila. This has been confirmed by the successful reproduction of tissue-specific DM1 hallmarks such as nuclear foci formation, muscleblind sequestration, missplicing, muscle atrophy and reduced lifespan in flies expressing a disease-associated CTG repeat tract [UAS-i(CTG)480 flies] (García-Alcover, 2014 and references therein).

Oculopharyngeal muscular dystrophy (OPMD) is another triplet expansion disease which results from short expansions of a GCN repeat in the gene encoding poly(A) binding protein nuclear 1 (PABPN1). OPMD is an autosomal dominant muscular dystrophy, which has a late onset and is characterized by progressive weakness and degeneration of specific muscles. Triplet expansion in PABPN1 leads to extension of a polyalanine tract from 10 alanines in the normal protein to a maximum of 17 alanines at the N-terminus of the protein. Nuclear aggregates in muscle fibres are a pathological hallmark of OPMD. These aggregates contain mutant insoluble PABPN1, ubiquitin, subunits of the proteasome, as well as poly(A) RNA. Polyalanine expansions in PABPN1 are thought to induce misfolding and formation of aggregates, which are targeted to the ubiquitin-proteasome degradation pathway. However, it is still unknown whether these nuclear aggregates have a pathological function, a protective role, or are a consequence of a cellular defence mechanism (Chartier, 2015 and references therein).

Lamins and Muscular dystrophy. Lamins belong to the type-V family of intermediate filament proteins that share the same tripartite structure; an α-helical rod domain flanked by N- and C-terminal head and tail domains. They are the basic subunits for assembling the stable lamina located between the inner nuclear membrane of the nuclear envelope and peripheral chromatin, and bind to chromosomes/chromatin, DNA, and various proteins of the inner nuclear membrane and nuclear pore complex to establish and maintain nuclear architecture. Lamins are present in all metazoans but not in unicellular organisms and plants and have been classified into A-type which is expressed in a subset of differentiated tissues and B-type which is constitutive during development. Studies demonstrate that mutations in both lamin types cause debilitating diseases in human, and developmental disorders in model animals, thus lamins have become recognized as having significant functions in tissue formation and maintenance during development (Uchino, 2013 and references therein).

Various mutations of the A-type lamin gene (LMNA) result in both dominant and recessive phenotypes that are associated with a number of different human disorders including both Hutchinson–Gilford premature aging syndrome and Emery–Dreifuss muscular dystrophy (EDMD). Analysis of mutated domains in the A-type lamin proteins provides some insights on how it functions in tissue organization, but the relationship between mutations and defective phenotypes of A-type lamin is very complex. In the mice model, LMNA null mutants show severe postnatal growth retardation and muscular dystrophy resembling EDMD, suggesting it will be a useful tool for the study of muscular dystrophy, however, the actual functions of A-type lamin in musculature have remained unclear (Uchino, 2013, see below).

Drosophila also has both types of lamins, which are the B-type lamin Dm0 and the A-type lamin C. Whereas in mammals, two types of B-type lamin genes, LMNB1 and LMNB2 are reported, Drosophila only has a single B-type lamin gene. Although both mammals and Drosophila have single A-type lamin genes, multiple translation products are generated by alternative splicing from mammalian LMNA, while Drosophila produces a single lamin C protein. Lamin Dm0 is ubiquitously expressed in almost all cell-types, lamin C is detected in differentiated tissues in a manner similar to LMNA in mammals. Drosophila A-type lamin C has been shown to be an essential gene, and mutation of lamin C causes nuclear and muscle defects, similar to those abnormalities in a mutation of mammalian LMNA. Although the exoskeleton system of Drosophila is different from the internal skeleton of vertebrates, adult Drosophila myogenesis is known to lead to the formation of multi-fiber muscles similar to those in vertebrates and the myotendinous systems are comparable between vertebrates and Drosophila. Thus Drosophila can serve as a useful model for lamin-related dystrophy studies (Uchino, 2013 and references therein).

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Relevant studies of Muscular dystrophy

García-Alcover, I., Colonques-Bellmunt, J., Garijo, R., Tormo, J.R., Artero, R., Álvarez-Abril, M.C., López Castel, A. and Pérez-Alonso, M. (2014). Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1. Dis Model Mech 7: 1297-1306. PubMed ID: 25239918

Abstract
Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1–MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, this study generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. Following this, an innovative 96-well plate screening platform was developed to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognize separate DM1-linked therapeutic traits for some of the hits, which corroborate the feasibility of the approach described in this study to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery (García-Alcover, 2014).

Highlights

  • Generation of Drosophila spliceosensors for DM1.
  • Characterization and validation of the best DM1 INSR spliceosensor line.
  • Drosophila-based platform set-up: HTS design.
  • Screening campaign results by using the DM1 INSR spliceosensor assay.
  • Evaluation of the effect of the hit compounds on well-known DM1 hallmarks.

Discussion
Manipulation of splicing by different means is considered a promising therapeutic approach to human disorders, such as for DM1 where target-based identification of active antisense oligonucleotides and rational design of small molecules are actively pursued strategies. A different strategy is the development of high-throughput screening assays using cell culture systems to search for chemical modulators of specific missplicing events, a technique hardly tested for DM1. Focusing on improving the often quite poor quality of hits resulting from traditional in vitro brute-force pharmacological screenings, this study established a novel in vivo screening methodology based on an automated platform for large-scale and cost-effective Drosophila-based evaluation of small molecules, pioneering the exploitation of a disease-linked alternative splicing reporter system. The method, initially devised for DM1 and potentially expandable to additional missplicing disorders, allows for the fast and reliable identification of splicing modulators after the successful validation of the first transgenic spliceosensor flies (García-Alcover, 2014).

For modeling purposes, the study took advantage of the competence of D. melanogaster both to allow a stable integration of human DNA in its genome and to mirror disease phenotypes. The ‘humanized’ spliceosensor flies accurately reproduce the alternative splicing deregulation described in DM1 patients in the presence of the disease mutation. Simultaneously, the study used the latest technology applied to small organisms to achieve in vivo HTS capabilities. For this, the standards for valid screening parameters (positive Z-factor and a z-score≥3) were determined on the use of the spliceosensor flies, thus allowing the assessment of chemical entities in a large-scale format. Importantly, some of the confirmed hits identified by positive modulation of the DM1 spliceosensor system were confirmed as molecules with positive roles in relevant independent DM1 assays, and for this reason they are currently the subject of further evaluation in additional DM1 systems (García-Alcover, 2014).

At this point, the evaluation of treatments that simultaneously combine use of compounds with different anti-DM1 features or chemical properties is an experimental strategy in need of exploration in order to improve the potential anti-DM1 response. Confirmatory data from this study suggest that the method presented is able to identify high-quality hits from screened compounds, one of the advantages anticipated from the use of whole animals in screening (García-Alcover, 2014).

The reliable use of a sensitive reporter-based system in an in vivo situation establishes a novel and interesting option for pharmacological evaluation in addition to previously established Drosophila-specific phenotypic outputs, such as behavioral assays, or assays of lethality or eye roughness, which are commonly limited to low- and medium-throughput assays, are difficult to miniaturize or automate and show higher heterogeneity in their final measurements. Furthermore, the use of the spliceosensor flies offers the possibility of establishing distinct types of mechanistic outputs from the compounds identified. The screening described in this study involves a splicing-phenotype-based approach, as induced by the expression of CUG repeats, without connecting the hit evaluation to a specific mechanism of action. That said, a screening approach that is closer to a target-based approach is also possible by using the spliceosensor flies alone and looking for direct modulators of the splicing event. Given that this approach entails a HTS format, the in vivo screening capabilities exhibited are very promising. Most pharmacological screens described in Drosophila are on the order of 500 to 1000 molecules tested per month (~15 to 30 daily). The approach presented in this study is around a 10-fold increase in the in vivo throughput because it can test up to 240 compounds daily (García-Alcover, 2014).

A limitation on the development of in vivo screening methods is the unfeasibility of traditional brute-force traditional methods that usually involve mass 384-, 1536- or 3456-well plate formats. In contrast, Drosophila-based screening methods offer the ability to test compound activity directly in a living animal with the simultaneous evaluation of toxicity and drug-like properties. Moreover, and of significance, the use of flies allows for an accurate control of the expression system, as demonstrated in this study by targeting the DM1 transgenes only in somatic muscles, a key tissue in DM1 progression (García-Alcover, 2014).

One potential drawback to the use of Drosophila is linked to the extent of genome and pathway conservation, although this organism does display a high degree of conservation in genes, structures and functional processes characterized in vertebrate skeletal muscle. This makes Drosophila particularly well-suited to modeling and studying muscular disorders. Regarding alternative splicing, although important functional conservation occurs between Drosophila and mammals, dissimilarities have also been described. Results obtained in this study from the DM1 spliceosensor flies suggest that in this specific condition, there are only slight alternative splicing machinery differences, allowing for robust disease mirroring. Noticeably, transgenes are strongly reliant on CUG repeat expression and successfully rescued by co-expression of human MBNL1. The best corroboration of the effectiveness of the screening method is the confirmation of positive activity on splicing-independent key DM1 features for some compounds, such as the ability to bind to the toxic CUG-RNA or to reduce the number of foci aggregates. Taken together, this suggests that the in vivo drug discovery approach could significantly reduce post-screening costs for identifying quality leads from the initial candidate pool (García-Alcover, 2014).

Success in the identification of novel valid compounds for the potential development of a DM1 treatment suggests that the method developed in this study could be adapted to any particular type of alternative splicing deregulation (exon skipping, intron retention and exon extension, among others) linked to human disease, such as in myotonic dystrophy type 2 (DM2), progeria, Alzheimer’s disease or cancer, where missplicing events are already well-described and for which key disease aspects are conserved in D. melanogaster. The versatile use of reporter-based platforms in whole organisms, where it is at the moment still very limited to cell culture, should serve to rapidly expand the kind of in vivo HTS screens for which D. melanogaster can be widely used (García-Alcover, 2014).

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Luu, L.M., Nguyen, L., Peng, S., Lee, J., Lee, H.Y., Wong, C.H., Hergenrother, P.J., Chan, H.Y. and Zimmerman, S.C. (2016). A potent inhibitor of protein sequestration by expanded triplet (CUG) repeats that shows phenotypic improvements in a Drosophila model of myotonic dystrophy.ChemMedChem [Epub ahead of print]. PubMed ID: 27245480

Abstract

Myotonic dystrophy is the most common form of adult-onset muscular dystrophy, originating in a CTG repeat expansion in the DMPK gene. The expanded CUG transcript sequesters MBNL1, a key regulator of alternative splicing, leading to the misregulation of numerous pre-mRNAs. This study reports an RNA-targeted agent as a possible lead compound for the treatment of myotonic dystrophy type 1 (DM1) that reveals both the promise and challenges for this type of small-molecule approach. The agent is a potent inhibitor of the MBNL1-rCUG complex with an inhibition constant (Ki ) of 25±8 nm, and is also relatively nontoxic to HeLa cells, able to dissolve nuclear foci, and correct the insulin receptor splicing defect in DM1 model cells. Moreover, treatment with this compound improves two separate disease phenotypes in a Drosophila model of DM1: adult external eye degeneration and larval crawling defect. However, the compound has a relatively low maximum tolerated dose in mice, and its cell uptake may be limited, providing insight into directions for future development (Luu, 2016).

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Yenigun, V. B., Sirito, M., Amcheslavky, A., Czernuszewicz, T., Colonques-Bellmunt, J., Garcia-Alcover, I., Wojciechowska, M., Bolduc, C., Chen, Z., Lopez Castel, A., Krahe, R. and Bergmann, A. (2017). (CCUG)n RNA toxicity in a Drosophila model for myotonic dystrophy type 2 (DM2) activates apoptosis. Dis Model Mech. PubMed ID: 28623239

Abstract

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions -- (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP. Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, a transgenic DM2 model was developed in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCTG)16, in muscle and retinal cells led to formation of (CCUG) ribonuclear inclusions and mis-splicing of genes implicated in the DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, while CUGBP1/CELF1 complementation did not. Flies with (CCUG)106 displayed strong disruption of the external eye morphology and the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, two chemical compounds were tested that have shown therapeutic potential in DM1 models. While treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. These data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. The Drosophila DM2 model may provide a convenient tool for in vivo drug screening (Yenigun, 2017).

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Cerro-Herreros, E., Fernandez-Costa, J.M., Sabater-Arcis, M., Llamusi, B. and Artero, R. (2016). Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila. Sci Rep 6: 36230. PubMed ID: 27805016

Abstract

Myotonic Dystrophy type 1 (DM1) originates from alleles of the DMPK gene with hundreds of extra CTG repeats in the 3' untranslated region (3' UTR). CUG repeat RNAs accumulate in foci that sequester Muscleblind-like (MBNL) proteins away from their functional target transcripts. Endogenous upregulation of MBNL proteins is, thus, a potential therapeutic approach to DM1. This study identifies two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate muscleblind RNA isoforms in miRNA sensor constructs. It was shown that their sequestration by sponge constructs derepresses endogenous muscleblind not only in a wild type background but also in a DM1 Drosophila model expressing non-coding CUG trinucleotide repeats throughout the musculature. Enhanced muscleblind expression results in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Rescued flies have improved muscle function in climbing and flight assays, and have longer lifespan compared to disease controls. These studies provide proof of concept for a similar potentially therapeutic approach to DM1 in humans (Cerro-Herreros, 2016).

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Vanhoutte, D., Schips, T. G., Kwong, J. Q., Davis, J., Tjondrokoesoemo, A., Brody, M. J., Sargent, M. A., Kanisicak, O., Yi, H., Gao, Q. Q., Rabinowitz, J. E., Volk, T., McNally, E. M. and Molkentin, J. D. (2016). Thrombospondin expression in myofibers stabilizes muscle membranes. Elife 5 [Epub ahead of print]. PubMed ID: 27669143

Abstract

Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. This study shows that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy (Vanhoutte, 2016).

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Dialynas, G., Shrestha, O.K., Ponce, J.M., Zwerger, M., Thiemann, D.A., Young, G.H., Moore, S.A., Yu, L., Lammerding, J. and Wallrath, L.L. (2015). Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway. PLoS Genet 11: e1005231. PubMed ID: 25996830

Abstract
Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, this study performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, these mutations were modeled in Drosophila Lamin C and the mutant lamins were expressed in muscle. It was found that the structural perturbations have minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology is not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles have cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlates with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of the Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis (Dialynas, 2015).

Highlights

  • Mutant lamins alter the tertiary structure of the Ig-fold domain.
  • Mutant lamins have minimal dominant effects on nuclear stiffness.
  • Mutant lamins alter muscle gene expression.
  • Mutant lamins cause reductive stress.
  • Mutant lamins activate the Nrf2/Keap-1 pathway.

Discussion
Structural studies of the lamin Ig-fold demonstrate that single amino acid substitutions in the loop regions perturb the tertiary structure, leaving the secondary structure of the folded domain largely intact. These data are consistent with single-molecule force spectroscopy showing that the lamin Ig-fold possessing an R453W substitution requires less force to unfold than the wild type Ig-fold domain. Structural data of this study are consistent with in silico modeling in which amino acid substitutions in the Ig-fold that cause muscular dystrophy were predicted to alter the structure, more so than those that cause lipodystrophy or progeria. NMR analysis of the mutant Ig-fold domains identifies surfaces on opposite sides of the Ig-fold barrel that are critical for muscle function. This finding predicts that substitution of other amino acids that comprise these surfaces might result in muscular dystrophy. Consistent with this prediction, amino acid substitutions in eight of the 21 amino acids that make up these surfaces cause muscular dystrophy (Dialynas, 2015).

It is interesting to note that the largest structural perturbations were observed for the G449V and W514R mutants, which correspond to the most severe patient phenotypes. The corresponding amino acid substitutions in Drosophila Lamin C cause the greatest percentage of lethality. The N456I mutant shows the least structural perturbations in the Ig-fold domain, though the relative severity of symptoms in this patient was not ascertained. Consistent with the structural data, the corresponding amino acid substitution in Drosophila Lamin C gives the least percentage of lethality. Thus, these data show an obvious correlation between the severity of the Ig-fold structural perturbations and phenotypic severity (Dialynas, 2015).

The structural perturbations within the Ig-fold might generate novel interaction surfaces that promote lamin aggregation. Both nuclear and cytoplasmic aggregation of mutant lamins have been reported, however, they are not commonly observed in human muscle biopsy tissue or tissue from a laminopathy mouse model. Cytoplasmic aggregation has been observed for a truncated form of A-type lamin that causes Hutchinson-Gilford progeria syndrome. Lamin aggregation is supported by X-ray crystallography studies of a R482W substitition in the A-type lamin Ig-fold domain that causes lipodystrophy. The R482W Ig-fold domain possesses unique interaction surfaces not present in the wild type Ig-fold that form a unique platform for tetramerization (Dialynas, 2015).

Cytoplasmic protein aggregation has been linked to reductive stress. This study shows that cytoplasmic lamin aggregation correlates with elevated levels of both GSH and NADPH, hallmarks of reductive stress. Elevated levels of isocitrate dehydrogenase enzyme activity contribute to the additional NADPH. In a similar manner, dominant negative forms of alphaB-crystallin (CryAB) result in cytoplasmic CryAB misfolding/aggregation and reductive stress in the mouse heart, ultimately leading to dilated cardiomyopathy. These findings suggest that reductive stress might contribute to the dilated cardiomyopathy in cases of lamin associated muscular dystrophy. Interestingly, mutations in the human CRYAB gene cause disease phenotypes that are strikingly similar to those observed for lamin associated muscular dystrophy, including skeletal muscle weakness and dilated cardiomyopathy in cases of lamin-associated muscular dystrophy. It is worthwhile to note that CryAB functions as a chaperone to prevent aggregation of intermediate filament proteins such as desmin, suggesting a common link between intermediate filament aggregation and reductive stress (Dialynas, 2015).

An imbalance in redox homeostasis can provide an environment that promotes protein misfolding and aggregation. The redox state influences aggregation of lamins; aggregation has been observed under both oxidative and reductive conditions. In fact, the formation of the novel tetramer generated by the R482W mutant Ig-fold domain requires a reductive environment. Reductive stress has also been observed in healthy individuals predisposed to Alzheimer disease, a disease of protein aggregation. Alzheimer disease is typically accompanied by oxidative stress, however, lymphocytes from patients carrying an ApoE4 allele that predisposes them to Alzheimer disease show reductive stress. It is hypothesized that continual activation of antioxidant defense systems, such as Nrf2/Keap-1 signaling, becomes exhausted over time, particularly later in life, resulting in the inability to properly defend against oxidative stress. This study analyzed the redox status in Drosophila muscle 24–48 hours post expression of the mutant lamins; their findings suggest reductive stress at the onset of pathology that could resolve into oxidative stress later in disease progression (Dialynas, 2015).

Typically lamins are thought to regulate gene expression from inside the nucleus, by interacting with transcription factors and organizing the genome. Data from this study support a novel model in which genes are misregulated as a consequence of mutant lamin aggregation in the cytoplasm. Cytoplasmic lamin aggregates have been found in high molecular weight complexes in cases of liver injury. Such complexes contain nuclear pore proteins, signaling mediators, transcription factors and ribosomal proteins, which are thought to disrupt the normal cellular physiology. Lamin aggregation might also serve a cytoprotective function by facilitating the coalescence of mutant lamin so that the contractile apparatus can properly function. A similar mechanism exists in Huntington’s disease, where sequestration of mutant huntingtin in inclusion bodies correlates with better neuron health (Dialynas, 2015).

Collectively, findings from this study continue to support this Drosophila model of laminopathies, as many of the phenotypes discovered in Drosophila have been validated in human muscle biopsies. It is now possible to use this rapid genetic model to (1) determine if mutations in other domains of lamin produce similar phenotypes and (2) if lamin mutations have similar effects in other tissues, such as the heart. Data suggest that cytoplasmic lamin aggregation contributes to muscle pathology. Consistent with this idea, increased rates of autophagy suppress phenotypes caused by mutant A-type lamin in cultured cells and mouse models. Furthermore, electron microscopy of skeletal muscle biopsies from patients with LMNA mutations show large perinuclear autophagosomes, similar to the localization of lamin aggregates and p62 foci in the Drosophila muscle. Thus, the regulation of autophagy, a process that removes both damaged organelles and proteins, might be central to the development of therapies. The Drosophila model will allow for genetic dissection of both the autophagy and reductive stress pathways to identify the key factors responsible for the muscle pathogenesis and its suppression (Dialynas, 2015).

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Chartier, A., Klein, P., Pierson, S., Barbezier, N., Gidaro, T., Casas, F., Carberry, S., Dowling, P., Maynadier, L., Bellec, M., Oloko, M., Jardel, C., Moritz, B., Dickson, G., Mouly, V., Ohlendieck, K., Butler-Browne, G., Trollet, C. and Simonelig, M. (2015). Mitochondrial dysfunction reveals the role of mRNA poly(A) tail regulation in oculopharyngeal muscular dystrophy pathogenesis. PLoS Genet 11: e1005092. PubMed ID: 25816335

Abstract
Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. This study shows, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. The study identifies a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. The study focuses on the deadenylation regulator Smaug and shows that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. The study proposes that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden the understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction (Chartier, 2015).

Highlights

  • Genes encoding mitochondrial proteins are down-regulated in Drosophila muscles expressing PABPN1-17ala.
  • Defects in mitochondrial function play an important role in OPMD physiopathology in the Drosophila model.
  • Role of mRNA poly(A) tail length regulation in the defects induced by PABPN1-17ala expression.
  • Affected nuclear cleavage/polyadenylation in the Drosophila model of OPMD.
  • Smg regulates mRNAs down-regulated in muscles expressing PABPN1-17ala.
  • Rescue of PABPN1-17ala-induced phenotypes by reducing deadenylation does not involve decreased PABPN1 aggregation.
  • Early down-regulation of mRNAs encoding mitochondrial proteins is conserved in the OPMD mouse model.
  • Mitochondrial protein levels are decreased in OPMD patient muscles.
     

Discussion
The molecular defects underlying OPMD pathology remain largely undetermined, although recent advances have implicated apoptosis and a general deregulation of the ubiquitin-proteasome system. However, these are downstream events in the pathogenesis. This study investigates the molecular mechanisms involved by analysing early defects in the disease. It was shown that specific mRNAs encoding proteins involved in mitochondrial activity are present at lower levels in pre-symptomatic OPMD muscles; this reduced expression results from the shortening of their poly(A) tails which leads to their destabilization. Poly(A) tail length regulation plays a key role in OPMD since muscle function is improved when deadenylation is decreased using mutants. It was further shown that nuclear cleavage/polyadenylation of pre-mRNAs is inefficient in PABPN1-17ala-expressing muscles. This defect occurs both on genes which are and on those which are not down-regulated, indicating that it does not per se systematically lead to reduced steady-state mRNA levels. The decreased levels of specific mRNAs result from their active deadenylation, itself dependent, at least in part, on the specific interaction of these mRNAs with the Smg RNA binding protein and the recruitment by Smg of the CCR4-NOT deadenylation complex. In addition, genetic data revealing the potential involvement of other RNA binding proteins suggest that OPMD pathogenesis is complex and probably involves additional mechanisms of RNA regulation (Chartier, 2015).

The function of PABPN1 during cleavage/polyadenylation has been documented. Nuclear polyadenylation occurs in two steps: first, cleavage of the pre-mRNA at the poly(A) site, which is co-transcriptional, and second, polyadenylation which potentially occurs after dissociation of the RNA from the RNA polymerase II. PABPN1 has been shown to be involved in the second step, polyadenylation, for the control of poly(A) tail lengths. More recent data have also implicated PABPN1 in the cleavage step for the regulation of weak poly(A) sites. This study shows impaired cleavage at poly(A) sites in the Pabp2 loss-of-function mutant, revealing a more general role of PABP2/PABPN1 in this step of the reaction. In the regulation of weak poly(A) sites, PABPN1 binds to non-canonical polyadenylation signals and prevents the binding of CPSF (Cleavage and polyadenylation specificity factor) required for cleavage. A general function of PABPN1 in cleavage would require other interactions, for example with proteins required for cleavage, such as the poly(A) polymerase known to associate with PABPN1. Although a global shift to proximal poly(A) sites has been reported in the mouse model of OPMD, this study shows that the down-regulation and poly(A) tail shortening of specific mRNAs participating in OPMD pathogenesis are independent of alternative poly(A) site utilization (Chartier, 2015).

The molecular defects observed in PABPN1-17ala-expressing muscles, reduction of mRNA poly(A) tail length and decreased efficiency of cleavage at poly(A) sites are similar to those observed in Pabp2 loss-of-function mutants. This suggests that part of the defects in OPMD could result from partial PABPN1 loss-of-function. However, the genetic suppression of wing posture phenotypes by reducing the dosage of Pabp2 does not favour a simple loss-of-function model. It has been proposed for polyglutamine diseases that the pathogenesis could result from both the gain-of-function and the loss-of-function of the same protein. The protein responsible for the disease would exist in two different conformations with two different yet normal functions. Extension of the polyglutamine tract would favor one conformer resulting in increased amounts of this conformer and the partial loss-of-function of the other conformer; the pathology would result from both these effects. In this model, the mutant protein would have the same function as the normal protein but would have the ability to alter the balance between both protein forms. Several properties of PABPN1 are consistent with this model for OPMD. It has been shown previously that the normal function of PABPN1 and more specifically its RNA binding activity is required for OPMD-like defects in the Drosophila model. In addition, PABPN1-17ala half-live has been reported to be longer than that of PABPN1 in cell models, leading to higher accumulation of PABPN1-17ala and protein aggregation. Thus, expansion of the polyalanine tract results in protein "overexpression" which contributes to the pathology. Given this data, overepression of the normal protein might be expected to induce similar defects as expression of the mutant protein, as it is the case in Drosophila models for other disorders. Consistent with this, it has been previously reported that PABPN1 expression in Drosophila muscles induces wing posture defects, although at lower levels than PABPN1-17ala expression. Finally, normal PABPN1 is also known to form oligomers during nuclear polyadenylation and can form nuclear aggregates that recruit ubiquitin and proteasomes under specific physiological conditions (Chartier, 2015).

Because the presence of nuclear aggregates and muscle defects can to some extent be uncoupled, it has been previously proposed that nuclear aggregates are not always pathological. This is consistent with results concerning polyglutamine diseases where aggregates can have a protective role. It was found that the improvement of muscle function when deadenylation is genetically reduced correlates with an increased number of PABPN1 aggregates, again strengthening the notion that the aggregates are not always causative of muscle defects. In that case, muscle protection that results from the reduction of molecular defects could allow the formation of more PABPN1 aggregates. Thus, these aggregates might not always be pathological, in particular during early stages of the disease, although they might become so at later stages, when their increased size could interfere with nuclear function (Chartier, 2015).

A major conclusion from this study is that the specificity of the defect in OPMD does not depend per se on PABPN1 defect in pre-mRNA cleavage, but on Smg-dependent regulation occurring in the cytoplasm. Because of the shift to proximal poly(A) sites that correlates with mRNA up-regulation, described in the OPMD mouse model, this study analyzed whether a similar mechanism could lead to increased Smg levels in Drosophila muscles expressing PABPN1-17ala and underlie increased deadenylation. However, the same poly(A) site was used in normal and PABPN1-17ala-expressing muscles, and a major deregulation of smg mRNA and protein levels in PABPN1-17ala-expressing muscles was not found. The study proposes that normal Smg-dependent deadenylation, following inefficient pre-mRNA cleavage, could lead to the reduced levels of specific mRNAs that were observed. In addition, other processes such as mRNP remodelling could contribute to enhanced mRNA decay in the course of the disease progression. Indeed, Smg forms cytoplasmic foci which are distinct, but related to other cytoplasmic RNA granules such as processing (P) bodies or stress granules, in which mRNAs are degraded or translationally repressed, and the regulation of which affects mRNA regulation. A recent study also revealed the implication of Smg/SAMD4A in Myotonic Dystrophy Type 1 (DM1). In that case, Smg mechanism of action appeared to be different, since overexpression of Smg decreased DM1 muscle defects by reducing unproductive CUGBP1-eIF2α translational complexes (Chartier, 2015).

Mitochondrial dysfunction has been shown to play a major role in most neurodegenerative diseases including Parkinson's, Alzheimer's, Huntington's and other polyglutamine diseases. More recent data have uncovered that aside mitochondrial function in energy production, mitochondrial dynamics including trafficking and quality control is instrumental in pathogenesis. Mitochondria also have a key role in muscle function. Drosophila mutants of pink1 and parkin, mutations of which cause Parkinson's disease in humans, lead to mitochondrial dysfunction and flight muscle degeneration. It was shown that mitochondrial dysfunction is also an important component of OPMD: Muscle function is improved when mitochondrial biogenesis and activity are genetically increased; in addition, mitochondrial proteins are down-regulated in OPMD muscle biopsies from patients. The molecular defects leading to early mitochondrial dysfunction in OPMD were identified: mRNAs encoding mitochondrial proteins are down-regulated due to their Smg-dependent deadenylation. Therefore, this study reveals Smg as a regulator of mRNAs involved in mitochondrial function. This finding might have important implications on the role of Smg in several neurodegenerative diseases that involve mitochondrial dysfunction and/or RNA toxicity (Chartier, 2015).

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Picchio, L., Plantie, E., Renaud, Y., Poovthumkadavil, P. and Jagla, K. (2013). Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression. Hum Mol Genet 22: 2795-2810. PubMed ID: 23525904

Abstract
Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant disorder characterized by myotonia and muscle degeneration. In DM1 patients, the mutant DMPK transcripts containing expanded CUG repeats form nuclear foci and sequester the Muscleblind-like 1 splicing factor, resulting in mis-splicing of its targets. However, several pathological defects observed in DM1 and their link with disease progression remain poorly understood. In an attempt to fill this gap, this study generated inducible transgenic Drosophila lines with increasing number of CTG repeats. Targeting the expression of these repeats to the larval muscles recapitulates in a repeat-size-dependent manner the major DM1 symptoms such as muscle hypercontraction, splitting of muscle fibers, reduced fiber size or myoblast fusion defects. Comparative transcriptional profiling performed on the generated DM1 lines and on the muscleblind (mbl)-RNAi line revealed that nuclear accumulation of toxic CUG repeats can affect gene expression independently of splicing or Mbl sequestration. Also, in mblRNAi contexts, the largest portion of deregulated genes corresponds to single-transcript genes, revealing an unexpected impact of the indirect influence of mbl on gene expression. Among the single-transcript Mbl targets is Muscle protein 20 involved in myoblast fusion and causing the reduced number of nuclei in muscles of mblRNAi larvae. Finally, by combining in silico prediction of Mbl targets with mblRNAi microarray data, the calcium pump dSERCA was found to be a Mbl splice target and it was shown that the membrane dSERCA isoform is sufficient to rescue a DM1-induced hypercontraction phenotype in a Drosophila model (Picchio, 2013).

Highlights

  • Expanded CUG repeats accumulate in nuclear foci and sequester Mbl in a new Drosophila larval model of DM1.
  • Reduced motility and affected muscle morphology of DM1 larvae.
  • Impaired muscle relaxation in a Drosophila larval model of DM1.
  • Transcriptional profiling of DM1 CTG-repeat lines and mblRNAi line reveals splice-independent gene deregulation.
  • Mp20, an indirect Mbl target involved in DM1-associated myoblast fusion defects.
  • In silico prediction of Mbl targets and microarray analyses identify transmembrane isoform of the Drosophila sarco endoplasmic reticulum calcium-ATPase (dSERCA) as required for proper muscle contraction in DM1.

Discussion
Drosophila has already proved to be a powerful tool for conducting genetic screening and global analyses on the effect of CTG repeats in DM1. So far, an inducible line expressing 480 interrupted CTG repeats has been used at the adult stage and shows age-dependent muscle degeneration. This study generated a set of three inducible site-specific lines expressing 240, 600 or 960 interrupted CTG repeats. In some cases of DM1, the existence of variant repeats interrupting the pure CTG expansions have been observed. Interestingly, interruptions allow either repeat stabilization or repeats contraction. If in one peculiar DM1 family CCG and CGG variants are associated with Charcot-Marie-Tooth symptoms, the role of interruptions remains unclear in other patients. The interrupted CTG repeats have already been used to generate different animal models of DM1. One could consider a possibility of additive toxic effect in all these models. However, the CTCGA interruption motif commonly used for these models is different from already described variants and its toxicity as well as unstability have not been not reported so far (Picchio, 2013).

This study used CTG size variation to compensate for age effect in third instar larvae. The study assessed larval muscles instead of adult muscle for three reasons: (i) segmentally repeated larval musculature is organized in a stereotyped network of muscle fibers and is easy to analyze at morphological and functional levels, (ii) establishing and characterizing larval DM1 model appears attractive for future genetic rescue approaches and molecular screening applications and (iii) adult lethality of the Mef>mblRNAi line prevents comparative analyses with DM1 lines in adult flies (Picchio, 2013). 

As observed in patients, it was found that expressing an increasing number of CTG repeats in larval somatic muscles leadd to the formation of nuclear foci and that these foci co-localize with Drosophila MBNL1 ortholog, Mbl. As the number of repeats positively influences the number of foci per nucleus and worsen muscle phenotypes, the new Drosophila model of DM1 presented here could be of interest for simulating disease progression and (or) severity (Picchio, 2013). 

Global analysis of muscle pattern in this model reveals a histopathological defect called ‘splitting fibers’ already observed in mbnl1 knockout mice as well as in DM1 patients. Here, splitting occurs during larval stages characterized by rapid muscle growth. As observed in dorsal oblique fibers, it is initiated at muscle endings at the level of interaction with tendon cells. This suggests that splitting results from affected muscle attachment to tendon cells and (or) abnormal sarcomeric organization that weaken the integrity of myofibrils. This latter hypothesis is supported by decreased expression of two sarcomere components (Mhc, up) in the DM1960 line. Surprisingly, the Mef>240CTG line which doesn not exhibit visible foci within muscle nuclei displays altered motility associated with muscle splitting but no fiber defects. This observation suggests that splitting is sufficient to alter motility (Picchio, 2013). 

Also, SBM and VL3 fiber examination shows reduced muscle size. So far, it has been shown in primary cell culture of myoblasts from DM1 patients that the ability of DM1-derived myoblasts to fuse is affected, consequently reducing myotube length. This study reports that expressing non-coding CTG repeats affects in vivo myoblast fusion. Interestingly, microarray data and RT–qPCR performed at embryonic and larval stages on mutant lines have shown decreased expression of Mp20 encoding an actin-binding protein involved in Drosophila myoblast fusion. Mp20 appears as an attractive candidate gene for myoblast fusion defects in DM1, since by overexpressing Mp20 during myogenesis, the number of nuclei per fiber was rescued in DM1 lines and in the mbl attenuated line. Surprisingly, Mp20 does not appear to undergo alternative splicing (single-transcript gene according to Flybase), suggesting that its Mbl-dependent down-regulation could occur through an indirect effect of Mbl. It is also noteworthy that one human counterpart of Mp20, the Calponin 3 gene, has been found to be involved in myoblast fusion in vitro. Thus, genes of the Mp20/Calponin family appear as attractive candidates to be tested for their role in DM1 muscle defects in humans (Picchio, 2013). 

Finally, this study reports that mutant larvae and in particular those from DM1960 and mbl attenuated lines display altered motility with affected complex movements. Interestingly, by measuring the contractility index and sarcomere size, it was found that both the lines exhibit hypercontraction, a phenotype related to myotonia. It has been previously shown that mbnl1 disruption in the mouse also leads to myotonia. In the Drosophila DM1 model, the effect of CTG repeat size on the severity of myotonia was observed, so that the DM1600 line exhibits intermediate hypercontraction phenotypes when compared with DM1240 and DM1960. As not only hypercontraction but also affected myoblast fusion account for a reduced muscle size in pathological lines, this study assume that both parameters need to be repaired to fully rescue muscle length (Picchio, 2013).

It was also found that the severity of several phenotypes is positively correlated with the size of the CTG repeats. This was followed by comparative transcriptional profiling on DM1600 and DM1960 lines. First, during validation of selected candidate genes from microarray analyses, repeat-size-dependent deregulation of genes involved in carbohydrate and nitrogen metabolism was observed. A more systematic classification of candidates deregulated in a repeat-size-dependent manner and having human orthologs was then performed based on the ratio of their fold-change between the two conditions and on their function. Data reveals that genes encoding transporter proteins are significantly enriched among gene categories down-regulated in larvae carrying high repeat numbers (Picchio, 2013). 

Among these, repeat-size-dependent deregulation of smvt, whose human orthologs (SLC5A3, SLC5A5, SLC5A8 and SLC5A12) encode myo-inositol transporters, and CG17597/SCP-2, involved in phosphatidylinositol transfer and signaling, was validated. It is known that phosphatidylinositol is a derivative of myo-inositol, suggesting that both transporters may work in the same pathway. However, how the alterations of transporters influence the accumulation of the inositol forms and how this is connected to muscle defects remain to be investigated. It was also found that two genes involved in the sarcomere structure Mhc and up were both down-regulated specifically in the DM1960 context. In DM1 patients, it has been shown that Mhc ortholog MYH14 and up orthologs TNNT2 and TNNT3 are mis-spliced. Besides, another report provides evidence that in a Drosophila mbl null mutant, up transcripts are mis-spliced as well. However, the link between the Mhc and up gene deregulations and DM1 muscle phenotypes and their impact on DM1 pathogenesis have not yet been investigated. This study speculate that down-regulation of Mhc and up might be involved in splitting fiber phenotype observed in DM1 larvae (Picchio, 2013). 

Comparative genomic analyses shows that a high percentage of genes with misregulated expression (∼70%) do not undergo alternative splicing but are sorted out in the Mef>mblRNAi context. As Mbl binds specifically to double-stranded RNA structures, the study hypothesized that it may influence transcript stability of this class of genes as already observed with MBNL1 in C2C12 cells. Alternatively, Mbl might play an indirect role on single-transcript genes via mis-splicing of transcription factors that regulate their expression. In order to gain insights into the second hypothesis, potential common regulators of Mbl-deregulated single-transcript genes were identified using the bioinformatics i-cisTarget approach. Interestingly, several transcription factors known to act in musclessuch as dMef2 and GATA factor Panier (Pnr) were found as potential transcriptional regulators of candidate genes. More importantly, the same transcription factors were found deregulated in transcriptional profiling experiments under all pathological conditions and most of them (including dMef2 and Pnr) were also predicted in silico to be targets of Mbl. Thus, these data reveal an important contribution of single-transcript gene deregulation in the Drosophila DM1 model and point to an indirect role of Mbl in the regulation of gene expression via mis-splicing of key myogenic factors. As a matter of fact, this mechanism may play a role in the regulation of Mp20 expression, one of dMef2 targets. Interestingly, both qPCR and microarray experiments showed that Mp20 expression is down-regulated in pathological contexts leading to myoblast fusion defects. Consequently, the study suggests an indirect role of Mbl in Mp20 expression through misregulation of dMef2 alternative splicing (Picchio, 2013). 

In DM1960 larvae, an Mbl-dependent muscle hypercontraction phenotype related to myotonia was observed. By associating microarray data with in silico prediction of Mbl direct targets, the dSERCA gene was identified as a putative candidate for Mbl-driven mis-splicing and hypercontraction phenotype. By RT–qPCR, it was confirmed that the isoforms B-H of dSERCA containing exons 8 or 11 encoding the transmembrane domain show decreased expression in mbl attenuated and in DM1960 lines. This indicates that in the Mbl-deficient context, the exons 8 or 11 of dSERCA are spliced out, leading to the production of dSERCA isoforms devoid of the transmembrane domain. This switch in dSERCA isoforms is consistent with the immunostaining of DM1 larval muscles, in which the membrane-associated dSERCA protein is barely detectable at muscle surface or even in sarcomere for the Mef>mblRNAi line, whereas the level of free dSERCA in nuclei appears to be enhanced in DM1 lines. It has been previously shown that in DM1 patient muscles, as a result of MBNL1 sequestration, SERCA1 exon 22 in the 3′ part of the transcript is excluded leading to the formation of a neonatal isoform of SERCA1. This isoform is expected to cause muscle degeneration but so far, no functional analysis has been performed to confirm this hypothesis. However, patients with Brody's disease, which is caused by different mutations in the SERCA1a gene, manifest impairment of skeletal muscle relaxation among other symptoms. In addition, it has been shown that dSERCA plays a key role in muscle contraction and heartbeat frequency and rythmicity in flies, suggesting that it might be involved in muscle hypercontraction phenotypes and myotonia in DM1 muscles (Picchio, 2013). 

To date, the only gene functionally implicated in myotonia in DM1 is the CIC-1 encoding a muscle-specific chloride channel. CIC-1 transcripts have been found to undergo MBNL1- and CUGBP1-dependent splice modifications causing muscle delayed relaxation and pathogenic muscle defects. However, analyses performed on HSA(LR) myotonic mice reveals that ClC-1 channels account for muscle hyperexcitability in young but not in old DM1 animals, suggesting alteration of conductance other than chloride currents. Consequently, this study tested if the loss of dSERCA function and in particular depletion in its isoforms carrying the transmembrane domain could indeed affect muscle contractility. By using a pharmacological tool, CPA, a highly specific inhibitor of SERCA, which binds to the entry channel, it was found that the contractility of CPA-treated larval muscles is severely affected. Next, by performing rescue experiments by overexpressing the transmembrane isoform of dSERCA in DM1 lines with hypercontracted phenotypes, it was found that the contractility index is significantly improved. Thus, these data provide the first evidence in an animal model of DM1 that SERCA mis-splicing is involved in muscle hypercontraction (Picchio, 2013).

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Pantoja, M., Fischer, K.A., Ieronimakis, N., Reyes, M. and Ruohola-Baker, H. (2013). Genetic elevation of sphingosine 1-phosphate suppresses dystrophic muscle phenotypes in Drosophila. Development 140: 136-146. PubMed ID: 23154413

Abstract
Duchenne muscular dystrophy is a lethal genetic disease characterized by the loss of muscle integrity and function over time. Using Drosophila, this study shows that dystrophic muscle phenotypes can be significantly suppressed by a reduction of wunen, a homolog of lipid phosphate phosphatase 3, which in higher animals can dephosphorylate a range of phospholipids. Suppression analyses include assessing the localization of Projectin protein, a titin homolog, in sarcomeres as well as muscle morphology and functional movement assays. The study hypothesizes that wunen-based suppression is through the elevation of the bioactive lipid Sphingosine 1-phosphate (S1P), which promotes cell proliferation and differentiation in many tissues, including muscle. The role of S1P in suppression is confirmed by genetically altering S1P levels via reduction of S1P lyase (Sply) and by upregulating the serine palmitoyl-CoA transferase catalytic subunit gene lace, the first gene in the de novo sphingolipid biosynthetic pathway and these manipulations also reduce muscle degeneration. Furthermore, it was shown that reduction of spinster (which encodes a major facilitator family transporter, homologs of which in higher animals have been shown to transport S1P) can also suppress dystrophic muscle degeneration. Finally, administration to adult flies of pharmacological agents reported to elevate S1P signaling significantly suppresses dystrophic muscle phenotypes. These data suggest that localized intracellular S1P elevation promotes the suppression of muscle wasting in flies (Pantoja, 2013).

Highlights

  • Establishment of a dystrophic myofibril phenotype.
  • Suppression of dystrophic muscle phenotypes by genetically reducing wunen.
  • Suppression of dystrophic muscle phenotypes by genetically increasing S1P levels.
  • Suppression of the dystrophic myofibril phenotype by genetically reducing spinster, a putative S1P transporter.
  • Suppression of dystrophic muscle function phenotypes as assessed by fly movement assays.
  • Suppression of dystrophic muscle phenotypes using pharmacological agents that increase S1P levels or S1P signaling.

Discussion
This study established an easy-to-score myofibril phenotype for dystrophic flies that can even be scored in relatively young flies in strong Dystrophin mutants. This assay complements the histological sections used previously to score gross morphological muscle degeneration. In using both assays, it was shown that a reduction of the LPP3 homolog wunen prevents, to a significant degree, Dystrophin-dependent muscle wasting. This is likely to occur through the increase of S1P levels as other avenues used to raise the level of S1P phenocopy this suppression. Both reduction of Sply, encoding the Drosophila S1P lyase, and overexpression of lace, encoding the catalytic subunit of serine palmitoyl CoA transferase in the de novo sphingolipid synthesis pathway, prevent muscle wasting. It was also shown that muscle function as assayed by fly movement is also improved in dystrophic animals when S1P is genetically elevated. Furthermore, S1P-based suppression is not occurring during development as feeding experiments utilizing adult flies revealed that S1P elevation in these animals can suppress dystrophic myofibril and activity phenotypes (Pantoja, 2013).

it was also found that minimal levels of S1P are necessary for viability in Drosophila as global reduction of Sphingosine Kinase 2 (SK2) results in lethality. Interestingly, global reduction of Sphingosine Kinase 1 (SK1) is not lethal in non-dystrophic flies yet is lethal in dystrophic flies, owing to exacerbation of the phenotype. These data indicate that S1P levels regulated by SK1 are crucial for Dystrophin mutant survival. These data argue for a precise requirement of minimal levels of S1P for muscle development and/or function. Future analyses of the activity of both sphingosine kinases in different tissues and cellular compartments might separate the roles of each kinase in homeostasis and muscle (Pantoja, 2013).

In mammals, there are five S1P receptors that share homology with G protein-coupled receptors. Though flies do have G protein-coupled receptors, they do not appear to have the S1P receptors seen in vertebrates, suggesting that S1P receptor-mediated signaling might have evolved later in higher organisms. S1P lyase mutants increase intracellular S1P levels and S1P is generated and has been shown to function inside the cells, indicating that the suppression of muscle wasting in Drosophila occurs intracellularly. With this in mind, this study hypothesized that if spinster, like its mammalian homolog spns2, is an S1P transporter, its reduction would prevent S1P from leaving the cytoplasmic compartment and it would then behave like reduced Sply and suppress muscle wasting. Data support this hypothesis yet more work is required to connect this transporter to S1P in Drosophila. Interestingly, Drosophila spinster has been reported to interact with genes of the cell death pathway and it is known that ceramide in the sphingolipid pathway can induce cell death. Perhaps spinster alters the cell death pathway by perturbing the equilibrium of sphingolipids, particularly S1P, in different subcellular compartments. Another report has revealed S1P epigenetic regulation of gene expression through direct intracellular interaction with histone deacetylases (HDACs). Through this mechanism, perhaps increasing intracellular S1P levels alters gene expression, which ultimately leads to elevated translation of muscle proteins, such as Projectin, which then reduces muscle wasting (Pantoja, 2013).

S1P has been shown to be necessary for the proliferation of satellite cells in mammals and is required for differentiation of myoblasts to myotubes. As there do not appear to be canonical satellite cells in Drosophila, i.e. muscle precursor cells located on the surface of muscle fibers, perhaps S1P-based suppression of muscle wasting occurs as a result of the requirement of S1P for proper differentiation. It has been reported in Drosophila that sarcomeres are formed by an assembly of latent protein complexes. It would be consistent with this if S1P elevates muscle protein synthesis (in turn increasing the level of latent protein complexes) so that after muscle contraction-induced damage these complexes can assemble and produce new myofibrils. Data from this study on small molecule effectors of S1P signaling indicate that the above mentioned possibilities for suppression occur in actively contracting adult muscle. S1P-based suppression of muscle wasting can be dissected further in Drosophila with the abundance of genetic tools available. Furthermore, given the observations with THI, THI oxime and FTY720, Drosophila may be used to screen small molecules for their efficacy in suppressing muscle wasting (Pantoja, 2013).

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Fernandez-Costa, J.M., Garcia-Lopez, A., Zuñiga, S., Fernandez-Pedrosa, V., Felipo-Benavent, A., Mata, M., Jaka, O., Aiastui, A., Hernandez-Torres, F., Aguado, B., Perez-Alonso, M., Vilchez, J.J., Lopez de Munain, A. and Artero, R.D. (2013). Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients. Hum Mol Genet 22: 704-716. PubMed ID: 23139243

Abstract
Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the 3′ untranslated region of the DMPK gene. Several missplicing events and transcriptional alterations have been described in DM1 patients. A large number of these defects have been reproduced in animal models expressing CTG repeats alone. Earlier studies have also reported miRNA dysregulation in DM1 patients. This study uses a Drosophila model to investigate miRNA transcriptome alterations in the muscle, specifically triggered by CTG expansions. Twenty miRNAs were found to be differentially expressed in CTG-expressing flies. Of these, 19 are down-regulated, whereas 1 is up-regulated. This trend was confirmed for those miRNAs conserved between Drosophila and humans (miR-1, miR-7 and miR-10) in muscle biopsies from DM1 patients. Consistently, at least seven target transcripts of these miRNAs are up-regulated in DM1 skeletal muscles. The mechanisms involved in dysregulation of miR-7 include a reduction of its primary precursor both in CTG-expressing flies and in DM1 patients. Additionally, a regulatory role for Muscleblind (Mbl) is also suggested for miR-1 and miR-7, as these miRNAs are down-regulated in flies where Mbl has been silenced. Finally, the physiological relevance of miRNA dysregulation is demonstrated for miR-10, since over-expression of this miRNA in Drosophila extends the lifespan of CTG-expressing flies. Taken together, these results contribute to our understanding of the origin and the role of miRNA alterations in DM1 (Fernandez-Costa, 2013).

Highlights

  • Expression of expanded CTG repeats in Drosophila causes reduction of defined miRNAs.
  • CTG-induced down-regulation of miR-1, miR-7 and miR-10 is conserved between Drosophila and DM1 patients.
  • Expanded CTG repeats alter the levels of defined pri-miRNA precursors.
  • Muscleblind is necessary for the regulation of miR-1 and miR-7 in Drosophila.
  • Over-expression of miR-10, but not miR-1 or miR-7, increases lifespan in CTG-expressing flies.

Discussion
Dysregulation of specific miRNAs in DM1 patients had been previously described. This study assessed the contribution of CTG expansions to miRNA defects in DM1, by analyzing changes in the muscle miRNome of a Drosophila model of CTG toxicity (2). Using SOLiD™ 3 sequencing, 20 miRNA alterations caused by expression of CTG repeats were identified. Of these, 19 are specifically down-regulated in the Drosophila model, whereas only 1 is up-regulated. Therefore, the alterations on miRNA regulation caused by CTG expression seem to trigger a reduction, rather than an increase, of miRNA expression levels. This effect is also observed in DM1 patients for all altered miRNAs that are conserved between Drosophila and humans: miR-1, an miRNA previously associated with DM1; and miR-7 and miR-10a, for which no previous link had been described (Fernandez-Costa, 2013).

Importantly, the conservation of miR-1, miR-7 and miR-10 defects between the fly model and DM1 patients confirms that: (i) the miRNA down-regulation found in Drosophila is specific, and not the consequence of a reduced contribution of the muscle transcriptome to the total transcriptome; and (ii) the dysregulation of these three miRNAs occurs in the presence of CUG-repeat transcripts devoid of additional DMPK sequences. Although it is possible that other coding or non-coding regions within the DMPK gene contribute to miRNA defects in DM1, this is the first demonstration that CTG expansions are directly linked to alterations in miRNA regulation. Of note, the fly model used in this work contains 480 CTG repeats interrupted every 20 units by the CTCGA sequence: i(CTG)480. The i(CUG)480 RNA is predicted to form a double-stranded structure that closely resembles the hairpin formed by 480 pure repeats, both of them having similar folding energies. The existence of complex repeat interruptions at the DM1 locus has been reported to attenuate the severity of symptoms in patients. Although the CTCGA interruption in the i(CTG)480 transgene does not resemble any of these variant repeat alleles, it is possible that its presence might also modify CTG-induced phenotypes in the flies. For example, the CUCGA interruption would determine the length of any putative repeat-associated non-ATG (RAN) translation products, should these be generated in Drosophila, as i(CAG)480 transcripts would produce polyS, polyA and polyQ peptides in consecutive tracts of 20 amino acids linked by 1–2 amino acids. It should be noted that RAN translation from pure CAG repeats produces individual polyS, polyA and polyQ peptides. Bearing all this in mind, the conservation of miR-1, miR-7 and miR-10 defects between the fly model and DM1 patients represents important evidence that dysregulation of at least these three miRNAs occurs independently of the CUCGA repeat interruption in the UAS-i(CTG)480 transgene (Fernandez-Costa, 2013).

By studying the expression levels of the predicted target genes of miR-1, miR-7 and miR-10 in skeletal muscles from DM1 patients, a total of 42 targets were found to be dysregulated, 41 of them being up-regulated and only 1 down-regulated. The up-regulation of these targets is consistent with a reduced degradation by their respective miRNA regulators. qRT–PCR analysis confirmed this general trend, and validated at least seven of these alterations in DM1 patients, which had not been previously described to be triggered by miRNA dysregulation. Affected genes do not fall into related functional categories, but instead involve multiple cellular processes. Moreover, miR-1, miR-7 and miR-10 down-regulation could have an even higher impact on gene expression, if it is taken into consideration that these miRNAs might also affect the translation of additional gene targets, without affecting the levels of their messenger transcripts. Therefore, these results highlight the wide number of cellular mechanisms potentially affected by CTG-mediated disruption of miRNA regulation (Fernandez-Costa, 2013).

A number of miRNAs found altered in DM1 to date are encoded in introns, thus suggesting a link between pre-mRNA splicing and miRNA processing. Given that splicing alterations are a hallmark of DM1, both defects could have a common origin. In this study, two Drosophila miRNAs affected by CTG expression, miR-1003 and miR-1006, are miRtrons. The precursor intron of miR-1006 is completely spliced out both in control and in DM1 flies, suggesting that miR-1006-reduced levels in CTG-expressing flies do not originate from defects in the splicing regulation of its host transcript, but would instead occur at a more downstream level. For miR-1003, it was found that its precursor intron is spliced out at higher levels in DM1 flies than in control individuals. However, mature miR-1003 levels are reduced in DM1 flies. Increased levels of spliced-out miR-1003 precursor could arise from a response mechanism triggered by the cells to compensate for the reduced levels of mature miR-1003, whereas the mature miRNA reduction itself would occur at a downstream level. In this study, altered miRNAs that belong to the same cluster (i.e. single-transcription units containing several miRNAs regulated by an upstream promoter) were also found. In Drosophila, the pri-miRNA levels of clusters miR-310–313 and miR-959–964 are reduced in CTG-expressing flies compared with controls. Additionally, the levels of pri-miRNA for miR-7, but not for miR-1 or miR-10, are down-regulated in CTG-expressing flies and in skeletal muscle of DM1 patients. Therefore, these results demonstrate that pri-miRNA transcription/stability is involved in at least part of the miRNA defects described in this work, supporting the idea of different origins for miRNA dysregulation in DM1 (Fernandez-Costa, 2013).

In the DM1 model flies, the CTG-mediated reduction of miR-1 seems to be dependent on Mbl, as over-expression of MblC in CTG-expressing flies rescues miR-1 levels. Moreover, mbl silencing in a wild-type background causes a strong reduction of miR-1. These results are consistent with previous reports that describe a direct implication of MBNL1 in the biogenesis of human miR-1. It has been reported that MBNL1 binds to a UGC motif located within the loop of the pre-miRNA, facilitating the Dicer processing that generates the mature miR-1. According to this model, MBNL1 sequestration by CUG repeats would lead to a reduction of miR-1 levels in DM1, which has been validated in cardiac muscle from DM1 patients (2.1-fold reduction), and is consistent with results in flies and DM1 muscle biopsies from this study. However, other reports have described a different situation for miR-1: miR-1 from biceps muscles of DM1 patients, a 1.9-fold up-regulation of this miRNA, together with an increase in eight of its predicted targets, has been found. This difference may be explained by the different types of muscles analyzed and/or their use of controls with suspected neuromuscular disorders. Intriguingly, another study reported no changes in miR-1 levels in the vastus lateralis muscle of DM1 patients. It is, therefore, possible that miR-1 dysregulation is particularly sensitive to cellular contexts, which could include factors such as the number of CTG repeats or the age of the patients (Fernandez-Costa, 2013).

It was found that mbl silencing also reduces miR-7 levels. However, this reduction is weaker than that observed for miR-1. Moreover, over-expression of MblC does not rescue the effect of CTG expansions on miR-7 levels. In the transdifferentiation cell model, miR-7 levels are reduced both before and after myogenesis, whereas miR-1 and miR-10 are only significantly affected after differentiation. In addition, pri-miRNA down-regulation occurrs for miR-7, but not for miR-1 or miR-10. These observations further suggest that miR-7 alterations in DM1 occur via a different mechanism, although further studies will be required to clarify the specific factors involved in each case (Fernandez-Costa, 2013).

The different behavior of miR-1, miR-7 and miR-10 in the presence of CTG expansions might translate into different consequences to the homeostasis of the cells. The pathological relevance of miRNA dysregulation in DM1 is unclear, as alterations previously described in miRNA levels could correspond either to a response mechanism or to a pathogenic consequence. It was shown that partial restoration of miR-10 levels by over-expression of this miRNA in the Drosophila muscles partially rescues the reduced lifespan phenotype of DM1 flies. This demonstrates that miR-10 down-regulation contributes to CTG-mediated toxicity. On the other hand, not all miRNA alterations triggered by CTG expression seem to have a phenotypic impact, as over-expression of miR-1 or miR-7 does not rescue the CTG-induced phenotype, and even reduce the survival of flies. For miR-7, this effect could originate from additive toxicity, as miR-7 over-expression alone affects the lifespan of flies. However, the case of miR-1 is more intriguing, since this miRNA is not toxic per se. Given that human MBNL1 has been described to bind to miR-1 directly, it would be possible that the CTG-specific detrimental effect observed for miR-1 over-expression results from a sequestration of Drosophila Mbl by excess of miR-1 (Fernandez-Costa, 2013). 

In summary, this study sheds light onto our understanding of the molecular mechanisms behind gene expression dysregulation in DM1 and CTG toxicity, providing a direct link between miRNA dysregulation and RNA toxicity in DM1, identifying a number of mechanisms and predicted target genes that are affected by CTG expansions and supporting the pathogenic potential of at least part of them (Fernandez-Costa, 2013).

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Marrone, A.K., Edeleva, E.V., Kucherenko, M.M., Hsiao, N.H. and Shcherbata, H.R. (2012). Dg-Dys-Syn1 signaling in Drosophila regulates the microRNA profile. BMC Cell Biol 13: 26. PubMed ID: 23107381

Abstract
The Dystrophin Glycoprotein Complex (DGC) is at the center of significant inheritable diseases, such as muscular dystrophies that can be fatal and impair neuronal function in addition to muscle degeneration. Recent evidence has shown that it can control cellular homeostasis and work via Dystrophin signaling to regulate microRNA gene expression which implies that disease phenotypes hide an entourage of regulatory and homeostatic anomalies. Uncovering these hidden processes could shed new light on the importance of proper DGC function for an organism’s overall welfare and bring forth new ideas for treatments. To better understand a role for the DGC in these processes, this study used the genetically advantageous Drosophila muscular dystrophy model to conduct a whole animal microarray screen. Since it has been shown earlier that dystrophic symptoms can be caused by stress even in wild type animals and are enhanced in mutants, this study screened stressed animals for microRNA misregulation as well. Data support the hypothesis that there is a Dystrophin and Dystroglycan dependent circuitry of processes linking stress response, dystrophic conditions and cellular signaling and that microRNAs play an important role in this network. Verification of a subset of the results was conducted via q-PCR and it was found that miR-956, miR-980 and miR-252 are regulated via a Dystroglycan-Dystrophin-Syntrophin dependent pathway. These results support the hypothesis that there is a Dystrophin and Dystroglycan dependent circuitry of processes that includes regulation of microRNAs. Dystrophin signaling has already been found to occur in mammalian musculature; however, data from this study reveals that this regulation is evolutionarily conserved and is also present in at least neuronal tissues. Also, Dystroglycan-Dystrophin-Syntrophin signaling, through control of multiple microRNAs, is involved in highly managed regulation of gene expression required to adapt cellular homeostasis that is compromised under stress and dystrophic conditions (Marrone, 2012).

Highlights

  • Stress and dystrophy alter the miRNA profile in Drosophila.
  • miRNA expression profiles link stress, dystrophy and DGC signaling.
  • Verification of microarray data points to miRNA regulation by stress.
  • Verification of microarray data points to miRNA regulation by the Dg-Dys-Syn1 pathway.
  • Putative mRNA targets of Dg-Dys-Syn1 regulated miRNAs are involved in general cellular processes, the nervous system, and muscle development and maintenance.
  • Dg-Dys-Syn1 regulated miRNAs are expressed in the nervous system and musculature.
  • miR-252 can regulate levels of DGC interacting components and is required for proper muscle development.
  • miR-980 regulates stress response in Drosophila.

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Uchino, R., Nonaka, Y.K., Horigome, T., Sugiyama, S. and Furukawa, K. (2013). Loss of Drosophila A-type lamin C initially causes tendon abnormality including disintegration of cytoskeleton and nuclear lamina in muscular defects. Dev Biol 373: 216-227. PubMed ID: 22982669

Abstract
Lamins are the major components of nuclear envelope architecture, being required for both the structural and informational roles of the nuclei. Mutations of lamins cause a spectrum of diseases in humans, including muscular dystrophy. This study reports that the loss of the A-type lamin gene, lamin C in Drosophila results in pupal metamorphic lethality caused by tendon defects, matching the characteristics of human A-type lamin revealed by Emery–Dreifuss muscular dystrophy (EDMD). In tendon cells lacking lamin C activity, overall cell morphology is affected and organization of the spectraplakin family cytoskeletal protein Shortstop which is prominently expressed in tendon cells gradually disintegrates, notably around the nucleus and in a manner correlating well with the degradation of musculature. Furthermore, lamin C null mutants are efficiently rescued by restoring lamin C expression to shortstop-expressing cells, which include tendon cells but exclude skeletal muscle cells. Thus the critical function of A-type lamin C proteins in Drosophila musculature is to maintain proper function and morphology of tendon cells (Uchino, 2013).

Highlights

  • Lethality of lamin C null alleles is associated with muscle defects during pupal metamorphic stages.
  • The primary defect of loss of lamin C is abnormality of the tendon cells.
  • Mutation of lamin C leads to aberrant tendon cell morphology during larval development.
  • Tendon cells lacking lamin C proteins exhibit changes in the organization of cytoskeleton.
  • The requirement for lamin C is in cells that express shortstop.

Discussion
Drosophila lamin C alleles have been described to show recessive pre-pupal lethality accompanied by aberrant nuclear morphology in imaginal discs and muscle cells. However, the direct cause of the lethality resulting from the loss of lamin C function had not been determined. This study reports the phenotypes of newly isolated and previously reported lamin C alleles. Under modified culture conditions, these alleles were found to develop to pharate adult stages and show head eversion defects. Although head eversion is known to be dependent on peristaltic contraction of the abdominal muscles and the abdominal muscle patterns in lamin C mutants are obviously abnormal, this defect is not rescued by lamin C transgene expression in muscle cells. However it could be rescued by expression in tendon cells, indicating that lamin C is critical in the tendons rather than the muscles (Uchino, 2013).

In contrast to these results, a direct effect of lamin C on muscle cells has been previously speculated, based on the observation that the expression of N-terminal truncated lamin C in muscle cells induces lethality accompanied by altered nuclear structures including lamin C aggregation. Similar head domain-less A-type lamins have been reported to aggregate in nucleoplasm in vertebrates. These aberrant lamin structures have been shown to disrupt the nuclear rim-distribution of endogenous lamins including B-type lamins, by causing them to co-aggregate, and result in the inhibition of replication and transcription. As N-terminal truncated A-type lamins interfere with lamin-nuclear function through an artificial dominant effect, the defects caused by their expression in Drosophila muscle cells could have been caused by their disruption of the normal localization of lamin Dm0 (B-type lamin). As endogenous lamin Dm0 retains localization at the nuclear rim in the lamin C null mutants, the recessive phenotypes caused by lamin C loss described in this study are probably different from, but not in conflict with the dominant negative phenotypes reported to be induced by head domain-less lamin C expression (Uchino, 2013).

The shot gene is essential in tendon cell differentiation. Its expression is detected in the tendon cells but not in the muscle cells from around stages 11 onward of embryonic development. Mutations of shot are reported to affect the EGF-receptor signaling pathway by inducing mislocalization of Vein activator at the MTJ and result in the disruption of somatic muscle pattern at stage 16 of embryonic development. Shot belongs to the spectraplakin family of cytoskeletal linker proteins and has also been shown to contribute to the mechanical strength of tendon cells. Thus, Shot has both structural and informational functions (Uchino, 2013).

It was found that the expression of a lamin C transgene with a shot-promoter driven GAL4 transgene is restricted to tendon cells in musculature, and its expression levels are similar between LamCL58 and wild type animals. Further, shot-GAL4 driver induced lamin C proteins are capable of effectively rescuing lamin C null mutants to adults. Although loss of lamin C induces disintegration of cytoskeletal and nuclear lamina structures in the tendon cells, they are relatively normal at the early larval stages in contrast to the muscular abnormality which occurs in shot alleles at embryonic stages. In lamin C null mutants, disintegration of the cytoskeleton which includes Shot is initiated before nuclear lamina deformation and progresses gradually during larval growth. This alteration eventually causes destruction of tendon cells and muscle detachment from epidermis in a manner similar to shot mutant phenotypes. A-type lamin is known to be major contributor to the mechanical properties of nuclei as well as the cytoskeleton and cytoskeleton-based processes, whereas B-type lamin contributes to nuclear integrity. Thus, these findings with lamin C alleles are coincident with these interpretations and strongly suggest that lamin C null mutation directly induces mechanical instability of the cytoskeleton and nuclear structures, which probably leads to weakening of tendon cell strength and function (Uchino, 2013).

Interactions between the cytoskeleton and the nuclear lamina in which A-type lamins are localized are generally mediated through the double layer lipid nuclear membrane by LINC complexes formed by Klarsicht/ANC-1/Syne homology (KASH) domain proteins directly linked to Sad1/UNC-84 (SUN) domain proteins in the nuclear envelope. In mammals, KASH domain proteins are divided into the three major groups by association with actin filaments, microtubules, and intermediate filaments (IF). In Drosophila, the nesprin-ortholog MSP-300 associated with actin filaments and Klarsicht associated with microtubules are the known KASH domain proteins, and Klaroid and testis specific Spag4 are the known SUN domain proteins. However, KASH domain deleted MSP-300 alleles and klarsicht, spag4 and klaroid null alleles have been reported not to result in lethality. Further, when the distribution of MSP-300 was studied in wild type and LamCLC58 muscles, it was detected in the sarcomere of both as previously described. In tendon cells its distribution could not be evaluated because of its low expression. Klarsicht and Klaroid were detected in the region of the nuclear envelope (NE) in wild type, and their localizations were similar to the distribution of lamin Dm0 at the NE in the tendon cells of LamCLC58 mutant larvae. Thus there is no indication that these known LINC complex proteins play a central role in the lamin C-depleted phenotypes of tendon cells (Uchino, 2013).

In humans, an IF-associating KASH domain protein Nesprin 3α has also been reported in addition to actin and microtubule associating KASH domain proteins, and its IF-binding is mediated by plectin protein containing a plakin (or plectin) repeat domain which can directly bind IF proteins. Although, interaction between Lamin C and Spectraplakin-related Shot which also has a plakin repeat domain has not been proven and an IF-associating type of KASH domain protein has not yet been reported in Drosophila, the mammalian spectraplakin; Bpag1/dystonin isoform has been reported to also interact with Nesprin 3α. Furthermore, for interaction of Shot with the nucleus, the actin–plakin and plakin domains of Shot have been shown to be targeted to the nucleus and to predominantly concentrate at the nuclear periphery in tendon cells. Thus, it is possible that lamin C connects to Shot mediated cytoskeletal complexes through a novel LINC complex protein with characteristics similar to Nesprin 3α and protects tendon cell and nuclei from forces produced by the contractions of the muscles (Uchino, 2013).

In human EDMD, mutations of A-type lamins are characterized by early onset contractures of the Achilles tendons and tendons of the elbows and neck, whereas muscle weakness and wasting shows slow progression compared to other types of muscular dystrophy. In LMNA null model mice, the interdigitation between muscle and tendon is reduced, and the muscle nuclei show abnormal morphology and tend to cluster at the MTJ. Thus tendon defects are strongly implicated in muscular dystrophy resulting from the loss of A-type lamin in mammals, which could be analogous to what this study reports in Drosophila (Uchino, 2013).

In vertebrates, tendons are morphologically diverse tissues and their strength depends mainly on extracellular collagen fibrils. However, well developed actin and myosin bundles are also observed in cytoplasm and around the nucleus in rabbit tendon cells. In Drosophila, the spectraplakin family protein Shot has a major role in formation and maintenance of cytoskeletal organization in tendon cells, but the mammalian spectraplakins, Bpag1/dystonin and MACF1, which are Shot orthologs, have not yet been analyzed in tendon cells. However, Bpag1 isoforms are known to be expressed in musculature, and show nuclear envelope targeting, nesprin 3α interaction, and functions in organizing the actin cytoskeleton around the nucleus in myogenic and other culture cells. As mature mammalian tendon cells are relatively large cells, with lengths up to 300 μm, and their volume is almost entirely taken up by a large nucleus. These cytoskeletal proteins might similarly protect the mammalian tendon cell and nucleus against mechanical stress from muscle contraction. The fact that the nuclear defects observed in LMNA model mice are concentrated at the MTJ structures suggests that the defects in the mouse and fly musculatures by loss of A-type lamins may be initially caused by similar mechanisms at sites of mechanical stress, namely tendon cells (Uchino, 2013).

In conclusion, this study hypothesizes that loss of A-type lamins causes a reduction in spectraplakin mediated cytoskeleton-integrity and cytoskeleton-based processes that perturbs tendon cell morphology and function leading to symptoms seen in human autosomal recessive EDMD. Further studies on the involvement of tendon cell-cytoskeletal structures in the progression of EDMD in both vertebrates and Drosophila are therefore warranted (Uchino, 2013).

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Marrone, A.K., Kucherenko, M.M., Wiek, R., Göpfert, M.C. and Shcherbata, H.R. (2011). Hyperthermic seizures and aberrant cellular homeostasis in Drosophila dystrophic muscles. Sci Rep 1: 47. PubMed ID: 22355566

Abstract
In humans, mutations in the Dystrophin Glycoprotein Complex (DGC) cause muscular dystrophies (MDs) that are associated with muscle loss, seizures and brain abnormalities leading to early death. Using Drosophila as a model to study MD, this study found that loss of Dystrophin (Dys) during development leads to heat-sensitive abnormal muscle contractions that are repressed by mutations in Dys's binding partner, Dystroglycan (Dg). Hyperthermic seizures are independent from dystrophic muscle degeneration and rely on neurotransmission, which suggests involvement of the DGC in muscle-neuron communication. Additionally, reduction of the Ca2+ regulator, Calmodulin or Ca2+ channel blockage rescues the seizing phenotype, pointing to Ca2+ mis-regulation in dystrophic muscles. Also, Dys and Dg mutants have antagonistically abnormal cellular levels of ROS, suggesting that the DGC has a function in regulation of muscle cell homeostasis. These data show that muscles deficient for Dys are predisposed to hypercontraction that may result from abnormal neuromuscular junction signaling (Marrone, 2012).

Highlights

  • Dystrophin mutants have elevated ROS and hyperthermic seizures.
  • Dystrophic seizures are dependent upon neuronal input that require Dys during development.
  • Loss of Dystroglycan, Coracle or Calmoduline represses the seizure phenotype of Dystrophin mutants.
  • Cora mutants have a mild seizure phenotype that does not look like and overrides the dystrophic phenotype.
  • Elevated Ca2+ from the sarcoplasmic reticulum (SR) leads to hyperthermic seizures.

Discussion
Muscle seizures accompany multiple human neurological and muscular disorders. Seizures can occur when a group of neurons becomes hyperexcited and discharge action potentials irregularly without suppression, which causes muscle fibers to contract inappropriately. The uncontrolled action potential is dependent upon K+ and Na+ channel permeability or Ca2+ mis-regulation. Ryanodine receptors that are regulators of fast Ca2+ release from the sarcoplasmic reticulum have been found to play a role in the disease pathology of seizures. Additionally, inositol 1,4,5-triphosphate receptors (IP3Rs) which regulate slow Ca2+ release from the SR are necessary in the central nervous system, causing epilepsy when mutated. Seizures can be alleviated by manipulation of ion channels or by blocking neurotransmission (Marrone, 2012).

It has been shown that Laminin binds directly to voltage gated Ca2+ channels (CaV) at the presynapse in mice, specifically P/Q- and N-type channels, and this binding induces vesicle clustering. Laminin also binds the transmembrane protein Dg providing a direct link between Dys and the presynaptic motoneuron in mammals. Importantly, not only does the motoneuron send signals to the muscle, but also retrograde signaling exists, where a signal travels from the muscle back to the presynaptic neuron. The current pathway by which synaptic retrograde signaling communicates is not known, however Dys has been implicated previously to play a role in the process. If it is assumed that hyperthermic seizures reported in this study are related to the role of Dys in retrograde signaling, then the CaV-Laminin-Dg signaling cascade could explain why the P/Q- and N-type, but not the L-type Ca2+ channel blocker affected Dys seizures. Similarly, the reduction of Dg or Cora reduces the seizure occurrence possibly by preventing propagation of signaling via loss of communication with Laminin. Another pathway that plays a role in retrograde signaling at the NMJ32 is TGF-β and interestingly, mutants of the TGF-β pathway have a hyperthermic seizure phenotype similar to Dys mutants. However, a genetic interaction between the DGC and TGF-β pathway components was not observed suggesting that they might act in parallel (Marrone, 2012).

Dys and Dg have been reported to have opposing functions in control of neurotransmitter release at the NMJ. Dg mutants show a decrease and Dys loss of function and heterozygous mutants an increase in release of neurotransmitter, however both mutants do not show a change in response to altered neurotransmitter levels. These opposing phenotypes are similar to what is reported in this study, where Dys mutants have seizures and Dg mutants do not. Additionally, Dys mutants have an increase in synaptic vesicle docking sites (T-bars) at larval NMJs, which could explain the developmental requirement for Dys. Once the NMJ is established with a normal number of active sites, animals would not be prone to seizures (Marrone, 2012).

The study also shows that Dys and Dg mutants have altered cellular homeostasis. In vertebrates, multiple metabolic disorders have been implicated in seizure activity; for example, mitochondrial encephalopathy, the most common neurometabolic disorder, presents various symptoms including seizures and mice that are partially deficient for mitochondrial superoxide dismutase have an increased incidence of spontaneous seizures. Additionally, mdx mice, a model for MD has sustained oxidative stress in skeletal muscle. In Drosophila, it has been shown that Dg mutant larvae have an altered state of cellular homeostasis and are sensitive to ambient temperature. A constant increase in mitochondrial oxidative metabolism, caused by a Dg hypomorphic mutation, results in a change in thermoregulatory behavior. In addition, it has been reported that suboptimal temperatures and energetic stress accelerate age-dependent muscular dystrophy in both, Dys and Dg mutants. This study shows that Dys and Dg mutants have antagonistically abnormal cellular levels of ROS (Marrone, 2012).

ROS are derived from elemental oxygen (O2), and ROS cascades begin with the superoxide anion radical. Sources for superoxide anion radicals include xanthine oxidase, prostanoid metabolism, catecholamine autooxidation, NAD(P)H oxidase activity and NO synthase. These radicals are generated in normal muscle, and the rate of generation is increased by muscle contraction. In Duchenne MD the absence of dystrophin at the sarcolemma delocalizes and downregulates neuronal nitric oxide synthase (nNOS), which in turn leads to increase in inducible nitric oxide synthase (nNOS) that generates excessive NO free radicals. This mechanism can explain extremely high ROS levels in Dys mutants. This high level of ROS in dystrophic Drosophila can be alleviated by transheterozygous interaction with Dg and Cam, which indicates a genetic interaction between Dys and these two genes in control of cellular homeostasis (Marrone, 2012).

Thus study provides the first in vivo measurements on dystrophic animals showing that they have hyperthermic seizures that are dependent upon neurotransmission. Dystrophin is required during development, since Dys downregulation in adulthood, after muscles and NMJs are already established precludes hyperthermic seizures. Data suggest that the DGC has a role in signaling at the NMJ: reduction of Dg, a protein that binds Dys and regulates localization of the NMJ specific proteins, prevents dystrophic seizure occurrence. Seizures are associated with abnormal Ca2+ release from the SR; introduction of a mutation of Ca2+ mediator Calmodulin and supply of calcium channel blockers reduce seizures. Taken together, these data show that the DGC acts at the muscle side of the NMJ to regulate muscle cell homeostasis in response to neuronal signaling and implies that Dys is involved in muscle-neuron communication (Marrone, 2012).

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Yu, Z., Tengm X. and Bonini, N.M. (2011). Triplet repeat-derived siRNAs enhance RNA-mediated toxicity in a Drosophila model for myotonic dystrophy. PLoS Genet 7: e1001340. PubMed ID: 21437269

Abstract
More than 20 human neurological and neurodegenerative diseases are caused by simple DNA repeat expansions; among these, non-coding CTG repeat expansions are the basis of myotonic dystrophy (DM1). Earlier work, however, has also revealed that many human genes have anti-sense transcripts, raising the possibility that human trinucleotide expansion diseases may be comprised of pathogenic activities due both to a sense expanded-repeat transcript and to an anti-sense expanded-repeat transcript. This study established a Drosophila model for DM1 and tested the role of interactions between expanded CTG transcripts and expanded CAG repeat transcripts. These studies reveal dramatically enhanced toxicity in flies co-expressing CTG with CAG expanded repeats. Expression of the two transcripts leads to novel pathogenesis with the generation of dcr-2 and ago2-dependent 21-nt triplet repeat-derived siRNAs. These small RNAs target the expression of CAG-containing genes, such as Ataxin-2 and TATA binding protein (TBP), which bear long CAG repeats in both fly and humans. These findings indicate that the generation of triplet repeat-derived siRNAs may dramatically enhance toxicity in human repeat expansion diseases in which anti-sense transcription occurs (Yu, 2011).

Highlights

  • Expression of expanded CUG RNA causes repeat-length dependent toxicity.
  • Enhancement of CTG-repeat toxicity by CAG-repeat transcripts.
  • CTG/CAG transcripts are processed into small RNAs.
  • Toxicity of co-expressed CTG/CAG transcripts is dependent on Dcr2 and Ago2.
  • Triplet repeat derived siRNAs compromise the expression of genes containing short CAG stretches.

Discussion
Like many genes within the mammalian genome, the DM1 gene displays bi-directional transcription, generating an anti-sense CAG repeat transcript in addition to the disease-associated CTG transcript. These transcripts have been shown to interact in human cells to generate small RNAs, with one effect being local gene silencing; however additional ways in which this may contribute to pathogenicity in disease is largely unknown. In order to provide new insight into DM1, this study generated a Drosophila model by expressing pure, uninterrupted CTG repeat expansions; fly models for various disorders have revealed critical insight into a number of human disease situations. Interestingly, targeted expression of the long CTG repeats in the fly eye causes a variable toxic effect. This has also been observed in a fly model of SCA8, which carries an uninterrupted CTG repeat expansion. In contrast, fly models generated using interrupted CTG repeats have not been reported to show variable phenotype. It is thus possible that phenotypic variability may be a feature of pure repeat sequences, which is in line with the fact that DM1 is among the most variable human disorders. To define potential effects of bi-directional transcription, the expanded CAG repeat transcripts were co-expressed with the DM1 CTG repeats. This resulted in dramatically enhanced toxicity concomitant with the generation of triplet repeat-derived siRNAs. These results are in striking contrast with previous findings that co-expression of CGG and CCG expansions in flies leads to mitigated toxicity in a ago2-dependent manner, suggesting that toxicity derived from interactions between sense and anti-sense repeat transcripts may be specific to CTG/CAG situations. Both CAG and CUG strands can be processed into ~21 nt small RNAs when coexpressed and small RNAs derived from both strands are methylated in a Hen1-dependent manner. These results suggest that both CAG and CUG small RNAs can be loaded into mature, holo-RISCs presumably due to the symmetrical thermodynamic properties of the repeat small RNA duplex. Although a direct cleavage of CAG containing transcripts was detected, however cleavage of CUG containing transcripts mediated by CAG small RNAs was not detected. Although underlying reasons for this differential effect remain unclear, CUG and CAG transcripts may have differential expression levels or translation efficiencies, and/or CUG-containing and CAG-containing transcripts may be associated with different RNA binding proteins of various affinities, making CUG-transcripts less accessible to the RISC complex than CAG-containing transcripts. A number of CUG-binding proteins have been defined, such as MBNL1, CUGBP1 and PKR. Interestingly, in-vitro gel retardation analysis indicated that MBNL1 has a much lower affinity for CAG repeat RNA than CUG repeat RNA. Moreover, expanded CAG transcripts, although co-localizing with MBNL1 in ribonuclear foci similarly to expanded CUG transcripts, do not appear to cause mis-regulation of alternative splicing in cells, further highlighting differential properties of these repeats in interacting with RNA binding proteins (Yu, 2011).

The toxicity caused by co-expression of expanded CAG and CTG was associated with deleterious effects on transcripts of other CAG containing genes within the genome; additional mechanisms that contribute to toxicity may also exist. A large number of genes contain CAG stretches in fly and human genomes. The enhanced toxicity observed in flies expressing expanded CAG and CTG may therefore be reflecting an additive effect of knockdown of multiple CAG-containing genes, with each individual gene contributing only partially to the overall outcome. Although further reducing atx2 dosage did not enhance toxicity of co-expressed CTG/CAG expansions, the compromised activities of many target genes may be involved and further compromising any single one has minimal impact. The toxic effects seen of the CAG/CTG situation may also be complicated by the later-onset and progressive nature of the toxicity. Further study will clarify the contribution of this mechanism, and key targets among all possible transcripts, to the overall phenotype of the disease. Moreover, the deleterious effects caused by triplet repeat derived small RNAs may be further exacerbated by the wide prevalence of CAG stretches in the human transcriptome and the relative low specificity of RNA interference when siRNAs and/or RNA targets contain simple repeats like CAG. Such interactions may represent a novel activity of endo-siRNAs that characterize disease situations where bi-directional transcription spanning the repeat region occurs (Yu, 2011).

Two CAG containing genes, atx2 and tbp, were conformed to be targets of the triplet repeat-derived siRNAs. Interestingly, CAG repeat expansions in ATXN2 (the human Ataxin-2 gene) and TBP define two of the CAG-repeat expansion diseases (SCA2 and SCA17, respectively). In such diseases, the expanded polyglutamine domain is thought to confer toxicity; however, increasing evidence suggests that the loss-of-function of gene activity, and not just dominant activities of the protein with an expanded polyglutamine region, occur in disease. These findings raise the possibility that bi-directional transcription of the repeat region in diseases like DM1 may confer additional components of pathogenicity due to deleterious interactions between the two overlapping repeat-containing transcripts through the generation and activity of triplet repeat-derived siRNAs (Yu, 2011).

Earlier studies indicate that bi-directionally transcribed RNAs, and presumably resultant endogenous double-stranded RNAs, are processed into ~21–23 nt small RNAs in human cells. This is despite the fact that in most mammalian cells, long exogenous double-stranded RNAs can elicit the interferon response. That response presumably occurs in a threshold-dependent manner; cells may also respond differentially to long exogenous double-stranded RNAs versus endogenous double-stranded RNAs. Thus, these findings suggest that the biogenesis pathway of small RNAs from endogenous double-stranded RNAs is conserved in mammalian cells. Many loci are bi-directionally transcribed throughout the mammalian genome, and among these are a number of human trinucleotide disease genes, including SCA8 and DM1. In SCA8, an anti-sense transcript is proposed to encode a polyglutamine protein, which itself may have deleterious actions. In DM1, the two transcripts interact to produce small RNAs that can have local effects on gene silencing. Data from this study raise another possibility that processing of co-expressed transcripts containing CUG/CAG expansions into triplet repeat-derived siRNAs in vivo, may contribute to toxicity with widespread deleterious effects. These effects may include downregulating the expression of other genes containing CAG repeats. Among the genes that could be targets are the polyglutamine disease genes themselves, one of which is TBP. Expansion of the TBP polyglutamine repeat underlies SCA17; intriguingly, general transcriptional compromise has been shown to be a component of repeat expansion diseases. Another reason why these diseases share transcriptional compromise may be that they share bi-directional transcript interactions that compromise common elements like TBP. This possibility underscores the idea of shared therapeutic targets and mechanisms in repeat expansion diseases (Yu, 2011).

It has been proposed that siCAG and siCUG may be used for therapy of triplet repeat expansion diseases based on findings in cell culture that these siRNAs seem to specifically target mutant transcripts with expanded repeats. This study's data suggest caution in designing such siRNA-based therapy, as in the intact organismal situation, pathogenic activities may be noted. Although previous findings suggest that expanded CUG alone can be processed into small RNAs, this study's data suggest that both expanded CAG and CTG are required for triplet repeat-derived siRNA generation and toxicity in vivo. Thus, co-expressed CAG and CTG expansions may contribute to DM1 pathogenesis through a fundamentally different mechanism from that of CTG expansions alone. Targeting human trinucleotide expansion diseases at the transcriptional level may therefore be a promising therapeutic approach that would minimize not only the effects of single expanded repeat transcripts, but deleterious interactions between sense and anti-sense repeat transcript domains (Yu, 2011).

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Goldstein, J.A., Kelly, S.M., LoPresti, P.P., Heydemann, A., Earley, J.U., Ferguson, E.L., Wolf, M.J. and McNally, E.M. (2011). SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy. Hum Mol Genet 20: 894-904. PubMed ID: 21138941

Abstract
Loss-of-function mutations in the genes encoding dystrophin and the associated membrane proteins, the sarcoglycans, produce muscular dystrophy and cardiomyopathy. The dystrophin complex provides stability to the plasma membrane of striated muscle during muscle contraction. Increased SMAD signaling due to activation of the transforming growth factor-β (TGFβ) pathway has been described in muscular dystrophy; however, it is not known whether this canonical TGFβ signaling is pathogenic in the muscle itself. Drosophila deleted for the γ/δ-sarcoglycan gene (Sgcd) develop progressive muscle and heart dysfunction and serve as a model for the human disorder. This study used dad-lacZ flies to demonstrate the signature of TGFβ activation in response to exercise-induced injury in Sgcd null flies, finding that those muscle nuclei immediately adjacent to muscle injury demonstrate high-level TGFβ signaling. To determine the pathogenic nature of this signaling, it was found that partial reduction of the co-SMAD Medea, homologous to SMAD4, or the r-SMAD, Smox, corrects both heart and muscle dysfunction in Sgcd mutants. Reduction in the r-SMAD, MAD, restores muscle function but interestingly not heart function in Sgcd mutants, consistent with a role for activin but not bone morphogenic protein signaling in cardiac dysfunction. Mammalian sarcoglycan null muscle was also found to exhibit exercise-induced SMAD signaling. These data demonstrate that hyperactivation of SMAD signaling occurs in response to repetitive injury in muscle and heart. Reduction of this pathway is sufficient to restore cardiac and muscle function and is therefore a target for therapeutic reduction (Goldstein, 2011).

Highlights

  • Exercise induces muscle disruption in Sgcd[840] mutants.
  • Exercise-induced muscle tears in the Sgcd[840] mutant lead to SMAD signaling.
  • TGFβ signaling is increased in Sgcd[840] mutant muscle.
  • Reducing TGFβ signaling rescues mobility in sarcoglycan mutant flies.
  • r-SMAD mutants rescue negative geotaxis in Sgcd[840] mutants.
  • Reducing TGFβ signaling rescues heart function in sarcoglycan mutant flies.
  • Exercise induces TGFβ signaling in Sgcg null mice.

Discussion
TGFβ signaling is mediated through canonical and non-canonical pathways. In mammalian muscle, pSMAD is detected in nuclei, and reduction of nuclear pSMAD has been reported in mdx mice treated with TGFβ-neutralizing antibodies. In dystrophic muscle, both myonuclei and those nuclei in non-muscle cells display enhanced nuclear pSMAD. This study shows that the orthologous SMAD-type signaling is increased in a Drosophila model of muscular dystrophy. The fly model was utilized for these studies because dystrophic fly muscle is characterized by a degenerative process. This is in contrast to dystrophic mammalian muscle where there is degeneration alongside regeneration and infiltration by non-muscle elements. Because in mammalian muscle these non-muscle elements, particularly the fibroblasts, have much greater pSMAD signaling, the simpler fly muscle allows examination of the myogenic component of injury response. With this approach, it was found that exercise enhances muscle disruption and the degree of β-gal activity from the dad-lacZ indicator allele in Sgcd[840] muscle. The myonuclei most proximal to the region of disruption display the greatest amount of β-gal activity, and this is consistent with a model of local TGFβ activity inducing response nearest to the area of injury. Injury alone, in the absence of disease, also elicits this signaling response, if the injury was severe. Another study profiled gene expression of mechanically injured Drosophila thorax and found elevated transcript levels for the BMP-like ligand gbb and the activin ligand daw at 1 and 6h after injury, suggesting that these ligands may be the TGFβ orthologs (Goldstein, 2011).

In addition to fibroblast activation and proliferation, mammalian muscular dystrophy is accompanied by an inflammatory response in muscle, in part mediated by mononuclear cells derived from the bone marrow. The equivalent in the fly is a hemocyte-like cell, which was very occasionally observed in sarcoglycan mutant flies. Hemocyte-derived cell lines are known to secrete TGFβ family ligands. However, this behavior has not been investigated in Drosophila adult muscle. In mammalian models of muscular dystrophy, immune cells are prominent and a plausible source of TGFβ and other cytokines. The use of the dad-lacZ indicator demonstrates that muscle cells clearly possess the capacity to respond to downstream signaling. In mammalian muscle, the finding of myofiber nuclei positive for pSMAD2/3 supports a parallel pathogenesis in mammalian muscle (Goldstein, 2011).

In mammalian muscle affected by muscular dystrophy, degeneration occurs concomitantly with regeneration. In this model reducing TGFβ signaling has been thought to primarily mediate its effect by improving muscle regeneration, suggesting that TGFβ signaling has a negative effect on satellite cell function. Although the Sgcd[840] model exhibits many features of mammalian muscular dystrophy, previous studies did not find evidence of muscle regeneration in this model. The absence of obvious regeneration in the fly model suggests that enhanced TGFβ signaling is exerting its effect directly by hastening muscle degeneration either by inhibiting membrane-mediated repair mechanisms or other cellular adaptations. Data suggest that the effects of inhibiting TGFβ signaling likely extend beyond the regenerative response, mitigating degeneration and enhancing repair. An additional possibility, raised by the improved climbing performance of normal flies with heterozygous SMAD alleles, is that reducing SMAD signaling developmentally increases muscle function, perhaps by antagonizing the myostatin homolog, myoglianin. Further understanding the genes regulated by nuclear pSMADs in injured muscle may help to define the precise pathways that are most critical for eliciting muscle dysfunction. Thus far, results point to the r-SMADs and co-SMADs as targets for therapeutic intervention (Goldstein, 2011).

It was found that exercising a mouse model of muscular dystrophy increases the pSMAD signaling in those nuclei positioned centrally within myofibers. Centrally positioned nuclei, as opposed to those in the normal peripheral position, are indicative of recent regeneration. However, increased pSMAD in central nuclei was detected immediately after exercise without sufficient time for myoblast fusion and regeneration. These findings indicate that all myonuclei, including those in the central position, are affected by pathogenic TGFβ signaling. Newly regenerated fibers are particularly important to protect and so targeting this pathway for therapy may offer additional protection (Goldstein, 2011).

It was found that heterozygous mutations of Medea, Smox or MAD are dominant suppressors of the loss of walking function in Sgcd[840] mutants. This finding implicates broad downstream signaling, including both the activin and BMP pathways. Curiously, the MAD[12] allele did not improve heart tube function while the Medea[13] and Smox[G0348] alleles did. MAD[12] is the r-SMAD for BMP, and therefore the differential response of the heart tube versus skeletal muscle implies that BMP is more important for muscle versus heart tube function. The Drosophila heart tube is a linear structure. During larval stages, the posterior region is lined with cells expressing cardiac-specific markers. Expression of the Hox gene AbdA is sufficient to determine the cardiac fate while the anterior portion is the aorta. In the adult, the more anterior segments, A1 through A4, assume cardiac function and are lined with cardiomyocytes derived from the larval cells. This developmental paradigm suggests a more intimate relationship between cardiac and vascular structures where activin but not BMP signaling may be essential. Although the SMAD family is more complex in mammals, there still may a differential response to SMAD reduction between heart and skeletal muscles, as inhibition of SMAD signaling may differ between vascular cells and skeletal muscle in mammals (Goldstein, 2011).

This study focused on canonical TGFβ signaling in the Sgcd[840] model. Non-canonical TGFβ signaling includes JNK, p38, ERK and Akt signaling. Notably, each of these pathways has been shown to be important for muscle regeneration and function. P38 MAP kinase and JNK2 signaling are induced by an exercise regimen in the mdx mouse. Akt mediates the muscle proliferative and differentiation functions of insulin-like growth factor-1. In addition, SMADs are not exclusively activated by TGFβ receptors, and the non-transcription factor roles of SMADs should be considered when targeting these pathways for therapeutic intervention (Goldstein, 2011).

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Reviews

Pantoja, M. and Ruohola-Baker, H. (2013). Drosophila as a starting point for developing therapeutics for the rare disease Duchenne Muscular Dystrophy. Rare Dis 1: e24995. PubMed ID: 25002997

Plantié, E., Migocka-Patrzałek, M., Daczewska, M. and Jagla, K. (2015). Model organisms in the fight against muscular dystrophy: lessons from Drosophila and Zebrafish. Molecules 20: 6237-6253. PubMed ID: 25859781

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Date revised: 20 June 2015

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