Publications by authors named "Rubén Artero"

56 Publications

Preclinical characterization of antagomiR-218 as a potential treatment for myotonic dystrophy.

Mol Ther Nucleic Acids 2021 Dec 29;26:174-191. Epub 2021 Jul 29.

University Research Institute for Biotechnology and Biomedicine (BIOTECMED), Universidad de Valencia, Dr. Moliner, 50, 46100 Burjasot, Valencia, Spain.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the gene. CUG expansions in mutant transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSA mouse model and patient-derived myotubes. In HSA, antagomiR-218 reached 40-60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target.
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http://dx.doi.org/10.1016/j.omtn.2021.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8413838PMC
December 2021

Neuroprotective properties of queen bee acid by autophagy induction.

Cell Biol Toxicol 2021 Aug 27. Epub 2021 Aug 27.

Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Avda de la Universidad s/n, 10003, Cáceres, Spain.

Autophagy is a conserved intracellular catabolic pathway that removes cytoplasmic components to contribute to neuronal homeostasis. Accumulating evidence has increasingly shown that the induction of autophagy improves neuronal health and extends longevity in several animal models. Therefore, there is a great interest in the identification of effective autophagy enhancers with potential nutraceutical or pharmaceutical properties to ameliorate age-related diseases, such as neurodegenerative disorders, and/or promote longevity. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and is found exclusively in, royal jelly, which has beneficial properties for human health. It is reported that QBA has antitumor, anti-inflammatory, and antibacterial activities and promotes neurogenesis and neuronal health; however, the mechanism by which QBA exerts these effects has not been fully elucidated. The present study investigated the role of the autophagic process in the protective effect of QBA. We found that QBA is a novel autophagy inducer that triggers autophagy in various neuronal cell lines and mouse and fly models. The beclin-1 (BECN1) and mTOR pathways participate in the regulation of QBA-induced autophagy. Moreover, our results showed that QBA stimulates sirtuin 1 (SIRT1), which promotes autophagy by the deacetylation of critical ATG proteins. Finally, QBA-mediated autophagy promotes neuroprotection in Parkinson's disease in vitro and in a mouse model and extends the lifespan of Drosophila melanogaster. This study provides detailed evidences showing that autophagy induction plays a critical role in the beneficial health effects of QBA.
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http://dx.doi.org/10.1007/s10565-021-09625-wDOI Listing
August 2021

Rabphilin silencing causes dilated cardiomyopathy in a Drosophila model of nephrocyte damage.

Sci Rep 2021 Jul 27;11(1):15287. Epub 2021 Jul 27.

INCLIVA Biomedical Research Institute, 46010, Valencia, Spain.

Heart failure (HF) and the development of chronic kidney disease (CKD) have a direct association. Both can be cause and consequence of the other. Many factors are known, such as diabetes or hypertension, which can lead to the appearance and/or development of these two conditions. However, it is suspected that other factors, namely genetic ones, may explain the differences in the manifestation and progression of HF and CKD among patients. One candidate factor is Rph, a gene expressed in the nervous and excretory system in mammals and Drosophila, encoding a Rab small GTPase family effector protein implicated in vesicular trafficking. We found that Rph is expressed in the Drosophila heart, and the silencing of Rph gene expression in this organ had a strong impact in the organization of fibers and functional cardiac parameters. Specifically, we observed a significant increase in diastolic and systolic diameters of the heart tube, which is a phenotype that resembles dilated cardiomyopathy in humans. Importantly, we also show that silencing of Rabphilin (Rph) expression exclusively in the pericardial nephrocytes, which are part of the flies' excretory system, brings about a non-cell-autonomous effect on the Drosophila cardiac system. In summary, in this work, we demonstrate the importance of Rph in the fly cardiac system and how silencing Rph expression in nephrocytes affects the Drosophila cardiac system.
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http://dx.doi.org/10.1038/s41598-021-94710-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316431PMC
July 2021

Practicing logical reasoning through Drosophila segmentation gene mutants.

Biochem Mol Biol Educ 2021 Sep 23;49(5):729-736. Epub 2021 Jun 23.

Department of Genetics, Faculty of Biology, University of Valencia, Valencia, Spain.

Laboratory practical sessions are critical to scientific training in biology but usually fail to promote logical and hypothesis-driven reasoning and rely heavily on the teacher's instructions. This paper describes a 2-day laboratory practicum in which students prepare and analyze larval cuticle preparations of Drosophila segmentation gene mutant strains. Embryonic segmentation involves three major classes of genes according to their loss-of-function phenotypes: the establishment of broad regions by gap genes, the specification of the segments by the pair-rule genes, and the compartments within segments by the segment polarity genes. Students are asked to sort undefined segmentation mutants into gap, pair-rule, or segment polarity categories based on their knowledge of the Drosophila segmentation process and the microscopic anatomical traits they are capable of finding in the sample preparations. This technically simple practicum prompts students to pay attention to detailed observation to detect anatomic markers of intrasegmental compartments and thorax versus abdomen cuticle, and promote their logical reasoning in hypothesizing to which segmentation type a given mutant fits best.
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http://dx.doi.org/10.1002/bmb.21554DOI Listing
September 2021

Bioengineered3D model of myotonic dystrophy type 1 human skeletal muscle.

Biofabrication 2021 04 26;13(3). Epub 2021 Apr 26.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), c/Baldiri Reixac 10-12, E08028 Barcelona, Spain.

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models andtwo-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.
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http://dx.doi.org/10.1088/1758-5090/abf6aeDOI Listing
April 2021

Myotonic dystrophy type 1 drug development: A pipeline toward the market.

Drug Discov Today 2021 Jul 31;26(7):1765-1772. Epub 2021 Mar 31.

University Institute for Biotechnology and Biomedicine (BIOTECMED), University of Valencia, Valencia, Spain; Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain; Joint Unit Incliva-CIPF, Valencia, Spain.

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular genetic disease with an estimated prevalence of approximately at least half a million individuals based on its vast ethnic variation. Building upon a well-known physiopathology and several proof-of-concept therapeutic approaches, herein we compile a comprehensive overview of the most recent drug development programs under preclinical and clinical evaluation. Specifically, close to two dozen drug developments, eight of which are already in clinical trials, explore a diversity of new chemical entities, drug repurposing, oligonucleotide, and gene therapy-based approaches. Of these, repurposing of tideglusib, mexiletine, or metformin appear to be therapies with the most potential to receive marketing authorization for DM1.
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http://dx.doi.org/10.1016/j.drudis.2021.03.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8372527PMC
July 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

The hallmarks of myotonic dystrophy type 1 muscle dysfunction.

Biol Rev Camb Philos Soc 2021 04 2;96(2):716-730. Epub 2020 Dec 2.

Translational Genomics Group, Incliva Health Research Institute, Avda. Menéndez Pelayo 4 acc., Valencia, 46010, Spain.

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3' untranslated region (3'UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin-proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.
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http://dx.doi.org/10.1111/brv.12674DOI Listing
April 2021

Therapeutic Potential of AntagomiR-23b for Treating Myotonic Dystrophy.

Mol Ther Nucleic Acids 2020 Sep 21;21:837-849. Epub 2020 Jul 21.

Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), Universidad de Valencia, 46100 Valencia, Spain; Translational Genomics Group, Incliva Health Research Institute, 46010 Valencia, Spain; Joint Unit Incliva-CIPF, Valencia, Spain. Electronic address:

Myotonic dystrophy type 1 (DM1) is a chronically debilitating, rare genetic disease that originates from an expansion of a noncoding CTG repeat in the dystrophia myotonica protein kinase (DMPK) gene. The expansion becomes pathogenic when DMPK transcripts contain 50 or more repetitions due to the sequestration of the muscleblind-like (MBNL) family of proteins. Depletion of MBNLs causes alterations in splicing patterns in transcripts that contribute to clinical symptoms such as myotonia and muscle weakness and wasting. We previously found that microRNA (miR)-23b directly regulates MBNL1 in DM1 myoblasts and mice and that antisense technology ("antagomiRs") blocking this microRNA (miRNA) boosts MBNL1 protein levels. Here, we show the therapeutic effect over time in response to administration of antagomiR-23b as a treatment in human skeletal actin long repeat (HSA) mice. Subcutaneous administration of antagomiR-23b upregulated the expression of MBNL1 protein and rescued splicing alterations, grip strength, and myotonia in a dose-dependent manner with long-lasting effects. Additionally, the effects of the treatment on grip strength and myotonia were still slightly notable after 45 days. The pharmacokinetic data obtained provide further evidence that miR-23b could be a valid therapeutic target for DM1.
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http://dx.doi.org/10.1016/j.omtn.2020.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452101PMC
September 2020

Rabphilin involvement in filtration and molecular uptake in nephrocytes suggests a similar role in human podocytes.

Dis Model Mech 2020 09 21;13(9). Epub 2020 Sep 21.

Hypertension Unit, Hospital Clínico Universitario, 46010 Valencia, Spain.

nephrocytes share functional, structural and molecular similarities with human podocytes. It is known that podocytes express the rabphilin 3A () complex, and its expression is altered in mouse and human proteinuric disease. Furthermore, we previously identified a polymorphism that suggested a role for RPH3A protein in the development of urinary albumin excretion. As endocytosis and vesicle trafficking are fundamental pathways for nephrocytes, the objective of this study was to assess the role of the orthologue in , (), in the structure and function of nephrocytes. We confirmed that is required for the correct function of the endocytic pathway in pericardial nephrocytes. Knockdown of reduced the expression of the and genes, which encode proteins that are involved in protein uptake and filtration. We also found that reduced expression resulted in a disappearance of the labyrinthine channel structure and a reduction in the number of endosomes, which ultimately leads to changes in the number and volume of nephrocytes. Finally, we demonstrated that the administration of retinoic acid to IR-Rph nephrocytes rescued some altered aspects, such as filtration and molecular uptake, as well as the maintenance of cell fate. According to our data, Rph is crucial for nephrocyte filtration and reabsorption, and it is required for the maintenance of the ultrastructure, integrity and differentiation of the nephrocyte.
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http://dx.doi.org/10.1242/dmm.041509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522021PMC
September 2020

Protective effects of mirtazapine in mice lacking the Mbnl2 gene in forebrain glutamatergic neurons: Relevance for myotonic dystrophy 1.

Neuropharmacology 2020 06 6;170:108030. Epub 2020 Apr 6.

Integrative Pharmacology and Systems Neuroscience, IMIM-Hospital del Mar Medical Research Institute, Barcelona, Spain; Department of Experimental and Health Sciences, Pompeu Fabra University (CEXS-UPF), Barcelona, Spain. Electronic address:

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder characterized by muscle weakness and wasting and by important central nervous system-related symptoms including impairments in executive functions, spatial abilities and increased anxiety and depression. The Mbnl2 gene has been implicated in several phenotypes consistent with DM1 neuropathology. In this study, we developed a tissue-specific knockout mouse model lacking the Mbnl2 gene in forebrain glutamatergic neurons to examine its specific contribution to the neurobiological perturbations related to DM1. We found that these mice exhibit long-term cognitive deficits and a depressive-like state associated with neuronal loss, increased microglia and decreased neurogenesis, specifically in the dentate gyrus (DG). Chronic treatment with the atypical antidepressant mirtazapine (3 and 10 mg/kg) for 21 days rescued these behavioral alterations, reduced inflammatory microglial overexpression, and reversed neuronal loss in the DG. We also show that mirtazapine re-established 5-HT and histaminergic H receptor gene expression in the hippocampus. Finally, metabolomics studies indicated that mirtazapine increased serotonin, noradrenaline, gamma-aminobutyric acid and adenosine production. These data suggest that loss of Mbnl2 gene in the glutamatergic neurons of hippocampus and cortex may underlie the most relevant DM1 neurobiological and behavioral features, and provide evidence that mirtazapine could be a novel potential candidate to alleviate these debilitating symptoms in DM1 patients.
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http://dx.doi.org/10.1016/j.neuropharm.2020.108030DOI Listing
June 2020

Drosophila SMN2 minigene reporter model identifies moxifloxacin as a candidate therapy for SMA.

FASEB J 2020 02 31;34(2):3021-3036. Epub 2019 Dec 31.

Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), University of Valencia, Valencia, Spain.

Spinal muscular atrophy is a rare and fatal neuromuscular disorder caused by the loss of alpha motor neurons. The affected individuals have mutated the ubiquitously expressed SMN1 gene resulting in the loss or reduction in the survival motor neuron (SMN) protein levels. However, an almost identical paralog exists in humans: SMN2. Pharmacological activation of SMN2 exon 7 inclusion by small molecules or modified antisense oligonucleotides is a valid approach to treat SMA. Here we describe an in vivo SMN2 minigene reporter system in Drosophila motor neurons that serves as a cost-effective, feasible, and stringent primary screening model for identifying chemicals capable of crossing the conserved Drosophila blood-brain barrier and modulating exon 7 inclusion. The model was used for the screening of 1100 drugs from the Prestwick Chemical Library, resulting in 2.45% hit rate. The most promising candidate drugs were validated in patient-derived fibroblasts where they proved to increase SMN protein levels. Among them, moxifloxacin modulated SMN2 splicing by promoting exon 7 inclusion. The recovery of SMN protein levels was confirmed by increased colocalization of nuclear gems with Cajal Bodies. Thus, a Drosophila-based drug screen allowed the discovery of an FDA-approved small molecule with the potential to become a novel therapy for SMA.
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http://dx.doi.org/10.1096/fj.201802554RRRDOI Listing
February 2020

miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy.

Mol Ther Nucleic Acids 2020 Mar 20;19:278-292. Epub 2019 Nov 20.

Translational Genomics Group, Incliva Health Research Institute, Valencia 46100, Spain; Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), University of Valencia, Valencia 46100, Spain; CIPF-INCLIVA Joint Unit, Valencia 46012, Spain.

Unstable CTG expansions in the 3' UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy- and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1.
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http://dx.doi.org/10.1016/j.omtn.2019.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926285PMC
March 2020

Increased Muscleblind levels by chloroquine treatment improve myotonic dystrophy type 1 phenotypes in in vitro and in vivo models.

Proc Natl Acad Sci U S A 2019 12 21;116(50):25203-25213. Epub 2019 Nov 21.

Translational Genomics Group, Incliva Health Research Institute, 46010 Valencia, Spain;

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3' UTR of the gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in and mouse (HSA) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSA mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.
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http://dx.doi.org/10.1073/pnas.1820297116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911202PMC
December 2019

MicroRNA-Based Therapeutic Perspectives in Myotonic Dystrophy.

Int J Mol Sci 2019 Nov 9;20(22). Epub 2019 Nov 9.

Translational Genomics Group, Incliva Health Research Institute, Burjassot, 46100 Valencia, Spain.

Myotonic dystrophy involves two types of chronically debilitating rare neuromuscular diseases: type 1 (DM1) and type 2 (DM2). Both share similarities in molecular cause, clinical signs, and symptoms with DM2 patients usually displaying milder phenotypes. It is well documented that key clinical symptoms in DM are associated with a strong mis-regulation of RNA metabolism observed in patient's cells. This mis-regulation is triggered by two leading DM-linked events: the sequestration of Muscleblind-like proteins (MBNL) and the mis-regulation of the CUGBP RNA-Binding Protein Elav-Like Family Member 1 (CELF1) that cause significant alterations to their important functions in RNA processing. It has been suggested that DM1 may be treatable through endogenous modulation of the expression of MBNL and CELF1 proteins. In this study, we analyzed the recent identification of the involvement of microRNA (miRNA) molecules in DM and focus on the modulation of these miRNAs to therapeutically restore normal MBNL or CELF1 function. We also discuss additional prospective miRNA targets, the use of miRNAs as disease biomarkers, and additional promising miRNA-based and miRNA-targeting drug development strategies. This review provides a unifying overview of the dispersed data on miRNA available in the context of DM.
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http://dx.doi.org/10.3390/ijms20225600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888406PMC
November 2019

RNA-mediated therapies in myotonic dystrophy.

Drug Discov Today 2018 12 4;23(12):2013-2022. Epub 2018 Aug 4.

University of Valencia, Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), Valencia, Spain; Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain; Joint Unit Incliva-CIPF, Valencia, Spain. Electronic address:

Myotonic dystrophy 1 (DM1) is a multisystemic neuromuscular disease caused by a dominantly inherited 'CTG' repeat expansion in the gene encoding DM Protein Kinase (DMPK). The repeats are transcribed into mRNA, which forms hairpins and binds with high affinity to the Muscleblind-like (MBNL) family of proteins, sequestering them from their normal function. The loss of function of MBNL proteins causes numerous downstream effects, primarily the appearance of nuclear foci, mis-splicing, and ultimately myotonia and other clinical symptoms. Antisense and other RNA-mediated technologies have been applied to target toxic-repeat mRNA transcripts to restore MBNL protein function in DM1 models, such as cells and mice, and in humans. This technique has had promising results in DM1 therapeutics by alleviating pathogenic phenotypes.
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http://dx.doi.org/10.1016/j.drudis.2018.08.004DOI Listing
December 2018

Modeling of Myotonic Dystrophy Cardiac Phenotypes in .

Front Neurol 2018 16;9:473. Epub 2018 Jul 16.

Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain.

After respiratory distress, cardiac dysfunction is the second most common cause of fatality associated with the myotonic dystrophy (DM) disease. Despite the prevalance of heart failure in DM, physiopathological studies on heart symptoms have been relatively scarce because few murine models faithfully reproduce the cardiac disease. Consequently, only a small number of candidate compounds have been evaluated in this specific phenotype. To help cover this gap combines the amenability of its invertebrate genetics with the possibility of quickly acquiring physiological parameters suitable for meaningful comparisons with vertebrate animal models and humans. Here we review available descriptions of cardiac disease in DM type 1 and type 2, and three recent papers reporting the cardiac toxicity of non-coding CUG (DM1) and CCUG (DM2) repeat RNA in flies. Notably, flies expressing CUG or CCUG RNA in their hearts developed strong arrhythmias and had reduced fractional shortening, which correlates with similar phenotypes in DM patients. Overexpression of Muscleblind, which is abnormally sequestered by CUG and CCUG repeat RNA, managed to strongly suppress arrhythmias and fractional shortening, thus demonstrating that Muscleblind depletion causes cardiac phenotypes in flies. Importantly, small molecules pentamidine and daunorubicin were able to rescue cardiac phenotypes by releasing Muscleblind from sequestration. Taken together, fly heart models have the potential to make important contributions to the understanding of the molecular causes of cardiac dysfunction in DM and in the quick assessment of candidate therapeutics.
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http://dx.doi.org/10.3389/fneur.2018.00473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054993PMC
July 2018

miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.

Nat Commun 2018 06 26;9(1):2482. Epub 2018 Jun 26.

Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), University of Valencia, Dr. Moliner 50, E46100, Burjassot, Valencia, Spain.

Functional depletion of the alternative splicing factors Muscleblind-like (MBNL 1 and 2) is at the basis of the neuromuscular disease myotonic dystrophy type 1 (DM1). We previously showed the efficacy of miRNA downregulation in Drosophila DM1 model. Here, we screen for miRNAs that regulate MBNL1 and MBNL2 in HeLa cells. We thus identify miR-23b and miR-218, and confirm that they downregulate MBNL proteins in this cell line. Antagonists of miR-23b and miR-218 miRNAs enhance MBNL protein levels and rescue pathogenic missplicing events in DM1 myoblasts. Systemic delivery of these "antagomiRs" similarly boost MBNL expression and improve DM1-like phenotypes, including splicing alterations, histopathology, and myotonia in the HSA DM1 model mice. These mammalian data provide evidence for therapeutic blocking of the miRNAs that control Muscleblind-like protein expression in myotonic dystrophy.
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http://dx.doi.org/10.1038/s41467-018-04892-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018771PMC
June 2018

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes.

Nat Commun 2018 05 23;9(1):2032. Epub 2018 May 23.

Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Lausanne and University of Geneva, Rue Michel-Servet 1, 1211, Geneva, Switzerland.

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.
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http://dx.doi.org/10.1038/s41467-018-04110-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966403PMC
May 2018

rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences.

Nat Commun 2018 05 22;9(1):2009. Epub 2018 May 22.

IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, 67404, Illkirch, France.

Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.
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http://dx.doi.org/10.1038/s41467-018-04370-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964235PMC
May 2018

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles.

J Vis Exp 2018 03 31(133). Epub 2018 Mar 31.

Department of Genetics and Interdisciplinary Research Structure for Biotechnology and Biomedicine (BIOTECMED), University of Valencia; Incliva Health Research Institute; Joint unit CIPF-Incliva.

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.
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http://dx.doi.org/10.3791/56179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933293PMC
March 2018

Daunorubicin reduces MBNL1 sequestration caused by CUG-repeat expansion and rescues cardiac dysfunctions in a model of myotonic dystrophy.

Dis Model Mech 2018 04 23;11(4). Epub 2018 Apr 23.

Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain.

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder caused by expression of mutant myotonin-protein kinase () transcripts containing expanded CUG repeats. Pathogenic RNA sequesters the muscleblind-like (MBNL) proteins, causing alterations in metabolism of various RNAs. Cardiac dysfunction represents the second most common cause of death in DM type 1 (DM1) patients. However, the contribution of MBNL sequestration in DM1 cardiac dysfunction is unclear. We overexpressed (), the orthologue, in cardiomyocytes of DM1 model flies and observed a rescue of heart dysfunctions, which are characteristic of these model flies and resemble cardiac defects observed in patients. We also identified a drug - daunorubicin hydrochloride - that directly binds to CUG repeats and alleviates sequestration in DM1 cardiomyocytes, resulting in mis-splicing rescue and cardiac function recovery. These results demonstrate the relevance of Mbl sequestration caused by expanded-CUG-repeat RNA in cardiac dysfunctions in DM1, and highlight the potential of strategies aimed at inhibiting this protein-RNA interaction to recover normal cardiac function.
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http://dx.doi.org/10.1242/dmm.032557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963859PMC
April 2018

The gene is functionally equivalent to its four mammalian counterparts and is a modifier of a Huntingtin poly-Q expansion and the Notch pathway.

Dis Model Mech 2018 01 17;11(1). Epub 2018 Jan 17.

Program in Molecular Mechanisms of Disease, Centro de Investigación Príncipe Felipe (CIPF), c/ Eduardo Primo Yúfera no. 3, 46012 Valencia, Spain

Members of the Junctophilin (JPH) protein family have emerged as key actors in all excitable cells, with crucial implications for human pathophysiology. In mammals, this family consists of four members (JPH1-JPH4) that are differentially expressed throughout excitable cells. The analysis of knockout mice lacking JPH subtypes has demonstrated their essential contribution to physiological functions in skeletal and cardiac muscles and in neurons. Moreover, mutations in the human gene are associated with hypertrophic and dilated cardiomyopathies; mutations in are responsible for the neurodegenerative Huntington's disease-like-2 (HDL2), whereas acts as a genetic modifier in Charcot-Marie-Tooth 2K peripheral neuropathy. has a single () gene, as is the case in all invertebrates, which might retain equivalent functions of the four homologous JPH genes present in mammalian genomes. Therefore, owing to the lack of putatively redundant genes, a model could provide an excellent platform to model the Junctophilin-related diseases, to discover the ancestral functions of the JPH proteins and to reveal new pathways. By up- and downregulation of Jp in a tissue-specific manner in , we show that altering its levels of expression produces a phenotypic spectrum characterized by muscular deficits, dilated cardiomyopathy and neuronal alterations. Importantly, our study has demonstrated that Jp modifies the neuronal degeneration in a model of Huntington's disease, and it has allowed us to uncover an unsuspected functional relationship with the Notch pathway. Therefore, this model has revealed new aspects of Junctophilin function that can be relevant for the disease mechanisms of their human counterparts.
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http://dx.doi.org/10.1242/dmm.029082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818072PMC
January 2018

Myotonic dystrophy: candidate small molecule therapeutics.

Drug Discov Today 2017 11 2;22(11):1740-1748. Epub 2017 Aug 2.

Department of Genetics and Interdisciplinary Research Structure for Biotechnology and Biomedicine (ERI BIOTECMED), Universitat de València, Valencia, Spain; Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain; Joint Unit Incliva-CIPF, Valencia, Spain. Electronic address:

Myotonic dystrophy type 1 (DM1) is a rare multisystemic neuromuscular disorder caused by expansion of CTG trinucleotide repeats in the noncoding region of the DMPK gene. Mutant DMPK transcripts are toxic and alter gene expression at several levels. Chiefly, the secondary structure formed by CUGs has a strong propensity to capture and retain proteins, like those of the muscleblind-like (MBNL) family. Sequestered MBNL proteins cannot then fulfill their normal functions. Many therapeutic approaches have been explored to reverse these pathological consequences. Here, we review the myriad of small molecules that have been proposed for DM1, including examples obtained from computational rational design, HTS, drug repurposing, and therapeutic gene modulation.
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http://dx.doi.org/10.1016/j.drudis.2017.07.011DOI Listing
November 2017

Expanded CCUG repeat RNA expression in Drosophila heart and muscle trigger Myotonic Dystrophy type 1-like phenotypes and activate autophagocytosis genes.

Sci Rep 2017 06 6;7(1):2843. Epub 2017 Jun 6.

Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain.

Myotonic dystrophies (DM1-2) are neuromuscular genetic disorders caused by the pathological expansion of untranslated microsatellites. DM1 and DM2, are caused by expanded CTG repeats in the 3'UTR of the DMPK gene and CCTG repeats in the first intron of the CNBP gene, respectively. Mutant RNAs containing expanded repeats are retained in the cell nucleus, where they sequester nuclear factors and cause alterations in RNA metabolism. However, for unknown reasons, DM1 is more severe than DM2. To study the differences and similarities in the pathogenesis of DM1 and DM2, we generated model flies by expressing pure expanded CUG ([250]×) or CCUG ([1100]×) repeats, respectively, and compared them with control flies expressing either 20 repeat units or GFP. We observed surprisingly severe muscle reduction and cardiac dysfunction in CCUG-expressing model flies. The muscle and cardiac tissue of both DM1 and DM2 model flies showed DM1-like phenotypes including overexpression of autophagy-related genes, RNA mis-splicing and repeat RNA aggregation in ribonuclear foci along with the Muscleblind protein. These data reveal, for the first time, that expanded non-coding CCUG repeat-RNA has similar in vivo toxicity potential as expanded CUG RNA in muscle and heart tissues and suggests that specific, as yet unknown factors, quench CCUG-repeat toxicity in DM2 patients.
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http://dx.doi.org/10.1038/s41598-017-02829-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460254PMC
June 2017

In silico discovery of substituted pyrido[2,3-d]pyrimidines and pentamidine-like compounds with biological activity in myotonic dystrophy models.

PLoS One 2017 5;12(6):e0178931. Epub 2017 Jun 5.

Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain.

Myotonic dystrophy type 1 (DM1) is a rare multisystemic disorder associated with an expansion of CUG repeats in mutant DMPK (dystrophia myotonica protein kinase) transcripts; the main effect of these expansions is the induction of pre-mRNA splicing defects by sequestering muscleblind-like family proteins (e.g. MBNL1). Disruption of the CUG repeats and the MBNL1 protein complex has been established as the best therapeutic approach for DM1, hence two main strategies have been proposed: targeted degradation of mutant DMPK transcripts and the development of CUG-binding molecules that prevent MBNL1 sequestration. Herein, suitable CUG-binding small molecules were selected using in silico approaches such as scaffold analysis, similarity searching, and druggability analysis. We used polarization assays to confirm the CUG repeat binding in vitro for a number of candidate compounds, and went on to evaluate the biological activity of the two with the strongest affinity for CUG repeats (which we refer to as compounds 1-2 and 2-5) in DM1 mutant cells and Drosophila DM1 models with an impaired locomotion phenotype. In particular, 1-2 and 2-5 enhanced the levels of free MBNL1 in patient-derived myoblasts in vitro and greatly improved DM1 fly locomotion in climbing assays. This work provides new computational approaches for rational large-scale virtual screens of molecules that selectively recognize CUG structures. Moreover, it contributes valuable knowledge regarding two compounds with desirable biological activity in DM1 models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178931PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459475PMC
September 2017

Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila.

Sci Rep 2016 11 2;6:36230. Epub 2016 Nov 2.

Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain.

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. Here we identify two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate muscleblind RNA isoforms in miRNA sensor constructs. We also show 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 resulted in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Rescued flies had improved muscle function in climbing and flight assays, and had longer lifespan compared to disease controls. These studies provide proof of concept for a similar potentially therapeutic approach to DM1 in humans.
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http://dx.doi.org/10.1038/srep36230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090246PMC
November 2016

Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster.

G3 (Bethesda) 2016 05 3;6(5):1427-37. Epub 2016 May 3.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, Pennsylvania 16802 Bioinformatics and Genomics Program, The Huck Institutes of of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802 Department of Anthropology, The Pennsylvania State University, University Park, Pennsylvania 16802

About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions.
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http://dx.doi.org/10.1534/g3.116.027060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856093PMC
May 2016

Six Serum miRNAs Fail to Validate as Myotonic Dystrophy Type 1 Biomarkers.

PLoS One 2016 26;11(2):e0150501. Epub 2016 Feb 26.

Translational Genomics Group, Incliva Health Research Institute, Valencia, Spain.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3' untranslated region of the DMPK gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, miR-103, miR-107, miR-21, miR-29a, miR-30c, and miR-652 all failed to show consistent differences in serum expression in subsequent validation experiments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150501PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769077PMC
July 2016

Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction.

Dis Model Mech 2015 Dec 29;8(12):1569-78. Epub 2015 Oct 29.

Translational Genomics Group, Incliva Health Research Institute, Avda. Menendez Pelayo 4 acc 46010, Valencia, Spain Department of Genetics and Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Universitat de València, Dr Moliner 50, Burjasot 46100, Spain.

Up to 80% of individuals with myotonic dystrophy type 1 (DM1) will develop cardiac abnormalities at some point during the progression of their disease, the most common of which is heart blockage of varying degrees. Such blockage is characterized by conduction defects and supraventricular and ventricular tachycardia, and carries a high risk of sudden cardiac death. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts, as revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in humans with DM1, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function, reduced heart tube diameters and reduced contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind from the CUG RNA repeats and reduced ribonuclear formation in the Drosophila heart, but also rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats.
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http://dx.doi.org/10.1242/dmm.021428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728315PMC
December 2015
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