Publications by authors named "Beatriz Llamusi"

25 Publications

  • Page 1 of 1

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

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

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

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

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

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

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

Increased autophagy and apoptosis contribute to muscle atrophy in a myotonic dystrophy type 1 Drosophila model.

Dis Model Mech 2015 Jul;8(7):679-90

Translational Genomics Group, Department of Genetics, University of Valencia, Burjassot 46100, Spain INCLIVA Health Research Institute, Valencia 46010, Spain

Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1) disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor) or muscleblind, or by RNA interference (RNAi)-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis.
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http://dx.doi.org/10.1242/dmm.018127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486854PMC
July 2015

The use of whole-mount in situ hybridization to illustrate gene expression regulation.

Biochem Mol Biol Educ 2014 Jul-Aug;42(4):339-47. Epub 2014 Jun 30.

Department of Genetics, Faculty of Biology, Universitat de València, Valencia, Spain; INCLIVA Health Research Institute, Valencia, Spain.

In situ hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the experiments, students analyzed the repressive effect of Snail over rhomboid expression using reporter lines containing different constructs of the rhomboid neuroectodermal enhancer fused to the lacZ gene. In the second experiment, the epistatic relationship between the cabut and decapentaplegic genes was analyzed. These simple experiments allowed students to (1) understand the role of transcription factors and cis-regulatory elements over gene expression regulation and (2) practice a widespread laboratory technique, in situ hybridization with nonradioactive labeled probes, to detect gene expression patterns. These experiments required 12 hr and were organized into four daily sessions that included the discussion of the results with students. Examples of the results obtained and their relevance are shown and discussed herein. The methods described in these laboratory exercises can be easily adapted to model organisms other than Drosophila.
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http://dx.doi.org/10.1002/bmb.20807DOI Listing
August 2015

Two enhancers control transcription of Drosophila muscleblind in the embryonic somatic musculature and in the central nervous system.

PLoS One 2014 25;9(3):e93125. Epub 2014 Mar 25.

Translational Genomics Group, Department of Genetics, University of Valencia, Valencia, Spain; INCLIVA Health Research Institute, Valencia, Spain.

The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2. Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord (NE, neural enhancer; 830 bp) and somatic (skeletal) musculature (ME, muscle enhancer; 3.3 kb). Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093125PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965525PMC
November 2014

Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model.

Dis Model Mech 2013 Jan 1;6(1):184-96. Epub 2012 Nov 1.

Translational Genomics Group, Department of Genetics, University of Valencia, Doctor Moliner 50, 46100 Burjasot, Valencia, Spain.

Myotonic dystrophy type 1 (DM1) is a genetic disease caused by the pathological expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene. In the DMPK transcripts, the CUG expansions sequester RNA-binding proteins into nuclear foci, including transcription factors and alternative splicing regulators such as MBNL1. MBNL1 sequestration has been associated with key features of DM1. However, the basis behind a number of molecular and histological alterations in DM1 remain unclear. To help identify new pathogenic components of the disease, we carried out a genetic screen using a Drosophila model of DM1 that expresses 480 interrupted CTG repeats, i(CTG)480, and a collection of 1215 transgenic RNA interference (RNAi) fly lines. Of the 34 modifiers identified, two RNA-binding proteins, TBPH (homolog of human TAR DNA-binding protein 43 or TDP-43) and BSF (Bicoid stability factor; homolog of human LRPPRC), were of particular interest. These factors modified i(CTG)480 phenotypes in the fly eye and wing, and TBPH silencing also suppressed CTG-induced defects in the flight muscles. In Drosophila flight muscle, TBPH, BSF and the fly ortholog of MBNL1, Muscleblind (Mbl), were detected in sarcomeric bands. Expression of i(CTG)480 resulted in changes in the sarcomeric patterns of these proteins, which could be restored by coexpression with human MBNL1. Epistasis studies showed that Mbl silencing was sufficient to induce a subcellular redistribution of TBPH and BSF proteins in the muscle, which mimicked the effect of i(CTG)480 expression. These results provide the first description of TBPH and BSF as targets of Mbl-mediated CTG toxicity, and they suggest an important role of these proteins in DM1 muscle pathology.
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http://dx.doi.org/10.1242/dmm.009563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529350PMC
January 2013

In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models.

Proc Natl Acad Sci U S A 2011 Jul 5;108(29):11866-71. Epub 2011 Jul 5.

Department of Genetics, University of Valencia, Burjassot E-46100, Spain.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of noncoding CTG repeats in the dystrophia myotonica-protein kinase gene. Mutant transcripts form CUG hairpins that sequester RNA-binding factors into nuclear foci, including Muscleblind-like-1 protein (MBNL1), which regulate alternative splicing and gene expression. To identify molecules that target toxic CUG transcripts in vivo, we performed a positional scanning combinatorial peptide library screen using a Drosophila model of DM1. The screen identified a D-amino acid hexapeptide (ABP1) that reduced CUG foci formation and suppressed CUG-induced lethality and muscle degeneration when administered orally. Transgenic expression of natural, L-amino acid ABP1 analogues reduced CUG-induced toxicity in fly eyes and muscles. Furthermore, ABP1 reversed muscle histopathology and splicing misregulation of MBNL1 targets in DM1 model mice. In vitro, ABP1 bound to CUG hairpins and induced a switch to a single-stranded conformation. Our findings demonstrate that ABP1 shows antimyotonic dystrophy activity by targeting the core of CUG toxicity.
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http://dx.doi.org/10.1073/pnas.1018213108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141925PMC
July 2011

Alternative splicing regulation by Muscleblind proteins: from development to disease.

Biol Rev Camb Philos Soc 2011 Nov 13;86(4):947-58. Epub 2011 Apr 13.

Department of Genetics, University of Valencia, Burjassot, Valencia, Spain.

Regulated use of exons in pre-mRNAs, a process known as alternative splicing, strongly contributes to proteome diversity. Alternative splicing is finely regulated by factors that bind specific sequences within the precursor mRNAs. Members of the Muscleblind (Mbl) family of splicing factors control critical exon use changes during the development of specific tissues, particularly heart and skeletal muscle. Muscleblind homologs are only found in metazoans from Nematoda to mammals. Splicing targets and recognition mechanisms are also conserved through evolution. In this recognition, Muscleblind CCCH-type zinc finger domains bind to intronic motifs in pre-mRNA targets in which the protein can either activate or repress splicing of nearby exons, depending on the localization of the binding motifs relative to the regulated alternative exon. In humans, the Muscleblind-like 1 (MBNL1) proteins play a critical role in hereditary diseases caused by microsatellite expansions, particularly myotonic dystrophy type 1 (DM1), in which depletion of MBNL1 activity through sequestration explains most misregulated alternative splicing events, at least in murine models. Because of the involvement of these proteins in human diseases, further understanding of the molecular mechanisms by which MBNL1 regulates splicing will help design therapies to revert pathological splicing alterations. Here we summarize the most relevant findings on this family of proteins in recent years, focusing on recently described functional motifs, transcriptional regulation of Muscleblind, regulatory activity on splicing, and involvement in human diseases.
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http://dx.doi.org/10.1111/j.1469-185X.2011.00180.xDOI Listing
November 2011

A GFP-tagged muscleblind C protein isoform reporter construct.

Fly (Austin) 2010 Oct-Dec;4(4):333-7. Epub 2010 Oct 1.

Department of Genetics, University of Valencia, Burjasot, Valencia, Spain.

Drosophila muscleblind (mbl), the ortholog of human Muscleblind-like 1 (MBNL1) gene involved in Myotonic Dystrophy (DM), gives raise to protein isoforms MblA to G. The specific functions and subcellular distribution of isoforms are still largely unknown. To overcome the lack of isoform-specific antibodies we generated transgenic flies that express a GFP:MblC fusion protein under the control of the Gal4/UAS system. The reporter fusion protein was able to functionally complement mbl loss of function mutations, demonstrating activity, and accumulated predominantly in adult muscle nuclei. The fluorescent nature of the reporter makes it appropriate for live imaging detection of MblC protein isoform.
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http://dx.doi.org/10.4161/fly.4.4.13259DOI Listing
April 2011

Chronic spinal injury repair by olfactory bulb ensheathing glia and feasibility for autologous therapy.

J Neuropathol Exp Neurol 2009 Dec;68(12):1294-308

Laboratory of Neural Regeneration, Institute of Biomedicine, Spanish National Research Council (CSIC), Valencia, Spain.

Olfactory bulb ensheathing glia (OB-OEG) promote repair of spinal cord injury (SCI) in rats after transplantation at acute or subacute (up to 45 days) stages. The most relevant clinical scenario in humans, however, is chronic SCI, in which no more major cellular or molecular changes occur at the injury site; this occurs after the third month in rodents. Whether adult OB-OEG grafts promote repair of severe chronic SCI has not been previously addressed. Rats with complete SCI that were transplanted with OB-OEG 4 months after injury exhibited progressive improvement in motor function and axonal regeneration from different brainstem nuclei across and beyond the SCI site. A positive correlation between motor outcome and axonal regeneration suggested a role for brainstem neurons in the recovery. Functional and histological outcomes did not differ after transplantation at subacute or chronic stages. Thus, autologous transplantation is a feasible approach as there is a time frame for patient stabilization and OEG preparation; moreover, the healing effects of OB-OEG on established injuries may offer new therapeutic opportunities for chronic SCI patients.
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http://dx.doi.org/10.1097/NEN.0b013e3181c34bbeDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875478PMC
December 2009

Molecular Effects of the CTG Repeats in Mutant Dystrophia Myotonica Protein Kinase Gene.

Curr Genomics 2008 Dec;9(8):509-16

Department of Genetics, University of Valencia, Doctor Moliner, 50, E46100 Burjasot, Valencia, Spain.

Myotonic Dystrophy type 1 (DM1) is a multi-system disorder characterized by muscle wasting, myotonia, cardiac conduction defects, cataracts, and neuropsychological dysfunction. DM1 is caused by expansion of a CTG repeat in the 3 untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. A body of work demonstrates that DMPK mRNAs containing abnormally expanded CUG repeats are toxic to several cell types. A core mechanism underlying symptoms of DM1 is that mutant DMPK RNA interferes with the developmentally regulated alternative splicing of defined pre-mRNAs. Expanded CUG repeats fold into ds(CUG) hairpins that sequester nuclear proteins including human Muscleblind-like (MBNL) and hnRNP H alternative splicing factors. DM1 cells activate CELF family member CUG-BP1 protein through hyperphosphorylation and stabilization in the cell nucleus. CUG-BP1 and MBNL1 proteins act antagonistically in exon selection in several pre-mRNA transcripts, thus MBNL1 sequestration and increase in nuclear activity of CUG-BP1 both act synergistically to missplice defined transcripts. Mutant DMPK-mediated effect on subcellular localization, and defective phosphorylation of cytoplasmic CUG-BP1, have additionally been linked to defective translation of p21 and MEF2A in DM1, possibly explaining delayed differentiation of DM1 muscle cells. Mutant DMPK transcripts bind and sequester transcription factors such as Specificity protein 1 leading to reduced transcription of selected genes. Recently, transcripts containing long hairpin structures of CUG repeats have been shown to be a Dicer ribonuclease target and Dicer-induced downregulation of the mutant DMPK transcripts triggers silencing effects on RNAs containing long complementary repeats. In summary, mutant DMPK transcripts alter gene transcription, alternative splicing, and translation of specific gene transcripts, and have the ability to trigger gene-specific silencing effects in DM1 cells. Therapies aimed at reversing these gene expression alterations should prove effective ways to treat DM1.
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http://dx.doi.org/10.2174/138920208786847944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2694559PMC
December 2008
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