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Kay E Davies - One of the best experts on this subject based on the ideXlab platform.
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The potential of Utrophin and dystrophin combination therapies for Duchenne muscular dystrophy.
Human molecular genetics, 2019Co-Authors: Simon Guiraud, Lee Moir, Sarah E Squire, Matthew J.a. Wood, Benjamin Edwards, Arran Babbs, Adam Berg, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Several therapeutic modalities are currently in clinical trials but none will achieve maximum functional rescue and full disease correction. Therefore, we explored the potential of combining the benefits of dystrophin with increases of Utrophin, an autosomal paralogue of dystrophin. Utrophin and dystrophin can be co-expressed and co-localized at the same muscle membrane. Wild-type (wt) levels of dystrophin are not significantly affected by a moderate increase of Utrophin whereas higher levels of Utrophin reduce wt dystrophin, suggesting a finite number of actin binding sites at the sarcolemma. Thus, Utrophin upregulation strategies may be applied to the more mildly affected Becker patients with lower dystrophin levels. Whereas increased dystrophin in wt animals does not offer functional improvement, overexpression of Utrophin in wt mice results in a significant supra-functional benefit over wt. These findings highlight an additive benefit of the combined therapy and potential new unique roles of Utrophin. Finally, we show a 30% restoration of wt dystrophin levels, using exon-skipping, together with increased Utrophin levels restores dystrophic muscle function to wt levels offering greater therapeutic benefit than either single approach alone. Thus, this combination therapy results in additive functional benefit and paves the way for potential future combinations of dystrophin- and Utrophin-based strategies.
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alternative Utrophin mrnas contribute to phenotypic differences between dystrophin deficient mice and duchenne muscular dystrophy
FEBS Letters, 2018Co-Authors: K Perkins, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a fatal disorder caused by absence of functional dystrophin protein. Compensation in dystrophin-deficient (mdx) mice may be achieved by overexpression of its fetal paralogue, Utrophin. Strategies to increase Utrophin levels by stimulating promoter activity using small compounds are therefore a promising pharmacological approach. Here, we characterise similarities and differences existing within the mouse and human Utrophin locus to assist in high-throughput screening for potential Utrophin modulator drugs. We identified five novel 5'-Utrophin isoforms (A',B',C,D and F) in adult and embryonic tissue. As the more efficient Utrophin-based response in mdx skeletal muscle appears to involve independent transcriptional activation of conserved, myogenic isoforms (A' and F), elevating their paralogues in DMD patients is an encouraging therapeutic strategy.
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Utrophin influences mitochondrial pathology and oxidative stress in dystrophic muscle
Skeletal Muscle, 2017Co-Authors: Tahnee L Kennedy, Kay E Davies, Lee Moir, Sarah Hemming, Ben Edwards, Sarah E Squire, Simon GuiraudAbstract:Duchenne muscular dystrophy (DMD) is a lethal X-linked muscle wasting disorder caused by the absence of dystrophin, a large cytoskeletal muscle protein. Increasing the levels of the dystrophin-related-protein Utrophin is a highly promising therapy for DMD and has been shown to improve pathology in dystrophin-deficient mice. One contributing factor to muscle wasting in DMD is mitochondrial pathology that contributes to oxidative stress and propagates muscle damage. The purpose of this study was to assess whether Utrophin could attenuate mitochondria pathology and oxidative stress. Skeletal muscles from wildtype C57BL/10, dystrophin-deficient mdx, dystrophin/Utrophin double knockout (dko) and dystrophin-deficient mdx/Utrophin over-expressing mdx-Fiona transgenic mice were assessed for markers of mitochondrial damage. Using transmission electron microscopy, we show that high levels of Utrophin ameliorate the aberrant structure and localisation of mitochondria in mdx mice whereas absence of Utrophin worsened these features in dko mice. Elevated Utrophin also reverts markers of protein oxidation and oxidative stress, elevated in mdx and dko mice, to wildtype levels. These changes were observed independently of a shift in oxidative phenotype. These findings show that Utrophin levels influence mitochondrial pathology and oxidative stress. While Utrophin deficiency worsens the pathology, Utrophin over-expression in dystrophic muscle benefits mitochondria and attenuates the downstream pathology associated with aberrant mitochondrial function.
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correlation of Utrophin levels with the dystrophin protein complex and muscle fibre regeneration in duchenne and becker muscular dystrophy muscle biopsies
PLOS ONE, 2016Co-Authors: Narinder Janghra, Jennifer E Morgan, Caroline Sewry, Kay E Davies, Francesco Muntoni, Francis Wilson, Jonathon M TinsleyAbstract:Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of Utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of Utrophin modulators in clinical trials, it is necessary to accurately measure Utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal Utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal Utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify Utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced Utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal Utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing Utrophin modulator activity in future clinical trials.
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biomarker development to support the clinical development of Utrophin modulators for duchenne muscular dystrophy therapy
Neuromuscular Disorders, 2015Co-Authors: Jonathon M Tinsley, Narinder Janghra, Jennifer E Morgan, Caroline Sewry, Francis Xavier Wilson, F Muntoni, D Elsey, Kay E DaviesAbstract:The continual expression of Utrophin protein by pharmacological maintenance of Utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). In order for proof of concept of Utrophin modulators to be demonstrated in DMD patients, a multicomponent biomarker strategy has been instigated to quantify Utrophin levels and demonstrate evidence of reduced muscle fibre regeneration. To demonstrate an increase in Utrophin derived from drug treatment above the natural levels resulting from regeneration, we aim to quantify both fibre numbers and levels of Utrophin protein localised to mature fibres in pre- and post-dose muscle biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of regenerating fibres, determined by the presence of neonatal and foetal myosin, will be calculated from biopsies. Serum samples will be analysed to quantify the levels of specific microRNAs (miRs) associated with fibre leakage and peptide markers of active fibrosis which characterise fibre damage and degeneration respectively. We will present data from these candidate biomarkers tested in DMD, Becker and normal samples looking to demonstrate quantifiable changes which are indicative of benefit. In future clinical trials of Utrophin modulators, and potentially other DMD therapeutic approaches, these biomarkers may be appropriate to confirm benefit to dystrophin deficient muscle.
Jonathon M Tinsley - One of the best experts on this subject based on the ideXlab platform.
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correlation of Utrophin levels with the dystrophin protein complex and muscle fibre regeneration in duchenne and becker muscular dystrophy muscle biopsies
PLOS ONE, 2016Co-Authors: Narinder Janghra, Jennifer E Morgan, Caroline Sewry, Kay E Davies, Francesco Muntoni, Francis Wilson, Jonathon M TinsleyAbstract:Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of Utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of Utrophin modulators in clinical trials, it is necessary to accurately measure Utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal Utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal Utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify Utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced Utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal Utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing Utrophin modulator activity in future clinical trials.
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biomarker development to support the clinical development of Utrophin modulators for duchenne muscular dystrophy therapy
Neuromuscular Disorders, 2015Co-Authors: Jonathon M Tinsley, Narinder Janghra, Jennifer E Morgan, Caroline Sewry, Francis Xavier Wilson, F Muntoni, D Elsey, Kay E DaviesAbstract:The continual expression of Utrophin protein by pharmacological maintenance of Utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). In order for proof of concept of Utrophin modulators to be demonstrated in DMD patients, a multicomponent biomarker strategy has been instigated to quantify Utrophin levels and demonstrate evidence of reduced muscle fibre regeneration. To demonstrate an increase in Utrophin derived from drug treatment above the natural levels resulting from regeneration, we aim to quantify both fibre numbers and levels of Utrophin protein localised to mature fibres in pre- and post-dose muscle biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of regenerating fibres, determined by the presence of neonatal and foetal myosin, will be calculated from biopsies. Serum samples will be analysed to quantify the levels of specific microRNAs (miRs) associated with fibre leakage and peptide markers of active fibrosis which characterise fibre damage and degeneration respectively. We will present data from these candidate biomarkers tested in DMD, Becker and normal samples looking to demonstrate quantifiable changes which are indicative of benefit. In future clinical trials of Utrophin modulators, and potentially other DMD therapeutic approaches, these biomarkers may be appropriate to confirm benefit to dystrophin deficient muscle.
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Non-toxic ubiquitous over-expression of Utrophin in the mdx mouse
Neuromuscular disorders : NMD, 2001Co-Authors: R Fisher, Jonathon M Tinsley, Sarah E Squire, S Phelps, Elizabeth R. Townsend, Jo E Martin, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is an inherited, severe muscle wasting disease caused by the loss of the cytoskeletal protein, dystrophin. Patients usually die in their late teens or early twenties of cardiac or respiratory failure. We have previously demonstrated that the dystrophin related protein, Utrophin is able to compensate for the loss of dystrophin in the mdx mouse, the mouse model of the disease. Expression of a Utrophin transgene under the control of an HSA promoter results in localization of Utrophin to the sarcolemma and prevents the muscle pathology. Here we show that the over-expression of full-length Utrophin in a broad range of tissues is not detrimental in the mdx mouse. These findings have important implications for the feasibility of the up-regulation of Utrophin in therapy for DMD since they suggest that tissue specific up-regulation may not be necessary.
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Prevention of the dystrophic phenotype in dystrophin/Utrophin-deficient muscle following adenovirus-mediated transfer of a Utrophin minigene.
Gene therapy, 2000Co-Authors: P M Wakefield, Jonathon M Tinsley, Rénald Gilbert, George Karpati, Matthew J.a. Wood, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by the lack of a subsarcolemmal protein, dystrophin. We have previously shown that the dystrophin-related protein, Utrophin is able to compensate for the lack of dystrophin in the mdx mouse, the mouse model for DMD. Here, we explore whether Utrophin delivered to the limb muscle of dystrophin/Utrophin-deficient double knockout (dko) neonatal mice can protect the muscle from subsequent dystrophic damage. Utrophin delivery may avoid the potential problems of an immune response associated with the delivery of dystrophin to a previously dystrophin-deficient host. Dko muscle (tibialis anterior) was injected with a first generation recombinant adenovirus containing a Utrophin minigene. Up to 95% of the fibres continued expressing the minigene 30 days after injection. Expression of Utrophin caused a marked reduction from 80% centrally nucleated fibres (CNFs) in the uninjected dko TA to 12% in the injected dko TA. Within the region of the TA expressing the Utrophin minigene, a significant decrease in the prevelance of necrosis was noted. These results demonstrate that the Utrophin minigene delivered using an adenoviral vector is able to afford protection to the dystrophin/Utrophin-deficient muscle of the dko mouse. Gene Therapy (2000) 7, 201-204.
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prevention of the dystrophic phenotype in dystrophin Utrophin deficient muscle following adenovirus mediated transfer of a Utrophin minigene
Gene Therapy, 2000Co-Authors: P M Wakefield, Jonathon M Tinsley, Rénald Gilbert, George Karpati, Matthew J.a. Wood, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by the lack of a subsarcolemmal protein, dystrophin. We have previously shown that the dystrophin-related protein, Utrophin is able to compensate for the lack of dystrophin in the mdx mouse, the mouse model for DMD. Here, we explore whether Utrophin delivered to the limb muscle of dystrophin/Utrophin-deficient double knockout (dko) neonatal mice can protect the muscle from subsequent dystrophic damage. Utrophin delivery may avoid the potential problems of an immune response associated with the delivery of dystrophin to a previously dystrophin-deficient host. Dko muscle (tibialis anterior) was injected with a first generation recombinant adenovirus containing a Utrophin minigene. Up to 95% of the fibres continued expressing the minigene 30 days after injection. Expression of Utrophin caused a marked reduction from 80% centrally nucleated fibres (CNFs) in the uninjected dko TA to 12% in the injected dko TA. Within the region of the TA expressing the Utrophin minigene, a significant decrease in the prevelance of necrosis was noted. These results demonstrate that the Utrophin minigene delivered using an adenoviral vector is able to afford protection to the dystrophin/Utrophin-deficient muscle of the dko mouse. Gene Therapy (2000) 7, 201-204.
Tejvir S. Khurana - One of the best experts on this subject based on the ideXlab platform.
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pmo based let 7c site blocking oligonucleotide sbo mediated Utrophin upregulation in mdx mice a therapeutic approach for duchenne muscular dystrophy dmd
Scientific Reports, 2020Co-Authors: Kasturi Sengupta, Emanuele Loro, Tejvir S. KhuranaAbstract:Upregulation of Utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3'UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating Utrophin. We used the C2C12UTRN5'luc3' reporter cell line in which the 5'- and 3'-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for Utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher Utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating Utrophin expression as a therapeutic approach for DMD.
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genome editing mediated Utrophin upregulation in duchenne muscular dystrophy stem cells
Molecular therapy. Nucleic acids, 2020Co-Authors: Kasturi Sengupta, Emanuele Loro, Manoj K. Mishra, Melissa J Spencer, April D Pyle, Tejvir S. KhuranaAbstract:Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate Utrophin expression by binding their cognate sites in the 3′ UTR. Previously we have shown that miRNA: UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve Utrophin upregulation and functional improvement in mdx mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3′ UTR, for achieving higher expression of endogenous Utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of Utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel Utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.
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High-throughput identification of post-transcriptional Utrophin up-regulators for Duchenne muscle dystrophy (DMD) therapy
Scientific Reports, 2020Co-Authors: Emanuele Loro, Kasturi Sengupta, Sasha Bogdanovich, Kanupriya Whig, David C. Schultz, Donna M. Huryn, Tejvir S. KhuranaAbstract:Upregulation of endogenous Utrophin offers great promise for treating DMD, as it can functionally compensate for the lack of dystrophin caused by DMD gene mutations, without the immunogenic concerns associated with delivering dystrophin. However, post-transcriptional repression mechanisms targeting the 5′ and 3′ untranslated regions (UTRs) of Utrophin mRNA significantly limit the magnitude of Utrophin upregulation achievable by promoter activation. Using a Utrophin 5′3′UTR reporter assay, we performed a high-throughput screen (HTS) for small molecules capable of relieving Utrophin post-transcriptional repression. We identified 27 hits that were ranked using an algorithm that we designed for hit prioritization that we call Hit to Lead Prioritization Score (H2LPS). The top 10 hits were validated using an orthogonal assay for endogenous Utrophin expression. Evaluation of the top scoring hit, Trichostatin A (TSA), demonstrated Utrophin upregulation and functional improvement in the mdx mouse model of DMD. TSA and the other small molecules identified here represent potential starting points for DMD drug discovery efforts.
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Utrophin expression in TA muscle of mdx mice treated with intraperitoneal injection of let7-SBOs.
2017Co-Authors: Manoj K. Mishra, Emanuele Loro, Steve D. Wilton, Kasturi Sengupta, Tejvir S. KhuranaAbstract:(A) Expression and localization of Utrophin in mdx mice treated with let7-SBOs. Frozen sections of the TA muscles immuno-labelled with anti-Utrophin antibodies and α-BTX. Utrophin labeling in regions of TA muscle with a paucity of neuromuscular junctions (demonstrated by lack of α-BTX staining). (Scale bar = 100 μm). (B) Relative fluorescence quantification of Utrophin expression in TA muscles with low and high dose let7-SBOs treatment compared with control oligonucleotides. Frozen 10μm thick sections of the TA muscles immuno-labelled with Utrophin antibodies. Bars represent mean ± SD (n = 3 mice per experimental group). Statistical comparison was analyzed by Mann-Whitney U test (*P ≤ 0.05).
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Therapeutic strategy for Utrophin upregulation validation in C2C12 cells.
2017Co-Authors: Manoj K. Mishra, Emanuele Loro, Steve D. Wilton, Kasturi Sengupta, Tejvir S. KhuranaAbstract:(A) Efficacy of let7-SBOs in C2C12-5’Luc3’ Utrophin reporter cells (cell line contains construct pGL4:50–5'Luc3' where reporter luciferase2 gene is flanked by the 5’- and 3’-UTRs of mouse Utrophin-A). Cells transiently transfected with let7-SBOs /control oligonucleotides and luciferase activity measured 24 hrs post-transfection. Results shows significant increase in luciferase activity in C2C12-5’Luc3’ Utrophin reporter cells 24 hrs of post-transfection with let7-SBOs compared to control oligonucleotides at various concentrations. Bars represent mean ± SD from 3 independent experiments. Statistical analysis was performed by 2-way ANOVA for multiple comparison followed by Bonferroni correction (***P ≤ 0.001). (B) Endogenous Utrophin protein expression in C2C12 cells 24 hrs after transfection with let7-SBOs or control oligonucleotides at various concentrations, assayed by western blotting. α-Tubulin staining was used to control for equal loading. (C) Quantification of Utrophin normalized to α-tubulin band density in western blot assay. Bars represent mean ± SD from 3 independent experiments and control. Control oligonucleotides treatment used as reference for Utrophin expression in each independent experiment. Statistical analysis was performed by 2-way ANOVA for multiple comparison followed by Bonferroni correction (*P ≤ 0.05, ***P ≤ 0.001).
Bernard J. Jasmin - One of the best experts on this subject based on the ideXlab platform.
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IRES-Mediated Translation of Utrophin A Is Enhanced by Glucocorticoid Treatment in Skeletal Muscle Cells
PloS one, 2008Co-Authors: Pedro Miura, Martin Holcik, Meghan Andrews, Bernard J. JasminAbstract:Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of Utrophin. Here, we show that treatment of myotubes with 6a2methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of Utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the Utrophin A 59UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the Utrophin A 59UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous Utrophin A mRNAs and reporter mRNAs harbouring the Utrophin A 59UTR. Additional experiments identified a distinct region within the Utrophin A 59UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of Utrophin A in muscle cells treated with glucocorticoids. Targeting the Utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.
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expression of Utrophin a mrna correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin nfat signaling
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Joe V Chakkalakal, Lindsay M. Angus, Mark A Stocksley, Maryann Harrison, Julie Deschenesfurry, Simon Stpierre, Lynn A Megeney, Eva R Chin, Robin N Michel, Bernard J. JasminAbstract:Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of Utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more Utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of Utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in Utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of Utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in Utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of Utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the Utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the Utrophin A promoter. Together, these results indicate that expression of Utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.
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Expression of the Utrophin gene during myogenic differentiation.
Nucleic acids research, 1999Co-Authors: Anthony O. Gramolini, Bernard J. JasminAbstract:The process of myogenic differentiation is known to be accompanied by large increases ( approximately 10-fold) in the expression of genes encoding cytoskeletal and membrane proteins including dystrophin and the acetylcholine receptor (AChR) subunits, via the effects of transcription factors belonging to the MyoD family. Since in skeletal muscle (i) Utrophin is a synaptic homolog to dystrophin, and (ii) the Utrophin promoter contains an E-box, we examined, in the present study, expression of the Utrophin gene during myogenic differentiation using the mouse C2 muscle cell line. We observed that in comparison to myoblasts, the levels of Utrophin and its transcript were approximately 2-fold higher in differentiated myotubes. In order to address whether a greater rate of transcription contributed to the elevated levels of Utrophin transcripts, we performed nuclear run-on assays. In these studies we determined that the rate of transcription of the Utrophin gene was approximately 2-fold greater in myotubes as compared to myoblasts. Finally, we examined the stability of Utrophin mRNAs in muscle cultures by two separate methods: following transcription blockade with actinomycin D and by pulse-chase experiments. Under these conditions, we determined that the half-life of Utrophin mRNAs in myoblasts was approximately 20 h and that it remained largely unaffected during myogenic differentiation. Altogether, these results show that in comparison to other synaptic proteins and to dystrophin, expression of the Utrophin gene is only moderately increased during myogenic differentiation.
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Induction of Utrophin gene expression by heregulin in skeletal muscle cells: Role of the N-box motif and GA binding protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Anthony O. Gramolini, Kay E Davies, Jonathon M Tinsley, Jean-pierre Changeux, Lindsay M. Angus, Laurent Schaeffer, Edward A. Burton, Bernard J. JasminAbstract:The modulation of Utrophin gene expression in muscle by the nerve-derived factor agrin plausibly involves the trophic factor ARIA/heregulin. Here we show that heregulin treatment of mouse and human cultured myotubes caused a ≈2.5-fold increase in Utrophin mRNA levels. Transient transfection experiments with Utrophin promoter-reporter gene constructs showed that this increase resulted from an enhanced transcription of the Utrophin gene. In the case of the nicotinic acetylcholine receptor δ and ɛ subunit genes, heregulin was previously reported to stimulate transcription via a conserved promoter element, the N-box, which binds the multimeric Ets-related transcription factor GA binding protein (GABP). Accordingly, site-directed mutagenesis of a single N-box motif in the Utrophin gene promoter abolished the transcriptional response to heregulin. In addition, overexpression of heregulin, or of the two GABP subunits in cultured myotubes, caused an N-box-dependent increase of the Utrophin promoter activity. In vivo, direct gene transfer into muscle confirmed that heregulin regulates Utrophin gene expression. Finally, electrophoretic mobility shift assays and supershift experiments performed with muscle extracts revealed that the N-box of the Utrophin promoter binds GABP. These findings suggest that the subsynaptic activation of transcription by heregulin via the N-box motif and GABP are conserved among genes expressed at the neuromuscular junction. Because Utrophin can functionally compensate for the lack of dystrophin, the elucidation of the molecular mechanisms regulating Utrophin gene transcription may ultimately lead to therapies based on Utrophin expression throughout the muscle fibers of Duchenne muscular dystrophy patients.
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Discordant expression of Utrophin and its transcript in human and mouse skeletal muscles.
Journal of neuropathology and experimental neurology, 1999Co-Authors: Anthony O. Gramolini, George Karpati, Bernard J. JasminAbstract:In order to determine the mechanisms regulating Utrophin expression in human skeletal muscle, we examined the expression and distribution of Utrophin and its transcript in biopsies from normal subjects as well as from Duchenne muscular dystrophy (DMD) and polymyositis (PM) patients. We first determined by immunoblotting that in comparison to biopsies from normal subjects, Utrophin levels were indeed higher in muscle samples from both DMD and PM patients as previously shown. By contrast, levels of Utrophin mRNAs as determined by both RT-PCR assays and in situ hybridization, were identical in muscle samples obtained from normal subjects versus DMD and PM patients. In these experiments, we also noted that while Utrophin transcripts had a clear tendency to accumulate within the postsynaptic sarcoplasm of normal human muscle fibers, the extent of synaptic accumulation was considerably less than that which we recently observed in mouse muscle fibers. The distribution of Utrophin transcripts in synaptic and extrasynaptic compartments of muscle fibers obtained from DMD and PM patients was similar to that seen along muscle fibers from normal subjects. Finally, we also monitored expression of Utrophin and its transcripts during regeneration of mouse muscle induced to degenerate by cardiotoxin injections. In these regenerating muscles, we observed by both immunoblotting and immunofluorescence, a large increase (4- to 7-fold) in the levels of Utrophin. In agreement with our results obtained with human muscle, the increase in Utrophin levels in regenerating mouse muscle was not accompanied by parallel changes in the abundance of Utrophin transcripts. Taken together, these results indicate that the levels of Utrophin and its transcript in muscle are discordantly regulated under certain conditions thereby highlighting the important contribution of post-transcriptional regulatory mechanisms in the control of Utrophin levels in skeletal muscle fibers.
Anthony O. Gramolini - One of the best experts on this subject based on the ideXlab platform.
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Expression of the Utrophin gene during myogenic differentiation.
Nucleic acids research, 1999Co-Authors: Anthony O. Gramolini, Bernard J. JasminAbstract:The process of myogenic differentiation is known to be accompanied by large increases ( approximately 10-fold) in the expression of genes encoding cytoskeletal and membrane proteins including dystrophin and the acetylcholine receptor (AChR) subunits, via the effects of transcription factors belonging to the MyoD family. Since in skeletal muscle (i) Utrophin is a synaptic homolog to dystrophin, and (ii) the Utrophin promoter contains an E-box, we examined, in the present study, expression of the Utrophin gene during myogenic differentiation using the mouse C2 muscle cell line. We observed that in comparison to myoblasts, the levels of Utrophin and its transcript were approximately 2-fold higher in differentiated myotubes. In order to address whether a greater rate of transcription contributed to the elevated levels of Utrophin transcripts, we performed nuclear run-on assays. In these studies we determined that the rate of transcription of the Utrophin gene was approximately 2-fold greater in myotubes as compared to myoblasts. Finally, we examined the stability of Utrophin mRNAs in muscle cultures by two separate methods: following transcription blockade with actinomycin D and by pulse-chase experiments. Under these conditions, we determined that the half-life of Utrophin mRNAs in myoblasts was approximately 20 h and that it remained largely unaffected during myogenic differentiation. Altogether, these results show that in comparison to other synaptic proteins and to dystrophin, expression of the Utrophin gene is only moderately increased during myogenic differentiation.
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Induction of Utrophin gene expression by heregulin in skeletal muscle cells: Role of the N-box motif and GA binding protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Anthony O. Gramolini, Kay E Davies, Jonathon M Tinsley, Jean-pierre Changeux, Lindsay M. Angus, Laurent Schaeffer, Edward A. Burton, Bernard J. JasminAbstract:The modulation of Utrophin gene expression in muscle by the nerve-derived factor agrin plausibly involves the trophic factor ARIA/heregulin. Here we show that heregulin treatment of mouse and human cultured myotubes caused a ≈2.5-fold increase in Utrophin mRNA levels. Transient transfection experiments with Utrophin promoter-reporter gene constructs showed that this increase resulted from an enhanced transcription of the Utrophin gene. In the case of the nicotinic acetylcholine receptor δ and ɛ subunit genes, heregulin was previously reported to stimulate transcription via a conserved promoter element, the N-box, which binds the multimeric Ets-related transcription factor GA binding protein (GABP). Accordingly, site-directed mutagenesis of a single N-box motif in the Utrophin gene promoter abolished the transcriptional response to heregulin. In addition, overexpression of heregulin, or of the two GABP subunits in cultured myotubes, caused an N-box-dependent increase of the Utrophin promoter activity. In vivo, direct gene transfer into muscle confirmed that heregulin regulates Utrophin gene expression. Finally, electrophoretic mobility shift assays and supershift experiments performed with muscle extracts revealed that the N-box of the Utrophin promoter binds GABP. These findings suggest that the subsynaptic activation of transcription by heregulin via the N-box motif and GABP are conserved among genes expressed at the neuromuscular junction. Because Utrophin can functionally compensate for the lack of dystrophin, the elucidation of the molecular mechanisms regulating Utrophin gene transcription may ultimately lead to therapies based on Utrophin expression throughout the muscle fibers of Duchenne muscular dystrophy patients.
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Discordant expression of Utrophin and its transcript in human and mouse skeletal muscles.
Journal of neuropathology and experimental neurology, 1999Co-Authors: Anthony O. Gramolini, George Karpati, Bernard J. JasminAbstract:In order to determine the mechanisms regulating Utrophin expression in human skeletal muscle, we examined the expression and distribution of Utrophin and its transcript in biopsies from normal subjects as well as from Duchenne muscular dystrophy (DMD) and polymyositis (PM) patients. We first determined by immunoblotting that in comparison to biopsies from normal subjects, Utrophin levels were indeed higher in muscle samples from both DMD and PM patients as previously shown. By contrast, levels of Utrophin mRNAs as determined by both RT-PCR assays and in situ hybridization, were identical in muscle samples obtained from normal subjects versus DMD and PM patients. In these experiments, we also noted that while Utrophin transcripts had a clear tendency to accumulate within the postsynaptic sarcoplasm of normal human muscle fibers, the extent of synaptic accumulation was considerably less than that which we recently observed in mouse muscle fibers. The distribution of Utrophin transcripts in synaptic and extrasynaptic compartments of muscle fibers obtained from DMD and PM patients was similar to that seen along muscle fibers from normal subjects. Finally, we also monitored expression of Utrophin and its transcripts during regeneration of mouse muscle induced to degenerate by cardiotoxin injections. In these regenerating muscles, we observed by both immunoblotting and immunofluorescence, a large increase (4- to 7-fold) in the levels of Utrophin. In agreement with our results obtained with human muscle, the increase in Utrophin levels in regenerating mouse muscle was not accompanied by parallel changes in the abundance of Utrophin transcripts. Taken together, these results indicate that the levels of Utrophin and its transcript in muscle are discordantly regulated under certain conditions thereby highlighting the important contribution of post-transcriptional regulatory mechanisms in the control of Utrophin levels in skeletal muscle fibers.
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muscle and neural isoforms of agrin increase Utrophin expression in cultured myotubes via a transcriptional regulatory mechanism
Journal of Biological Chemistry, 1998Co-Authors: Anthony O. Gramolini, Kay E Davies, Jonathon M Tinsley, Edward A. Burton, John A. Lunde, Jean Cartaud, Michael J Ferns, Annie Cartaud, Bernard J. JasminAbstract:Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that Utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over Utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on Utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the Utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997)J. Biol. Chem. 272, 8117–8120). In the present study, we have therefore focused on the effect of agrin on Utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in Utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of Utrophin transcripts. Western blotting experiments also revealed that the increase in Utrophin mRNAs was accompanied by an increase in the levels of Utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the Utrophin gene, we transfected muscle cells with a 1.3-kilobase pair Utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24–48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of β-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the Utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair Utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5′ from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the Utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the Utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the Utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of Utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.
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Local Transcriptional Control of Utrophin Expression at the Neuromuscular Synapse
The Journal of biological chemistry, 1997Co-Authors: Anthony O. Gramolini, Kay E Davies, Jonathon M Tinsley, Jean Cartaud, Carina L. Dennis, George S. Robertson, Bernard J. JasminAbstract:Abstract Recently, the use of a transgenic mouse model system for Duchenne muscular dystrophy has demonstrated the ability of Utrophin to functionally replace dystrophin and alleviate the muscle pathology (see Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). However, there is currently a clear lack of information concerning the regulatory mechanisms presiding over Utrophin expression during normal myogenesis and synaptogenesis. Using in situ hybridization, we show that Utrophin mRNAs selectively accumulate within the postsynaptic sarcoplasm of adult muscle fibers. In addition, we demonstrate that a 1.3-kilobase fragment of the human Utrophin promoter is sufficient to confer synapse-specific expression to a reporter gene. Deletion of 800 base pairs from this promoter fragment reduces the overall expression of the reporter gene and abolishes its synapse-specific expression. Finally, we also show that Utrophin is present at the postsynaptic membrane of ectopic synapses induced to form at sites distant from the original neuromuscular junctions. Taken together, these results indicate that nerve-derived factors regulate locally the transcriptional activation of the Utrophin gene in skeletal muscle fibers and that myonuclei located in extrasynaptic regions are capable of expressing Utrophin upon receiving appropriate neuronal cues.
