The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Kay Ohlendieck - One of the best experts on this subject based on the ideXlab platform.
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subproteomic profiling of sarcolemma from dystrophic mdx 4cv skeletal muscle
Data in Brief, 2018Co-Authors: Sandra Murphy, Paula Meleady, Michael Henry, Margit Zweyer, Rustam R Mundegar, Dieter Swandulla, Kay OhlendieckAbstract:Abstract The proteomic data presented in this article provide supporting information to the related research article "Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle" (Murphy et al., 2018) [1] . In the associated research article, the sarcolemma from normal versus dystrophic skeletal muscle was analyzed by mass spectrometry-based proteomics. Sarcolemma vesicles were enriched by a lectin agglutination method and then analyzed by liquid chromatography tandem mass spectrometry. Here we provide additional datasets on proteins with decreased versus increased abundance in Dystrophin-deficient muscle plasma membranes.
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Proteomic profiling of the Dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin
Journal of Muscle Research and Cell Motility, 2017Co-Authors: Sandra Murphy, Heinrich Brinkmeier, Paula Meleady, Michael Henry, Mirjam Krautwald, Kay OhlendieckAbstract:The almost complete loss of the membrane cytoskeletal protein Dystrophin and concomitant drastic reduction in Dystrophin-associated glycoproteins are the underlying mechanisms of the highly progressive neuromuscular disorder Duchenne muscular dystrophy. In order to identify new potential binding partners of Dystrophin or proteins in close proximity to the sarcolemmal Dystrophin complex, proteomic profiling of the isolated Dystrophin–glycoprotein complex was carried out. Subcellular membrane fractionation and detergent solubilisation, in combination with ion exchange, lectin chromatography and density gradient ultracentrifugation, was performed to isolate a Dystrophin complex-enriched fraction. Following gradient gel electrophoresis and on-membrane digestion, the protein constituents of the Dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which act as cytolinker proteins and possibly exist in close proximity to the Dystrophin complex in the sarcolemma membrane. Interestingly, comparative immunoblotting showed a significant reduction in desmoglein in Dystrophin-deficient mdx skeletal muscles, reminiscent of the pathobiochemical fate of the Dystrophin-associated core proteins in muscular dystrophy. Comparative membrane proteomics was used to correlate this novel finding to large-scale changes in the dystrophic phenotype. A drastic increase in the extracellular stabilizers biglycan and fibronectin was shown by both mass spectrometric analysis and immunoblotting. The reduced expression of desmoglein in Dystrophin-deficient skeletal muscles, and simultaneous increase in components of the extracellular matrix, suggest that muscular dystrophy is associated with plasmalemmal disintegration, loss of cellular linkage and reactive myofibrosis.
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proteomic analysis of Dystrophin deficiency and associated changes in the aged mdx 4cv heart model of Dystrophinopathy related cardiomyopathy
Journal of Proteomics, 2016Co-Authors: Sandra Murphy, Paula Meleady, Michael Henry, Paul Dowling, Margit Zweyer, Rustam R Mundegar, Dieter Swandulla, Kay OhlendieckAbstract:Abstract Cardiomyopathy is a serious complication in Duchenne muscular dystrophy, an X-linked neuromuscular disease of childhood that is triggered by primary abnormalities in the Dystrophin gene. In order to directly correlate the deficiency in the membrane cytoskeletal protein Dystrophin to secondary abnormalities in the dystrophic heart, this study has used label-free mass spectrometry to compare protein expression patterns in the aged mdx-4cv heart model of Dystrophinopathy versus wild type heart. This report is the first successful identification of members of the cardiac Dystrophin–glycoprotein complex by comparative whole tissue proteomics. The mass spectrometric analysis confirmed the loss of Dystrophin and concomitant reduction of syntrophin and sarcoglycans in the Dystrophin-deficient heart. Proteomic profiling of secondary changes identified distinct alterations in the basal lamina component laminin, the Ca 2 + -binding protein sarcalumenin, the matricellular protein periostin, the proteoglycans asporin and lumican, the cardiac-specific myosin light chain kinase, heat shock proteins and a large number of mitochondrial and glycolytic enzymes. The proteomic findings indicate that the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy is highly complex and involves impairments, modulations and/or adaptations of mitochondrial metabolism, glycolysis, protein chaperoning and ion homeostasis, as well as the maintenance of the contractile apparatus, the intracellular cytoskeleton and the extracellular matrisome. Significance The X-linked inherited disorder Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood. Primary abnormalities in the Dystrophin gene trigger progressive skeletal muscle wasting and impaired cardiorespiratory functions. In order to improve our general understanding of the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy and to identify new marker candidates of cardiac changes in Dystrophinopathy, we have carried out a comparative proteomic study of the mdx-4cv mouse model of Duchenne muscular dystrophy. The mass spectrometric profiling of whole heart preparations has identified the reduction in the Dystrophin–glycoprotein complex and a large variety of secondary changes in the dystrophic heart. Cardiac proteins with a changed abundance were shown to be involved in fibre contraction, energy metabolism, cellular signalling, the cytoskeletal network, the extracellular matrix and the stress response. In the future, the newly identified cardiac proteins may be useful to improve predictive, diagnostic, prognostic or therapy-monitoring approaches in the field of muscular dystrophy and cardiomyopathy.
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new pathobiochemical insights into Dystrophinopathy from the proteomics of senescent mdx mouse muscle
Frontiers in Aging Neuroscience, 2014Co-Authors: Ashling Holland, Paul Dowling, Kay OhlendieckAbstract:Primary abnormalities in the Dystrophin gene cause X-linked muscular dystrophy, a highly progressive muscle wasting disorder of childhood. A spontaneous animal model of Duchenne muscular dystrophy is the mdx mouse, which presents a highly interesting phenotype that exhibits considerable variations in the degree of fibre degeneration in different subtypes of muscles. The idea that aging exacerbates the dystrophic mdx phenotype, as previously indicated by a large number of biochemical and cell biological studies, was clearly confirmed by comparative muscle proteomics. Here we outline recent findings of age-dependent changes in the Dystrophin-deficient muscle proteome and contrast these results with the previously established proteomic profile of sarcopenic muscle. Besides comparable perturbations of various biochemical functions, especially striking are similarities in the cellular stress response associated with a drastic up-regulation of small αB-crystallin-like heat shock proteins. Hence, the comparison of large-scale proteomic data sets of natural muscle aging with dystrophic sarcopenia promises to shed light on the differential effect of sarcopenia of old age versus senescent abnormalities on a mutant dystrophic background.
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proteomics of the Dystrophin glycoprotein complex and Dystrophinopathy
Current Protein & Peptide Science, 2013Co-Authors: Ashling Holland, Steven Carberry, Kay OhlendieckAbstract:The largest human gene is represented by the X-chromosomal Dystrophin gene of 2.4 million bases, which encodes for the membrane cytoskeletal protein Dystrophin. The Dystrophin isoform Dp427 has a subsarcolemmal location and forms a supramolecular membrane assembly with a variety of glycoproteins. In healthy muscle fibres, Dystrophin acts as an actin-binding protein that links the cytoskeleton via the α/β-dystroglycan complex to the extracellular matrix protein laminin. This trans-sarcolemmal complex is believed to stabilize the muscle surface and thus prevents membrane rupturing during excitation-contraction-relaxation cycles. In the highly progressive muscle wasting disease Duchenne muscular dystrophy, the primary deficiency in Dystrophin causes a drastic reduction in Dystrophin-associated glycoproteins, which renders muscle fibres more susceptible to necrosis. Following the biochemical and cell biological characterization of the Dystrophin-glycoprotein complex, several mass spectrometry-based proteomic studies have investigated global changes in Dystrophin-deficient muscle tissues. This review briefly outlines the basic domain structure of Dp427 and the composition of the Dystrophin-associated glycoprotein complex from skeletal muscle. A detailed discussion of recent proteomic analyses of the purified Dystrophin-glycoprotein complex is included, as well as a summary of mass spectrometric surveys of dystrophic specimens. The study of these new areas of muscle proteomics tends to improve our understanding of the normal function of Dystrophin in contractile fibres and better define the molecular mechanism of X-linked muscular dystrophy.
Kay E Davies - One of the best experts on this subject based on the ideXlab platform.
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embryonic myosin is a regeneration marker to monitor utrophin based therapies for dmd
Human Molecular Genetics, 2018Co-Authors: Simon Guiraud, Lee Moir, Sarah E Squire, Matthew J.a. Wood, Benjamin Edwards, Adam Berg, Arran Babbs, Nesrine Ramadan, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein Dystrophin. Constitutive utrophin expression, a structural and functional paralogue of Dystrophin, can successfully prevent the dystrophic pathology in the Dystrophin-deficient mdx mouse model. In dystrophic muscles, utrophin is increased as part of the repair process and localized at the sarcolemma of regenerating myofibers. The presence of developmental myosin such as embryonic myosin (MyHC-emb) and neonatal represents a useful marker of muscle regeneration and a meaningful indicator of muscle damage, which correlates with the clinical severity of milder Becker muscular dystrophy and DMD patients. In the present study, we demonstrate that MyHC-emb is a robust marker of regeneration at different ages and in different skeletal muscles. We also evaluate the correlation between utrophin, Dystrophin and MyHC-emb in wild-type (wt) and regenerating dystrophic muscles. Restoration of Dystrophin significantly reduced MyHC-emb levels. Similarly, overexpression of utrophin in the transgenic mdx-Fiona mice reduced the number of MyHC-emb positive fibers to wt level, prevented the regenerative process and rescued the muscle function. In contrast, the absence of utrophin in the Dystrophin-deficient double-knockout mice resulted in a higher MyHC-emb content and in a more severe dystrophic pathophysiology than in mdx mice. These data illustrate the importance of monitoring utrophin and MyHC-emb levels in the preclinical evaluation of therapies and provide translational support for the use of developmental myosin as a disease biomarker in DMD clinical trials.
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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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aav genome loss from dystrophic mouse muscles during aav u7 snrna mediated exon skipping therapy
Molecular Therapy, 2013Co-Authors: Kay E Davies, Maeva Le Hir, Aurelie Goyenvalle, Cecile Peccate, Guillaume Precigout, Thomas Voit, L Garcia, Stephanie LorainAbstract:In the context of future adeno-associated viral (AAV)–based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened Dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal Dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of Dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment.
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prevention of dystrophic pathology in severely affected Dystrophin utrophin deficient mice by morpholino oligomer mediated exon skipping
Molecular Therapy, 2010Co-Authors: Aurelie Goyenvalle, Steve D. Wilton, Arran Babbs, S Fletcher, D Powell, Ryszard Kole, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the Dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore Dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of Dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/Dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of Dystrophin pre-mRNA in dKO mice induce a near-normal level of Dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.
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duchenne muscular dystrophy and Dystrophin pathogenesis and opportunities for treatment
EMBO Reports, 2004Co-Authors: Kristen J Nowak, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is caused by mutations in the gene that encodes the 427‐kDa cytoskeletal protein Dystrophin. Increased knowledge of the function of Dystrophin and its role in muscle has led to a greater understanding of the pathogenesis of DMD. This, together with advances in the genetic toolkit of the molecular biologist, are leading to many different approaches to treatment. Gene therapy can be achieved using plasmids or viruses, mutations can be corrected using chimaeraplasts and short DNA fragments, exon skipping of mutations can be induced using oligonucleotides and readthrough of nonsense mutations can be achieved using aminoglycoside antibiotics. Blocking the proteasome degradation pathway can stabilize any truncated Dystrophin protein, and upregulation of other proteins can also prevent the dystrophic process. Muscle can be repopulated with myoblasts or stem cells. All, or a combination, of these approaches hold great promise for the treatment of this devastating disease.
Stephanie Lorain - One of the best experts on this subject based on the ideXlab platform.
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aav genome loss from dystrophic mouse muscles during aav u7 snrna mediated exon skipping therapy
Molecular Therapy, 2013Co-Authors: Kay E Davies, Maeva Le Hir, Aurelie Goyenvalle, Cecile Peccate, Guillaume Precigout, Thomas Voit, L Garcia, Stephanie LorainAbstract:In the context of future adeno-associated viral (AAV)–based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened Dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal Dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of Dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment.
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muscle function recovery in golden retriever muscular dystrophy after aav1 u7 exon skipping
Molecular Therapy, 2012Co-Authors: Adeline Vulin, Maeva Le Hir, Aurelie Goyenvalle, Ines Barthelemy, Jeanlaurent Thibaud, Cyriaque Beley, Graziella Griffith, Rachid Benchaouir, Yves Unterfinger, Stephanie LorainAbstract:Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding Dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to Dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of Dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue Dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated Dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.
Paul Dowling - One of the best experts on this subject based on the ideXlab platform.
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proteomic analysis of Dystrophin deficiency and associated changes in the aged mdx 4cv heart model of Dystrophinopathy related cardiomyopathy
Journal of Proteomics, 2016Co-Authors: Sandra Murphy, Paula Meleady, Michael Henry, Paul Dowling, Margit Zweyer, Rustam R Mundegar, Dieter Swandulla, Kay OhlendieckAbstract:Abstract Cardiomyopathy is a serious complication in Duchenne muscular dystrophy, an X-linked neuromuscular disease of childhood that is triggered by primary abnormalities in the Dystrophin gene. In order to directly correlate the deficiency in the membrane cytoskeletal protein Dystrophin to secondary abnormalities in the dystrophic heart, this study has used label-free mass spectrometry to compare protein expression patterns in the aged mdx-4cv heart model of Dystrophinopathy versus wild type heart. This report is the first successful identification of members of the cardiac Dystrophin–glycoprotein complex by comparative whole tissue proteomics. The mass spectrometric analysis confirmed the loss of Dystrophin and concomitant reduction of syntrophin and sarcoglycans in the Dystrophin-deficient heart. Proteomic profiling of secondary changes identified distinct alterations in the basal lamina component laminin, the Ca 2 + -binding protein sarcalumenin, the matricellular protein periostin, the proteoglycans asporin and lumican, the cardiac-specific myosin light chain kinase, heat shock proteins and a large number of mitochondrial and glycolytic enzymes. The proteomic findings indicate that the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy is highly complex and involves impairments, modulations and/or adaptations of mitochondrial metabolism, glycolysis, protein chaperoning and ion homeostasis, as well as the maintenance of the contractile apparatus, the intracellular cytoskeleton and the extracellular matrisome. Significance The X-linked inherited disorder Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood. Primary abnormalities in the Dystrophin gene trigger progressive skeletal muscle wasting and impaired cardiorespiratory functions. In order to improve our general understanding of the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy and to identify new marker candidates of cardiac changes in Dystrophinopathy, we have carried out a comparative proteomic study of the mdx-4cv mouse model of Duchenne muscular dystrophy. The mass spectrometric profiling of whole heart preparations has identified the reduction in the Dystrophin–glycoprotein complex and a large variety of secondary changes in the dystrophic heart. Cardiac proteins with a changed abundance were shown to be involved in fibre contraction, energy metabolism, cellular signalling, the cytoskeletal network, the extracellular matrix and the stress response. In the future, the newly identified cardiac proteins may be useful to improve predictive, diagnostic, prognostic or therapy-monitoring approaches in the field of muscular dystrophy and cardiomyopathy.
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new pathobiochemical insights into Dystrophinopathy from the proteomics of senescent mdx mouse muscle
Frontiers in Aging Neuroscience, 2014Co-Authors: Ashling Holland, Paul Dowling, Kay OhlendieckAbstract:Primary abnormalities in the Dystrophin gene cause X-linked muscular dystrophy, a highly progressive muscle wasting disorder of childhood. A spontaneous animal model of Duchenne muscular dystrophy is the mdx mouse, which presents a highly interesting phenotype that exhibits considerable variations in the degree of fibre degeneration in different subtypes of muscles. The idea that aging exacerbates the dystrophic mdx phenotype, as previously indicated by a large number of biochemical and cell biological studies, was clearly confirmed by comparative muscle proteomics. Here we outline recent findings of age-dependent changes in the Dystrophin-deficient muscle proteome and contrast these results with the previously established proteomic profile of sarcopenic muscle. Besides comparable perturbations of various biochemical functions, especially striking are similarities in the cellular stress response associated with a drastic up-regulation of small αB-crystallin-like heat shock proteins. Hence, the comparison of large-scale proteomic data sets of natural muscle aging with dystrophic sarcopenia promises to shed light on the differential effect of sarcopenia of old age versus senescent abnormalities on a mutant dystrophic background.
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subproteomics analysis of ca2 binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle
FEBS Journal, 2004Co-Authors: Philip Doran, Paul Dowling, James Lohan, Karen Mcdonnell, Stephan Poetsch, Kay OhlendieckAbstract:Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render Dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca(2+) buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca(2+) reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye 'Stains-All' was performed in order to evaluate the fate of major Ca(2+)-binding proteins in Dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca(2+)-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca(2+)-shuttle element, sarcalumenin. In contrast, the 'Stains-All'-positive protein spot, representing the cytosolic Ca(2+)-binding component, calmodulin, was not changed in Dystrophin-deficient fibres. The reduced 2D 'Stains-All' pattern of luminal Ca(2+)-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca(2+) buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca(2+)-binding proteins, including calsequestrin.
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drastic reduction of calsequestrin like proteins and impaired calcium binding in dystrophic mdx muscle
Journal of Applied Physiology, 2002Co-Authors: Kevin Culligan, Paul Dowling, Niamh Banville, Kay OhlendieckAbstract:Although the reduction in Dystrophin-associated glycoproteins is the primary pathophysiological consequence of the deficiency in Dystrophin, little is known about the secondary abnormalities leading to x-linked muscular dystrophy. As abnormal Ca2+ handling may be involved in myonecrosis, we investigated the fate of key Ca2+ regulatory membrane proteins in dystrophic mdx skeletal muscle membranes. Whereas the expression of the ryanodine receptor, the dihydropyridine receptor, the Ca2+-ATPase, and calsequestrin was not affected, a drastic decline in calsequestrin-like proteins of 150–220 kDa was observed in dystrophic microsomes using one-dimensional immunoblotting, two-dimensional immunoblotting with isoelectric focusing, diagonal two-dimensional blotting technique, and immunoprecipitation. In analogy, overall Ca2+ binding was reduced in the sarcoplasmic reticulum of dystrophic muscle. The reduction in Ca2+ binding proteins might be directly involved in triggering impaired Ca2+ sequestration within the lumen of the sarcoplasmic reticulum. Thus disturbed sarcolemmal Ca2+ fluxes seem to influence overall Ca2+homeostasis, resulting in distinct changes in the expression profile of a subset of Ca2+ handling proteins, which might be an important factor in the progressive functional decline of dystrophic muscle fibers.
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brain Dystrophin glycoprotein complex persistent expression of beta dystroglycan impaired oligomerization of dp71 and up regulation of utrophins in animal models of muscular dystrophy
BMC Cell Biology, 2001Co-Authors: Kevin Culligan, Louise Glover, Paul Dowling, Kay OhlendieckAbstract:Aside from muscle, brain is also a major expression site for Dystrophin, the protein whose abnormal expression is responsible for Duchenne muscular dystrophy. Cognitive impairments are frequently associated with this genetic disease, we therefore studied the fate of brain and skeletal muscle Dystrophins and dystroglycans in dystrophic animal models. All Dystrophin-associated glycoproteins investigated were reduced in dystrophic muscle fibres. In Dp427-deficient mdx brain and Dp71-deficient mdx-3cv brain, the expression of α-dystroglycan and laminin was reduced, utrophin isoforms were up-regulated and β-dystroglycan was not affected. Immunofluorescence localization of β-dystroglycan in comparison with glial, endothelial and neuronal cell markers revealed co-localization of von Willebrand factor with β-dystroglycan. Its expression at the endothelial-glial interface was preserved in Dystrophin isoform-deficient brain from mdx and mdx-3cv mice. In addition, chemical crosslinking revealed that the Dp71 isoform exists in mdx brain predominantly as a monomer. This suggests an association of β-dystroglycan with membranes at the vascular-glial interface in the forebrain. In contrast to dystrophic skeletal muscle fibres, Dystrophin deficiency does not trigger a reduction of all dystroglycans in the brain, and utrophins may partially compensate for the lack of brain Dystrophins. Abnormal oligomerization of the Dystrophin isoform Dp71 might be involved in the pathophysiological mechanisms underlying abnormal brain functions.
Matthew J.a. Wood - One of the best experts on this subject based on the ideXlab platform.
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embryonic myosin is a regeneration marker to monitor utrophin based therapies for dmd
Human Molecular Genetics, 2018Co-Authors: Simon Guiraud, Lee Moir, Sarah E Squire, Matthew J.a. Wood, Benjamin Edwards, Adam Berg, Arran Babbs, Nesrine Ramadan, Kay E DaviesAbstract:Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein Dystrophin. Constitutive utrophin expression, a structural and functional paralogue of Dystrophin, can successfully prevent the dystrophic pathology in the Dystrophin-deficient mdx mouse model. In dystrophic muscles, utrophin is increased as part of the repair process and localized at the sarcolemma of regenerating myofibers. The presence of developmental myosin such as embryonic myosin (MyHC-emb) and neonatal represents a useful marker of muscle regeneration and a meaningful indicator of muscle damage, which correlates with the clinical severity of milder Becker muscular dystrophy and DMD patients. In the present study, we demonstrate that MyHC-emb is a robust marker of regeneration at different ages and in different skeletal muscles. We also evaluate the correlation between utrophin, Dystrophin and MyHC-emb in wild-type (wt) and regenerating dystrophic muscles. Restoration of Dystrophin significantly reduced MyHC-emb levels. Similarly, overexpression of utrophin in the transgenic mdx-Fiona mice reduced the number of MyHC-emb positive fibers to wt level, prevented the regenerative process and rescued the muscle function. In contrast, the absence of utrophin in the Dystrophin-deficient double-knockout mice resulted in a higher MyHC-emb content and in a more severe dystrophic pathophysiology than in mdx mice. These data illustrate the importance of monitoring utrophin and MyHC-emb levels in the preclinical evaluation of therapies and provide translational support for the use of developmental myosin as a disease biomarker in DMD clinical trials.
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antisense pre treatment increases gene therapy efficacy in dystrophic muscles
Human Molecular Genetics, 2016Co-Authors: Cecile Peccate, Graham Mcclorey, Maeva Le Hir, Amedee Mollard, Laura Julien, Susan Jarmin, Anita Le Heron, George Dickson, Sofia Benkhelifaziyyat, Matthew J.a. WoodAbstract:In preclinical models for Duchenne muscular dystrophy, Dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary Dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-Dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of Dystrophin and the use of lower and thus safer vector doses for Duchenne patients.
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cell penetrating peptide conjugated antisense oligonucleotides restore systemic muscle and cardiac Dystrophin expression and function
Human Molecular Genetics, 2008Co-Authors: Hong M Moulton, Yiqi Seow, Corinne Boyd, Jordan Boutilier, Patrick L Iverson, Matthew J.a. WoodAbstract:: Antisense oligonucleotides (AOs) have the potential to induce functional Dystrophin protein expression via exon skipping by restoring in-frame transcripts in the majority of patients suffering from Duchenne muscular dystrophy (DMD). AOs of morpholino phosphoroamidate (PMO) and 2'-O-methyl phosphorothioate RNA (2'Ome RNA) chemistry have been shown to restore Dystrophin expression in skeletal muscle but not in heart, following high-dose systemic delivery in murine models of muscular dystrophy (mdx). Exploiting the cell transduction properties of two basic arginine-rich cell penetrating peptides, we demonstrate widespread systemic correction of Dystrophin expression in body-wide muscles and cardiac tissue in adult dystrophic mdx mice, with a single low-dose injection of peptide-conjugated PMO AO. This approach was sufficient to restore uniform, high-level Dystrophin protein expression in peripheral muscle and cardiac tissue, with robust sarcolemmal relocalization of the Dystrophin-associated protein complex and functional improvement in muscle. Peptide-conjugated AOs therefore have significant potential for systemic correction of the DMD phenotype.
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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.