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Scientific Reports Jan 2021Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches...
Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.
Topics: Adolescent; Aged, 80 and over; Animals; Child; Dystrophin; Female; Fluorescent Antibody Technique; Gadolinium; Humans; Immunohistochemistry; Male; Mass Spectrometry; Mice; Muscle Fibers, Skeletal; Muscular Dystrophy, Duchenne; Mutation; Quadriceps Muscle
PubMed: 33441839
DOI: 10.1038/s41598-020-80495-8 -
Journal of Cachexia, Sarcopenia and... Apr 2022Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is...
BACKGROUND
Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not.
METHODS
Functional outcome data from 387 DMD boys aged 4-15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild-type and DMD models). Grip strength and rotarod running test were studied in wild-type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms).
RESULTS
In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four-limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD-null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild-type, mdx52 or DMD-null mice.
CONCLUSIONS
Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.
Topics: Animals; Dystrophin; Humans; Mice; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Protein Isoforms
PubMed: 35083887
DOI: 10.1002/jcsm.12914 -
Disease Models & Mechanisms Mar 2022Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin...
Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterisation of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs, including reduced attention, problem solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, quantitative PCR and RNAScope in situ hybridisation, we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full-length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.
Topics: Animals; Brain; Dogs; Dystrophin; Exons; Muscular Dystrophy, Duchenne; Phenotype
PubMed: 35019137
DOI: 10.1242/dmm.049291 -
Traffic (Copenhagen, Denmark) Mar 2007The assembly, processing and translocation of proteins occur constantly in all cells, and these processes also take place during the genesis, maintenance and repair of... (Review)
Review
The assembly, processing and translocation of proteins occur constantly in all cells, and these processes also take place during the genesis, maintenance and repair of skeletal muscle. Skeletal muscle fibers are composed of myofibrils and are surrounded by a muscle plasma membrane, the sarcolemma. The sarcolemma serves as a docking location for many proteins. These proteins are important for establishing the physical connection between the extracellular matrix and the cytoskeleton and play a role in transmitting force related to muscle contraction. This physical connection is maintained through a myriad of proteins including the dystrophin glycoprotein complex (DGC). Normal sarcolemmal function requires proper DGC synthesis and positioning, and perturbation of the DGC leads to muscle membrane instability and disease.
Topics: Animals; Dystrophin; Dystrophin-Associated Proteins; Glycoproteins; Humans; Membrane Proteins; Muscle Fibers, Skeletal; Nitric Oxide Synthase Type I; Protein Processing, Post-Translational; Sarcoglycans; Sarcolemma
PubMed: 17274800
DOI: 10.1111/j.1600-0854.2006.00519.x -
BMC Genomics Jan 2007Dystrophins and dystrobrevins are distantly related proteins with important but poorly understood roles in the function of metazoan muscular and neuronal tissues....
BACKGROUND
Dystrophins and dystrobrevins are distantly related proteins with important but poorly understood roles in the function of metazoan muscular and neuronal tissues. Defects in them and their associated proteins cause a range of neuromuscular disorders. Members of this superfamily have been discovered in a relatively serendipitous way; we set out to compile a comprehensive description of dystrophin- and dystrobrevin-related sequences from available metazoan genome sequences, validated in representative organisms by RT-PCR, or acquired de novo from key species.
RESULTS
Features of the superfamily revealed by our survey include: a) Dystrotelin, an entirely novel branch of the superfamily, present in most vertebrates examined. Dystrotelin is expressed in the central nervous system, and is a possible orthologue of Drosophila DAH. We describe the preliminary characterisation of its function, evolution and expression. b) A novel vertebrate member of the dystrobrevin family, gamma-dystrobrevin, an ancient branch now extant only in fish, but probably present in our own ancestors. Like dystrophin, zebrafish gamma-dystrobrevin mRNA is localised to myosepta. c) The extent of conservation of alternative splicing and alternative promoter use in the dystrophin and dystrobrevin genes; alternative splicing of dystrophin exons 73 and 78 and alpha-dystrobrevin exon 13 are conserved across vertebrates, as are the use of the Dp116, Dp71 and G-utrophin promoters; the Dp260 and Dp140 promoters are tetrapod innovations. d) The evolution of the unique N-terminus of DRP2 and its relationship to Dp116 and G-utrophin. e) A C-terminally truncated common ancestor of dystrophin and utrophin in cyclostomes. f) A severely restricted repertoire of dystrophin complex components in ascidians.
CONCLUSION
We have refined our understanding of the evolutionary history and isoform diversity of the five previously reported vertebrate superfamily members and describe two novel members, dystrotelin and gamma-dystrobrevin. Dystrotelins, dystrophins and dystrobrevins are roughly equally related to each other. Vertebrates therefore have a repertoire of seven superfamily members (three dystrophins, three dystrobevins, and one dystrotelin), with one lost in tetrapods. Most invertebrates studied have one member from each branch. Although the basic shared function which is implied by the common architecture of these distantly related proteins remains unclear, it clearly permeates metazoan biology.
Topics: Amino Acid Sequence; Animals; Drosophila; Drosophila Proteins; Dystrophin; Dystrophin-Associated Proteins; Expressed Sequence Tags; Humans; Membrane Proteins; Mice; Molecular Sequence Data; Multigene Family; Promoter Regions, Genetic; Protein Isoforms; Sequence Homology, Amino Acid; Zebrafish
PubMed: 17233888
DOI: 10.1186/1471-2164-8-19 -
Biochimica Et Biophysica Acta Feb 2007Duchenne muscular dystrophy is the most prevalent and severe form of human muscular dystrophy. Investigations into the molecular basis for Duchenne muscular dystrophy... (Review)
Review
Duchenne muscular dystrophy is the most prevalent and severe form of human muscular dystrophy. Investigations into the molecular basis for Duchenne muscular dystrophy were greatly facilitated by seminal studies in the 1980s that identified the defective gene and its major protein product, dystrophin. Biochemical studies revealed its tight association with a multi-subunit complex, the so-named dystrophin-glycoprotein complex. Since its description, the dystrophin-glycoprotein complex has emerged as an important structural unit of muscle and also as a critical nexus for understanding a diverse array of muscular dystrophies arising from defects in several distinct genes. The dystrophin homologue utrophin can compensate at the cell/tissue level for dystrophin deficiency, but functions through distinct molecular mechanisms of protein-protein interaction.
Topics: Animals; Dystrophin; Glycoproteins; Humans; Muscular Dystrophy, Duchenne; Protein Interaction Mapping
PubMed: 16829057
DOI: 10.1016/j.bbadis.2006.05.010 -
Archivum Immunologiae Et Therapiae... Aug 2022Duchenne muscular dystrophy (DMD) is a lethal disease caused by X-linked mutations in the dystrophin gene. Dystrophin deficiency results in progressive degeneration of...
Duchenne muscular dystrophy (DMD) is a lethal disease caused by X-linked mutations in the dystrophin gene. Dystrophin deficiency results in progressive degeneration of cardiac, respiratory and skeletal muscles leading to premature death due to cardiopulmonary complications. Currently, no cure exists for DMD. Based on our previous reports confirming a protective effect of human dystrophin expressing chimeric (DEC) cell therapy on cardiac, respiratory, and skeletal muscle function after intraosseous administration, now we assessed long-term safety and biodistribution of human DEC therapy for potential clinical applications in DMD patients. Safety of different DEC doses (1 × 10 and 5 × 10) was assessed at 180 days after systemic-intraosseous administration to mdx/scid mice, a model of DMD. Assessments included: single cell gel electrophoresis assay (COMET assay) to confirm lack of genetic toxicology, magnetic resonance imaging (MRI) for tumorigenicity, and body, muscle and organ weights. Human DEC biodistribution to the target (heart, diaphragm, gastrocnemius muscle) and non-target (blood, bone marrow, lung, liver, spleen) organs was detected by flow cytometry assessment of HLA-ABC markers. Human origin of dystrophin was verified by co-localization of dystrophin and human spectrin by immunofluorescence. No complications were observed after intraosseous transplant of human DEC. COMET assay of donors and fused DEC cells confirmed lack of DNA damage. Biodistribution analysis of HLA-ABC expression revealed dose-dependent presence of human DEC cells in target organs, whereas negligible presence was detected in non-target organs. Human origin of dystrophin in the heart, diaphragm and gastrocnemius muscle was confirmed by co-localization of dystrophin expression with human spectrin. MRI revealed no evidence of tumor formation. Body mass and muscle and organ weights were stable and comparable to vehicle controls, further confirming DEC safety at 180 days post- transplant. This preclinical study confirmed long-term local and systemic safety of human DEC therapy at 180 days after intraosseous administration. Thus, DEC can be considered as a novel myoblast based advanced therapy medicinal product for DMD patients.
Topics: Animals; Cell- and Tissue-Based Therapy; Dystrophin; Humans; Mice; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Spectrin; Tissue Distribution
PubMed: 35978142
DOI: 10.1007/s00005-022-00656-7 -
Annals of Clinical and Translational... Dec 2020The novel morpholino antisense oligonucleotide viltolarsen targets exon 53 of the dystrophin gene, and could be an effective treatment for patients with Duchenne...
OBJECTIVE
The novel morpholino antisense oligonucleotide viltolarsen targets exon 53 of the dystrophin gene, and could be an effective treatment for patients with Duchenne muscular dystrophy (DMD). We investigated viltolarsen's ability to induce dystrophin expression and examined its safety in DMD patients.
METHODS
In this open-label, multicenter, parallel-group, phase 1/2, exploratory study, 16 ambulant and nonambulant males aged 5-12 years with DMD received viltolarsen 40 or 80 mg/kg/week via intravenous infusion for 24 weeks. Primary endpoints were dystrophin expression and exon 53 skipping levels.
RESULTS
In western blot analysis, mean changes in dystrophin expression (% normal) from baseline to Weeks 12 and 24 were - 1.21 (P = 0.5136) and 1.46 (P = 0.1636), respectively, in the 40 mg/kg group, and 0.76 (P = 0.2367) and 4.81 (P = 0.0536), respectively, in the 80 mg/kg group. The increase in mean dystrophin level at Weeks 12 and 24 was significant in the 80 mg/kg group (2.78%; P = 0.0364). Patients receiving 80 mg/kg showed a higher mean exon 53 skipping level (42.4%) than those receiving 40 mg/kg (21.8%). All adverse events were judged to be mild or moderate in intensity and none led to study discontinuation.
INTERPRETATION
Treatment with viltolarsen 40 or 80 mg/kg elicited an increasing trend in dystrophin expression and exon 53 skipping levels, and was safe and well tolerated. The decline in motor function appeared less marked in patients with higher dystrophin levels; this may warrant further investigation. This study supports the potential clinical benefit of viltolarsen.
Topics: Child; Child, Preschool; Dystrophin; Humans; Japan; Male; Muscular Dystrophy, Duchenne; Oligonucleotides; Outcome Assessment, Health Care
PubMed: 33285037
DOI: 10.1002/acn3.51235 -
Skeletal Muscle Jan 2023The dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM)...
Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways.
BACKGROUND
The dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdx) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression.
METHODS
The tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdx mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the ingenuity pathway analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators.
RESULTS
Through our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdx muscle, including those that were (1) unrestored (significantly different from wild type, but not from mdx), (2) restored (significantly different from mdx, but not from wild type), and (3) compensatory (significantly different from both wild type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM-optimized proteomics revealed an increased abundance of collagens II, V, and XI, along with β-spectrin in mdx samples. Using ingenuity pathway analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Yap1, Sox9, Rho, RAC, and Wnt.
CONCLUSIONS
Our findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.
Topics: Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Mechanotransduction, Cellular; Multiomics; Mice, Inbred mdx; Muscle, Skeletal; Cytoskeleton; Membrane Proteins; Neoplasm Proteins
PubMed: 36609344
DOI: 10.1186/s13395-022-00311-x -
FASEB Journal : Official Publication of... Jun 2013Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by the lack of functional dystrophin. There is no cure, but several clinical trials aimed to...
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by the lack of functional dystrophin. There is no cure, but several clinical trials aimed to restore the synthesis of functional dystrophin are underway. The dystrophin levels needed for improvement of muscle pathology, function, and overall vitality are not known. Here, we describe the mdx/utrn(-/-)/Xist(Δhs) mouse model, which expresses a range of low dystrophin levels, depending on the degree of skewing of X inactivation in a utrophin-negative background. Mdx/utrn(-/-) mice develop severe muscle weakness, kyphosis, respiratory and heart failure, and premature death closely resembling DMD pathology. We show that at dystrophin levels < 4%, survival and motor function in these animals are greatly improved. In mice expressing >4% dystrophin, histopathology is ameliorated, as well. These findings suggest that the dystrophin levels needed to benefit vitality and functioning of patients with DMD might be lower than those needed for full protection against muscle damage.
Topics: Animals; Biomarkers; Dystrophin; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Mice, Knockout; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Animal; Muscular Dystrophy, Duchenne; Phenotype; Utrophin
PubMed: 23460734
DOI: 10.1096/fj.12-224170