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Proceedings of the National Academy of... Jan 2023Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial...
Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.
Topics: Female; Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Muscles; Gene Editing; Treatment Outcome; Mice, Inbred mdx; Muscle, Skeletal; Disease Models, Animal
PubMed: 36595689
DOI: 10.1073/pnas.2206324120 -
Proceedings of the National Academy of... Jun 2021Recent advances in gene editing technologies are enabling the potential correction of devastating monogenic disorders through elimination of underlying genetic... (Review)
Review
Recent advances in gene editing technologies are enabling the potential correction of devastating monogenic disorders through elimination of underlying genetic mutations. Duchenne muscular dystrophy (DMD) is an especially severe genetic disorder caused by mutations in the gene encoding dystrophin, a membrane-associated protein required for maintenance of muscle structure and function. Patients with DMD succumb to loss of mobility early in life, culminating in premature death from cardiac and respiratory failure. The disease has thus far defied all curative strategies. CRISPR gene editing has provided new opportunities to ameliorate the disease by eliminating DMD mutations and thereby restore dystrophin expression throughout skeletal and cardiac muscle. Proof-of-concept studies in rodents, large mammals, and human cells have validated the potential of this approach, but numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene editing components throughout the musculature, and mitigation of possible immune responses. This paper provides an overview of recent work from our laboratory and others toward the genetic correction of DMD and considers the opportunities and challenges in the path to clinical translation. Lessons learned from these studies will undoubtedly enable further applications of gene editing to numerous other diseases of muscle and other tissues.
Topics: Animals; CRISPR-Cas Systems; Dystrophin; Gene Editing; Genetic Therapy; Humans; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Myocardium
PubMed: 34074727
DOI: 10.1073/pnas.2004840117 -
Biochimica Et Biophysica Acta.... Jan 2021The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is...
The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is poorly understood. In this study, we characterized lenses derived from the dystrophin-deficient mdx mouse model. Expression of Dp71, a predominant isoform of dystrophin in the lens, was induced during lens fiber cell differentiation. Dp71 was found to co-distribute with dystroglycan, connexin-50 and 46, aquaporin-0, and NrCAM as a large cluster at the center of long arms of the hexagonal fibers. Although mdx mouse lenses exhibited dramatically reduced levels of Dp71, only older lenses revealed punctate nuclear opacities compared to littermate wild type (WT) lenses. The levels of dystroglycan, syntrophin, and dystrobrevin which comprise the dystrophin-associated protein complex (DAPC), and NrCAM, connexin-50, and aquaporin-0, were significantly lower in the lens membrane fraction of adult mdx mice compared to WT mice. Additionally, decreases were observed in myosin light chain phosphorylation and lens stiffness together with a significant elevation in the levels of utrophin, a functional homolog of dystrophin in mdx mouse lenses compared to WT lenses. The levels of perlecan and laminin (ligands of α-dystroglycan) remained normal in dystrophin-deficient lens fibers. Taken together, although mdx mouse lenses exhibit only minor defects in lens clarity possibly due to a compensatory increase in utrophin, the noted disruptions of DAPC, stability, and organization of membrane integral proteins of fibers, and stiffness of mdx lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.
Topics: Animals; Dystrophin; Eye Proteins; Gene Expression Regulation; Lens, Crystalline; Mice; Mice, Inbred mdx
PubMed: 33127476
DOI: 10.1016/j.bbadis.2020.165998 -
Life Science Alliance Oct 2021Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy....
Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.
Topics: Actins; Animals; Cell Line; Dystrophin; Mice; Myoblasts, Skeletal; Serum Response Factor; Signal Transduction; Utrophin; rhoA GTP-Binding Protein
PubMed: 34389686
DOI: 10.26508/lsa.202101014 -
Biochimica Et Biophysica Acta Feb 2014Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the... (Review)
Review
Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.
Topics: Animals; Dystrophin; Humans; Multiprotein Complexes; Muscle Contraction; Signal Transduction
PubMed: 24021238
DOI: 10.1016/j.bbamem.2013.08.023 -
Cellular Oncology (Dordrecht) Feb 2021Mutation of the Duchenne muscular dystrophy (DMD) gene causes Duchenne and Becker muscular dystrophy, degenerative neuromuscular disorders that primarily affect... (Review)
Review
BACKGROUND
Mutation of the Duchenne muscular dystrophy (DMD) gene causes Duchenne and Becker muscular dystrophy, degenerative neuromuscular disorders that primarily affect voluntary muscles. However, increasing evidence implicates DMD in the development of all major cancer types. DMD is a large gene with 79 exons that codes for the essential muscle protein dystrophin. Alternative promotor usage drives the production of several additional dystrophin protein products with roles that extend beyond skeletal muscle. The importance and function(s) of these gene products outside of muscle are not well understood.
CONCLUSIONS
We highlight a clear role for DMD in the pathogenesis of several cancers, including sarcomas, leukaemia's, lymphomas, nervous system tumours, melanomas and various carcinomas. We note that the normal balance of DMD gene products is often disrupted in cancer. The short dystrophin protein Dp71 is, for example, typically maintained in cancer whilst the full-length Dp427 gene product, a likely tumour suppressor, is frequently inactivated in cancer due to a recurrent loss of 5' exons. Therefore, the ratio of short and long gene products may be important in tumorigenesis. In this review, we summarise the tumours in which DMD is implicated and provide a hypothesis for possible mechanisms of tumorigenesis, although the question of cause or effect may remain. We hope to stimulate further study into the potential role of DMD gene products in cancer and the development of novel therapeutics that target DMD.
Topics: Animals; Dystrophin; Genetic Predisposition to Disease; Humans; Models, Biological; Muscular Dystrophy, Duchenne; Neoplasms
PubMed: 33188621
DOI: 10.1007/s13402-020-00572-y -
Neuroscience Letters Mar 2020The Dystrophin Glycoprotein Complex (DGC) is a large multi-protein complex that links cytoskeleton actin to the extracellular matrix. This complex is critical in... (Review)
Review
The Dystrophin Glycoprotein Complex (DGC) is a large multi-protein complex that links cytoskeleton actin to the extracellular matrix. This complex is critical in maintaining the structural integrity of muscle fibers and the stability of the neuromuscular synapse. The DGC consists of dystrophin and its utrophin homolog, as well as dystroglycans, sarcoglycans, sarcospan, syntrophins, and dystrobrevins. Deficiencies in DGC proteins result in several forms of muscular dystrophy with varying symptoms and degrees of severity in addition to structurally abnormal neuromuscular junctions (NMJs). This mini-review highlights current knowledge regarding the role of the DGC on the molecular dynamics of acetylcholine receptors (AChRs) as it relates to the formation and maintenance of the mammalian NMJ.
Topics: Animals; Dystrophin; Glycoproteins; Humans; Neuromuscular Junction; Receptors, Cholinergic
PubMed: 32057921
DOI: 10.1016/j.neulet.2020.134833 -
Circulation Research Sep 2017Gene editing is moving rapidly from a highly useful laboratory-based tool towards human clinical application. gene editing has been documented in mouse models of...
Gene editing is moving rapidly from a highly useful laboratory-based tool towards human clinical application. gene editing has been documented in mouse models of Duchenne Muscular Dystrophy, where skeletal and cardiac muscle editing produces internally-truncated dystrophin. A recent report now demonstrates that editing the dystrophin gene in the heart improves cardiac function, paving the path to application of cardiac gene correction.
Topics: Animals; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Dystrophin; Gene Editing; Mice; Mutation
PubMed: 28963182
DOI: 10.1161/CIRCRESAHA.117.311865 -
Methods in Molecular Biology (Clifton,... 2023Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder, caused by mutations in the DMD gene coding dystrophin. Applying clustered regularly interspaced...
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder, caused by mutations in the DMD gene coding dystrophin. Applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) for therapeutic gene editing represents a promising technology to correct this devastating disease through elimination of underlying genetic mutations. Adeno-associated virus (AAV) has been widely used for gene therapy due to its low immunogenicity and high tissue tropism. In particular, CRISPR-Cas9 gene editing components packaged by self-complementary AAV (scAAV) demonstrate robust viral transduction and efficient gene editing, enabling restoration of dystrophin expression throughout skeletal and cardiac muscle in animal models of DMD. Here, we describe protocols for cloning CRISPR single guide RNAs (sgRNAs) into a scAAV plasmid and procedures for systemic delivery of AAVs into a DMD mouse model. We also provide methodologies for quantification of dystrophin restoration after systemic CRISPR-Cas9-mediated correction of DMD.
Topics: Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Dependovirus; CRISPR-Cas Systems; Exons; Muscle, Skeletal
PubMed: 36401041
DOI: 10.1007/978-1-0716-2772-3_21 -
International Journal of Molecular... Oct 2021Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the gene encoding diverse isoforms of dystrophin.... (Review)
Review
Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease.
Topics: Calcium; Calcium Signaling; Dystrophin; Endoplasmic Reticulum; Humans; Mitochondria; Muscle, Skeletal; Muscular Dystrophy, Duchenne
PubMed: 34681707
DOI: 10.3390/ijms222011040