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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 -
Molecular Neurobiology Mar 2020Duchenne muscular dystrophy (DMD) is caused by frameshift mutations in the DMD gene that prevent the body-wide translation of its protein product, dystrophin. Besides a... (Review)
Review
Duchenne muscular dystrophy (DMD) is caused by frameshift mutations in the DMD gene that prevent the body-wide translation of its protein product, dystrophin. Besides a severe muscle phenotype, cognitive impairment and neuropsychiatric symptoms are prevalent. Dystrophin protein 71 (Dp71) is the major DMD gene product expressed in the brain and mutations affecting its expression are associated with the DMD neuropsychiatric syndrome. As with dystrophin in muscle, Dp71 localises to dystrophin-associated protein complexes in the brain. However, unlike in skeletal muscle; in the brain, Dp71 is alternatively spliced to produce many isoforms with differential subcellular localisations and diverse cellular functions. These include neuronal differentiation, adhesion, cell division and excitatory synapse organisation as well as nuclear functions such as nuclear scaffolding and DNA repair. In this review, we first describe brain involvement in DMD and the abnormalities observed in the DMD brain. We then review the gene expression, RNA processing and functions of Dp71. We review genotype-phenotype correlations and discuss emerging cellular/tissue evidence for the involvement of Dp71 in the neuropathophysiology of DMD. The literature suggests changes observed in the DMD brain are neurodevelopmental in origin and that their risk and severity is associated with a cumulative loss of distal DMD gene products such as Dp71. The high risk of neuropsychiatric syndromes in Duchenne patients warrants early intervention to achieve the best possible quality of life. Unravelling the function and pathophysiological significance of dystrophin in the brain has become a high research priority to inform the development of brain-targeting treatments for Duchenne.
Topics: Animals; Brain; Dystrophin; Gene Expression; Humans; Muscular Dystrophy, Duchenne; Synapses
PubMed: 31836945
DOI: 10.1007/s12035-019-01845-w -
Skeletal Muscle May 2017Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the DMD gene and loss of the protein dystrophin. The absence of dystrophin leads to... (Review)
Review
Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the DMD gene and loss of the protein dystrophin. The absence of dystrophin leads to myofiber membrane fragility and necrosis, with eventual muscle atrophy and contractures. Affected boys typically die in their second or third decade due to either respiratory failure or cardiomyopathy. Despite extensive attempts to develop definitive therapies for DMD, the standard of care remains prednisone, which has only palliative benefits. Animal models, mainly the mdx mouse and golden retriever muscular dystrophy (GRMD) dog, have played a key role in studies of DMD pathogenesis and treatment development. Because the GRMD clinical syndrome is more severe than in mice, better aligning with the progressive course of DMD, canine studies may translate better to humans. The original founder dog for all GRMD colonies worldwide was identified in the early 1980s before the discovery of the DMD gene and dystrophin. Accordingly, analogies to DMD were initially drawn based on similar clinical features, ranging from the X-linked pattern of inheritance to overlapping histopathologic lesions. Confirmation of genetic homology between DMD and GRMD came with identification of the underlying GRMD mutation, a single nucleotide change that leads to exon skipping and an out-of-frame DMD transcript. GRMD colonies have subsequently been established to conduct pathogenetic and preclinical treatment studies. Simultaneous with the onset of GRMD treatment trials, phenotypic biomarkers were developed, allowing definitive characterization of treatment effect. Importantly, GRMD studies have not always substantiated findings from mdx mice and have sometimes identified serious treatment side effects. While the GRMD model may be more clinically relevant than the mdx mouse, usage has been limited by practical considerations related to expense and the number of dogs available. This further complicates ongoing broader concerns about the poor rate of translation of animal model preclinical studies to humans with analogous diseases. Accordingly, in performing GRMD trials, special attention must be paid to experimental design to align with the approach used in DMD clinical trials. This review provides context for the GRMD model, beginning with its original description and extending to its use in preclinical trials.
Topics: Animals; Disease Models, Animal; Dogs; Dystrophin; Muscular Dystrophy, Duchenne
PubMed: 28526070
DOI: 10.1186/s13395-017-0124-z -
Journal of Cellular Physiology Jul 2018Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell... (Review)
Review
Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signaling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signaling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signaling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role, and functioning of DGC and may lead to a deeper understanding of its role in several human diseases.
Topics: Cell Membrane; Dystrophin; Dystrophin-Associated Protein Complex; Glycoproteins; Humans; Multiprotein Complexes; Muscle, Skeletal; Signal Transduction
PubMed: 28464259
DOI: 10.1002/jcp.25982 -
Gene May 2023Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic... (Review)
Review
Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.
Topics: Humans; Muscular Dystrophy, Duchenne; Dystrophin; CRISPR-Cas Systems; Gene Editing; Genetic Therapy
PubMed: 36914142
DOI: 10.1016/j.gene.2023.147358 -
Journal of Cachexia, Sarcopenia and... Aug 2023Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular...
BACKGROUND
Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho-associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown.
METHODS
Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine.
RESULTS
Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P < 0.05) and in mice (+25%, P < 0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P < 0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P < 0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles.
CONCLUSIONS
Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.
Topics: Animals; Mice; Dystrophin; Mice, Inbred mdx; Muscle Weakness; Muscle, Skeletal; Muscular Dystrophy, Duchenne
PubMed: 37311604
DOI: 10.1002/jcsm.13278 -
Neuroscience Letters Oct 2020The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the protein dystrophin and is characterized as a neuromuscular... (Review)
Review
The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the protein dystrophin and is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but data show that both human patients and animal models for DMD present with fragmented neuromuscular junction (NMJ) morphology. In addition to pre- and post-synaptic abnormalities, studies indicate increased susceptibility of the NMJ to contraction-induced injury, with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyographic activity. Such findings suggest that alterations in the NMJ of dystrophic muscle may play a role in muscle weakness via impairment of neuromuscular transmission. Further work is needed to fully understand the role of the NMJ in the weakness, susceptibility to injury, and progressive wasting associated with DMD.
Topics: Animals; Dystrophin; Humans; Mice; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Neuromuscular Junction
PubMed: 32818587
DOI: 10.1016/j.neulet.2020.135304 -
Acta Physiologica (Oxford, England) Apr 2021Loss of dystrophin causes oxidative stress and affects nitric oxide synthase-mediated vascular function in striated muscle. Because tetrahydrobiopterin is an antioxidant...
AIM
Loss of dystrophin causes oxidative stress and affects nitric oxide synthase-mediated vascular function in striated muscle. Because tetrahydrobiopterin is an antioxidant and co-factor for nitric oxide synthase, we tested the hypothesis that tetrahydrobiopterin would be low in mdx mice and humans deficient for dystrophin.
METHODS
Tetrahydrobiopterin and its metabolites were measured at rest and in response to exercise in Duchenne and Becker muscular dystrophy patients, age-matched male controls as well as wild-type, mdx and mdx mice transgenically overexpressing skeletal muscle-specific dystrophins. Mdx mice were also supplemented with tetrahydrobiopterin and pathophysiology was assessed.
RESULTS
Duchenne muscular dystrophy patients had lower urinary dihydrobiopterin + tetrahydrobiopterin/specific gravity compared to unaffected age-matched males and Becker muscular dystrophy patients. Mdx mice had low urinary and skeletal muscle dihydrobiopterin + tetrahydrobiopterin compared to wild-type mice. Overexpression of dystrophins that localize neuronal nitric oxide synthase restored dihydrobiopterin + tetrahydrobiopterin in mdx mice to wild-type levels while utrophin overexpression did not. Mdx mice and Duchenne muscular dystrophy patients did not increase tetrahydrobiopterin during exercise and in mdx mice tetrahydrobiopterin deficiency was likely because of lower levels of sepiapterin reductase in skeletal muscle. Tetrahydrobiopterin supplementation improved skeletal muscle strength, resistance to fatiguing and injurious contractions in vivo, increased utrophin and capillary density of skeletal muscle and lowered cardiac muscle fibrosis and left ventricular wall thickness in mdx mice.
CONCLUSION
These data demonstrate that impaired tetrahydrobiopterin synthesis is associated with dystrophin loss and treatment with tetrahydrobiopterin improves striated muscle histopathology and skeletal muscle function in mdx mice.
Topics: Animals; Biopterins; Dystrophin; Humans; Male; Mice; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Utrophin
PubMed: 33580591
DOI: 10.1111/apha.13627 -
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 -
Current Gene Therapy 2024
Topics: Humans; Muscular Dystrophy, Duchenne; Genetic Therapy; Dystrophin; Genetic Vectors
PubMed: 36959131
DOI: 10.2174/1566523223666230320120839