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1.
Zool Res ; 42(5): 650-659, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34472226

RESUMO

Phosphatidylserine (PS) is distributed asymmetrically in the plasma membrane of eukaryotic cells. Phosphatidylserine flippase (P4-ATPase) transports PS from the outer leaflet of the lipid bilayer to the inner leaflet of the membrane to maintain PS asymmetry. The ß subunit TMEM30A is indispensable for transport and proper function of P4-ATPase. Previous studies have shown that the ATP11A and TMEM30A complex is the molecular switch for myotube formation. However, the role of Tmem30a in skeletal muscle regeneration remains elusive. In the current study, Tmem30a was highly expressed in the tibialis anterior (TA) muscles of dystrophin-null ( mdx) mice and BaCl 2-induced muscle injury model mice. We generated a satellite cell (SC)-specific Tmem30a conditional knockout (cKO) mouse model to investigate the role of Tmem30a in skeletal muscle regeneration. The regenerative ability of cKO mice was evaluated by analyzing the number and diameter of regenerated SCs after the TA muscles were injured by BaCl 2-injection. Compared to the control mice, the cKO mice showed decreased Pax7 + and MYH3 + SCs, indicating diminished SC proliferation, and decreased expression of muscular regulatory factors (MYOD and MYOG), suggesting impaired myoblast proliferation in skeletal muscle regeneration. Taken together, these results demonstrate the essential role of Tmem30a in skeletal muscle regeneration.


Assuntos
Proteínas de Membrana/metabolismo , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Proliferação de Células , Distrofina/genética , Distrofina/metabolismo , Antagonistas de Estrogênios/toxicidade , Regulação da Expressão Gênica/fisiologia , Genótipo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Tamoxifeno/toxicidade
2.
Cell Mol Life Sci ; 78(14): 5447-5468, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34091693

RESUMO

Dystrophin is a large protein serving as local scaffolding repetitively bridging cytoskeleton and the outside of striated muscle cell. As such dystrophin is a critical brick primarily in dystrophin-associated protein complex (DAGC) and in a larger submembranous unit, costamere. Accordingly, the lack of functional dystrophin laying at the root of Duchenne muscular dystrophy (DMD) drives sarcolemma instability. From this point on, the cascade inevitably leading to the death of myocyte begins. In cardiomyocytes, intracellular calcium overload and related mitochondrial-mediated cell death mainly contribute to myocardial dysfunction and dilation while other protein dysregulation and/or mislocalization may affect electrical conduction system and favor arrhythmogenesis. Although clinically DMD manifests as progressive muscle weakness and skeletal muscle symptoms define characteristic of DMD, it is the heart problem the biggest challenge that most often develop in the form of dilated cardiomyopathy (DCM). Current standards of treatment and recent progress in respiratory care, introduced in most settings in the 1990s, have improved quality of life and median life expectancy to 4th decade of patient's age. At the same time, cardiac causes of death related to DMD increases. Despite preventive and palliative cardiac treatments available, the prognoses remain poor. Direct therapeutic targeting of dystrophin deficiency is critical, however, hindered by the large size of the dystrophin cDNA and/or stochastic, often extensive genetic changes in DMD gene. The correlation between cardiac involvement and mutations affecting specific dystrophin isoforms, may provide a mutation-specific cardiac management and novel therapeutic approaches for patients with CM. Nonetheless, the successful cardiac treatment poses a big challenge and may require combined therapy to combat dystrophin deficiency and its after-effects (critical in DMD pathogenesis). This review locates the multifaceted heart problem in the course of DMD, balancing the insights into basic science, translational efforts and clinical manifestation of dystrophic heart disease.


Assuntos
Arritmias Cardíacas/patologia , Cardiomiopatias/patologia , Distrofina/metabolismo , Distrofia Muscular de Duchenne/complicações , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Humanos
3.
Nat Commun ; 12(1): 3719, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140489

RESUMO

Recent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx4cv mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases.


Assuntos
Sistemas CRISPR-Cas , Distrofina/genética , Edição de Genes/métodos , Terapia Genética/métodos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Animais , Linhagem Celular , Dependovirus , Modelos Animais de Doenças , Distrofina/metabolismo , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mutação , RNA Guia/genética , RNA-Seq
4.
FASEB J ; 35(6): e21628, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33982338

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in dystrophin gene, affecting 1/3000 males. Gene therapy has been proven to ameliorate dystrophic pathology. To investigate therapeutic benefits from long-term effect of human mini-dystrophin and functional outcomes, transgenic mdx mice (Tg-mdx) containing a single copy of human mini-dystrophin (∆hDys3849) gene, five rods (Rods1-2, Rods22-24), and two hinges (H1 and H4) driven by a truncated creatine-kinase promoter (dMCK) in a recombinant adeno-associated viral vector (rAAV) backbone, were generated and used to determine gene expression and improvement of muscle function. Human mini-dystrophin gene expression was found in a majority of the skeletal muscles, but no expression in cardiac muscle. Dystrophin-associated glycoproteins (DAGs) such as sarcoglycans and nNOS were restored at the sarcolemma and coincided with human mini-dystrophin gene expression at the ages of 6, 10, and 20 months; Morphology of dystrophic muscle expressing the human mini-dystrophin gene was improved and central nuclei were reduced. Myofiber membrane integrity was improved by Evans blue dye test. Improvement in treadmill running and grip force was observed in transgenic mice at 6 months. Tetanic force and specific force of tibialis anterior (TA) muscle were significantly increased at the ages of 6, 10, and 20 months. Pseudohypertrophy was not found in TA muscle at 10 and 20 months when compared with wild-type C57 (WT) group. This study demonstrated that the long-term effects of human mini-dystrophin effectively ameliorated pathology and improved the functions of the dystrophic muscles in the transgenic DMD mouse model.


Assuntos
Distrofina/metabolismo , Terapia Genética , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Animais , Distrofina/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/citologia , Distrofia Muscular Animal/etiologia , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/etiologia , Distrofia Muscular de Duchenne/patologia
5.
Biochem Biophys Res Commun ; 560: 152-158, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33989907

RESUMO

Dp71 and Dp40 are the main products of the DMD gene in the central nervous system, and they are developmentally regulated from the early stages of embryonic development to adulthood. To further study the roles of Dp71 and Dp40 during cell proliferation and neural differentiation, we analyzed Dp71/Dp40 isoform expression at the mRNA level by RT-PCR assays to identify alternative splicing (AS) in the isoforms expressed in rat neural stem/progenitor cells (NSPCs) and in differentiated cells (neurons and glia). We found that proliferating NSPCs expressed Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71, Dp71dΔ74 and Dp40, as well as two Dp40 isoforms: Dp40Δ63,64 and Dp40Δ64-67. In differentiated cells we also found the expression of Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71 and Dp40. However, the expression frequencies were different in both stages. In addition, in differentiated cells, we found Dp71fΔ71-74, and interestingly, we did not find the expression of Dp71dΔ74 or the newly identified Dp40 isoforms. In this work we show that NSPC differentiation is accompanied by changes in Dp71/Dp40 isoform expression, suggesting different roles for these isoforms in NSPCs proliferation and neuronal differentiation, and we describe, for the first time, alternative splicing of Dp40.


Assuntos
Processamento Alternativo , Distrofina/genética , Células-Tronco Neurais/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Distrofina/metabolismo , Células-Tronco Neurais/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de RNA/metabolismo , Ratos Wistar
6.
Nutrients ; 13(3)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808773

RESUMO

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited neuromuscular disorder that causes loss of muscle mass and motor skills. In the era of genomic medicine, there is still no known cure for DMD. In clinical practice, there is a growing awareness of the possible importance of nutrition in neuromuscular diseases. This is mostly the result of patients' or caregivers' empirical reports of how active substances derived from food have led to improved muscle strength and, thus, better quality of life. In this report, we investigate several nutraceutical principles in the sapje strain of zebrafish, a validated model of DMD, in order to identify possible natural products that, if supplemented in the diet, might improve the quality of life of DMD patients. Gingerol, a constituent of fresh ginger, statistically increased the locomotion of mutant larvae and upregulated the expression of heme oxygenase 1, a target gene for therapy aimed at improving dystrophic symptoms. Although three other compounds showed a partial positive effect on locomotor and muscle structure phenotypes, our nutraceutical screening study lent preliminary support to the efficacy and safety only of gingerol. Gingerol could easily be proposed as a dietary supplement in DMD.


Assuntos
Catecóis/administração & dosagem , Suplementos Nutricionais , Álcoois Graxos/administração & dosagem , Distrofia Muscular de Duchenne/dietoterapia , Animais , Fibras na Dieta , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Feminino , Heme Oxigenase-1 , Larva , Locomoção , Masculino , Força Muscular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Qualidade de Vida , Peixe-Zebra
7.
Cell Mol Life Sci ; 78(11): 4867-4891, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33825942

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating chromosome X-linked disease that manifests predominantly in progressive skeletal muscle wasting and dysfunctions in the heart and diaphragm. Approximately 1/5000 boys and 1/50,000,000 girls suffer from DMD, and to date, the disease is incurable and leads to premature death. This phenotypic severity is due to mutations in the DMD gene, which result in the absence of functional dystrophin protein. Initially, dystrophin was thought to be a force transducer; however, it is now considered an essential component of the dystrophin-associated protein complex (DAPC), viewed as a multicomponent mechanical scaffold and a signal transduction hub. Modulating signal pathway activation or gene expression through epigenetic modifications has emerged at the forefront of therapeutic approaches as either an adjunct or stand-alone strategy. In this review, we propose a broader perspective by considering DMD to be a disease that affects myofibers and muscle stem (satellite) cells, as well as a disorder in which abrogated communication between different cell types occurs. We believe that by taking this systemic view, we can achieve safe and holistic treatments that can restore correct signal transmission and gene expression in diseased DMD tissues.


Assuntos
Comunicação Celular , Distrofina/metabolismo , Distrofia Muscular de Duchenne/patologia , Transdução de Sinais , Animais , Osso e Ossos/metabolismo , Distrofina/química , Distrofina/genética , Humanos , Microvasos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Neurônios/metabolismo , Transdução de Sinais/genética
8.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805378

RESUMO

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.


Assuntos
Distrofina/genética , Guanidina/química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Linhagem Celular , Distrofina/metabolismo , Éxons , Marcação de Genes/métodos , Humanos , Ácidos Nucleicos/química , Oligonucleotídeos/síntese química , Splicing de RNA , Temperatura , Transfecção
9.
Am J Physiol Cell Physiol ; 320(6): C956-C965, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33729835

RESUMO

The dystrophin-glycoprotein complex (DGC) is a multiprotein structure required to maintain muscle fiber membrane integrity, transmit force by linking the actin cytoskeleton with the extracellular matrix, and maintain muscle homeostasis. Membrane localization of dystrophin is perturbed in muscles wasting as a consequence of cancer cachexia, tenotomy, and advanced aging, which are all associated with low level, chronic inflammation. Strategies to preserve dystrophin expression at the sarcolemma might therefore combat muscle wasting. Phosphorylation of dystrophin serine 3059 (S3059) enhances the interaction between dystrophin and ß-dystroglycan. To test the contribution of amino acid phosphorylation to muscle fiber size changes, dystrophin constructs with phospho-null and phosphomimetic mutations were transfected into C2C12 muscle cells or AAV-293 cells in the presence or absence of kinase inhibitors/activators to assess effects on myotube diameter and protein function. Overexpression of a dystrophin construct with a phospho-null mutation at S3059 in vitro reduced myotube size in healthy C2C12 cells. Conversely overexpression of a phosphomimetic mutation at S3059 attenuated inflammation-induced myotube atrophy. Increased ERK activation by addition of phorbol myristate acetate (PMA) also reduced inflammation-associated myotube atrophy and increased the interaction between dystrophin and ß-dystroglycan. These findings demonstrate a link between increased ERK activation, dystrophin S3059 phosphorylation, stabilization of the DGC, and the regulation of muscle fiber size. Interventions that increase dystrophin S3059 phosphorylation to promote stronger binding of dystrophin to ß-dystroglycan may have therapeutic potential for attenuation of inflammation-associated muscle wasting.


Assuntos
Distrofina/metabolismo , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Fosforilação/fisiologia , Animais , Caquexia/metabolismo , Membrana Celular/metabolismo , Distroglicanas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Sarcolema/metabolismo
10.
Biochemistry ; 60(10): 765-779, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33656846

RESUMO

Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein. That is one possible therapeutic aim, but it can also be used to restore the reading frame for diseases caused by frameshift mutations, which is the case for Duchenne muscular dystrophy (DMD). DMD most commonly arises as a result of large exonic deletions that create a frameshift and abolish protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is restoration of protein expression by reading frame correction. However, the therapeutically expressed protein is missing both the region of the underlying genetic defect and the therapeutically skipped exon. How removing some region from the middle of a protein affects its structure and function is unclear. Many different underlying deletions are known, and exon skipping can be applied in many ways, in some cases in different ways to the same defect. These vary in how severely perturbative they are, with possible clinical consequences. In this study, we examine a systematic, comprehensive panel of exon edits in a region of dystrophin and identify for the first time exon edits that are minimally perturbed and appear to keep the structural stability similar to that of wild-type protein. We also identify factors that appear to be correlated with how perturbative an edit is.


Assuntos
Distrofina/química , Endopeptidase K/metabolismo , Éxons , Distrofina/genética , Distrofina/metabolismo , Humanos , Conformação Proteica , Estabilidade Proteica , Proteólise
11.
Biochem Biophys Res Commun ; 545: 40-45, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33540285

RESUMO

Duchenne muscular dystrophy (DMD), the most common lethal muscular disorder, affects 1 in 5000 male births. It is caused by mutations in the X-linked dystrophin gene (DMD), and there is no effective treatment currently. Gene addition is a promising strategy owing to its universality for patients with all gene mutations types. In this study, we describe a site-specific gene addition strategy in induced pluripotent stem cells (iPSCs) derived from a DMD patient with exon 50 deletion. By using transcription activator-like effector nickases (TALENickases), the mini-dystrophin cassette was precisely targeted at the ribosomal RNA gene (rDNA) locus via homologous recombination with high targeting efficiency. The targeted clone retained the main pluripotent properties and was differentiated into cardiomyocytes. Significantly, the dystrophin expression and membrane localization were restored in the genetic corrected iPSCs and their derived cardiomyocytes. More importantly, the enhanced spontaneous contraction was observed in modified cardiomyocytes. These results provide a proof of principle for an efficient targeted gene addition for DMD gene therapy and represents a significant step toward precisely therapeutic for DMD.


Assuntos
DNA Ribossômico/genética , Distrofina/genética , Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Diferenciação Celular , Linhagem Celular , Técnicas de Reprogramação Celular , Distrofina/metabolismo , Éxons , Expressão Gênica , Marcação de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mutação com Perda de Função , Masculino , Distrofia Muscular de Duchenne/urina , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estudo de Prova de Conceito , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Urina/citologia
12.
Am J Pathol ; 191(4): 730-747, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33497702

RESUMO

Duchenne muscular dystrophy (DMD) is a genetic, degenerative, striated muscle disease exacerbated by chronic inflammation. Mdx mice in the genotypic DMD model poorly represent immune-mediated pathology observed in patients. Improved understanding of innate immunity in dystrophic muscles is required to develop specific anti-inflammatory treatments. Here, inflammation in mdx mice and the more fibrotic utrn+/-;mdx Het model was comprehensively investigated. Unbiased analysis showed that mdx and Het mice contain increased levels of numerous chemokines and cytokines, with further increased in Het mice. Chemokine and chemokine receptor gene expression levels were dramatically increased in 4-week-old dystrophic quadriceps muscles, and to a lesser extent in diaphragm during the early injury phase, and had a small but consistent increase at 8 and 20 weeks. An optimized direct immune cell isolation method prevented loss of up to 90% of macrophages with density-dependent centrifugation previously used for mdx flow cytometry. Het quadriceps contain higher proportions of neutrophils and infiltrating monocytes than mdx, and higher percentages of F4/80Hi, but lower percentages of F4/80Lo cells and patrolling monocytes compared with Het diaphragms. These differences may restrict regenerative potential of dystrophic diaphragms, increasing pathologic severity. Fibrotic and inflammatory gene expression levels are higher in myeloid cells isolated from Het compared with mdx quadriceps, supporting Het mice may represent an improved model for testing therapeutic manipulation of inflammation in DMD.


Assuntos
Distrofina/metabolismo , Inflamação/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/patologia , Animais , Inflamação/patologia , Macrófagos/metabolismo , Camundongos Transgênicos , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculos Respiratórios/metabolismo , Músculos Respiratórios/patologia
13.
Ann Neurol ; 89(2): 280-292, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33159473

RESUMO

OBJECTIVE: This study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD mutations. METHODS: We performed a retrospective multicentric cohort study by using molecular and clinical data from patients with DMD mutations registered in the Universal Mutation Database-DMD France database. Patients with intronic, splice site, or nonsense DMD mutations, with available muscle biopsy Western blot data, were included irrespective of whether they presented with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD). Patients were separated into 3 groups based on dystrophin protein levels. Clinical outcomes were ages at appearance of first symptoms; loss of ambulation; fall in vital capacity and left ventricular ejection fraction; interventions such as spinal fusion, tracheostomy, and noninvasive ventilation; and death. RESULTS: Of 3,880 patients with DMD mutations, 90 with mutations of interest were included. Forty-two patients expressed no dystrophin (group A), and 31 of 42 (74%) developed DMD. Thirty-four patients had dystrophin quantities < 5% (group B), and 21 of 34 (61%) developed BMD. Fourteen patients had dystrophin quantities ≥ 5% (group C), and all but 4 who lost ambulation beyond 24 years of age were ambulant. Dystrophin quantities of <5%, as low as <0.5%, were associated with milder phenotype for most of the evaluated clinical outcomes, including age at loss of ambulation (p < 0.001). INTERPRETATION: Very low residual dystrophin protein quantity can cause a shift in disease phenotype from DMD toward BMD. ANN NEUROL 2021;89:280-292.


Assuntos
Distrofina/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Adolescente , Corticosteroides/uso terapêutico , Adulto , Idade de Início , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Western Blotting , Criança , Estudos de Coortes , Progressão da Doença , Distrofina/genética , Humanos , Masculino , Limitação da Mobilidade , Mortalidade , Distrofia Muscular de Duchenne/terapia , Ventilação não Invasiva/estatística & dados numéricos , Oxidiazóis/uso terapêutico , Fenótipo , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Índice de Gravidade de Doença , Fusão Vertebral/estatística & dados numéricos , Volume Sistólico , Traqueostomia/estatística & dados numéricos , Capacidade Vital , Adulto Jovem
14.
Biochim Biophys Acta Mol Basis Dis ; 1867(1): 165998, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127476

RESUMO

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 mdx3cv 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 mdx3cv 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 mdx3cv 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 mdx3cv 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 mdx3cv 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 mdx3cv lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.


Assuntos
Distrofina/deficiência , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica , Cristalino/metabolismo , Animais , Distrofina/metabolismo , Proteínas do Olho/genética , Camundongos , Camundongos Endogâmicos mdx
15.
Aging (Albany NY) ; 12(23): 23525-23547, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33276344

RESUMO

Some genes are essential for survival, while other genes play modulatory roles on health and survival. Genes that play modulatory roles may promote an organism's survival and health by fine-tuning physiological processes. An unbiased search for genes that alter an organism's ability to maintain aspects of health may uncover modulators of lifespan and healthspan. From an unbiased screen for Caenorhabditis elegans mutants that show a progressive decline in motility, we aimed to identify genes that play a modulatory role in maintenance of locomotor healthspan. Here we report the involvement of hda-3, encoding a class I histone deacetylase, as a genetic factor that contributes in the maintenance of general health and locomotion in C. elegans. We identified a missense mutation in HDA-3 as the causative mutation in one of the isolated strains that show a progressive decline in maximum velocity and travel distance. From transcriptome analysis, we found a cluster of genes on Chromosome II carrying BATH domains that were downregulated by hda-3. Furthermore, downregulation of individual bath genes leads to significant decline in motility. Our study identifies genetic factors that modulate the maintenance of locomotor healthspan and may reveal potential targets for delaying age-related locomotor decline.


Assuntos
Envelhecimento/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Histona Desacetilases/genética , Locomoção/genética , Mutação de Sentido Incorreto , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/metabolismo , Distrofina/genética , Distrofina/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Transcriptoma
16.
Ann Clin Transl Neurol ; 7(9): 1738-1752, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33325654

RESUMO

To evaluate the effect of pharmacological treatments that increase the synthesis of dystrophin in Duchenne muscular dystrophy (DMD). Systematic searches were carried out in MEDLINE, EMBASE, and Web of Science, and in gray literature from inception to December 2019. Clinical trials addressing the effect of restorative treatments of dystrophin expression in children and adolescents with DMD on functional outcomes {(6-minute walking distance [6MWD], other timed functional tests [TFTs], The North Star Ambulatory Assessment)}, dystrophin expression, cardiorespiratory function, and biochemical tests were included. The DerSimonian-Laird method was used to calculate the pooled estimates for functional outcomes. Eleven studies were included in the systematic review and five in the meta-analysis. Eteplirsen showed a significant effect on 6MWD, Δ6MWD = 67.3 m (95% CI: 27.32, 107.28), and Δ6MWD = 151.0 m (95% CI: 36.15, 265.85) at 48 weeks and 3 years, respectively. In the systematic review, analyzing individually the clinical trials using Ataluren and Drisapersen showed a nonsignificant effect on 6MWD. However, the meta-analysis showed a significant effect on 6MWD for Ataluren and Drisapersen, Δ6MWD = 18.3 m (95% CI: 1.0, 35.5) and Δ6MWD = 21.5 m (95% CI: 4.7, 38.3), respectively. There were no significant differences according to baseline age for Drisapersen. Similarly, the meta-analysis showed effect in TFT with Ataluren. All drugs induced a partial synthesis of dystrophin, and exon skipping was obtained with Eteplirsen and Drisapersen. Eteplirsen also improved forced vital capacity (Δ%pFVC = 1.8%) and maximal inspiratory pressure (Δ%pMIP = 4.4%). Eteplirsen and Ataluren could modestly reduce disease progression. However, more trials are needed to confirm its efficacy, as well as quality of life and cost-utility studies.


Assuntos
Distrofina/metabolismo , Morfolinos/farmacologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos/farmacologia , Avaliação de Resultados em Cuidados de Saúde , Oxidiazóis/farmacologia , Humanos
17.
PLoS One ; 15(12): e0244215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362201

RESUMO

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.


Assuntos
Modelos Animais de Doenças , Distrofina/genética , Deleção de Genes , Distrofia Muscular de Duchenne/genética , Fenótipo , Transgenes , Animais , Fenômenos Biomecânicos , Distrofina/metabolismo , Éxons , Marcha , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/patologia
18.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228026

RESUMO

Caveolae are the cholesterol-rich small invaginations of the plasma membrane present in many cell types including adipocytes, endothelial cells, epithelial cells, fibroblasts, smooth muscles, skeletal muscles and cardiac muscles. They serve as specialized platforms for many signaling molecules and regulate important cellular processes like energy metabolism, lipid metabolism, mitochondria homeostasis, and mechano-transduction. Caveolae can be internalized together with associated cargo. The caveolae-dependent endocytic pathway plays a role in the withdrawal of many plasma membrane components that can be sent for degradation or recycled back to the cell surface. Caveolae are formed by oligomerization of caveolin proteins. Caveolin-3 is a muscle-specific isoform, whose malfunction is associated with several diseases including diabetes, cancer, atherosclerosis, and cardiovascular diseases. Mutations in Caveolin-3 are known to cause muscular dystrophies that are collectively called caveolinopathies. Altered expression of Caveolin-3 is also observed in Duchenne's muscular dystrophy, which is likely a part of the pathological process leading to muscle weakness. This review summarizes the major functions of Caveolin-3 in skeletal muscles and discusses its involvement in the pathology of muscular dystrophies.


Assuntos
Arritmias Cardíacas/genética , Cardiomegalia/genética , Caveolina 3/genética , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Junção Neuromuscular/genética , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Cavéolas/metabolismo , Caveolina 3/química , Caveolina 3/metabolismo , Distrofina/genética , Distrofina/metabolismo , Endocitose , Regulação da Expressão Gênica , Humanos , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/fisiopatologia , Distrofias Musculares/metabolismo , Distrofias Musculares/fisiopatologia , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo
19.
BMC Med ; 18(1): 343, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33208172

RESUMO

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. METHODS: To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). RESULTS: Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. CONCLUSIONS: nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.


Assuntos
Distrofia Muscular de Duchenne/genética , Esfingomielina Fosfodiesterase/metabolismo , Animais , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Distrofina/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout
20.
PLoS One ; 15(10): e0237138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002037

RESUMO

In Duchenne muscular dystrophy, a lack of dystrophin leads to extensive muscle weakness and atrophy that is linked to cellular metabolic dysfunction and oxidative stress. This dystrophinopathy results in a loss of tethering between microtubules and the sarcolemma. Microtubules are also believed to regulate mitochondrial bioenergetics potentially by binding the outer mitochondrial membrane voltage dependent anion channel (VDAC) and influencing permeability to ADP/ATP cycling. The objective of this investigation was to determine if a lack of dystrophin causes microtubule disorganization concurrent with mitochondrial dysfunction in skeletal muscle, and whether this relationship is linked to altered binding of tubulin to VDAC. In extensor digitorum longus (EDL) muscle from 4-week old D2.mdx mice, microtubule disorganization was observed when probing for α-tubulin. This cytoskeletal disorder was associated with a reduced ability of ADP to stimulate respiration and attenuate H2O2 emission relative to wildtype controls. However, this was not associated with altered α-tubulin-VDAC2 interactions. These findings reveal that microtubule disorganization in dystrophin-deficient EDL is associated with impaired ADP control of mitochondrial bioenergetics, and suggests that mechanisms alternative to α-tubulin's regulation of VDAC2 should be examined to understand how cytoskeletal disruption in the absence of dystrophin may cause metabolic dysfunctions in skeletal muscle.


Assuntos
Distrofina/metabolismo , Mitocôndrias , Músculo Esquelético , Distrofia Muscular de Duchenne/metabolismo , Tubulina (Proteína)/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Metabolismo Energético , Camundongos , Camundongos Endogâmicos mdx , Microtúbulos/metabolismo , Microtúbulos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia
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