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1.
Cell ; 163(5): 1204-1213, 2015 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-26582133

RESUMEN

Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner. PAPERCLIP.


Asunto(s)
Proteínas de Unión al Calcio/genética , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Distrofia Muscular de Duchenne/genética , Animales , Proliferación Celular , Enfermedades de los Perros/genética , Perros , Distrofina/deficiencia , Distrofina/genética , Femenino , Estudio de Asociación del Genoma Completo , Proteína Jagged-1 , Masculino , Ratones , Distrofia Muscular Animal/genética , Linaje , Penetrancia , Proteínas Serrate-Jagged , Transcriptoma , Pez Cebra , Proteínas de Pez Cebra
2.
Hum Mol Genet ; 33(13): 1107-1119, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38507070

RESUMEN

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.


Asunto(s)
Proteínas Asociadas a la Distrofina , Distrofina , Mitocondrias , Retículo Sarcoplasmático , Animales , Ratones , Distrofina/genética , Distrofina/metabolismo , Distrofina/deficiencia , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas/genética , Glicoproteínas/deficiencia , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitocondrias/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestructura
3.
Proc Natl Acad Sci U S A ; 120(2): e2206324120, 2023 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-36595689

RESUMEN

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 (DmdEGFP), 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 Dmd 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 Dmd 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.


Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Femenino , Ratones , Animales , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Músculos/metabolismo , Edición Génica , Resultado del Tratamiento , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Modelos Animales de Enfermedad
4.
Circulation ; 149(17): 1375-1390, 2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38214189

RESUMEN

BACKGROUND: Cardiac transverse tubules (T-tubules) are anchored to sarcomeric Z-discs by costameres to establish a regular spaced pattern. One of the major components of costameres is the dystrophin-glycoprotein complex (DGC). Nevertheless, how the assembly of the DGC coordinates with the formation and maintenance of T-tubules under physiological and pathological conditions remains unclear. METHODS: Given the known role of Ptpn23 (protein tyrosine phosphatase, nonreceptor type 23) in regulating membrane deformation, its expression in patients with dilated cardiomyopathy was determined. Taking advantage of Cre/Loxp, CRISPR/Cas9, and adeno-associated virus 9 (AAV9)-mediated in vivo gene editing, we generated cardiomyocyte-specific Ptpn23 and Actn2 (α-actinin-2, a major component of Z-discs) knockout mice. We also perturbed the DGC by using dystrophin global knockout mice (DmdE4*). MM 4-64 and Di-8-ANEPPS staining, Cav3 immunofluorescence, and transmission electron microscopy were performed to determine T-tubule structure in isolated cells and intact hearts. In addition, the assembly of the DGC with Ptpn23 and dystrophin loss of function was determined by glycerol-gradient fractionation and SDS-PAGE analysis. RESULTS: The expression level of Ptpn23 was reduced in failing hearts from dilated cardiomyopathy patients and mice. Genetic deletion of Ptpn23 resulted in disorganized T-tubules with enlarged diameters and progressive dilated cardiomyopathy without affecting sarcomere organization. AAV9-mediated mosaic somatic mutagenesis further indicated a cell-autonomous role of Ptpn23 in regulating T-tubule formation. Genetic and biochemical analyses showed that Ptpn23 was essential for the integrity of costameres, which anchor the T-tubule membrane to Z-discs, through interactions with α-actinin and dystrophin. Deletion of α-actinin altered the subcellular localization of Ptpn23 and DGCs. In addition, genetic inactivation of dystrophin caused similar T-tubule defects to Ptpn23 loss-of-function without affecting Ptpn23 localization at Z-discs. Last, inducible Ptpn23 knockout at 1 month of age showed Ptpn23 is also required for the maintenance of T-tubules in adult cardiomyocytes. CONCLUSIONS: Ptpn23 is essential for cardiac T-tubule formation and maintenance along Z-discs. During postnatal heart development, Ptpn23 interacts with sarcomeric α-actinin and coordinates the assembly of the DGC at costameres to sculpt T-tubule spatial patterning and morphology.

5.
Mass Spectrom Rev ; 43(1): 90-105, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-36420714

RESUMEN

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.


Asunto(s)
Complejo de Proteínas Asociado a la Distrofina , Distrofina , Distrofina/análisis , Distrofina/genética , Distrofina/metabolismo , Complejo de Proteínas Asociado a la Distrofina/análisis , Complejo de Proteínas Asociado a la Distrofina/metabolismo , Laminina/análisis , Laminina/metabolismo , Sarcolema/química , Sarcolema/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Glicoproteínas/análisis
6.
FASEB J ; 38(11): e23718, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38847487

RESUMEN

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.


Asunto(s)
Estrógenos , Ratones Endogámicos mdx , Músculo Esquelético , Proteínas de Unión al ARN , Animales , Femenino , Ratones , Estrógenos/metabolismo , Estrógenos/farmacología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/genética , Ratones Endogámicos C57BL , Ovariectomía , Mitocondrias/metabolismo , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/efectos de los fármacos
7.
Mol Ther ; 32(8): 2604-2623, 2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-38910327

RESUMEN

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRPP448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes.


Asunto(s)
Dependovirus , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos , Fuerza Muscular , Músculo Esquelético , Pentosiltransferasa , Animales , Ratones , Terapia Genética/métodos , Fuerza Muscular/genética , Dependovirus/genética , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Expresión Génica , Caminata , Humanos , Regulación de la Expresión Génica
8.
Proc Natl Acad Sci U S A ; 119(35): e2122004119, 2022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-35994666

RESUMEN

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).


Asunto(s)
Codón sin Sentido , Enfermedades Genéticas Congénitas , Guanidinas , Quinazolinas , Línea Celular , Codón sin Sentido/efectos de los fármacos , Codón sin Sentido/genética , Codón de Terminación/efectos de los fármacos , Codón de Terminación/genética , Evaluación Preclínica de Medicamentos , Genes Reporteros/efectos de los fármacos , Enfermedades Genéticas Congénitas/tratamiento farmacológico , Enfermedades Genéticas Congénitas/genética , Gentamicinas/farmacología , Guanidinas/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Quinazolinas/farmacología
9.
BMC Biol ; 22(1): 214, 2024 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-39334101

RESUMEN

BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.


Asunto(s)
Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Ratones Transgénicos , Distrofia Muscular de Duchenne , Transgenes , Animales , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Ratones , Humanos , Edición Génica/métodos , Distrofina/genética , Duplicación de Gen , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética
10.
Am J Physiol Cell Physiol ; 326(4): C1011-C1026, 2024 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-38145301

RESUMEN

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.


Asunto(s)
Distrofia Muscular de Duchenne , Animales , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Adiponectina/genética , Modelos Animales de Enfermedad , Interleucina-6/metabolismo , Ratones Endogámicos C57BL , Peróxido de Hidrógeno/metabolismo , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Ratones Endogámicos DBA , Músculo Esquelético/metabolismo , Diafragma/metabolismo , Fibrosis , Inflamación/metabolismo , Progresión de la Enfermedad , Atrofia/metabolismo , Atrofia/patología
11.
J Biol Chem ; 299(2): 102847, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36587764

RESUMEN

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.


Asunto(s)
Fosforilación , Utrofina , Animales , Distrofina/genética , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Utrofina/química , Utrofina/genética , Bacterias , Insectos , Ratones
12.
Neurogenetics ; 25(3): 201-213, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38850354

RESUMEN

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.


Asunto(s)
Distrofina , Exones , Distrofia Muscular de Duchenne , Fenotipo , Humanos , Distrofia Muscular de Duchenne/genética , Turquía , Masculino , Distrofina/genética , Niño , Femenino , Adolescente , Preescolar , Exones/genética , Estudios de Asociación Genética/métodos , Mutación , Adulto , Genotipo , Adulto Joven , Reacción en Cadena de la Polimerasa Multiplex
13.
Neurobiol Dis ; 199: 106586, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38950712

RESUMEN

OBJECTIVE: The glymphatic system serves as a perivascular pathway that aids in clearing liquid and solute waste from the brain, thereby enhancing neurological function. Disorders in glymphatic drainage contribute to the development of vasogenic edema following cerebral ischemia, although the molecular mechanisms involved remain poorly understood. This study aims to determine whether a deficiency in dystrophin 71 (DP71) leads to aquaporin-4 (AQP4) depolarization, contributing to glymphatic dysfunction in cerebral ischemia and resulting in brain edema. METHODS: A mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation. RESULTS: During the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level. CONCLUSION: AQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.


Asunto(s)
Acuaporina 4 , Edema Encefálico , Isquemia Encefálica , Distrofina , Sistema Glinfático , Animales , Masculino , Ratones , Acuaporina 4/metabolismo , Acuaporina 4/genética , Astrocitos/metabolismo , Edema Encefálico/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Distrofina/metabolismo , Distrofina/deficiencia , Sistema Glinfático/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Ratones Endogámicos C57BL
14.
Eur J Neurosci ; 2024 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-39415418

RESUMEN

Patients with Duchenne muscular dystrophy (DMD) commonly show specific cognitive deficits in addition to a severe muscle impairment caused by the absence of dystrophin expression in skeletal muscle. These cognitive deficits have been related to the absence of dystrophin in specific regions of the central nervous system, notably cerebellar Purkinje cells (PCs). Dystrophin has recently been involved in GABAA receptors clustering at postsynaptic densities, and its absence, by disrupting this clustering, leads to decreased inhibitory input to PC. We performed an in vivo electrophysiological study of the dystrophin-deficient muscular dystrophy X-linked (mdx) mouse model of DMD to compare PC firing and local field potential (LFP) in alert mdx and control C57Bl/10 mice. We found that the absence of dystrophin is associated with altered PC firing and the emergence of fast (~160-200 Hz) LFP oscillations in the cerebellar cortex of alert mdx mice. These abnormalities were not related to the disrupted expression of calcium-binding proteins in cerebellar PC. We also demonstrate that cerebellar long-term depression is altered in alert mdx mice. Finally, mdx mice displayed a force weakness, mild impairment of motor coordination and balance during behavioural tests. These findings demonstrate the existence of cerebellar dysfunction in mdx mice. A similar cerebellar dysfunction may contribute to the cognitive deficits observed in patients with DMD.

15.
Biochem Biophys Res Commun ; 727: 150323, 2024 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-38945065

RESUMEN

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.


Asunto(s)
Saponinas , Trehalosa , Saponinas/química , Trehalosa/química , Inmunoprecipitación/métodos , Animales , Detergentes/química , Unión Proteica , Humanos , Octoxinol/química
16.
J Transl Med ; 22(1): 664, 2024 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-39014470

RESUMEN

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive and devastating muscle disease, resulting from the absence of dystrophin. This leads to cell membrane instability, susceptibility to contraction-induced muscle damage, subsequent muscle degeneration, and eventually disability and early death of patients. Currently, there is no cure for DMD. Our recent studies identified that lipin1 plays a critical role in maintaining myofiber stability and integrity. However, lipin1 gene expression levels are dramatically reduced in the skeletal muscles of DMD patients and mdx mice. METHODS: To identify whether increased lipin1 expression could prevent dystrophic pathology, we employed unique muscle-specific mdx:lipin1 transgenic (mdx:lipin1Tg/0) mice in which lipin1 was restored in the dystrophic muscle of mdx mice, intramuscular gene delivery, as well as cell culture system. RESULTS: We found that increased lipin1 expression suppressed muscle degeneration and inflammation, reduced fibrosis, strengthened membrane integrity, and resulted in improved muscle contractile and lengthening force, and muscle performance in mdx:lipin1Tg/0 compared to mdx mice. To confirm the role of lipin1 in dystrophic muscle, we then administered AAV1-lipin1 via intramuscular injection in mdx mice. Consistently, lipin1 restoration inhibited myofiber necroptosis and lessened muscle degeneration. Using a cell culture system, we further found that differentiated primary mdx myoblasts had elevated expression levels of necroptotic markers and medium creatine kinase (CK), which could be a result of sarcolemmal damage. Most importantly, increased lipin1 expression levels in differentiated myoblasts from mdx:lipin1Tg/0 mice substantially inhibited the elevation of necroptotic markers and medium CK levels. CONCLUSIONS: Overall, our data suggest that lipin1 is a promising therapeutic target for the treatment of dystrophic muscles.


Asunto(s)
Ratones Endogámicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne , Fosfatidato Fosfatasa , Animales , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/metabolismo , Fosfatidato Fosfatasa/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Ratones Transgénicos , Ratones , Contracción Muscular , Terapia Molecular Dirigida , Ratones Endogámicos C57BL , Terapia Genética , Masculino
17.
Int J Exp Pathol ; 105(2): 75-85, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38477495

RESUMEN

Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to a deficiency in dystrophin production and consequent progressive degeneration of skeletal muscle fibres, through oxidative stress and an exacerbated inflammatory process. The flavonoid trilobatin (TLB) demonstrates antioxidant and anti-inflammatory potential. Its high safety profile and effective action make it a potent therapy for the process of dystrophic muscle myonecrosis. Thus, we sought to investigate the action of TLB on damage in a DMD model, the mdx mouse. Eight-week-old male animals were treated with 160 mg/kg/day of trilobatin for 8 weeks. Control animals were treated with saline. Following treatment, muscle strength, serum creatine kinase (CK) levels, histopathology (necrotic myofibres, regenerated fibres/central nuclei, Feret's diameter and inflammatory area) and the levels of catalase and NF-κB (western blotting) of the quadriceps (QUA), diaphragm (DIA) and tibialis anterior (TA) muscles were measured. TLB was able to significantly increase muscle strength and reduce serum CK levels in dystrophic animals. The QUA of mdx mice showed a reduction in catalase and the number of fibres with a centralized nucleus after treatment with TLB. In the DIA of dystrophic animals, TLB reduced the necrotic myofibres, inflammatory area and NF-κB and increased the number of regenerated fibres and the total fibre diameter. In TA, TLB increased the number of regenerated fibres and reduced catalase levels in these animals. It is concluded that in the mdx experimental model, treatment with TLB was beneficial in the treatment of DMD.


Asunto(s)
Flavonoides , Distrofia Muscular de Duchenne , Polifenoles , Ratones , Animales , Masculino , Distrofia Muscular de Duchenne/tratamiento farmacológico , Catalasa , Ratones Endogámicos mdx , FN-kappa B , Músculo Esquelético/patología
18.
Muscle Nerve ; 69(1): 93-98, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37577753

RESUMEN

INTRODUCTION/AIMS: Delandistrogene moxeparvovec is indicated in the United States for the treatment of ambulatory pediatric patients aged 4 through 5 years with Duchenne muscular dystrophy (DMD) with a confirmed mutation in the DMD gene. Long-term delandistrogene moxeparvovec microdystrophin protein (a shortened dystrophin that retains key functional domains of the wild-type protein) expression may positively alter disease progression in patients with DMD. We evaluated long-term safety and functional outcomes of delandistrogene moxeparvovec in patients with DMD. METHODS: An open-label, phase 1/2a, nonrandomized controlled trial (Study 101; NCT03375164) enrolled ambulatory males, ≥4 to <8 years old, with DMD. Patients received a single intravenous infusion (2.0 × 1014 vg/kg by supercoiled quantitative polymerase chain reaction) of delandistrogene moxeparvovec and prednisone (1 mg/kg/day) 1 day before to 30 days after treatment. The primary endpoint was safety. Functional outcomes were change from baseline in North Star Ambulatory Assessment (NSAA) and timed function tests. RESULTS: Four patients (mean age, 5.1 years) were enrolled. There were 18 treatment-related adverse events; all occurred within 70 days posttreatment and resolved. Mean NSAA total score increased from 20.5 to 27.5, baseline to year 4, with a mean (standard deviation) change of +7.0 (2.9). Post hoc analysis demonstrated a statistically significant and clinically meaningful 9-point difference in NSAA score, relative to a propensity-score-weighted external control cohort (least-squares mean [standard error] = 9.4 [3.4]; P = .0125). DISCUSSION: Gene transfer therapy with delandistrogene moxeparvovec treatment is well tolerated, with a favorable safety profile. Functional improvements are sustained through 4 years, suggesting delandistrogene moxeparvovec may positively alter disease progression.


Asunto(s)
Distrofia Muscular de Duchenne , Niño , Preescolar , Humanos , Masculino , Progresión de la Enfermedad , Terapia Genética/efectos adversos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Prednisona/uso terapéutico
19.
Behav Brain Funct ; 20(1): 21, 2024 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-39182120

RESUMEN

BACKGROUND: The Duchenne and Becker muscular dystrophies (DMD, BMD) are neuromuscular disorders commonly associated with diverse cognitive and behavioral comorbidities. Genotype-phenotype studies suggest that severity and risk of central defects in DMD patients increase with cumulative loss of different dystrophins produced in CNS from independent promoters of the DMD gene. Mutations affecting all dystrophins are nevertheless rare and therefore the clinical evidence on the contribution of the shortest Dp71 isoform to cognitive and behavioral dysfunctions is limited. In this study, we evaluated social, emotional and locomotor functions, and fear-related learning in the Dp71-null mouse model specifically lacking this short dystrophin. RESULTS: We demonstrate the presence of abnormal social behavior and ultrasonic vocalization in Dp71-null mice, accompanied by slight changes in exploratory activity and anxiety-related behaviors, in the absence of myopathy and alterations of learning and memory of aversive cue-outcome associations. CONCLUSIONS: These results support the hypothesis that distal DMD gene mutations affecting Dp71 may contribute to the emergence of social and emotional problems that may relate to the autistic traits and executive dysfunctions reported in DMD. The present alterations in Dp71-null mice may possibly add to the subtle social behavior problems previously associated with the loss of the Dp427 dystrophin, in line with the current hypothesis that risk and severity of behavioral problems in patients increase with cumulative loss of several brain dystrophin isoforms.


Asunto(s)
Distrofina , Ratones Noqueados , Conducta Social , Animales , Distrofina/genética , Distrofina/deficiencia , Ratones , Masculino , Emociones/fisiología , Miedo/fisiología , Miedo/psicología , Conducta Animal/fisiología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Ansiedad/genética , Ansiedad/psicología
20.
EMBO Rep ; 23(6): e53955, 2022 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-35393769

RESUMEN

Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.


Asunto(s)
Distrofina , Metformina , Animales , Distrofina/genética , Terapia Genética/métodos , Glicina/uso terapéutico , Humanos , Metformina/uso terapéutico , Ratones , Ratones Endogámicos mdx , Morfolinos/genética , Morfolinos/uso terapéutico , Músculo Esquelético , Utrofina/genética
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