Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Hum Mol Genet ; 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30986853

RESUMO

Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific-adapters for CUL3 E3 ubiquitin ligase to regulate protein turn-over through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM, however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric protein contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

2.
Acta Neuropathol ; 137(3): 501-519, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30701273

RESUMO

The identification of genes implicated in myopathies is essential for diagnosis and for revealing novel therapeutic targets. Here we characterize a novel subclass of congenital myopathy at the morphological, molecular, and functional level. Through exome sequencing, we identified de novo ACTN2 mutations, a missense and a deletion, in two unrelated patients presenting with progressive early-onset muscle weakness and respiratory involvement. Morphological and ultrastructural analyses of muscle biopsies revealed a distinctive pattern with the presence of muscle fibers containing small structured cores and jagged Z-lines. Deeper analysis of the missense mutation revealed mutant alpha-actinin-2 properly localized to the Z-line in differentiating myotubes and its level was not altered in muscle biopsy. Modelling of the disease in zebrafish and mice by exogenous expression of mutated alpha-actinin-2 recapitulated the abnormal muscle function and structure seen in the patients. Motor deficits were noted in zebrafish, and muscle force was impaired in isolated muscles from AAV-transduced mice. In both models, sarcomeric disorganization was evident, while expression of wild-type alpha-actinin-2 did not result in muscle anomalies. The murine muscles injected with mutant ACTN2 displayed cores and Z-line defects. Dominant ACTN2 mutations were previously associated with cardiomyopathies, and our data demonstrate that specific mutations in the well-known Z-line regulator alpha-actinin-2 can cause a skeletal muscle disorder.

3.
Hum Mol Genet ; 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30307508

RESUMO

Facioscapulohumeral dystrophy Type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of approximately 1 in 8,000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germline, primate specific transcription factor DUX4-fl (double homeo-box 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4 inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein we introduce a hypothesis that DUX4 expression during developmental stages, is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.

4.
NPJ Genom Med ; 3: 21, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30131872

RESUMO

Despite major progress in defining the genetic basis of Mendelian disorders, the molecular etiology of many cases remains unknown. Patients with these undiagnosed disorders often have complex presentations and require treatment by multiple health care specialists. Here, we describe an integrated clinical diagnostic and research program using whole-exome and whole-genome sequencing (WES/WGS) for Mendelian disease gene discovery. This program employs specific case ascertainment parameters, a WES/WGS computational analysis pipeline that is optimized for Mendelian disease gene discovery with variant callers tuned to specific inheritance modes, an interdisciplinary crowdsourcing strategy for genomic sequence analysis, matchmaking for additional cases, and integration of the findings regarding gene causality with the clinical management plan. The interdisciplinary gene discovery team includes clinical, computational, and experimental biomedical specialists who interact to identify the genetic etiology of the disease, and when so warranted, to devise improved or novel treatments for affected patients. This program effectively integrates the clinical and research missions of an academic medical center and affords both diagnostic and therapeutic options for patients suffering from genetic disease. It may therefore be germane to other academic medical institutions engaged in implementing genomic medicine programs.

5.
PLoS One ; 13(6): e0199712, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29944715

RESUMO

Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s-1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18-24% faster Umax of adult males vs. females, and a 14-20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.

6.
PLoS Genet ; 14(3): e1007226, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29518074

RESUMO

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.


Assuntos
RNA Helicases DEAD-box/metabolismo , Músculo Esquelético/fisiologia , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Proliferação de Células/genética , RNA Helicases DEAD-box/genética , Embrião não Mamífero , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos/citologia , Mioblastos/fisiologia , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , RNA Ribossômico/genética , Regeneração/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
PLoS One ; 12(2): e0172648, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28241031

RESUMO

Merosin deficient congenital muscular dystrophy (MDC1A) is a severe neuromuscular disorder with onset in infancy that is associated with severe morbidities (particularly wheelchair dependence) and early mortality. It is caused by recessive mutations in the LAMA2 gene that encodes a subunit of the extracellular matrix protein laminin 211. At present, there are no treatments for this disabling disease. The zebrafish has emerged as a powerful model system for the identification of novel therapies. However, drug discovery in the zebrafish is largely dependent on the identification of phenotypes suitable for chemical screening. Our goal in this study was to elucidate novel, early onset abnormalities in the candyfloss (caf) zebrafish, a model of MDC1A. We uncovered and characterize abnormalities in spontaneous coiling, the earliest motor movement in the zebrafish, as a fully penetrant change specific to caf mutants that is ideal for future drug testing.


Assuntos
Modelos Animais de Doenças , Laminina/genética , Distrofias Musculares/genética , Animais , Animais Geneticamente Modificados , Heterozigoto , Laminina/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Mutação , Faloidina/biossíntese , Fenótipo , Peixe-Zebra/metabolismo
8.
Ann Neurol ; 79(6): 959-69, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27074222

RESUMO

OBJECTIVE: Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. METHODS: We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. RESULTS: Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. INTERPRETATION: These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.


Assuntos
Citoesqueleto/genética , Proteínas Musculares/genética , Doenças Musculares/genética , Doenças Musculares/fisiopatologia , Sarcômeros/genética , Actinas/genética , Animais , Estudos de Casos e Controles , Citoesqueleto/fisiologia , Humanos , Camundongos Knockout , Contração Muscular/genética , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Mutação , Sarcômeros/fisiologia
9.
Hum Genet ; 135(1): 21-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26541337

RESUMO

Myopathies are heterogeneous disorders characterized clinically by weakness and hypotonia, usually in the absence of gross dystrophic changes. Mitochondrial dysfunction is a frequent cause of myopathy. We report a simplex case born to consanguineous parents who presented with muscle weakness, lactic acidosis, and muscle changes suggestive of mitochondrial dysfunction. Combined autozygome and exome analysis revealed a missense variant in the SLC25A42 gene, which encodes an inner mitochondrial membrane protein that imports coenzyme A into the mitochondrial matrix. Zebrafish slc25a42 knockdown morphants display severe muscle disorganization and weakness. Importantly, these features are rescued by normal human SLC25A42 RNA, but not by RNA harboring the patient's variant. Our data support a potentially causal link between SLC25A42 mutation and mitochondrial myopathy in humans.


Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Miopatias Mitocondriais/genética , Mutação , Adolescente , Animais , Feminino , Humanos , Masculino , Modelos Animais , Linhagem , RNA Mensageiro/genética , Peixe-Zebra
11.
J Clin Invest ; 124(11): 4693-708, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25250574

RESUMO

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.


Assuntos
Proteínas Musculares/genética , Miofibrilas/patologia , Miopatias da Nemalina/genética , Actinas/química , Animais , Células Cultivadas , Análise Mutacional de DNA , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Masculino , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Miopatias da Nemalina/patologia , Multimerização Proteica , Peixe-Zebra
12.
Hum Mol Genet ; 23(24): 6584-93, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25055871

RESUMO

Lethal congenital contracture syndrome (LCCS) is a lethal autosomal recessive form of arthrogryposis multiplex congenita (AMC). LCCS is genetically heterogeneous with mutations in five genes identified to date, all with a role in the innervation or contractile apparatus of skeletal muscles. In a consanguineous Saudi family with multiple stillbirths presenting with LCCS, we excluded linkage to all known LCCS loci and combined autozygome analysis and whole-exome sequencing to identify a novel homozygous variant in ZBTB42, which had been shown to be enriched in skeletal muscles, especially at the neuromuscular junction. Knockdown experiments of zbtb42 in zebrafish consistently resulted in grossly abnormal skeletal muscle development and myofibrillar disorganization at the microscopic level. This severe muscular phenotype is successfully rescued with overexpression of the human wild-type ZBTB42 gene, but not with the mutant form of ZBTB42 that models the human missense change. Our data assign a novel muscular developmental phenotype to ZBTB42 in vertebrates and establish a new LCCS6 type caused by ZBTB42 mutation.


Assuntos
Artrogripose/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Mutação de Sentido Incorreto , Junção Neuromuscular/metabolismo , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Artrogripose/metabolismo , Artrogripose/patologia , Consanguinidade , Exoma , Feminino , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Junção Neuromuscular/patologia , Linhagem , Arábia Saudita , Natimorto , Peixe-Zebra
13.
Skelet Muscle ; 4: 11, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24959344

RESUMO

Our understanding of genes that cause skeletal muscle disease has increased tremendously over the past three decades. Advances in approaches to genetics and genomics have aided in the identification of new pathogenic mechanisms in rare genetic disorders and have opened up new avenues for therapeutic interventions by identification of new molecular pathways in muscle disease. Recent studies have identified mutations of several Kelch proteins in skeletal muscle disorders. The Kelch superfamily is one of the largest evolutionary conserved gene families. The 66 known family members all possess a Kelch-repeat containing domain and are implicated in diverse biological functions. In skeletal muscle development, several Kelch family members regulate the processes of proliferation and/or differentiation resulting in normal functioning of mature muscles. Importantly, many Kelch proteins function as substrate-specific adaptors for Cullin E3 ubiquitin ligase (Cul3), a core component of the ubiquitin-proteasome system to regulate the protein turnover. This review discusses the emerging roles of Kelch proteins in skeletal muscle function and disease.

14.
Hum Mol Genet ; 23(21): 5781-92, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24925318

RESUMO

Dystroglycan is a transmembrane glycoprotein whose interactions with the extracellular matrix (ECM) are necessary for normal muscle and brain development, and disruptions of its function lead to dystroglycanopathies, a group of congenital muscular dystrophies showing extreme genetic and clinical heterogeneity. Specific glycans bound to the extracellular portion of dystroglycan, α-dystroglycan, mediate ECM interactions and most known dystroglycanopathy genes encode glycosyltransferases involved in glycan synthesis. POMK, which was found mutated in two dystroglycanopathy cases, is instead involved in a glycan phosphorylation reaction critical for ECM binding, but little is known about the clinical presentation of POMK mutations or of the function of this protein in the muscle. Here, we describe two families carrying different truncating alleles, both removing the kinase domain in POMK, with different clinical manifestations ranging from Walker-Warburg syndrome, the most severe form of dystroglycanopathy, to limb-girdle muscular dystrophy with cognitive defects. We explored POMK expression in fetal and adult human muscle and identified widespread expression primarily during fetal development in myocytes and interstitial cells suggesting a role for this protein during early muscle differentiation. Analysis of loss of function in the zebrafish embryo and larva showed that pomk function is necessary for normal muscle development, leading to locomotor dysfuction in the embryo and signs of muscular dystrophy in the larva. In summary, we defined diverse clinical presentations following POMK mutations and showed that this gene is necessary for early muscle development.


Assuntos
Estudos de Associação Genética , Desenvolvimento Muscular/genética , Mutação , Doenças Neuromusculares/diagnóstico , Doenças Neuromusculares/genética , Fenótipo , Proteínas Quinases/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Distroglicanas/metabolismo , Exoma , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Estudo de Associação Genômica Ampla , Glicosilação , Humanos , Imagem por Ressonância Magnética , Masculino , Dados de Sequência Molecular , Linhagem , Proteínas Quinases/química , Alinhamento de Sequência , Adulto Jovem , Peixe-Zebra
15.
FASEB J ; 28(7): 2955-69, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24687993

RESUMO

Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in differentiating human muscle cells and regenerating muscle tissue. In vitro, human muscle cells were treated with histone-deacetylase (HDAC) inhibitors, and expression of Fam65b and interacting proteins was studied. Nontreated cells were used as controls. In vivo, expression of Fam65b was down-regulated in developing zebrafish to determine the effects on muscle development. Fam65b binds to HDAC6 and dysferlin, the protein mutated in limb girdle muscular dystrophy 2B. The tricomplex Fam65b-HDAC6-dysferlin is transient, and Fam65b expression is necessary for the complex to form. Treatment of myogenic cells with pan-HDAC or HDAC6-specific inhibitors alters Fam65b expression, while dysferlin expression does not change. Inhibition of Fam65b expression in developing zebrafish results in abnormal muscle, with low birefringence, tears at the myosepta, and increased embryo lethality. Fam65b is an essential component of the HDAC6-dysferlin complex. Down-regulation of Fam65b in developing muscle causes changes consistent with muscle disease.-Balasubramanian, A., Kawahara, G., Gupta, V. A., Rozkalne, A., Beauvais, A., Kunkel, L. M., Gussoni, E. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.


Assuntos
Diferenciação Celular/genética , Histona Desacetilases/metabolismo , Proteínas de Membrana/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteínas Musculares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Regulação para Baixo/genética , Disferlina , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Ligação Proteica/genética , Alinhamento de Sequência , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Peixe-Zebra
16.
Hum Mol Genet ; 23(13): 3566-78, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24549043

RESUMO

Autosomal recessive centronuclear myopathy (CNM2), caused by mutations in bridging integrator 1 (BIN1), is a mildly progressive neuromuscular disorder characterized by abnormally centralized myonuclei and muscle weakness. BIN1 is important for membrane sensing and remodeling in vitro in different cell types. However, to fully understand the biological roles of BIN1 in vivo and to answer critical questions concerning the muscle-specific function of BIN1 in vertebrates, robust small animal models are required. In this study, we create and characterize a novel zebrafish model of CNM2 using antisense morpholinos. Immunofluorescence and histopathological analyses of Bin1-deficient zebrafish skeletal muscle reveal structural defects commonly reported in human CNM2 biopsies. Live imaging of zebrafish embryos shows defective calcium release in bin1 morphants, linking the presence of abnormal triads to impairments in intracellular signaling. RNA-mediated rescue assays demonstrate that knockdown of zebrafish bin1 can reliably examine the pathogenicity of novel BIN1 mutations in vivo. Finally, our results strongly suggest that the phosphoinositide-binding domain of BIN1, present only in skeletal muscle isoforms, may be more critical for muscle maturation and maintenance than for early muscle development. Overall, our data support that BIN1 plays an important role in membrane tubulation and may promote skeletal muscle weakness in CNM2 by disrupting machinery necessary for excitation-contraction coupling in vertebrate organisms. The reproducible phenotype of Bin1-deficient zebrafish, together with the generalized advantages of the teleost system, makes this model readily adaptable to high-throughput screening strategies and may be used to identify therapies for CNM2 and related neuromuscular diseases.


Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Miopatias Congênitas Estruturais/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas de Drosophila/deficiência , Músculo Esquelético/metabolismo , Miopatias Congênitas Estruturais/genética , Fosfatidilinositóis/metabolismo , Fatores de Transcrição/deficiência , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
17.
Am J Hum Genet ; 93(6): 1108-17, 2013 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-24268659

RESUMO

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.


Assuntos
Mutação , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/genética , Transdução de Sinais , Ubiquitinação , Adolescente , Animais , Criança , Pré-Escolar , Evolução Fatal , Feminino , Expressão Gênica , Ordem dos Genes , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miopatias da Nemalina/diagnóstico , Conformação Proteica , Proteínas/química , Peixe-Zebra
18.
PLoS Genet ; 9(6): e1003583, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23818870

RESUMO

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs"). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.


Assuntos
Miopatias Congênitas Estruturais/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas/genética , Peixe-Zebra/genética , Animais , Catálise , Linhagem Celular , Humanos , Camundongos , Músculo Esquelético , Músculos/metabolismo , Músculos/fisiopatologia , Mutação , Miopatias Congênitas Estruturais/fisiopatologia , Estabilidade Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas/química , Proteínas/metabolismo
19.
J Vis Exp ; (82): e50925, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24378748

RESUMO

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies, congenital myopathies, and disruptions in sarcomeric assembly, due to high genomic and structural conservation with mammals. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral). Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres. The touch-evoked escape assay involves observing an embryo's swimming behavior in response to tactile stimulation. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.


Assuntos
Modelos Animais de Doenças , Reação de Fuga/fisiologia , Músculo Esquelético/anormalidades , Músculo Esquelético/química , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Natação/fisiologia , Animais , Birrefringência , Feminino , Larva , Masculino , Peixe-Zebra
20.
PLoS One ; 7(8): e43794, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952766

RESUMO

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.


Assuntos
Laminina/genética , Distrofias Musculares/genética , Mutação , Sítios de Splice de RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Sequência de Bases , Matriz Extracelular/metabolismo , Humanos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA