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1.
J Cardiovasc Dev Dis ; 10(10)2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37887855

RESUMO

Dilated cardiomyopathy (DCM) is a common heart muscle disorder that frequently leads to heart failure, arrhythmias, and death. While DCM is often heritable, disease-causing mutations are identified in only ~30% of cases. In a forward genetic mutagenesis screen, we identified a novel zebrafish mutant, heart and head (hahvcc43), characterized by early-onset cardiomyopathy and craniofacial defects. Linkage analysis and next-generation sequencing identified a nonsense variant in the highly conserved scfd1 gene, also known as sly1, that encodes sec1 family domain-containing 1. Sec1/Munc18 proteins, such as Scfd1, are involved in membrane fusion regulating endoplasmic reticulum (ER)/Golgi transport. CRISPR/Cas9-engineered scfd1vcc44 null mutants showed severe cardiac and craniofacial defects and embryonic lethality that recapitulated the phenotype of hahvcc43 mutants. Electron micrographs of scfd1-depleted cardiomyocytes showed reduced myofibril width and sarcomere density, as well as reticular network disorganization and fragmentation of Golgi stacks. Furthermore, quantitative PCR analysis showed upregulation of ER stress response and apoptosis markers. Both heterozygous hahvcc43 mutants and scfd1vcc44 mutants survived to adulthood, showing chamber dilation and reduced ventricular contraction. Collectively, our data implicate scfd1 loss-of-function as the genetic defect at the hahvcc43 locus and provide new insights into the role of scfd1 in cardiac development and function.

2.
J Ophthalmic Vis Res ; 18(1): 60-67, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36937198

RESUMO

Purpose: Mutations in TCP-1 ring complex (TRiC) have been associated with Leber Congenital Amaurosis (LCA). TRiC is involved in protein folding and has 8 essential subunits including CCT5. Herein, we studied the retina of TRiC mutant zebrafish to evaluate the possible role of impaired actin and tubulin folding in LCA. Methods: The cct5 t f 212 b retina was histologically studied using Toluidine Blue staining as well as TUNEL, BrdU-labeling, and Phalloidin assays. Retinal organisation was assessed by quantification of the cellularity utilising DAPI. Results: Laminar organization of cct5 t f 212 b retinas was intact. Enhanced apoptosis throughout the cct5 t f 212 b retina was not compensated by higher proliferation rates, leaving the cct5 t f 212 b retina smaller in size. Quantification of retinal layer cellularity demonstrated that specifically the numbers of the amacrine and the retinal ganglion cells were depleted, suggesting that the cct5 t f 212 b retina was not uniformly affected by the reduced actin folding. Conclusion: Whereas the current literature suggests that LCA is predominantly affecting retinal photoreceptor cells and the retinal pigment epithelium, cct5 t f 212 b analyses demonstrated the important role of folding of actin by TRiC, suggesting that cct5 t f 212 b is a useful tool to specifically analyze the role of F-actin filaments in the context of LCA.

3.
PLoS Genet ; 18(6): e1010287, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35737712

RESUMO

Myofibrils of the skeletal muscle are comprised of sarcomeres that generate force by contraction when myosin-rich thick filaments slide past actin-based thin filaments. Surprisingly little is known about the molecular processes that guide sarcomere assembly in vivo, despite deficits within this process being a major cause of human disease. To overcome this knowledge gap, we undertook a forward genetic screen coupled with reverse genetics to identify genes required for vertebrate sarcomere assembly. In this screen, we identified a zebrafish mutant with a nonsense mutation in mob4. In Drosophila, mob4 has been reported to play a role in spindle focusing as well as neurite branching and in planarians mob4 was implemented in body size regulation. In contrast, zebrafish mob4geh mutants are characterised by an impaired actin biogenesis resulting in sarcomere defects. Whereas loss of mob4 leads to a reduction in the amount of myofibril, transgenic expression of mob4 triggers an increase. Further genetic analysis revealed the interaction of Mob4 with the actin-folding chaperonin TRiC, suggesting that Mob4 impacts on TRiC to control actin biogenesis and thus myofibril growth. Additionally, mob4geh features a defective microtubule network, which is in-line with tubulin being the second main folding substrate of TRiC. We also detected similar characteristics for strn3-deficient mutants, which confirmed Mob4 as a core component of STRIPAK and surprisingly implicates a role of the STRIPAK complex in sarcomerogenesis.


Assuntos
Miofibrilas , Peixe-Zebra , Actinas/genética , Actinas/metabolismo , Animais , Chaperoninas/metabolismo , Microtúbulos/genética , Miofibrilas/metabolismo , Sarcômeros/metabolismo , Peixe-Zebra/genética
4.
PLoS Genet ; 18(2): e1010066, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35148320

RESUMO

Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.


Assuntos
Proteína de Capeamento de Actina CapZ/metabolismo , Doenças Musculares , Miofibrilas , Actinas/genética , Actinas/metabolismo , Animais , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Miofibrilas/genética , Miofibrilas/metabolismo , Tropomodulina/genética , Tropomodulina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
5.
J Cell Mol Med ; 24(12): 6680-6689, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32343037

RESUMO

Duchenne muscular dystrophy is a severe muscle wasting disease caused by mutations in the dystrophin gene (dmd). Ataluren has been approved by the European Medicines Agency for treatment of Duchenne muscular dystrophy. Ataluren has been reported to promote ribosomal read-through of premature stop codons, leading to restoration of full-length dystrophin protein. However, the mechanism of Ataluren action has not been fully described. To evaluate the efficacy of Ataluren on all three premature stop codons featuring different termination strengths (UAA > UAG > UGA), novel dystrophin-deficient zebrafish were generated. Pathological assessment of the muscle by birefringence quantification, a tool to directly measure muscle integrity, did not reveal a significant effect of Ataluren on any of the analysed dystrophin-deficient mutants at 3 days after fertilization. Functional analysis of the musculature at 6 days after fertilization by direct measurement of the generated force revealed a significant improvement by Ataluren only for the UAA-carrying mutant dmdta222a . Interestingly however, all other analysed dystrophin-deficient mutants were not affected by Ataluren, including the dmdpc3 and dmdpc2 mutants that harbour weaker premature stop codons UAG and UGA, respectively. These in vivo results contradict reported in vitro data on Ataluren efficacy, suggesting that Ataluren might not promote read-through of premature stop codons. In addition, Ataluren had no effect on dystrophin transcript levels, but mild adverse effects on wild-type larvae were identified. Further assessment of N-terminally truncated dystrophin opened the possibility of Ataluren promoting alternative translation codons within dystrophin, thereby potentially shifting the patient cohort applicable for Ataluren.


Assuntos
Distrofina/genética , Mutação/genética , Oxidiazóis/farmacologia , Animais , Códon sem Sentido/genética , Éxons/genética , Homozigoto , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Oxidiazóis/efeitos adversos , Fenótipo , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética
6.
J Anat ; 233(6): 687-695, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30277257

RESUMO

Klippel-Feil syndrome is a congenital vertebral anomaly, which is characterised by the fusion of at least two cervical vertebrae and a clinically broad set of symptoms, including congenital scoliosis and elevated scapula (Sprengel's deformity). Klippel-Feil syndrome is associated with mutations in MEOX1. The zebrafish mutant choker (cho) carries a mutation in its orthologue, meox1. Although zebrafish is being increasingly employed as fidelitous models of human spinal disease, the vertebral column of Meox1-deficient fish has not been assessed for defects. Here, we describe the skeletal defects of meox1cho mutant zebrafish utilising alizarin red to stain bones and chemical maceration of soft tissue to detect fusions in an unbiased manner. Obtained data reveal that meox1cho mutants feature aspects of a number of described symptoms of patients who suffer from Klippel-Feil syndrome and have mutations in MEOX1. These include vertebral fusion, congenital scoliosis and an asymmetry of the pectoral girdle, which resembles Sprengel's deformity. Thus, the meox1cho mutant zebrafish may serve as a useful tool to study the pathogenesis of the symptoms associated with Klippel-Feil syndrome.


Assuntos
Osso e Ossos/anormalidades , Modelos Animais de Doenças , Proteínas de Peixe-Zebra/deficiência , Animais , Animais Geneticamente Modificados , Técnicas de Inativação de Genes , Proteínas de Homeodomínio , Humanos , Síndrome de Klippel-Feil/genética , Síndrome de Klippel-Feil/patologia , Peixe-Zebra
7.
Int J Mol Sci ; 19(3)2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518972

RESUMO

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated.


Assuntos
Proteína ADAMTS5/genética , Diferenciação Celular , Somitos/embriologia , Somitos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteína ADAMTS5/metabolismo , Animais , Embrião não Mamífero , Espaço Extracelular , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas Hedgehog/metabolismo , Morfogênese/genética , Desenvolvimento Muscular/genética , Transdução de Sinais
8.
Cell Rep ; 22(2): 313-322, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29320728

RESUMO

The TCP-1 ring complex (TRiC) is a multi-subunit group II chaperonin that assists nascent or misfolded proteins to attain their native conformation in an ATP-dependent manner. Functional studies in yeast have suggested that TRiC is an essential and generalized component of the protein-folding machinery of eukaryotic cells. However, TRiC's involvement in specific cellular processes within multicellular organisms is largely unknown because little validation of TRiC function exists in animals. Our in vivo analysis reveals a surprisingly specific role of TRiC in the biogenesis of skeletal muscle α-actin during sarcomere assembly in myofibers. TRiC acts at the sarcomere's Z-disk, where it is required for efficient assembly of actin thin filaments. Binding of ATP specifically by the TRiC subunit Cct5 is required for efficient actin folding in vivo. Furthermore, mutant α-actin isoforms that result in nemaline myopathy in patients obtain their pathogenic conformation via this function of TRiC.


Assuntos
Actinas/metabolismo , Chaperonina com TCP-1/metabolismo , Chaperoninas/química , Sarcômeros/metabolismo , Animais , Humanos , Peixe-Zebra
9.
Endocrine ; 58(2): 246-252, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28914407

RESUMO

OBJECTIVE: The liver-derived plasma protein fetuin B is associated with nonalcoholic fatty liver disease (NAFLD) and impaired glucose homeostasis in mice. However, its association with non-invasive ultrasound- and magnetic resonance (MR)-based markers of liver fibrosis and steatosis, the enhanced liver fibrosis (ELF) score, liver biopsy, as well as rs738409 in PNPLA3, has not been elucidated in NAFLD, so far. DESIGN AND METHODS: The association of circulating fetuin B and transient elastography (TE), controlled attenuation parameter (CAP), 1H-MR-spectroscopy, the ELF score, liver biopsy, as well as risk alleles in rs738409 in PNPLA3, was studied in 101 NAFLD patients as compared to 15 healthy controls. RESULTS: Serum fetuin B levels did not differ between NAFLD patients and controls (p = 0.863). Fetuin B was independently and negatively associated with transient elastography liver stiffness measurement (LSM) (p = 0.002), but not with the steatosis markers CAP or 1H-MR-spectroscopy. Fetuin B serum concentrations were significantly lower in individuals with LSM > 7.0 kPa as compared to patients with LSM < 7.0 kPa (p = 0.024). Furthermore, the ELF score and histologically proven fibrosis were independent and negative predictors of circulating fetuin B. Moreover, serum fetuin B significantly depended on number of rs738409 risk alleles (p = 0.026). CONCLUSIONS: Fetuin B is independently and negatively associated with non-invasive markers of liver fibrosis and PNPLA3 status in NAFLD patients but does not show a correlation with the hepatic lipid content. Future studies need to elucidate the pathophysiological significance of fetuin B in NAFLD and its potential value as predictor for disease severity.


Assuntos
Fetuína-B/análise , Cirrose Hepática/sangue , Fígado/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/sangue , Adolescente , Adulto , Idoso , Alelos , Biomarcadores , Técnicas de Imagem por Elasticidade , Feminino , Humanos , Fígado/patologia , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/genética , Cirrose Hepática/patologia , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Ultrassonografia , Adulto Jovem
10.
Hum Mol Genet ; 26(6): 1146-1156, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28104788

RESUMO

Congenital myopathies are muscle degenerative disorders with a broad clinical spectrum. A number of myopathies have been associated with molecular defects within sarcomeres, the force-generating component of the muscle cell. Whereas the highly regular organization of the myofibril has been studied in detail, in vivo assembly of sarcomeres remains a poorly understood process. Therefore, a more detailed knowledge of sarcomere assembly is crucial to better understand the pathogenic mechanisms within myopathies. Recently, mutations in myosin XVIIIB (MYO18B) have been associated with cases of myopathies, although the underlying mechanism for the resulting pathology remains to be defined. To analyze the role of myosin XVIIIB in skeletal muscle disease, zebrafish mutants for myo18b were generated. Full loss of myo18b function results in a complete lack of sarcomeric structure, revealing a highly surprising and essential role for myo18b in sarcomere assembly. Importantly, scattered thin and thick filaments assemble throughout the sarcoplasm; but fail to organize into recognizable sarcomeric structures in myo18b null mutants. In myo18b partial loss-of-function mutants sarcomeric structures are assembled, but thin and thick filaments remain misaligned within these structures. These observations suggest a novel model of sarcomere assembly where Myo18b coordinates the integration of preformed thick and thin filaments into the sarcomere. Disruption of this highly coordinated process results in a block in sarcomere biogenesis and the onset of myopathic pathology.


Assuntos
Músculo Esquelético/metabolismo , Miopatias Congênitas Estruturais/genética , Miosinas/genética , Sarcômeros/genética , Proteínas Supressoras de Tumor/genética , Peixe-Zebra/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Humanos , Músculo Esquelético/patologia , Proteínas Mutantes/genética , Miopatias Congênitas Estruturais/patologia , Miosinas/biossíntese , Sarcômeros/metabolismo , Sarcômeros/patologia , Proteínas Supressoras de Tumor/biossíntese , Peixe-Zebra/fisiologia
11.
Science ; 353(6295): aad9969, 2016 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-27198673

RESUMO

Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare "immortal" stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.


Assuntos
Divisão Celular/fisiologia , Rastreamento de Células/métodos , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Animais Geneticamente Modificados , Divisão Celular/genética , Células Clonais , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/lesões , Mutação , Fator Regulador Miogênico 5/genética , Miogenina/genética , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Transgenes , Peixe-Zebra
12.
Front Biosci (Landmark Ed) ; 21(4): 744-56, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26709803

RESUMO

In skeletal muscle, the dystrophin-associated glycoprotein complex forms a link between the actin cytoskeleton and the extracellular matrix that is critical for muscle integrity. Within this complex resides the sarcoglycan subcomplex, which consists of four transmembrane glycoproteins (alpha-, beta-, gamma-, and delta-sarcoglycan). During assembly, beta-sarcoglycan tightly associates with delta-sarcoglycan to form a functional core that then recruits gamma- and alpha-sarcoglycan to form the sarcoglycan complex. Together with sarcospan, the sarcoglycan complex binds other components of the dystrophin-associated glycoprotein complex and integrates into the myofibre's membrane. Once integrated, the sarcoglycan complex plays a pivotal role in mechanically stabilising the sarcolemma as well as the dystrophin-associated glycoprotein complex. Additionally, the sarcoglycan complex undergoes chemical modifications in response to muscle contractions, thereby transducing mechanical information into a cellular signal. Mutations in the sarcoglycans induce limb girdle muscular dystrophy, and several animal models have been established to study the molecular biology and function of the sarcoglycan complex. This review discusses the role of the sarcoglycan complex in skeletal muscle and describes the functional deficiencies that lead to muscular dystrophies.


Assuntos
Músculo Esquelético/metabolismo , Sarcoglicanas/metabolismo , Humanos , Filogenia , Sarcoglicanas/genética
13.
World J Gastroenterol ; 21(16): 4894-902, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25945002

RESUMO

AIM: To compare ultrasound-based acoustic structure quantification (ASQ) with established non-invasive techniques for grading and staging fatty liver disease. METHODS: Type 2 diabetic patients at risk of non-alcoholic fatty liver disease (n = 50) and healthy volunteers (n = 20) were evaluated using laboratory analysis and anthropometric measurements, transient elastography (TE), controlled attenuation parameter (CAP), proton magnetic resonance spectroscopy ((1)H-MRS; only available for the diabetic cohort), and ASQ. ASQ parameters mode, average and focal disturbance (FD) ratio were compared with: (1) the extent of liver fibrosis estimated from TE and non-alcoholic fatty liver disease (NAFLD) fibrosis scores; and (2) the amount of steatosis, which was classified according to CAP values. RESULTS: Forty-seven diabetic patients (age 67.0 ± 8.6 years; body mass index 29.4 ± 4.5 kg/m²) with reliable CAP measurements and all controls (age 26.5 ± 3.2 years; body mass index 22.0 ± 2.7 kg/m²) were included in the analysis. All ASQ parameters showed differences between healthy controls and diabetic patients (P < 0.001, respectively). The ASQ FD ratio (logarithmic) correlated with the CAP (r = -0.81, P < 0.001) and (1)H-MRS (r = -0.43, P = 0.004) results. The FD ratio [CAP < 250 dB/m: 107 (102-109), CAP between 250 and 300 dB/m: 106 (102-114); CAP between 300 and 350 dB/m: 105 (100-112), CAP ≥ 350 dB/m: 102 (99-108)] as well as mode and average parameters, were reduced in cases with advanced steatosis (ANOVA P < 0.05). However, none of the ASQ parameters showed a significant difference in patients with advanced fibrosis, as determined by TE and the NAFLD fibrosis score (P > 0.08, respectively). CONCLUSION: ASQ parameters correlate with steatosis, but not with fibrosis in fatty liver disease. Steatosis estimation with ASQ should be further evaluated in biopsy-controlled studies.


Assuntos
Técnicas de Imagem por Elasticidade , Cirrose Hepática/diagnóstico por imagem , Fígado/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Fígado/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Espectroscopia de Prótons por Ressonância Magnética , Índice de Gravidade de Doença , Adulto Jovem
15.
Dis Model Mech ; 7(12): 1407-15, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25288681

RESUMO

Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4(trg) mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4(trg) mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4(trg) mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4(trg) mutant will be a valuable tool to study human muscle disorders.


Assuntos
Mutação , Tropomodulina/genética , Tropomodulina/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Actinina/química , Actinas/química , Alelos , Animais , Animais Geneticamente Modificados , Citoplasma/metabolismo , Modelos Animais de Doenças , Ligação Genética , Masculino , Músculos/patologia , Doenças Musculares/genética , Doenças Musculares/patologia , Miofibrilas/metabolismo , Doenças Neuromusculares/metabolismo , Faloidina/química , Fenótipo , Sarcômeros/metabolismo , Peixe-Zebra
16.
PLoS One ; 8(6): e64810, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840306

RESUMO

Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain.


Assuntos
Lesões Encefálicas/genética , Modelos Animais de Doenças , Camundongos Transgênicos , Síndromes Neurotóxicas/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Animais , Lesões Encefálicas/induzido quimicamente , Dosagem de Genes , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurotoxinas , Espermina/metabolismo , Poliamina Oxidase
17.
Hum Mol Genet ; 22(22): 4562-78, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23804750

RESUMO

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.


Assuntos
Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Citocromos c/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Dinâmica Mitocondrial , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Morte Celular , Ciclofilinas/antagonistas & inibidores , Citocromos c/genética , Técnicas de Silenciamento de Genes , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Metazolamida/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Filogenia , Homologia de Sequência , Peixe-Zebra/embriologia , Peixe-Zebra/genética
18.
Genesis ; 51(6): 443-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23444339

RESUMO

The muscle-specific UNC-45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc-45b upstream region revealed that unc-45b promoter fragments reliably drive GFP expression after germline transmission. The muscle-specific 503-bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(-503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc-45b expression pattern. We propose that the 503unc promoter is a small and muscle-specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Chaperonas Moleculares/genética , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas , Deleção de Sequência , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Desenvolvimento Muscular , Proteínas Musculares , Músculo Esquelético/crescimento & desenvolvimento
19.
Dis Model Mech ; 5(6): 726-32, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23115202

RESUMO

Muscular dystrophies are a group of genetic disorders that specifically affect skeletal muscle and are characterized by progressive muscle degeneration and weakening. To develop therapies and treatments for these diseases, a better understanding of the molecular basis of muscular dystrophies is required. Thus, identification of causative genes mutated in specific disorders and the study of relevant animal models are imperative. Zebrafish genetic models of human muscle disorders often closely resemble disease pathogenesis, and the optical clarity of zebrafish embryos and larvae enables visualization of dynamic molecular processes in vivo. As an adjunct tool, morpholino studies provide insight into the molecular function of genes and allow rapid assessment of candidate genes for human muscular dystrophies. This unique set of attributes makes the zebrafish model system particularly valuable for the study of muscle diseases. This review discusses how recent research using zebrafish has shed light on the pathological basis of muscular dystrophies, with particular focus on the muscle cell membrane and the linkage between the myofibre cytoskeleton and the extracellular matrix.


Assuntos
Modelos Animais de Doenças , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Peixe-Zebra/fisiologia , Animais , Humanos , Distrofia Muscular Animal/congênito
20.
Biochem Biophys Res Commun ; 423(4): 785-8, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22713473

RESUMO

Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in an otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.


Assuntos
Birrefringência , Modelos Animais de Doenças , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Peixe-Zebra , Animais , Larva/ultraestrutura , Proteínas de Membrana/genética , Proteínas Musculares/genética , Distrofia Muscular de Duchenne/genética , Mutação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
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