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Purpose: Lignin is the most abundant source of aromatic biopolymers and has gained interest in industrial and biomedical applications due to the reported biocompatibility and defense provided against bacterial and fungal pathogens, besides antioxidant and UV-blocking properties. Especially in the form of nanoparticles (NPs), lignin may display also antioxidant and anti-inflammatory activities. Methods: To evaluate these characteristics, sonochemically nano-formulated pristine lignin (LigNPs) and enzymatically-phenolated one (PheLigNPs) were used to expose zebrafish embryos, without chorion, at different concentrations. Furthermore, two different zebrafish inflammation models were generated, by injecting Pseudomonas aeruginosa lipopolysaccharide (LPS) and by provoking a wound injury in the embryo caudal fin. The inflammatory process was investigated in both models by qPCR, analyzing the level of genes as il8, il6, il1ß, tnfα, nfkbiaa, nfk2, and ccl34a.4, and by the evaluation of neutrophils recruitment, taking advantage of the Sudan Black staining, in the presence or not of LigNPs and PheLigNPs. Finally, the Wnt/ß-catenin pathway, related to tissue regeneration, was investigated at the molecular level in embryos wounded and exposed to NPs. Results: The data obtained demonstrated that the lignin-based NPs showed the capacity to induce a positive response during an inflammatory event, increasing the recruitment of cytokines to accelerate their chemotactic function. Moreover, the LigNPs and PheLigNPs have a role in the resolution of wounds, favoring the regeneration process. Conclusion: In this paper, we used zebrafish embryos within 5 days post fertilization (hpf). Despite being an early-stage exemplary, the zebrafish embryos have proven their potential as predicting models. Further long-term experiments in adults will be needed to explore completely the biomedical capabilities of lignin NPs. The results underlined the safety of both NPs tested paved the way for further evaluations to exploit the anti-inflammatory and pro-healing properties of the lignin nanoparticles examined.
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Inflamación , Lignina , Nanopartículas , Pez Cebra , Animales , Lignina/química , Lignina/farmacología , Nanopartículas/química , Lipopolisacáridos/farmacología , Antiinflamatorios/farmacología , Antiinflamatorios/química , Modelos Animales de Enfermedad , Citocinas/metabolismo , Citocinas/genética , Embrión no Mamífero/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
During the twenty-first century, engineered nanomaterials (ENMs) have attracted rising interest, globally revolutionizing all industrial sectors. The expanding world population and the implementation of new global policies are increasingly pushing society toward a bioeconomy, focused on fostering the adoption of bio-based nanomaterials that are functional, cost-effective, and potentially secure to be implied in different areas, the medical field included. This research was focused on silica nanoparticles (SiO2-NPs) of bio-based and synthetic origin. SiO2-NPs are composed of silicon dioxide, the most abundant compound on Earth. Due to their characteristics and biocompatibility, they are widely used in many applications, including the food industry, synthetic processes, medical diagnosis, and drug delivery. Using zebrafish embryos as in vivo models, we evaluated the effects of amorphous silica bio-based NPs from rice husk (SiO2-RHSK NPs) compared to commercial hydrophilic fumed silica NPs (SiO2-Aerosil200). We evaluated the outcomes of embryo exposure to both nanoparticles (NPs) at the histochemical and molecular levels to assess their safety profile, including developmental toxicity, neurotoxicity, and pro-inflammatory potential. The results showed differences between the two silica NPs, highlighting that bio-based SiO2-RHSK NPs do not significantly affect neutrophils, macrophages, or other innate immune system cells.
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Materiales Biocompatibles , Embrión no Mamífero , Nanopartículas , Dióxido de Silicio , Pez Cebra , Pez Cebra/embriología , Animales , Dióxido de Silicio/química , Nanopartículas/química , Embrión no Mamífero/efectos de los fármacos , Materiales Biocompatibles/química , Desarrollo Embrionario/efectos de los fármacos , Ensayo de MaterialesRESUMEN
Classical dynamins (DNMs) are GTPase proteins engaged in endocytosis, a fundamental process for cargo internalization from the plasma membrane. In mammals, three DNM genes are present with different expression patterns. DNM1 is expressed at high levels in neurons, where it takes place in the recycling of synaptic vesicles; DNM2 is ubiquitously expressed, while DNM3 is found in the brain and in the testis. Due to the conservation of genes in comparison to mammals, we took advantage of a zebrafish model for functional characterization of dnm1a, ortholog of mammalian DNM1. Our data strongly demonstrated that dnm1a has a nervous tissue-specific expression pattern and plays a role in the formation of both axon and synapse. This is the first in vivo study that collects evidence about the effects of dnm1a loss of function in zebrafish, thus providing a new excellent model to be used in different scientific fields.
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Tejido Nervioso , Pez Cebra , Animales , Masculino , Axones , Neuronas/metabolismo , Sinapsis/metabolismo , MamíferosRESUMEN
Zinc oxide (ZnO) is the most efficient curing activator employed in the industrial rubber production. However, ZnO and Zn(II) ions are largely recognized as an environmental hazard being toxic to aquatic organisms, especially considering Zn(II) release during tire lifecycle. In this context, aiming at reducing the amount of microcrystalline ZnO, a novel activator was recently synthetized, constituted by ZnO nanoparticles (NPs) anchored to silica NPs (ZnO-NP@SiO2-NP). The objective of this work is to define the possible hazards deriving from the use of ZnO-NP@SiO2-NP compared to ZnO and SiO2 NPs traditionally used in the tire industry. The safety of the novel activators was assessed by in vitro testing, using human lung epithelial (A549) and immune (THP-1) cells, and by the in vivo model zebrafish (Danio rerio). The novel manufactured nanomaterial was characterized morphologically and structurally, and its effects evaluated in vitro by the measurement of the cell viability and the release of inflammatory mediators, while in vivo by the Fish Embryo Acute Toxicity (FET) test. Resulting data demonstrated that ZnO-NP@SiO2-NP, despite presenting some subtoxic events, exhibits the lack of acute effects both in vitro and in vivo, supporting the safe-by-design development of this novel material for the rubber industry.
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Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.
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Diferenciación Celular , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos , Indoles , Desarrollo de Músculos , Músculo Esquelético , Distrofia Muscular de Duchenne , Proteínas Represoras , Proteínas de Pez Cebra , Animales , Humanos , Acetilación , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/enzimología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Procesamiento Proteico-Postraduccional , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Mutations in the acidic alpha-glucosidase (GAA) coding gene cause Pompe disease. Late-onset Pompe disease (LOPD) is characterized by progressive proximal and axial muscle weakness and atrophy, causing respiratory failure. Enzyme replacement therapy (ERT), based on recombinant human GAA infusions, is the only available treatment; however, the efficacy of ERT is variable. Here we address the question whether proteins at variance in LOPD muscle of patients before and after 1 year of ERT, compared withhealthy age-matched subjects (CTR), reveal a specific signature. Proteins extracted from skeletal muscle of LOPD patients and CTR were analyzed by combining gel based (two-dimensional difference gel electrophoresis) and label-free (liquid chromatography-mass spectrometry) proteomic approaches, and ingenuity pathway analysis. Upstream regulators targeting autophagy and lysosomal tethering were assessed by immunoblotting. 178 proteins were changed in abundance in LOPD patients, 47 of them recovered normal level after ERT. Defects in oxidative metabolism, muscle contractile protein regulation, cytoskeletal rearrangement, and membrane reorganization persisted. Metabolic changes, ER stress and UPR (unfolded protein response) contribute to muscle proteostasis dysregulation with active membrane remodeling (high levels of LC3BII/LC3BI) and accumulation of p62, suggesting imbalance in the autophagic process. Active lysosome biogenesis characterizes both LOPD PRE and POST, unparalleled by molecules involved in lysosome tethering (VAMP8, SNAP29, STX17, and GORASP2) and BNIP3. In conclusion this study reveals a specific signature that suggests ERT prolongation and molecular targets to ameliorate patient's outcome.
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Terapia de Reemplazo Enzimático/métodos , Glucano 1,4-alfa-Glucosidasa/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Músculo Esquelético/metabolismo , Proteómica/métodos , Adulto , Autofagia , Cromatografía Liquida/métodos , Electroforesis en Gel Bidimensional/métodos , Femenino , Glucano 1,4-alfa-Glucosidasa/genética , Humanos , Lisosomas/metabolismo , Masculino , Microscopía Electrónica de Transmisión , Proteínas Musculares/metabolismo , Músculo Esquelético/ultraestructura , Proteoma/metabolismo , Proteínas Recombinantes/uso terapéutico , Espectrometría de Masas en Tándem/métodosRESUMEN
3,4-Diaminopyridine (3,4-DAP) and its phosphate form, 3,4-DAPP have been used efficiently in the past years to treat muscular weakness in myasthenic syndromes with neuromuscular junctions (NMJs) impairment. Pompe disease (PD), an autosomal recessive metabolic disorder due to a defect of the lysosomal enzyme α-glucosidase (GAA), presents some secondary symptoms that are related to neuromuscular transmission dysfunction, resulting in endurance and strength failure. In order to evaluate whether 3,4-DAPP could have a beneficial effect on this pathology, we took advantage of a transient zebrafish PD model that we previously generated and characterized. We investigated presynaptic and postsynaptic structures, NMJs at the electron microscopy level, and zebrafish behavior, before and after treatment with 3,4-DAPP. After drug administration, we observed an increase in the number of acetylcholine receptors an increment in the percentage of NMJs with normal structure and amelioration in embryo behavior, with recovery of typical movements that were lost in the embryo PD model. Our results revealed early NMJ impairment in Pompe zebrafish model with improvement after administration of 3,4-DAPP, suggesting its potential use as symptomatic drug in patients with Pompe disease.
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Amifampridina/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Unión Neuromuscular/efectos de los fármacos , Animales , Conducta Animal , Embrión no Mamífero , Actividad Motora/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Receptores Colinérgicos/efectos de los fármacos , Pez Cebra , alfa-Glucosidasas/metabolismoRESUMEN
Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, due to an enzymatic error of lipid metabolism. Patients present always with skeletal muscle myopathy and variable cardiac and hepatic involvement. NLSDM is caused by mutations in the PNPLA2 gene, which encodes the adipose triglyceride lipase (ATGL). Here we report the molecular characterization and clinical findings of two NLSDM siblings carrying the novel c.187+1G > C homozygous PNPLA2 mutation, localized in the splice site of intron 2. Molecular analyses revealed that neither aberrant PNPLA2 mRNA isoforms, nor ATGL mutated protein were detectable in patient's cells. Clinically, both patients presented early onset muscle weakness, in particular of proximal upper limb muscles. In almost 15 years, muscle damage affected also distal upper limbs. This is a NLSDM family, displaying a severe PNPLA2 mutation in two siblings with clinical presentation characterized by an early onset, but a slowly evolution of severe myopathy.
RESUMEN
Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.
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Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Glucógeno/metabolismo , Hexoquinasa/antagonistas & inhibidores , Piruvatos/farmacología , Animales , Animales Modificados Genéticamente , Autofagia/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Técnicas de Silenciamiento del Gen , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Glucólisis/efectos de los fármacos , Hexoquinasa/metabolismo , Humanos , Lisosomas , Microscopía Electrónica , Morfolinos/administración & dosificación , Morfolinos/genética , Actividad Motora/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Piruvatos/uso terapéutico , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismoRESUMEN
Cornelia de Lange syndrome (CdLS), which is reported to affect â¼1 in 10 000 to 30 000 newborns, is a multisystem organ developmental disorder with relatively mild to severe effects. Among others, intellectual disability represents an important feature of this condition. CdLS can result from mutations in at least five genes: nipped-B-like protein, structural maintenance of chromosomes 1A, structural maintenance of chromosomes 3, RAD21 cohesin complex component and histone deacetylase 8 (HDAC8). It is believed that mutations in these genes cause CdLS by impairing the function of the cohesin complex (to which all the aforementioned genes contribute to the structure or function), disrupting gene regulation during critical stages of early development. Since intellectual disorder might result from alterations in neural development, in this work, we studied the role of Hdac8 gene in mouse neural stem cells (NSCs) and in vertebrate (Danio rerio) brain development by knockdown and chemical inhibition experiments. Underlying features of Hdac8 deficiency is an increased cell death in the developing neural tissues, either in mouse NSCs or in zebrafish embryos.
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Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Síndrome de Cornelia de Lange/genética , Histona Desacetilasas/genética , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas Cromosómicas no Histona/genética , Síndrome de Cornelia de Lange/fisiopatología , Regulación de la Expresión Génica/genética , Histona Desacetilasas/metabolismo , Histona Desacetilasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Células-Madre Neurales/fisiología , Neuronas/fisiología , Fenotipo , Proteínas Represoras/genética , Pez Cebra , Proteínas de Pez Cebra , CohesinasRESUMEN
Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.
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Histona Desacetilasas/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Proteínas Represoras/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Diferenciación Celular/fisiología , Humanos , Ratones , Músculo Esquelético/citología , Mioblastos/metabolismo , Pez CebraRESUMEN
Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis.
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Exosomas/genética , MicroARNs/genética , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Regulación hacia Arriba , Actinas/genética , Actinas/metabolismo , Animales , Cardiotoxinas/efectos adversos , Comunicación Celular , Proliferación Celular , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Exosomas/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/inducido químicamente , Distrofia Muscular de Duchenne/metabolismoRESUMEN
Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc.
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Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neoplasias Mamarias Animales/metabolismo , Células Madre Neoplásicas/metabolismo , Organoides/metabolismo , Animales , Perros , Femenino , Neoplasias Mamarias Animales/patología , Células Madre Neoplásicas/patología , Organoides/patología , Células Tumorales CultivadasRESUMEN
Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down the zebrafish dnm2a, producing defects in muscle morphology. Finally, we expressed two mutated DNM2 mRNA by injecting zebrafish embryos with human mRNAs carrying the R522H mutation, causing CNM, or the G537C mutation, causing CMT. Defects arose especially in secondary motor neuron formation, with incorrect branching in embryos injected with CNM-mutated mRNA, and total absence of branching in those injected with CMT-mutated mRNA. Muscle morphology in embryos injected with CMT-mutated mRNA appeared less regularly organized than in those injected with CNM-mutated mRNA. Our results showing, a continuum between CNM and CMTDIB phenotypes in zebrafish, similarly to the human conditions, confirm this animal model to be a powerful tool to investigate mutations of DNM2 in vivo.
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Enfermedad de Charcot-Marie-Tooth/patología , Modelos Animales de Enfermedad , Dinamina II/deficiencia , Dinamina II/genética , Dinaminas/genética , Miopatías Estructurales Congénitas/patología , Pez Cebra/metabolismo , Empalme Alternativo , Animales , Animales Modificados Genéticamente , Enfermedad de Charcot-Marie-Tooth/genética , Dinamina II/metabolismo , Dinaminas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Células Musculares/metabolismo , Células Musculares/patología , Mutación , Miopatías Estructurales Congénitas/genética , Homología de Secuencia de Ácido Nucleico , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
UNLABELLED: Mutations in SEPN1 cause selenoprotein N (SEPN)-related myopathy (SEPN-RM) characterized by early-onset axial and neck weakness, spinal rigidity, respiratory failure and histopathological features, ranging from mild dystrophic signs to a congenital myopathy pattern with myofibrillar disorganization. We report on clinical and instrumental features in three patients affected with a congenital myopathy characterized by prevalent neck weakness starting at different ages and mild myopathy, in whom we performed diagnosis of SEPN-RM. The patients presented myopathic signs since their first years of life, but the disease remained unrecognized because of a relatively benign myopathic course. In two cases, myopathic features were stable after 2 years of follow-up, but respiratory involvement worsened. The muscle MRI and muscle biopsy showed a typical pattern of SEPN-RM. Molecular diagnosis revealed two novel homozygous mutations in SEPN1, c.1176delA and c.726_727InsTCC. CONCLUSION: This report underlines the clinical diagnostic clues of early neck and axial weakness to suspect a SEPN-RM and the usefulness of muscle MRI in conjunction with clinical features to achieve the diagnosis. Our data confirm the slow progression of respiratory involvement in spite of the relatively stable course of myopathy. We report two previously undescribed mutations in SEPN1. WHAT IS KNOWN: ⢠Mutations in SEPN1 cause myopathy characterized by early-onset axial and neck weakness spinal rigidity and respiratory failure. ⢠SEPN-related myopathies have been initially associated with four distinct histopathological entities that however appear more mixed in recently described cases. What is New: ⢠SEPN-related myopathies can remain unrecognized because of the normal early motor development and relatively benign myopathic course of the disease. ⢠Our study adds two novel homozygous mutations to the number of reported pathogenic SEPN1 variants.
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Proteínas Musculares/genética , Mutación , Miopatías Estructurales Congénitas/diagnóstico , Músculos del Cuello/anomalías , Selenoproteínas/genética , Adolescente , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Electrocardiografía , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Insuficiencia Respiratoria/etiologíaRESUMEN
AIMS: Tissue fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the end point of diseases affecting the kidney, bladder, liver, lung, gut, skin, heart and muscle. In Duchenne muscular dystrophy (DMD), connective fibrotic tissue progressively substitutes muscle fibers. So far no specific pharmacological treatment is available for muscle fibrosis. Among promising anti-fibrotic molecules, pirfenidone has shown anti-fibrotic and anti-inflammatory activity in animal and cell models, and has already been employed in clinical trials. Therefore we tested pirfenidone anti-fibrotic properties in an in vitro model of muscle fibrosis. MAIN METHODS: We evaluated effect of pirfenidone on fibroblasts isolated from DMD muscle biopsies. These cells have been previously characterized as having a pro-fibrotic phenotype. We tested cell proliferation and migration, secretion of soluble collagens, intracellular levels of collagen type I and fibronectin, and diameter of 3D fibrotic nodules. KEY FINDINGS: We found that pirfenidone significantly reduced proliferation and cell migration of control and DMD muscle-derived fibroblasts, decreased extracellular secretion of soluble collagens by control and DMD fibroblasts, as well as levels of collagen type I and fibronectin, and, in DMD fibroblasts only, reduced synthesis and deposition of intracellular collagen. Furthermore, pirfenidone was able to reduce the diameter of fibrotic-nodules in our 3D model of in vitro fibrosis. SIGNIFICANCE: These pre-clinical results indicate that pirfenidone has potential anti-fibrotic effects also in skeletal muscle fibrosis, urging further studies in in vivo animal models of muscular dystrophy in order to translate the drug into the treatment of muscle fibrosis in DMD patients.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Músculos/efectos de los fármacos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Piridonas/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Preescolar , Colágeno/análisis , Fibroblastos/patología , Fibrosis , Humanos , Músculos/patología , Distrofia Muscular de Duchenne/patologíaRESUMEN
Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. To date, mutation in 7 different genes has been reported to cause CNMs but 30 % of cases still remain genetically undefined. Genetic investigations are often expensive and time consuming. Clinical and morphological clues are needed to facilitate genetic tests and to choose the best approach for genetic screening. We aimed to describe genotype-phenotype correlation in an Italian cohort of patients affected by CNMs, to define the relative frequencies of its defined genetic forms and to draw a diagnostic algorithm to address genetic investigations. We recruited patients with CNMs from all the Italian tertiary neuromuscular centers following clinical and histological criteria. All selected patients were screened for the four 'canonical' genes related to CNMs: MTM1, DNM2, RYR1 and BIN1. Pathogenetic mutations were found in 38 of the 54 screened patients (70 %), mostly in patients with congenital onset (25 of 30 patients, 83 %): 15 in MTM1, 6 in DNM2, 3 in RYR1 and one in TTN. Among the 13 patients with a childhood-adolescence onset, mutations were found in 6 patients (46 %), all in DNM2. In the group of the 11 patients with adult onset, mutations were identified in 7 patients (63 %), again in DNM2, confirming that variants in this gene are relatively more common in late-onset phenotypes. The present study provides the relative molecular frequency of centronuclear myopathy and of its genetically defined forms in Italy and also proposes a diagnostic algorithm to be used in clinical practice to address genetic investigations.
Asunto(s)
Conectina/genética , Dinamina II/genética , Predisposición Genética a la Enfermedad/genética , Mutación/genética , Miopatías Estructurales Congénitas/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Niño , Preescolar , Estudios de Cohortes , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Recién Nacido , Italia/epidemiología , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/fisiopatología , Fenotipo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Adulto JovenRESUMEN
BACKGROUND: Mutations in the PTRF gene, coding for cavin-1, cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. In CGL4, symptoms are variable comprising, in addition to myopathy, smooth and skeletal muscle hypertrophy, cardiac arrhythmias, and skeletal abnormalities. Secondary features are atlantoaxial instability, acanthosis nigricans, hepatomegaly, umbilical prominence and metabolic abnormalities related to insulin resistance, such as diabetes mellitus, hyperlipidemia and hepatic steatosis. CASE PRESENTATION: We describe a 3 year-old child of Moroccan origin with mild muscle phenotype, mainly characterized by mounding, muscle pain, hyperCKemia and mild caveolin 3 reduction on muscle biopsy. No CAV3 gene mutation was detected; instead we found a novel mutation, a homozygous single base pair deletion, in the PTRF gene. Only after detection of this mutation a mild generalized loss of subcutaneous fat, at first underestimated, was noticed and the diagnosis of lipodystrophy inferred. CONCLUSIONS: The PTRF gene should be investigated in patients with hyperCKemia, mild myopathy associated with spontaneous or percussion-induced muscle contractions like rippling or mounding, and no CAV3 mutation. The analysis should be performed even if cardiac or metabolic alterations are absent, particularly in young patients in whom lipodystrophy may be difficult to ascertain.
Asunto(s)
Lipodistrofia Generalizada Congénita/genética , Enfermedades Musculares/genética , Proteínas de Unión al ARN/genética , Caveolina 1/metabolismo , Caveolina 3/genética , Caveolina 3/metabolismo , Preescolar , Eliminación de Gen , Homocigoto , Humanos , Lipodistrofia Generalizada Congénita/complicaciones , Lipodistrofia Generalizada Congénita/patología , Masculino , Contracción Muscular , Enfermedades Musculares/complicaciones , Mutación , Grasa Subcutánea/metabolismo , Tomografía Computarizada por Rayos XRESUMEN
Mutations in dynamin 2 (DNM2) gene cause autosomal dominant centronuclear myopathy and occur in around 50% of patients with centronuclear myopathy. We report clinical, morphological, muscle imaging and genetic data of 10 unrelated Italian patients with centronuclear myopathy related to DNM2 mutations. Our results confirm the clinical heterogeneity of this disease, underlining some peculiar clinical features, such as severe pulmonary impairment and jaw contracture that should be considered in the clinical follow-up of these patients. Muscle MRI showed a distinct pattern of involvement, with predominant involvement of soleus and tibialis anterior in the lower leg muscles, followed by hamstring muscles and adductor magnus at thigh level and gluteus maximus. The detection of three novel DNM2 mutations and the first case of somatic mosaicism further expand the genetic spectrum of the disease.