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.
Asunto(s)
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
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the corticospinal tract and leading to motor neuron death. According to a recent study, magnetic resonance imaging-visible changes suggestive of neurodegeneration seem absent in the motor cortex of G93A-SOD1 ALS mice. However, it has not yet been ascertained whether the cortical neural activity is intact, or alterations are present, perhaps even from an early stage. Here, cortical neurons from this model were isolated at post-natal day 1 and cultured on multielectrode arrays. Their activity was studied with a comprehensive pool of neurophysiological analyses probing excitability, criticality and network architecture, alongside immunocytochemistry and molecular investigations. Significant hyperexcitability was visible through increased network firing rate and bursting, whereas topological changes in the synchronization patterns were apparently absent. The number of dendritic spines was increased, accompanied by elevated transcriptional levels of the DLG4 gene, NMDA receptor 1 and the early pro-apoptotic APAF1 gene. The extracellular Na+, Ca2+, K+ and Cl- concentrations were elevated, pointing to perturbations in the culture micro-environment. Our findings highlight remarkable early changes in ALS cortical neuron activity and physiology. These changes suggest that the causative factors of hyperexcitability and associated toxicity could become established much earlier than the appearance of disease symptoms, with implications for the discovery of new hypothetical therapeutic targets.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Corteza Motora/patología , Neuronas Motoras/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Ratones Transgénicos , Enfermedades Neurodegenerativas/patología , Superóxido Dismutasa/metabolismoRESUMEN
Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-ß1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition.
Asunto(s)
MicroARNs/genética , Distrofia Muscular de Duchenne/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Fibrosis/genética , Fibrosis/metabolismo , Humanos , Lactante , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos mdx , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Mioblastos/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismoRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease. RESULTS: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls. CONCLUSIONS: Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Encéfalo/metabolismo , Ciclo Celular , MicroARNs/genética , Neuronas/metabolismo , Regulación hacia Arriba/genética , Animales , Recuento de Células , Diferenciación Celular , Progresión de la Enfermedad , Hipocampo/patología , Ratones , Ratones Transgénicos , MicroARNs/metabolismo , Células-Madre Neurales/metabolismo , Neuroglía/metabolismo , Neuronas/patología , Especificidad de Órganos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Superóxido Dismutasa/genéticaRESUMEN
Severe muscle fibrosis is the endpoint of many chronic myopathies. Identification of factors that regulate fibrosis is important for understanding its pathogenesis and for developing anti-fibrotic treatments that prevent muscle destruction. We have developed an in vitro model for screening potential anti-fibrotic agents. The model consists of three-dimensional clusters (nodules) of fibroblasts derived from Duchenne muscular dystrophy (DMD) muscle. The primary fibroblasts spontaneously and quickly form nodules resembling fibrotic foci (cells plus extracellular matrix) when grown on a solid substrate. We tested the anti-fibrotic action of suramin, decorin, and spironolactone (all with established anti-fibrotic activity) on the model. All three agents significantly reduced nodule number, and spironolactone and suramin significantly reduced nodule diameter. Nodule secretion of soluble collagen was also significantly reduced by decorin and spironolactone treatment, whereas suramin had no significant effect. Collagen I and fibronectin protein expression was significantly reduced in the culture medium of control and DMD fibroblasts by spironolactone treatment, but not by decorin and suramin treatment. Finally, in DMD fibroblast monolayers, collagen deposition was significantly reduced by all three agents. Spironolactone significantly reduced collagen I and fibronectin transcript levels, whereas decorin reduced only fibronectin. Our in vitro model of fibrogenesis has thus revealed differing anti-fibrotic effects in the three anti-fibrotic agents tested. It therefore appears as a useful and sensitive system for the testing of anti-fibrotic drugs and could be adapted for the high-throughput screening of new anti-fibrotic molecules.