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1.
Cells ; 9(2)2020 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-32053901

RESUMEN

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

2.
Matrix Biol ; 2019 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-31422156

RESUMEN

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2-/- (Mmrn2-/-) mice. Although Mmrn2-/- mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2-/- mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2-/- vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2-/- mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

3.
Skelet Muscle ; 9(1): 14, 2019 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-31133047

RESUMEN

BACKGROUND: Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients. CASE PRESENTATION: A consanguineous family with congenital to adult-onset muscle weakness and hanging big toe was reported. Muscle biopsy showed minimal changes with internal nuclei, type 1 fiber predominance, and ultrastructural defects of Z line. Muscle CT imaging showed marked hypodensity of the sartorius bilaterally and MRI scattered abnormal high-intensity areas in the internal tongue muscle and in the posterior cervical muscles. Cardiac involvement was demonstrated by magnetic resonance imaging and late gadolinium enhancement. Whole exome sequencing analysis identified a homozygous loss of function single nucleotide deletion in the exon 11 of the MYPN gene in two siblings. Full-length MYPN protein was undetectable on immunoblotting, and on immunofluorescence, its localization at the Z line was missed. CONCLUSIONS: This report extends the phenotypic spectrum of recessive MYPN-related myopathies showing: (1) the two patients had hanging big toe and the oldest one developed spine and hand contractures, none of these signs observed in the previously reported patients, (2) specific ultrastructural changes consisting in Z line fragmentation, but (3) no nemaline or caps on muscle pathology.

4.
Autophagy ; 15(8): 1438-1454, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30806141

RESUMEN

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

5.
Cardiovasc Res ; 115(4): 739-751, 2019 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-30304392

RESUMEN

AIMS: Arrhythmogenic cardiomyopathy (AC) is one of the most common inherited cardiomyopathies, characterized by progressive fibro-fatty replacement in the myocardium. Clinically, AC manifests itself with ventricular arrhythmias, syncope, and sudden death and shows wide inter- and intra-familial variability. Among the causative genes identified so far, those encoding for the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are the most commonly mutated. So far, little is known about the molecular mechanism(s) behind such a varied spectrum of phenotypes, although it has been shown that the causative mutations not only lead to structural abnormalities but also affect the miRNA profiling of cardiac tissue. Here, we aimed at studying the pathogenic effects of a nonsense mutation of the desmoglein-2 gene, both at the structural level and in terms of miRNA expression pattern. METHODS AND RESULTS: We generated transgenic mice with cardiomyocyte-specific overexpression of a FLAG-tagged human desmoglein-2 harbouring the Q558* nonsense mutation found in an AC patient. The hearts of these mice showed signs of fibrosis, decrease in desmosomal size and number, and reduction of the Wnt/ß-catenin signalling. Genome-wide RNA-Seq performed in Tg-hQ hearts and non-transgenic hearts revealed that 24 miRNAs were dysregulated in transgenic animals. Further bioinformatic analyses for selected miRNAs suggested that miR-217-5p, miR-499-5p, and miR-708-5p might be involved in the pathogenesis of the disease. CONCLUSION: Down-regulation of the canonical Wnt/ß-catenin signalling might be considered a common key event in the AC pathogenesis. We identified the miRNA signature in AC hearts, with miR-708-5p and miR-217-5p being the most up-regulated and miR-499-5p the most down-regulated miRNAs. All of them were predicted to be involved in the regulation of the Wnt/ß-catenin pathway and might reveal the potential pathophysiology mechanisms of AC, as well as be useful as therapeutic targets for the disease.

6.
Matrix Biol ; 81: 34-49, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30408617

RESUMEN

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1-/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9ß1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain.

7.
Acta Neuropathol ; 136(3): 483-499, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29752552

RESUMEN

The synaptic cleft of the neuromuscular junction (NMJ) consists of a highly specialized extracellular matrix (ECM) involved in synapse maturation, in the juxtaposition of pre- to post-synaptic areas, and in ensuring proper synaptic transmission. Key components of synaptic ECM, such as collagen IV, perlecan and biglycan, are binding partners of one of the most abundant ECM protein of skeletal muscle, collagen VI (ColVI), previously never linked to NMJ. Here, we demonstrate that ColVI is itself a component of this specialized ECM and that it is required for the structural and functional integrity of NMJs. In vivo, ColVI deficiency causes fragmentation of acetylcholine receptor (AChR) clusters, with abnormal expression of NMJ-enriched proteins and re-expression of fetal AChRγ subunit, both in Col6a1 null mice and in patients affected by Ullrich congenital muscular dystrophy (UCMD), the most severe form of ColVI-related myopathies. Ex vivo muscle preparations from ColVI null mice revealed altered neuromuscular transmission, with electrophysiological defects and decreased safety factor (i.e., the excess current generated in response to a nerve impulse over that required to reach the action potential threshold). Moreover, in vitro studies in differentiated C2C12 myotubes showed the ability of ColVI to induce AChR clustering and synaptic gene expression. These findings reveal a novel role for ColVI at the NMJ and point to the involvement of NMJ defects in the etiopathology of ColVI-related myopathies.


Asunto(s)
Colágeno Tipo VI/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Unión Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Esclerosis/metabolismo , Animales , Colágeno Tipo VI/genética , Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Noqueados , Distrofias Musculares/genética , Esclerosis/genética
8.
Nucleus ; 9(1): 292-304, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-29693488

RESUMEN

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF ß2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF ß2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF ß2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF ß2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF ß2-dependent mechanisms were also determined in laminopathic cells, where TGF ß2 activated AKT/mTOR phosphorylation. These data show that TGF ß2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF ß2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.


Asunto(s)
Diferenciación Celular , Células Musculares/patología , Distrofia Muscular de Emery-Dreifuss/sangre , Distrofia Muscular de Emery-Dreifuss/patología , Tenocitos/patología , Factor de Crecimiento Transformador beta2/sangre , Adulto , Animales , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Células Musculares/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Tenocitos/metabolismo , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Adulto Joven
9.
Front Genet ; 9: 723, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30740127

RESUMEN

Only a few genes involved in teeth development and morphology are known to be responsible for tooth abnormalities in Mendelian-inherited diseases. We studied an inbred family of Pakistani origin in which two first-cousin born brothers are affected by early tooth loss with peculiar teeth abnormalities characterized by the absence of cementum formation. Whole exome sequencing revealed a H2665L homozygous sequence variant in the VCAN gene. Dominant splicing mutations in VCAN are known to cause Wagner syndrome or vitreoretinopathy. We explored teeth morphology in these two patients, while versican expression was assessed by western blot analysis. Early signs of vitreoretinopathy were found in the elder brother while the parents were completely negative. Our findings suggest that the homozygous recessive H2665L missense sequence variant impairs the normal morphology of the teeth roots via loss of cementum synthesis, and is also associated with early onset, recessive, Wagner syndrome, thus expanding both the phenotype mutation scenario and the inheritance mode of VCAN mutations.

10.
Pharmacol Res ; 125(Pt B): 122-131, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28899790

RESUMEN

Duchenne muscular dystrophy (DMD) is a severe muscle disease of known etiology without effective, or generally applicable therapy. Mitochondria are affected by the disease in animal models but whether mitochondrial dysfunction is part of the pathogenesis in patients remains unclear. We show that primary cultures obtained from muscle biopsies of DMD patients display a decrease of the respiratory reserve, a consequence of inappropriate opening of the permeability transition pore (PTP). Treatment with the cyclophilin inhibitor alisporivir - a cyclosporin A derivative that desensitizes the PTP but does not inhibit calcineurin - largely restored the maximal respiratory capacity without affecting basal oxygen consumption in cells from patients, thus reinstating a normal respiratory reserve. Treatment with alisporivir, but not with cyclosporin A, led to a substantial recovery of respiratory function matching improved muscle ultrastructure and survival of sapje zebrafish, a severe model of DMD where muscle defects are close to those of DMD patients. Alisporivir was generally well tolerated in HCV patients and could be used for the treatment of DMD.


Asunto(s)
Ciclosporina/farmacología , Mitocondrias/efectos de los fármacos , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Ciclosporina/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/fisiología , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Consumo de Oxígeno/efectos de los fármacos , Pez Cebra
11.
Autophagy ; 12(12): 2484-2495, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27656840

RESUMEN

A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients.


Asunto(s)
Autofagia , Colágeno Tipo VI/genética , Dieta con Restricción de Proteínas , Enfermedades Musculares/dietoterapia , Adulto , Alanina/metabolismo , Biomarcadores/metabolismo , Biopsia , Composición Corporal , Contractura/metabolismo , Contractura/patología , Contractura/fisiopatología , Femenino , Humanos , Ácido Láctico/metabolismo , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Músculos/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/fisiopatología , Distrofias Musculares/congénito , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofias Musculares/fisiopatología , Proyectos Piloto , Esclerosis/metabolismo , Esclerosis/patología , Esclerosis/fisiopatología , Caminata , Adulto Joven
12.
Front Aging Neurosci ; 8: 131, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27375477

RESUMEN

Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies.

13.
Matrix Biol ; 55: 90-105, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-26944560

RESUMEN

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI-NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI-NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor ß1 (TGFß1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFß1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair.


Asunto(s)
Antígenos/metabolismo , Colágeno Tipo VI/fisiología , Fibroblastos/metabolismo , Proteoglicanos/metabolismo , Adolescente , Movimiento Celular , Transdiferenciación Celular , Células Cultivadas , Humanos , Persona de Mediana Edad , Unión Proteica , Transporte de Proteínas , Tendones/citología , Factor de Crecimiento Transformador beta1/fisiología , Adulto Joven
14.
J Cell Sci ; 129(8): 1671-84, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26945058

RESUMEN

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.


Asunto(s)
Factores de Transcripción ARNTL/genética , Relojes Circadianos/fisiología , Colágeno Tipo VI/genética , Contractura/genética , Mitocondrias/fisiología , Distrofias Musculares/congénito , Mutación/genética , Esclerosis/genética , Animales , Autofagia/genética , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Análisis por Micromatrices , Distrofias Musculares/genética , ARN/análisis
15.
BMC Neurol ; 15: 153, 2015 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-26306629

RESUMEN

BACKGROUND: Currently, the most promising therapies for Duchenne muscular dystrophy (DMD) are exon skipping and stop codon read-through, two strategies aimed at restoring the expression of dystrophin. A phase 3 clinical trial with drisapersen, a drug designed to induce exon 51-skipping, has failed to show significant improvement of the primary outcome measure, the six-minute walk test. DISCUSSION: Here, we review some key points that should be considered when designing clinical trials for these new therapies. First, younger patients have more functional abilities and more muscle fibers to preserve than older patients and therefore are better subjects for trials designed to demonstrate the success of new treatments. Second, the inclusion of patients on corticosteroids both in the treatment and placebo groups is of concern because the positive effect of corticosteroids might mask the effect of the treatment being tested. Additionally, the reasonable expectation from these therapies is the slowing of disease progression rather than improvement. Therefore, the appropriate clinical endpoints are the prolongation of the ability to stand from the floor, climb stairs, and walk, not an increase in muscle strength or function. Hence, the time frames for the detection of new dystrophin, which occurs within months, and the ability to demonstrate a slowing of disease progression, which requires years, are strikingly different. Finally, placebo-controlled trials are difficult to manage if years of blindness are required to demonstrate a slowing of disease progression. Thus, accelerated/conditional approval for new therapies should be based on surrogate biochemical outcomes: the demonstration of de novo dystrophin production and of its beneficial effect on the functional recovery of muscle fiber. These data suggest that clinical trials for DMD patients must be adapted to the particular characteristics of the disease in order to demonstrate the expected positive effect of new treatments.


Asunto(s)
Corticoesteroides/uso terapéutico , Ensayos Clínicos como Asunto/métodos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Oligonucleótidos/uso terapéutico , Mejoramiento de la Calidad , Proyectos de Investigación , Progresión de la Enfermedad , Quimioterapia Combinada , Distrofina/genética , Exones , Humanos , Masculino , Fuerza Muscular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatología , Resultado del Tratamiento , Caminata
17.
Front Aging Neurosci ; 6: 324, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25477819

RESUMEN

Ullrich congenital muscular dystrophy and Bethlem myopathy are caused by mutations in collagen VI (ColVI) genes, which encode an extracellular matrix protein; yet, mitochondria play a major role in disease pathogenesis through a short circuit caused by inappropriate opening of the permeability transition pore, a high-conductance channel, which causes a shortage in ATP production. We find that melanocytes do not produce ColVI yet they bind it at the cell surface, suggesting that this protein may play a trophic role and that its absence may cause lesions similar to those seen in skeletal muscle. We show that mitochondria in melanocytes of Ullrich congenital muscular dystrophy and Bethlem myopathy patients display increased size, reduced matrix density, and disrupted cristae, findings that suggest a functional impairment. In keeping with this hypothesis, mitochondria (i) underwent anomalous depolarization after inhibition of the F-ATP synthase with oligomycin, and (ii) displayed decreased respiratory reserve capacity. The non-immunosuppressive cyclophilin inhibitor NIM811 prevented mitochondrial depolarization in response to oligomycin in melanocytes from both Ullrich congenital muscular dystrophy and Bethlem myopathy patients, and partially restored the respiratory reserve of melanocytes from one Bethlem myopathy patient. These results match our recent findings on melanocytes from patients affected by Duchenne muscular dystrophy (Pellegrini et al., 2013), and suggest that skin biopsies may represent a minimally invasive tool to investigate mitochondrial dysfunction and to evaluate drug efficacy in ColVI-related myopathies and possibly in other muscle wasting conditions like aging sarcopenia.

18.
Front Aging Neurosci ; 6: 215, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25191266

RESUMEN

The four-and-half LIM domain protein 1 (FHL1) is highly expressed in skeletal and cardiac muscle. Mutations of the FHL1 gene have been associated with diverse chronic myopathies including reducing body myopathy, rigid spine syndrome (RSS), and Emery-Dreifuss muscular dystrophy. We investigated a family with a mutation (p.C150R) in the second LIM domain of FHL1. In this family, a brother and a sister were affected by RSS, and their mother had mild lower limbs weakness. The 34-year-old female had an early and progressive rigidity of the cervical spine and severe respiratory insufficiency. Muscle mass evaluated by DXA was markedly reduced, while fat mass was increased to 40%. CT scan showed an almost complete substitution of muscle by fibro-adipose tissue. Muscle biopsy showed accumulation of FHL1 throughout the cytoplasm and around myonuclei into multiprotein aggregates with aggresome/autophagy features as indicated by ubiquitin, p62, and LC3 labeling. DNA deposits, not associated with nuclear lamina components and histones, were also detected in the aggregates, suggesting nuclear degradation. Ultrastructural analysis showed the presence of dysmorphic nuclei, accumulation of tubulofilamentous and granular material, and perinuclear accumulation of autophagic vacuoles. These data point to involvement of the aggresome-autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation.

19.
Hum Mol Genet ; 23(20): 5353-63, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24852368

RESUMEN

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.


Asunto(s)
Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Ciclosporina/administración & dosificación , Distrofias Musculares/tratamiento farmacológico , Distrofias Musculares/patología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Ciclosporina/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Ratones , Mitocondrias/metabolismo , Fuerza Muscular/efectos de los fármacos , Distrofias Musculares/congénito , Distrofias Musculares/genética , Pez Cebra
20.
J Cell Physiol ; 229(7): 878-86, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24356950

RESUMEN

Cell-extracellular matrix interaction plays a major role in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. Collagen VI is a widely expressed non-fibrillar collagen, which regulates tissues homeostasis. The objective of the present investigation was to extend our understanding of the role of collagen VI in human ACL. This study shows that collagen VI is associated both in vivo and in vitro to the cell membrane of knee ACL fibroblasts, contributing to the constitution of a microfibrillar pericellular matrix. In cultured cells the localization of collagen VI at the cell surface correlated with the expression of NG2 proteoglycan, a major collagen VI receptor. The treatment of ACL fibroblasts with anti-NG2 antibody abolished the localization of collagen VI indicating that collagen VI pericellular matrix organization in ACL fibroblasts is mainly mediated by NG2 proteoglycan. In vitro mechanical strain injury dramatically reduced the NG2 proteoglycan protein level, impaired the association of collagen VI to the cell surface, and promoted cell cycle withdrawal. Our data suggest that the injury-induced alteration of specific cell-ECM interactions may lead to a defective fibroblast self-renewal and contribute to the poor regenerative ability of ACL fibroblasts.


Asunto(s)
Ligamento Cruzado Anterior/metabolismo , Membrana Celular/metabolismo , Colágeno Tipo VI/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Ligamento Cruzado Anterior/ultraestructura , Comunicación Celular , Membrana Celular/ultraestructura , Colágeno Tipo VI/ultraestructura , Tejido Conectivo/metabolismo , Tejido Conectivo/ultraestructura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Estrés Mecánico
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