Non-invasive evaluation of murine models for genetic muscle diseases

Novel therapeutic approaches are being introduced for genetic muscle diseases such as muscle dystrophies and congenital myopathies, all of them having remained without cure so far. These recent developments have motivated a renewed and augmented interest in non-invasive methods for muscle characterization and monitoring, particularly during and after therapeutic intervention. In this context, animal models are essential to better understand the disease mechanisms and to test new therapies. Recently, significant advances in the non-invasive evaluation of mouse models for genetic muscle diseases have been achieved. Nevertheless, there were still several mouse strains not characterized non-invasively, and it was necessary to develop sensitive methods to identify subtle alterations in the murine affected muscle. The purpose of this thesis was to apply non-invasive techniques in the study of murine models for genetic muscle diseases with variable phenotypes. Three mouse models for muscle dystrophy (mdx, Large_myd, mdx/Large_myd) and one mouse model for congenital myopathy (KI-Dnm2_R465W) were studied with Nuclear Magnetic Resonance (NMR) methods. Two dystrophic strains (Large_myd, mdx/Large_myd) and normal mice after injury were studied through micro-Computed Tomography (micro-CT). On NMR, all affected mouse strains presented increased muscle T2, which could be related to variable features in the histological evaluation, including necrosis and inflammation, but also to clusters of fibers under regeneration or with altered cytoarchitecture. The combination of NMR and texture analyses allowed the unambiguous differential identification of all the dystrophic strains, although it was not feasible when comparing the muscle T2 measurements only. Mdx mice showed functional and morphological alterations of vascular network. In the KI-Dnm2_R465W mice, a pilot study revealed tendencies of functional impairment. Finally, micro-CT images were unable to detect differences in muscle´s content in dystrophic mice. Altogether, these results not only increased the number of murine models for genetic muscle diseases non-invasively characterized, it also demonstrated some degree of specificity of the imaging anomalies, as revealed by texture analysis. It also showed that non-invasive NMR methods can be sensitive enough to identify subtle alterations in murine muscle phenotype, even in early stages. This thesis was developed under an international joint supervision between France and Brazil, and comprised an important transfer of technology, with the first non-invasive studies of murine muscles performed in Brazil.De nouvelles options thérapeutiques sont en cours d'introduction pour les maladies musculaires génétiques telles que les dystrophies musculaires et les myopathies congénitales, maladies jusque là sans traitement causal. Ces développements récents ont suscité un intérêt renouvelé et croissant pour les méthodes atraumatiques en vue de caractériser et de suivre les muscles atteints, en particulier pendant et après une intervention thérapeutique. Dans ce contexte, les modèles animaux sont essentiels pour mieux comprendre les mécanismes des maladies et pour tester des nouvelles thérapies. Récemment, il y a eu des avancées significatives dans l'évaluation atraumatique de modèles murins de maladies musculaires génétiques. Néanmoins, nombre de lignées de souris n'ont pas encore été caractérisées de façon atraumatique et il reste à mettre au point des méthodes plus sensibles pour identifier précocement des altérations subtiles dans le muscle des souris malades. L'objectif de cette thèse est d'appliquer des techniques atraumatiques innovantes à l'étude du muscle de modèles murins de maladies musculaires génétiques avec des phénotypes variés. Trois lignées de souris modèles de dystrophies musculaires (mdx, Large_myd et mdx/Large_myd) et une lignée de souris modèle de la myopathie congénitale (KI-Dnm2_R465W) ont été étudiées par des méthodes de Résonance Magnétique Nucléaire (RMN). Deux lignées dystrophiques (Large_myd et mdx/Large_myd) plus des souris normales après une blessure ont été étudiées par micro-tomographie (micro-CT). En RMN, toutes les souches de souris affectées ont présenté un T2 musculaire augmenté, en relation avec une gamme d'anomalies histologiques, y comprises nécrose et inflammation, mais aussi des groupes de fibres en régénération ou des fibres avec altérations de l'architecture. Avec la combinaison de la RMN et de l'analyse de la texture, il a été possible d'identifier sans ambiguïté toutes les lignées dystrophiques, alors que la seule mesure du T2 ne permettait pas de les différencier. Les souris mdx ont présenté des altérations fonctionnelles et morphologiques du réseau vasculaire musculaire. Pour les souris KI-Dnm2_R465W, des études préliminaires ont révélé une tendance à développer des altérations fonctionnelles musculaires. Finalement, les images de micro-CT n'ont pas pu détecter des différences du contenu musculaire dans les souris dystrophiques. L'ensemble des résultats non seulement enrichit le panel de modèles murins de maladies génétiques musculaires caractérisés de manière atraumatique, il révèle également un certain degré de spécificité des anomalies dans l'imagerie, comme l'a montré l'analyse de texture. Les résultats démontrent aussi que des méthodes de RMN non-invasives peuvent être assez sensibles pour identifier des altérations subtiles dans le phénotype musculaire murin, même à des stades précoces. Cette thèse a été développée dans le cadre d'une co-tutelle internationale entre la France et le Brésil, et elle a comporté un important transfert de compétence, qui a permis de réaliser les premières explorations atraumatiques du muscle murin effectuées au Brésil

Dmdmdx/Largemyd: a new mouse model of neuromuscular diseases useful for studying physiopathological mechanisms and testing therapies

Although muscular dystrophies are among the most common human genetic disorders, there are few treatment options available. Animal models have become increasingly important for testing new therapies prior to entering human clinical trials. The Dmdmdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD), presenting the same molecular and protein defect as seen in humans with the disease. However, this mouse is not useful for clinical trials because of its very mild phenotype. The mouse model for congenital myodystrophy type 1D, Largemyd, harbors a mutation in the glycosyltransferase Large gene and displays a severe phenotype. To help elucidate the role of the proteins dystrophin and LARGE in the organization of the dystrophin-glycoprotein complex in muscle sarcolemma, we generated double-mutant mice for the dystrophin and LARGE proteins. The new Dmdmdx/Largemyd mouse model is viable and shows a severe phenotype that is associated with the lack of dystrophin in muscle. We tested the usefulness of our new mouse model for cell therapy by systemically injecting them with normal murine mesenchymal adipose stem cells (mASCs). We verified that the mASCs were hosted in the dystrophic muscle. The new mouse model has proven to be very useful for the study of several other therapies, because injected cells can be screened both through DNA and protein analysis. Study of its substantial muscle weakness will also be very informative in the evaluation of functional benefits of these therapies.FAPESP - CEPIDInstituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular (INCTC) - CNPqFINEPABDIMCAPES / COFECU

Globalização económica e fragmentação geopolítica : a caminho de um mundo de equilíbrios instáveis e temporários?

Este texto explora a ideia de que a evolução mundial nos próximos anos vai ser marcada pela interacção complexa entre, por um lado as tensões associadas à Globalização da Economia Mundial, e por outro as Incertezas em torno da Fragmentação Geopolítica Mundial. Começa por identificar os grandes processos envolvidos na primeira "força motriz" - uma ampliação e "regionalização" da Economia Mundial; uma dinâmica de Globalização económica; uma competição acesa entre "Modelos de Capitalísmo"; uma mutação tecnológica abrangente, que modifica as estruturas económicas e a posição relativa das economias; e por último uma regulação económica global que procura responder à acumulação de tensões geradas pela interacção dos processos anteriores. Seguidamente identifica alguns processos chave que organizam a segunda" força motriz", como sejam o avanço da democratização, decorrendo em paralelo com a sobreposição de crises profundas em diversos Estados; um processo de fragmentação e "regionalização" em termos geopolíticos e de segurança; uma alteração na relação de forças entre potências, que está ainda numa fase inconclusiva; uma mutação tecnológica militar que pode influenciar decisivamente essa alteração; e a manifestação de dificuldades na regulação estratégica e geopolítica mundial, pela interacção dos processos anteriores e no contexto da ultrapassagem dos mecanismos de regulação típicos da guerra fria. Por último o texto ilustra algumas das interacções que se podem estabelecer entre as dinâmicas das duas "forças motrizes" sem explorar em profundidade o tema

Mutation in the FUS nuclear localisation signal domain causes neurodevelopmental and systemic metabolic alterations

Variants in the ubiquitously expressed DNA/RNA-binding protein FUS cause aggressive juvenile forms of amyotrophic lateral sclerosis (ALS). Most FUS mutation studies have focused on motor neuron degeneration; little is known about wider systemic or developmental effects. We studied pleiotropic phenotypes in a physiological knock-in mouse model carrying the pathogenic FUSDelta14 mutation in homozygosity. RNA sequencing of multiple organs aimed to identify pathways altered by the mutant protein in the systemic transcriptome, including metabolic tissues, given the link between ALS-frontotemporal dementia and altered metabolism. Few genes were commonly altered across all tissues, and most genes and pathways affected were generally tissue specific. Phenotypic assessment of mice revealed systemic metabolic alterations related to the pathway changes identified. Magnetic resonance imaging brain scans and histological characterisation revealed that homozygous FUSDelta14 brains were smaller than heterozygous and wild-type brains and displayed significant morphological alterations, including a thinner cortex, reduced neuronal number and increased gliosis, which correlated with early cognitive impairment and fatal seizures. These findings show that the disease aetiology of FUS variants can include both neurodevelopmental and systemic alterations

Phenotypic and genetic associations of quantitative magnetic susceptibility in UK Biobank brain imaging

A key aim in epidemiological neuroscience is identification of markers to assess brain health and monitor therapeutic interventions. Quantitative susceptibility mapping (QSM) is an emerging magnetic resonance imaging technique that measures tissue magnetic susceptibility and has been shown to detect pathological changes in tissue iron, myelin and calcification. We present an open resource of QSM-based imaging measures of multiple brain structures in 35,273 individuals from the UK Biobank prospective epidemiological study. We identify statistically significant associations of 251 phenotypes with magnetic susceptibility that include body iron, disease, diet and alcohol consumption. Genome-wide associations relate magnetic susceptibility to 76 replicating clusters of genetic variants with biological functions involving iron, calcium, myelin and extracellular matrix. These patterns of associations include relationships that are unique to QSM, in particular being complementary to T2* signal decay time measures. These new imaging phenotypes are being integrated into the core UK Biobank measures provided to researchers worldwide, creating the potential to discover new, non-invasive markers of brain health

Study of muscular dystrophy in mdx mice with nuclear magnetic resonance

Atualmente, a espectroscopia de Ressonância Magnética Nuclear (RMN) in vitro tem sido extensivamente empregada para estudar tecidos biológicos, atuando como uma poderosa ferramenta de análise química. Em particular, a RMN de próton (1H) e de fósforo (31P) vem sendo utilizada para estudar o metabolismo muscular de animais portadores de deficiências genéticas, como os camundongos com distrofia muscular mdx, modelos para a distrofia muscular Duchenne (DMD). A DMD, que afeta humanos, é um distúrbio recessivo ligado ao cromossomo-X e ocorre em 1 para cada 3500 nascidos vivos do sexo masculino. A DMD é caracterizada pela ausência da proteína distrofina, o que provoca um processo progressivo e rápido de degeneração muscular. Atualmente, o acompanhamento da evolução da doença e de benefícios de tratamentos é feito através de biópsias do tecido muscular. Neste estudo foram realizadas medidas de RMN de 1H em amostras de diafragma e do músculo quadríceps femural de camundongos mdx e de controle com 3 e 6 meses de idade. Os resultados foram comparados com a análise histológica dos mesmos tecidos. O objetivo deste trabalho é monitorar o desenvolvimento normal dos músculos de animais de controle e o progresso da distrofia nos músculos de animais mdx, através da análise dos espectros de RMN. Foi possível identificar diferenças entre os grupos de animais a partir das integrais dos picos observados, mostrando que a distrofia acarreta alterações em diversas vias metabólicas nos camundongos mdx. Estes resultados formam a base para estudos da doença in vivo, para que então seja possível diferenciar músculos distróficos de músculos sadios e caracterizar diferentes estágios de evolução da doença de maneira não invasiva.Currently, Nuclear Magnetic Resonance spectroscopy in vitro has been extensively used to study biological tissues, acting as a powerful tool for chemical analysis. In particular, NMR of proton (1H) and phosphorus (31P) has been used to study muscle metabolism in animals with genetic diseases, such as mice with muscular dystrophy mdx, models for Duchenne muscular dystrophy (DMD). The DMD, which affects humans, is a recessive disorder linked to X-chromosome and occurs in 1 each 3,500 live births male. The DMD is characterized by the absence of dystrophin protein, which causes a progressive and rapid degeneration. Currently, the monitoring of disease progression and benefits of treatments is made by biopsy of muscle tissue. In this study, 1H NMR spectrum were acquired from samples of diaphragm and quadriceps muscle of mdx and control mice 3 or 6 months-old. Results were compared with histological analysis of the same tissues. The objective of this study is to monitor the normal development of the muscles of control animals and the progress of dystrophy in the muscles of mdx animals by analyzing the NMR spectra. Differences were found between the groups of animals comparing the integrals of the observed peaks, showing that dystrophy leads to alterations in several methabolic pathways in the mdx mouse. These results form the basis for studies of the disease in vivo, so then it can be possible to distinguish dystrophic muscles from healthy muscles and characterize different stages of the disease noninvasively

Evaluation atraumatique de modèles murins de maladies musculaires génétiques

De nouvelles options thérapeutiques sont en cours d'introduction pour les maladies musculaires génétiques telles que les dystrophies musculaires et les myopathies congénitales, maladies jusque là sans traitement causal. Ces développements récents ont suscité un intérêt renouvelé et croissant pour les méthodes atraumatiques en vue de caractériser et de suivre les muscles atteints, en particulier pendant et après une intervention thérapeutique. Dans ce contexte, les modèles animaux sont essentiels pour mieux comprendre les mécanismes des maladies et pour tester des nouvelles thérapies. Récemment, il y a eu des avancées significatives dans l'évaluation atraumatique de modèles murins de maladies musculaires génétiques. Néanmoins, nombre de lignées de souris n'ont pas encore été caractérisées de façon atraumatique et il reste à mettre au point des méthodes plus sensibles pour identifier précocement des altérations subtiles dans le muscle des souris malades. L'objectif de cette thèse est d'appliquer des techniques atraumatiques innovantes à l'étude du muscle de modèles murins de maladies musculaires génétiques avec des phénotypes variés. Trois lignées de souris modèles de dystrophies musculaires (mdx, Large_myd et mdx/Large_myd) et une lignée de souris modèle de la myopathie congénitale (KI-Dnm2_R465W) ont été étudiées par des méthodes de Résonance Magnétique Nucléaire (RMN). Deux lignées dystrophiques (Large_myd et mdx/Large_myd) plus des souris normales après une blessure ont été étudiées par micro-tomographie (micro-CT). En RMN, toutes les souches de souris affectées ont présenté un T2 musculaire augmenté, en relation avec une gamme d'anomalies histologiques, y comprises nécrose et inflammation, mais aussi des groupes de fibres en régénération ou des fibres avec altérations de l'architecture. Avec la combinaison de la RMN et de l'analyse de la texture, il a été possible d'identifier sans ambiguïté toutes les lignées dystrophiques, alors que la seule mesure du T2 ne permettait pas de les différencier. Les souris mdx ont présenté des altérations fonctionnelles et morphologiques du réseau vasculaire musculaire. Pour les souris KI-Dnm2_R465W, des études préliminaires ont révélé une tendance à développer des altérations fonctionnelles musculaires. Finalement, les images de micro-CT n'ont pas pu détecter des différences du contenu musculaire dans les souris dystrophiques. L'ensemble des résultats non seulement enrichit le panel de modèles murins de maladies génétiques musculaires caractérisés de manière atraumatique, il révèle également un certain degré de spécificité des anomalies dans l'imagerie, comme l'a montré l'analyse de texture. Les résultats démontrent aussi que des méthodes de RMN non-invasives peuvent être assez sensibles pour identifier des altérations subtiles dans le phénotype musculaire murin, même à des stades précoces. Cette thèse a été développée dans le cadre d'une co-tutelle internationale entre la France et le Brésil, et elle a comporté un important transfert de compétence, qui a permis de réaliser les premières explorations atraumatiques du muscle murin effectuées au Brésil.Novel therapeutic approaches are being introduced for genetic muscle diseases such as muscle dystrophies and congenital myopathies, all of them having remained without cure so far. These recent developments have motivated a renewed and augmented interest in non-invasive methods for muscle characterization and monitoring, particularly during and after therapeutic intervention. In this context, animal models are essential to better understand the disease mechanisms and to test new therapies. Recently, significant advances in the non-invasive evaluation of mouse models for genetic muscle diseases have been achieved. Nevertheless, there were still several mouse strains not characterized non-invasively, and it was necessary to develop sensitive methods to identify subtle alterations in the murine affected muscle. The purpose of this thesis was to apply non-invasive techniques in the study of murine models for genetic muscle diseases with variable phenotypes. Three mouse models for muscle dystrophy (mdx, Large_myd, mdx/Large_myd) and one mouse model for congenital myopathy (KI-Dnm2_R465W) were studied with Nuclear Magnetic Resonance (NMR) methods. Two dystrophic strains (Large_myd, mdx/Large_myd) and normal mice after injury were studied through micro-Computed Tomography (micro-CT). On NMR, all affected mouse strains presented increased muscle T2, which could be related to variable features in the histological evaluation, including necrosis and inflammation, but also to clusters of fibers under regeneration or with altered cytoarchitecture. The combination of NMR and texture analyses allowed the unambiguous differential identification of all the dystrophic strains, although it was not feasible when comparing the muscle T2 measurements only. Mdx mice showed functional and morphological alterations of vascular network. In the KI-Dnm2_R465W mice, a pilot study revealed tendencies of functional impairment. Finally, micro-CT images were unable to detect differences in muscle´s content in dystrophic mice. Altogether, these results not only increased the number of murine models for genetic muscle diseases non-invasively characterized, it also demonstrated some degree of specificity of the imaging anomalies, as revealed by texture analysis. It also showed that non-invasive NMR methods can be sensitive enough to identify subtle alterations in murine muscle phenotype, even in early stages. This thesis was developed under an international joint supervision between France and Brazil, and comprised an important transfer of technology, with the first non-invasive studies of murine muscles performed in Brazil

Metabolic profile of dystrophic mdx mouse muscles analyzed with in vitro magnetic resonance spectroscopy (MRS)

Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy characterized by progressive and irreversible degeneration of the muscles. The mdx mouse is the classical animal model for DMD, showing similar molecular and protein defects. The mdx mouse, however, does not show significant muscle weakness, and the diaphragm muscle is significantly more degenerated than skeletal muscles. In this work, magnetic resonance spectroscopy (MRS) was used to study the metabolic profile of quadriceps and diaphragm muscles from mdx and control mice. Using principal components analysis (PCA), the animals were separated into groups according to age and lineages. The classification was compared to histopathological analysis. Among the 24 metabolites identified from the nuclear MR spectra, only 19 were used by the PCA program for classification purposes. These can be important key biomarkers associated with the progression of degeneration in mdx muscles and with natural aging in control mice. Glutamate, glutamine, succinate, isoleucine, acetate, alanine and glycerol were increased in mdx samples as compared to control mice, in contrast to carnosine, taurine, glycine, methionine and creatine that were decreased. These results suggest that MRS associated with pattern recognition analysis can be a reliable tool to assess the degree of pathological and metabolic alterations in the dystrophic tissue, thereby affording the possibility of evaluation of beneficial effects of putative therapies. (C) 2012 Elsevier Inc. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Cientifico e TecnologicoCoordenacao de Aperfeicoamento de Pessoal de Nivel SuperiorCoordenacao de Aperfeicoamento de Pessoal de Nivel Superio

Nuclear defects in skeletal muscle from a Dynamin 2-linked centronuclear myopathy mouse model

Abstract Dynamin 2 (DNM2) is a key protein of the endocytosis and intracellular membrane trafficking machinery. Mutations in the DNM2 gene cause autosomal dominant centronuclear myopathy (CNM) and a knock-in mouse model expressing the most frequent human DNM2 mutation in CNM (Knock In-Dnm2 R465W/+) develops a myopathy sharing similarities with human disease. Using isolated muscle fibres from Knock In-Dnm2 R465W/+ mice, we investigated number, spatial distribution and morphology of myonuclei. We showed a reduction of nuclear number from 20 weeks of age in Tibialis anterior muscle from heterozygous mice. This reduction is associated with a decrease in the satellite cell content in heterozygous muscles. The concomitant reduction of myonuclei number and cross-section area in the heterozygous fibres contributes to largely maintain myonuclear density and volume of myonuclear domain. Moreover, we identified signs of impaired spatial nuclear distribution including alteration of distance from myonuclei to their nearest neighbours and change in orientation of the nuclei. This study highlights reduction of number of myonuclei, a key regulator of the myofiber size, as a new pathomechanism underlying muscle atrophy in the dominant centronuclear myopathy. In addition, this study opens a new line of investigation which could prove particularly important on satellite cells in dominant centronuclear myopathy

Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice.

International audienceDuchenne muscular dystrophy (DMD) is a progressive neuromuscular disease, caused by an absence of dystrophin, inevitably leading to death. Although muscle lesions are well characterized, blood vessel alterations that may have a major impact on muscle regeneration remain poorly understood. Our aim was to elucidate alterations of the vascular network organization, taking advantage of Flk1(GFP/+) crossed with mdx mice (model for human DMD where all blood vessels express green fluorescent protein) and functional repercussions using in vivo nuclear magnetic resonance, combining arterial spin-labeling imaging of perfusion, and (31)P-spectroscopy of phosphocreatine kinetics. For the first time, our study focused on old (12-month-old) mdx mice, displaying marked chronic muscle lesions, similar to the lesions observed in human DMD, in comparison to young-adult (3-month-old) mdx mice displaying only mild muscle lesions with no fibrosis. By using an original approach combining a specific animal model, state-of-the-art histology/morphometry techniques, and functional nuclear magnetic resonance, we demonstrated that the microvascular system is almost normal in young-adult in contrast to old mdx mice, displaying marked microvessel alterations, and the functional repercussions on muscle perfusion and bioenergetics after a hypoxic stress vary depending on stage of pathology. This original approach clarifies disease evolution and paves the way for setting up new diagnostic markers or therapeutic strategies