Distrofia muscular de Duchenne é uma desordem neuromuscular causada pela mutação ou deleção do gene da distrofina, a qual é ligada ao cromossomo X. Estudos recentes têm demonstrado o importante papel da distrofina no SNC, sendo sua deficiência relacionada com uma variedade de anormalidades na função do SNC, como comportamento e disfunção cognitiva. Os modelos animais mais adequados para esses estudos são os que apresentam o quadro clinico mais semelhante ao da DMD encontrada em humanos, como cães Golden Retriever com distrofia muscular (GRMD). Por não haver ainda estudos a respeito do SNC de animais GRMD, o objetivo deste trabalho foi analisar a morfologia do encéfalo dos GRMD e o de animais não distróficos, através de análise macroscópica, utilizando métodos de medição e registro fotográfico, e análise microscópica, utilizando a técnica de coloração de violeta cresil modificada. Entretanto, usando a metodologia proposta, não foi possível verificar diferenças significativas no encéfalo quando comparados os animais distróficos e os não distróficos, o que está em concordância com a literatura para a DMD usando os mesmos parâmetros. Em tempo, existe uma variação individual na morfologia do encéfalo do cão, independente de serem animais do grupo de distróficos ou controles. Outras técnicas devem ser aplicadas a fim de elucidar as consequências da ausência total ou parcial da distrofina no SNC / Duchenne muscular dystrophy is a neuromuscular disorder caused by the mutation or deletion of the dystrophin gene, which is linked to chromosome X. Recent studies have shown the important role of dystrophin in the CNS, and its related defect with a variety of abnormalities in the function of CNS, such as behavior and cognitive dysfunction. The most suitable animals models for these studies are those with the most similar clinical picture to DMD found in humans, as Golden Retriever dogs with muscular dystrophy (GRMD). There are no further studies on the GRMD animal CNS, and the aim of this study was to analyze the morphology of the brain of GRMD and not dystrophic animals through macroscopic analysis using measurement and photographic registration methods, and microscopic analysis using the modified cresyl violet staining technique. However, using the proposed methodology, we could not find significant differences in the brain when comparing the dystrophic animals and non-dystrophic, which is in agreement with the literature for DMD using the same parameters. In time, there is individual variation in dog brain morphology, whether they are animals of the dystrophic group or controls. Other techniques should be applied in order to elucidate the consequences of the total or partial absence of dystrophin in the CNS

The synapse is the primary locus of cell-cell communication in the nervous system. The elaboration of a functional synapse requires both a specialized structure and an efficient communication system. For my thesis work, I studied proteins implicated in each of these functions: the structural molecules dystroglycan and dystrophin, and the signaling elements Insulin Receptor Substrate p58/53 and insulin receptor. The α/β-dystroglycan complex, believed to be the heart of cellmatrix adhesion in muscle and other tissues, provides a link between dystrophin, a cytoskeletal protein at the base of the muscle cell's Dystrophin Associated Protein Complex, and the extracellular matrix. In addition, dystrophin is found at central synapses, tightly associated with the postsynaptic density. The absence of dystrophin and the secondary loss of its associated proteins causes the genetic disease Duchenne Muscular Dystrophy. DMD affects both muscle and brain, causing a severe muscular dystrophy and lower IQs than control groups. In the first portion of my thesis work, I sought to determine the role of dystroglycan, dystrophin's peripheral partner, at central synapses. I probed Northern blots of brain regions to delineate the distribution of brain β-dystroglycan mRNA and to uncover any β-dystroglycan-related transcripts in brain. Then, using subcellular brain fractions, and cultured hippocampal neurons, I determined that whereas α-dystroglycan is associated with central synapses, β-dystroglycan is not. This discovery is surprising, and differs from the finding that dystrophin and α- and β-dystroglycan colocalize at the presynaptic membrane of retinal photoreceptors. In the course of the above mentioned work, using the anti-β-dystroglycan antiserum Ab98, I discovered a pair of proteins that were tightly associated with the postsynaptic density. These polypeptides of 58 kDa and 53 kDa (p58/53) were highly enriched in postsynaptic density (PSD) fractions from rat cerebral cortex, hippocampus, and cerebellum. In pursuit of a potential synapse-specific dystroglycan relative, I purified p58 and p53 by a combination of hydrophobic interaction chromatography and two-dimensional gel electrophoresis. Mass spectroscopy and peptide microsequencing revealed that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Whereas IRSp58/53 has no significant homology to β-dystroglycan other than the one span of peptides that confers its antibody cross-reactivity, its localization to the PSD newly implicates insulin signaling at synapses. Analysis of IRSp58/53 mass profiles, peptides, and mRNA indicated that IRSp58 and IRSp53 are the product of the same coding sequence. Immunolocalization showed that IRSp58/53 is expressed in the synapserich molecular layer of the cerebellum. Immunostaining of cultured hippocampal neurons showed that both IRSp58/53 and insulin receptor are highly concentrated at synapses. Like IRSp58/53, insulin receptors are a component of the PSD fraction. Together, these data suggest that the synapse is a specialized site for insulin signaling in the brain.

Distrofia muscular de Duchenne é uma desordem neuromuscular causada pela mutação ou deleção do gene da distrofina, a qual é ligada ao cromossomo X. Estudos recentes têm demonstrado o importante papel da distrofina no SNC, sendo sua deficiência relacionada com uma variedade de anormalidades na função do SNC, como comportamento e disfunção cognitiva. Os modelos animais mais adequados para esses estudos são os que apresentam o quadro clinico mais semelhante ao da DMD encontrada em humanos, como cães Golden Retriever com distrofia muscular (GRMD). Por não haver ainda estudos a respeito do SNC de animais GRMD, o objetivo deste trabalho foi analisar a morfologia do encéfalo dos GRMD e o de animais não distróficos, através de análise macroscópica, utilizando métodos de medição e registro fotográfico, e análise microscópica, utilizando a técnica de coloração de violeta cresil modificada. Entretanto, usando a metodologia proposta, não foi possível verificar diferenças significativas no encéfalo quando comparados os animais distróficos e os não distróficos, o que está em concordância com a literatura para a DMD usando os mesmos parâmetros. Em tempo, existe uma variação individual na morfologia do encéfalo do cão, independente de serem animais do grupo de distróficos ou controles. Outras técnicas devem ser aplicadas a fim de elucidar as consequências da ausência total ou parcial da distrofina no SNC / Duchenne muscular dystrophy is a neuromuscular disorder caused by the mutation or deletion of the dystrophin gene, which is linked to chromosome X. Recent studies have shown the important role of dystrophin in the CNS, and its related defect with a variety of abnormalities in the function of CNS, such as behavior and cognitive dysfunction. The most suitable animals models for these studies are those with the most similar clinical picture to DMD found in humans, as Golden Retriever dogs with muscular dystrophy (GRMD). There are no further studies on the GRMD animal CNS, and the aim of this study was to analyze the morphology of the brain of GRMD and not dystrophic animals through macroscopic analysis using measurement and photographic registration methods, and microscopic analysis using the modified cresyl violet staining technique. However, using the proposed methodology, we could not find significant differences in the brain when comparing the dystrophic animals and non-dystrophic, which is in agreement with the literature for DMD using the same parameters. In time, there is individual variation in dog brain morphology, whether they are animals of the dystrophic group or controls. Other techniques should be applied in order to elucidate the consequences of the total or partial absence of dystrophin in the CNS

A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença / The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient\'s life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease

Abstract The function of skeletal muscle is to generate force and produce movement. These tasks are carried out by long multinucleated cells, the skeletal myofibres. The membrane system and the cytoskeleton of these cells are uniquely organized to respond rapidly to neuronal stimuli and to achieve efficient contraction. In the present study the organization and distribution of selected protein/lipid based microdomains that reside in the plasma membrane and sarcoplasmic reticulum of isolated rat skeletal myofibres, were investigated. Aquaporin 4 (AQP4) water channels are arranged as higher order oligomers of several sizes in the sarcolemma and in the T tubules. These oligomers, however, were absent from many specialized micro- and- macrodomains. The distribution of AQP4 coincided with that of a highly organized protein assembly, the dystrophin glycoprotein complex (DGC), in the sarcolemma. A chimaeric venus-AQP4 was equally mobile in the T tubules and sarcolemma, but the anchoring mechanisms of the protein appeared to be different. In contrast to AQP4, the proteins resident in cholesterol and sphingolipid-based microdomains, known as rafts, also occupied DGC deficient areas, which surround the T tubule openings. Indeed, flotillin-1 rafts were located in the neck portions of the T tubules. The rafts defined by the influenza haemagglutinin (HA) also resided in DGC deficient areas, but at the borders of the DGC area. Importantly, of the raft proteins, only the localization of caveolin 3 (CAV3) was dependent on the cholesterol enriched lipid environment, as evidenced by cholesterol depletion experiments and localization studies on a non-raft associated variant of HA. The organization and distribution of membrane associated rough ER (RER) proteins were also analysed. Biochemical detergent extraction analyses and immunofluorescence staining indicated that the ER proteins were assembled as microdomains within the sarcoplasmic reticulum (SR). The microdomains were distributed throughout the SR network and they were capable of protein translocation. Taken together, skeletal myofibres comprise visually distinct microdomains both in the plasma membrane and in the SR. In the plasma membrane, different types of microdomains are not homogenously distributed and function in diverse locations. This may have important physiological implications concerning, among other things, local regulation of ion concentrations and cell signalling cascades. Different constraints ranging from protein-protein interactions to the surrounding lipid environment are important for dictating the observed distribution patterns. / Tiivistelmä Luustolihaksen toimintojen perustana ovat supistumiskykyiset lihassolut, joiden kalvorakenne on järjestynyt erityisellä tavalla ohjaamaan supistusta. Tässä tutkimuksessa analysoitiin proteiini- ja lipidiperustaisten mikroalueiden järjestäytymistä ja tähän vaikuttavia tekijöitä luustolihassolun solukalvolla sekä lihassolun sisäisessä kalvojärjestelmässä, sarkoplasmisessa verkossa (SR). Ensin analysoitiin vesikanavatyyppiä 4 (AQP4), joka oligomerisoituessaan muodostaa erikokoisia mikroalueita. Havaittiin, että AQP4-mikroalueita esiintyy kaikkialla solukalvolla lukuun ottamatta eräitä erilaistuneita mikro- ja makroalueita. AQP4-oligomeerien jakauma solukalvon lateraalisessa osassa, sarkolemmalla, noudatti dystrofiini-glykoproteiinikompleksin jakaumaa. Fluoresoivan venus-AQP4-proteiinin avulla osoitettiin, että proteiinin liikkuvuus oli samanlainen solun sisään ulottuvissa poikkiputkistoissa ja sarkolemmalla, mutta liikkuvuutta rajoittavat tekijät olivat erilaisia näissä solukalvon osissa. Toiseksi analysoitiin kolesteroli- ja sfingolipidipitoisia mikroalueita, kalvolauttoja. Flotilliini-1- ja influenssaviruksen hemagglutiniini (HA) -proteiinia sisältäviä lauttoja esiintyi vain poikkiputkien suuaukkojen alueella, mutta lauttojen jakauma oli erilainen. Lauttojen lipidiympäristöllä ei ollut vaikutusta proteiinien sijaintiin. Tämä osoitettiin kolesterolin poistokokeilla sekä kokeilla, joissa käytettiin mutatoitua HA-proteiinia, joka ei hakeudu kolesteroliympäristöön. Kaveoliini-3-proteiinin sijainti poikkeaa edellä mainituista, ja kolesterolin poisto vaikutti merkittävästi sijainnin määräytymiseen. Kolmanneksi analysoitiin, miten karkean endoplasmakalvoston proteiinit ovat järjestäytyneet SR:ssä. Havaittiin, että endoplasmiset kalvoproteiinit eivät ole homogeenisesti levittäytyneet SR-kalvostoon vaan muodostavat pieniä mikroalueita. Detergenttiuuttoanalyysit osoittivat lisäksi, että näissä mikroalueissa on erilainen lipidikoostumus kuin SR:ssä yleensä. Huomattavaa oli myös, että mikroalueet olivat toiminnallisia kaikkialla SR-kalvostossa. Tulosten perusteella luustolihassolujen kalvojärjestelmä sisältää mikroalueita, joiden jakautuminen vaikuttaa hyvin organisoituneelta. Erityisesti solukalvon mikroalueet esiintyvät tietyillä spesifeillä alueilla, joissa niiden voidaan olettaa toimivan mm. erilaisissa solusignalointitapahtumissa ja paikallisessa ionipitoisuuksien säätelyssä. Eräissä tapauksissa lipidiympäristöllä on merkitystä mikroalueiden sijainnin määräytymisessä, mutta proteiinien sitoutuminen solukalvo- tai solukalvon alaisiin rakenteisiin saattaa myös olla määräävä tekijä.

La Dystrophie Musculaire de Duchenne (DMD) est la plus fréquente et la plus sévère des maladies dégénératives du muscle. Elle se caractérise par une dégénérescence progressive des fibres musculaires due à l’absence de dystrophine fonctionnelle dans les muscles. Actuellement, le rôle physiologique de la dystrophine n’est pas clairement établi et il n’existe pas encore de traitement curatif pour cette maladie. La difficulté de mettre en évidence la fonction de la dystrophine et la physiopathologie de la DMD est en partie expliquée par la complexité moléculaire et cellulaire du muscle des modèles vertébrés utilisés dans les études actuelles. Notre équipe de recherche a développé un modèle de DMD chez le nématode Caenorhabditis elegans. Dans ce modèle, la mutation du gène de la dystrophine, provoque une dégénérescence progressive des muscles conduisant à une paralysie des animaux adultes. Nous utilisons ce modèle afin d’étudier la fonction de la dystrophine et les mécanismes impliqués dans la dégénérescence musculaire chez le nématode. Ce travail de thèse porte sur deux nouveaux acteurs de la dégénérescence musculaire dépendante de la dystrophine : la protéine DYC‐1 et son principal partenaire ZYX‐1. Ce travail présente la caractérisation de ces deux protéines et étudie leurs fonctions dans le muscle. Par ailleurs, ce travail de thèse présente les premiers résultats d’un projet de microscopie électronique ayant pour but de caractériser en détail les évènements subcellulaires du processus dégénératif au cours du cycle de vie du nématode dystrophique. À plus long terme, les études chez le nématode permettront de proposer de nouvelles hypothèses quant aux mécanismes moléculaires et cellulaires de la dégénérescence musculaire / Duchenne Muscular Dystrophy (DMD) is the most prevalent and one of the most severe muscular dystrophy. DMD is due to the absence of functional dystrophin in cardiac and skeletal muscle cells, this lack leads to a progressive muscle degeneration of contractile fibres. Currently, the physiological role of dystrophin is not yet clearly established and curative treatments for DMD are not yet available. The lack of knowledge about dystrophin function and DMD physiopathology can be partly attributed to the complexity of vertebrate muscle, and the absence of a simple model that emulates the human pathology. Our research team developed a model of muscle degeneration in the nematode Caenorhabditis elegans. In this model, the mutation of the dystrophin gene produces a progressive muscle degeneration leading to the paralysis of the adult worms. We use this model for investigating the role of dystrophin and the mechanisms of muscle degeneration in C. elegans. This PhD work concerns two new actors of dystrophin‐dependant muscle degeneration: The DYC‐1 protein and its main interactor ZYX‐1. This study aims to characterise these proteins and to study their muscle functions. Moreover, this PhD work presents preliminary results of an in depth characterisation of subcellular processes of muscle degeneration in dystrophic worms by electron microscopy. Our aim is to visualise first events and to observe the progression of degeneration until the death of muscle cell. These molecular and cellular approaches aims to get new insights in the mechanisms underlying muscle degeneration in order to propose new hypotheses for the understanding of DMD

La dystrophie musculaire de Duchenne (DMD) est un syndrome neuromusculaire dû à des mutations dans le gène dmd qui conduisent à la perte d’expression des dystrophines, protéines normalement exprimée dans différents tissus y compris le cerveau. Le profil cognitif des patients est hétérogène et la présence d’une déficience intellectuelle dépend de la position des mutations dans le gène. Cette variabilité s’explique par la complexité du gène dmd qui comprend plusieurs promoteurs internes permettant l’expression cérébrale de plusieurs dystrophines de tailles différentes. Dans ce travail de thèse, nous nous sommes intéressés à deux dystrophines : la dystrophine complète (Dp427), normalement exprimée dans le muscle et le cerveau et absente chez tous les patients DMD, et la forme la plus courte de dystrophine, la Dp71, produit cérébral majeur du gène dmd absente dans un sous-groupe de patients. Ces deux dystrophines ont des fonctions cellulaires différentes : La Dp427, normalement exprimée dans les synapses inhibitrices en interaction avec les récepteurs du GABA, joue un rôle dans la plasticité synaptique, l’apprentissage et la mémoire. Sa perte conduit à des déficits cognitifs modérés. La Dp71, majoritairement exprimée dans les astrocytes périvasculaires, contribue à l’ancrage de canaux ioniques impliqués dans l’homéostasie cérébrale et joue aussi un rôle dans la synapse glutamatergique. La perte de Dp71 aggrave fortement les déficits associés à la perte de Dp427 chez les patients et conduit à une déficience intellectuelle sévère. Les relations génotypes-phénotypes restent à préciser et on suppose qu’au-delà de la sévérité des déficits, la nature même des altérations cognitives, ainsi que que la présence de troubles sensoriels, cognitifs, exécutifs et neuropsychiatriques, dépendent des formes de dystrophines touchées. Pour étudier le rôle de ces deux dystrophines, nous avons utilisé deux modèles murins : la souris mdx uniquement déficiente en Dp427, et la souris Dp71-null uniquement déficiente en Dp71. Une étude comportementale à large spectre nous a permis de mieux caractériser le phénotype associé à la perte de Dp427 et de Dp71, en précisant l’intégrité de la perception et du traitement des stimuli sensoriels auditifs, des réponses émotionnelles et de la réactivité au stress, des performances d’apprentissage, ainsi que de certaines composantes des fonctions exécutives, comme la mémoire de travail spatiale et la flexibilité comportementale. Ce travail a été complété par des études collaboratives visant à caractériser le rôle de la Dp71 dans la plasticité corticale et à développer une approche de thérapie génique pour restaurer la fonction de la Dp427 chez la souris mdx. Nous montrons que la perte de Dp427 perturbe les fonctions GABAergiques, les réponses émotionnelles induites par un stress ainsi que la mémoire émotionnelle et la mémoire à long terme, sans altération majeure des fonctions sensorielles et exécutives. Nous montrons aussi qu’une thérapie génique basée sur des injections systémiques d’oligonucléotides antisens, porteurs de chimies spécifiques et passant la barrière hémato-encéphalique, est capable de restaurer une Dp427 fonctionnelle par la technique du saut d’exon et de compenser les altérations émotionnelles des souris mdx. La perte de Dp71 a un impact différent : Elle altère la balance excitation/inhibition et la plasticité synaptique corticale et perturbe l’apprentissage, la flexibilité comportementale et la mémoire de travail dans des tâches d’apprentissage spatial. Notre étude de ces modèles murins a donc permis de clarifier les relations génotype-phénotype et les bases neurobiologiques de cette maladie, et d’identifier des phénotypes utiles pour valider l’efficacité de traitements ciblant le cerveau dans des études précliniques. / Duchenne muscular dystrophy (DMD) is a neuromuscular syndrome caused by mutations in the dmd gene, leading to the loss of dystrophin proteins, which are normally expressed in various tissues including the brain. Patients exhibit heterogenous cognitive profiles and the presence of intellectual disability depends on the location of the mutation within the gene. This variability can be explained by the complexity of the dmd gene, which includes several internal promoters leading to the cerebral expression of several dystrophins of different sizes. In this thesis work, we focused on two dystrophins : the full-length dystrophin (Dp427) normally expressed in muscle and brain and lost by all DMD patients, and the shortest dystrophin, Dp71, major cerebral product of the dmd gene that is absent in a subgroup of patients. These two dystrophins have distinct cellular functions : Dp427, normally interacting with GABA receptors in inhibitory synapses, plays a role in synaptic plasticity, learning and memory. Its loss leads to mild cognitive deficits. Dp71, mostly expressed in perivascular astrocytes, contributes to the anchoring of ionic channels involved in brain homeostasis and also plays a role in glutamatergic synapses. Dp71 loss strongly aggravate the deficits associated with the loss of Dp427 in patients and lead to severe intellectual disability. Genotype-phenotype relationships need be further specified and it is assumed that beyond deficits severity, the actual nature of cognitive alterations, as well as the presence of sensorial, cognitive, executive and neuropsychiatric disturbances, depend on the specific forms of dystrophin affected by mutations. To study the role of these two dystrophins, we used two mouse models : the mdx mouse that only lacks Dp427, and the Dp71-null mouse that only lacks Dp71. A extensive behavioral study allowed us to better characterize the phenotype associated with the loss of Dp427 and Dp71, detailing integrity of perception and processing of auditory sensory stimuli, of emotional responses and stress reactivity, of learning performance, and of components of executive functions, such like spatial working memory and behavioral flexibility. The work has been completed by collaborative studies aimed at characterizing the role of Dp71 in cortical plasticity and at developing gene therapy approaches to rescue Dp427 function in the mdx mouse. We demonstrate that Dp427 loss perturbs GABAergic functions, stress-induced emotional responses, as well as emotional and long-term memories, without major alterations of sensory and executive functions. We also show that a gene therapy based on systemic injections of antisens oligonucleotides holding specific chemistries and crossing the blood-brain barrier enables Dp427 functional rescue by exon-skipping strategy and alleviates emotional disturbances in mdx mice. The loss of Dp71 has a distinct impact : It alters cortical excitation/inhibition balance and plasticity and disrupt learning, behavioral flexibility and working memory in spatial learning tasks. Our study of these mouse models therefore enabled to clarify the genotype-phenotype relationships and neurobiological bases of this disease, and identified valuable phenotypes to validate treatment efficacy in future brain-targeting preclinical studies.

Hintergrund und Zielsetzung: DMD ist die häufigste Form der Muskeldystrophie im Kindesalter und bis heute unheilbar. Sie wird durch das Fehlen des Proteins Dystrophin verursacht, welches verschiedene Signaltransduktionswege beeinflusst. Das Anliegen der Arbeit ist die Untersuchung und Modulation von Signaltransduktionswegen, die als alternative Therapiestrategie den Verlust von Dystrophin kompensieren könnten. Experimentelle Strategie: Für die Charakterisierung von Dystrophin nachgeschalteten Prozessen wurden mRNA-Expressionsanalysen in Muskelgeweben von DMD-Patienten und einem DMD-Brüderpaar mit einem infrafamiliär unterschiedlichen Verlauf der DMD durchgeführt. Aus diesen Expressionsdaten wurde erstmalig ein Petri-Netz entwickelt, welches Dystrophin mit in diesem Zusammenhang bisher unbekannten Signaltransduktionswegen verknüpft. Das Petri-Netz wurde auf Netzwerkintegrität und –verhalten mittels Invarianten- (INA) und theoretischen Knockout- (Mauritius Maps) Analysen untersucht. Durch beide Methoden läßt sich der maßgebliche Teilsignalweg bestimmen. In diesem Signalweg wurden die Proteinaktivität und die Genexpression durch siRNA, Vektor-DNA und chemische Substanzen in humanen SkMCs moduliert. Anschließend wurden die Proliferation und die Vitalität der Zellen sowie auch die Expression auf mRNA- und Protein-Niveau untersucht. Ergebnisse: RAP2B und CSNK1A1 waren in dem DMD-Brüderpaar differentiell exprimiert und konnten erstmalig in einem neuen, komplexen Signalweg in Zusammenhang mit Dystrophin nachgeschalteten Prozessen dargestellt werden. Mittelpunkt dieses Signalweges ist die De- und Aktivierung des Transkriptionsfaktors NFATc. Seine Zielgene umfassen neben anderen den negativen Proliferationsfaktor p21, das Dystrophin homologe UTRN und den Differenzierungsfaktor MYF5. Folglich würde ein Anstieg von UTRN eine unerwünschte Reduktion der Proliferationsrate von Myoblasten implizieren. Letzteres konnte bereits nachgewiesen werden und stellte das Motiv für weitere Studien dar. Jedoch zeigten siRNA- und Vektor-DNA-Experimente, daß NFATc nicht der ausschlaggebende Faktor für diese Zielgene ist. Die Substanzen Deflazacort (DFZ) und Cyclosporin A (CsA) wurden dagegen beschrieben, die Aktivierung von NFATc zu beeinflussen. Die Ergebnisse zeigten, daß beide Substanzen die Proliferation von Myoblasten erhöhen können. Die gleichzeitige Applikation von DFZ und CsA führte zu einem Anstieg der UTRN-Expression. Schlußfolgerung: Die Modulation der Proliferation und UTRN-Expression ist unabhängig von einander möglich. Entsprechend der Grundidee der Arbeit zeichnet sich eine neue Therapiestrategie ab, welche Dystrophin nachgeschaltete Prozesse einbezieht. / Background and aim: DMD is the most common muscular dystrophy in childhood and incurable to date. It is caused by the absence of dystrophin, what influences several signal transduction pathways. The thesis is interested in the investigation and modulation of signal transduction pathways that may compensate the lack of dystrophin as an alternative therapy strategy. Experimental strategy: To study Dystrophin downstream pathways the mRNA expression of DMD patients and two DMD siblings with an intra-familially different course of DMD were analysed in muscle tissue. On the basis of these expression data a Petri net was first developed implicating signal transduction pathways and Dystrophin downstream cascades. Invariant (INA) and theoretical knockout (Mauritius Maps) analyses were applied for studying network integrity and behaviour. Both methods provide information about the most relevant part of the network. In this part modulation of protein activity and of gene expression using siRNA, vector-DNA, and chemical substances were performed on human SkMCs. Subsequently, the cells were studied by proliferation and vitality tests as well as expression analyses at mRNA and protein level. Results: RAP2B and CSNK1A1 were differently expressed in two DMD siblings, and first are part of a signal transduction pathway implicating Dystrophin downstream processes. The central point of this pathway is the de- and activation of the transcription factor NFATc. Its target genes are, among others, the negative proliferation factor p21, the Dystrophin homologue UTRN, and the differentiation factor MYF5. Consequently, an increase in UTRN implicates an undesirably reduced myoblast proliferation rate. Latter was found in DMD patients and was target for further studies. But, siRNA and vector DNA experiments showed that NFATc is not the decisive factor for the target genes. Deflazacort and cyclosporin A are known to influence the activation of NFATc. The results first showed that both substances do induce myoblast proliferation. The use of deflazacort in combination with cyclosporin A resulted in an increase of UTRN expression. Conclusion: The modulation of proliferation and UTRN-expression independently of each other is possible. According to the basic idea of this study, a new therapeutic strategy becomes apparent, which considers Dystrophin downstream processes.

O complexo distrofina-glicoproteínas associadas (DGC) localiza-se no sarcolema das células musculares esqueléticas e cardíacas e tem como função principal proporcionar ligação mecânica entre o citoesqueleto intracelular e a matriz extracelular. Estudos prévios realizados em nosso laboratório, focalizando o complexo DGC, demonstraram perda de proteínas importantes desse complexo. As situações avaliadas anteriormente foram: infecção experimental por Trypanosoma cruzi (T. cruzi) e sépsis experimental. Em ambas as situações verificou-se a perda da distrofina acompanhada por disfunção contrátil e aumento nos níveis da calpaína, protease dependente de cálcio implicada na proteólise da distrofina. Todavia, o mecanismo responsável pela ativação das calpaínas e proteólise da distrofina na infecção experimental por T. cruzi e na sépsis experimental não está totalmente definido. O objetivo desse trabalho foi avaliar in vitro o mecanismo responsável pela ativação das calpaínas nas culturas de cardiomiócitos desafiadas com o soro dos animais infectados experimentalmente com T. cruzi ou com o soro dos animais submetidos à sépsis grave experimental. Camundongos C57BL/6 foram submetidos à sépsis grave ou infectados com a cepa Y de T. cruzi. No pico de expressão das citocinas pró-inflamatórias, 12 dias após inoculação do parasito ou 6 horas após a indução da sépsis, o sangue foi coletado e o soro separado. Corações de camundongos recém-nascidos foram isolados para o cultivo dos cardiomiócitos. No quinto dia após o início das culturas, as células foram estimuladas com 10% do soro de animais infectados com T. cruzi ou o soro de animais submetidos à sépsis grave durante 24 horas. Após, as células foram coletadas para análises de Western blotting e imunofluorescência para verificar a expressão da distrofina e calpaína-1. Avaliou-se também, por imunofluorescência, a expressão do NF-B. Os cardiomiócitos foram estimulados e tratados com o dantrolene, inibidor da liberação de cálcio do retículo sarcoplasmático, ou ALLN, inibidor da calpaína-1, e após coletados para verificar a expressão da distrofina e calpaína-1 por Western blotting e imunofluorescência. Nossos resultados mostraram uma redução significativa na expressão da distrofina com desarranjo das miofibrilas contráteis e formação de bolhas citoplasmáticas, além de um aumento nos níveis da calpaína-1 e do NF-B. O tratamento com dantrolene nas culturas estimuladas com o soro de animais infectados experimentalmente com T. cruzi ou com o soro dos animais submetidos à sépsis grave, recuperou a expressão da distrofina e reduziu os níveis da calpaína-1. O tratamento com ALLN nos cardiomiócitos estimulados com o soro de animais infectados experimentalmente com T. cruzi recuperou a expressão da distrofina e não alterou os níveis da calpaína-1. Nas culturas estimuladas com o soro dos animais submetidos à sépsis grave, o tratamento com o ALLN recuperou a expressão da distrofina e reduziu os níveis da calpaína-1. Nossos resultados demonstraram que citocinas pró-inflamatórias presentes no soro dos animais infectados experimentalmente com T. cruzi como também no soro dos animais submetidos à sépsis grave induziriam um aumento no influxo de cálcio com consequente ativação das calpaínas, as quais atuariam na ativação do NF-B e na degradação da distrofina. Esse mecanismo poderia ser responsável pela proteólise da distrofina cardíaca observada na infecção experimental por Trypanosoma cruzi como também sépsis experimental. Mais estudos são necessários para elucidar este mecanismo, principalmente em relação a inibidores dos canais de cálcio, das citocinas pró-inflamatórias e das calpaínas, com o objetivo de fornecer novas vias de intervenção na prevenção de alterações cardíacas observadas na doença de Chagas e na sépsis. / The dystrophin-glycoprotein complex (DGC), located in the sarcolemma of cardiac and skeletal muscle cells and concentrated along the plasma membrane in costameric structures provides a framework that connects the intracellular cytoskeleton to the extracellular matrix. Previous studies from our laboratory clearly demonstrated disruption of DGC proteins in experimentally-induced T. cruzi infection and experimental sepsis. Both situation presented dystrophin disruption associated with contractile dysfunction and increased calpain levels, calcium dependent protease responsible for dystrophin proteolysis. However, the mechanism responsible for calpain activation and dystrophin proteolysis in experimentally-induced T. cruzi infection and experimental sepsis is not totally understood. The aim of this study was to evaluate in vitro the mechanism responsible for calpain activation in cultured cardiomyocytes challenged with serum from animals experimentally infected with T. cruzi or subjected to severe sepsis. Mice C57BL/6 were subjected to sepsis induction or infected with Y strain from T. cruzi. At the peak of proinflammatory cytokines expression, 12 days after parasite inoculation or 6 hours after sepsis induction, the blood was collected and the serum separated. Hearts from newborn mice were isolated for culture of cardiomyocytes. After 5 days of incubation, the cardiac cells were stimulated with 10% of serum from animals experimentally infected with T. cruzi or subjected to severe sepsis during 24 hours, and collected for Western blotting and immunofluorescence analysis to verify dystrophin and calpain-1 expression. The expression of NF-B was evaluated by immunofluorescence. The treatments with dantrolene, inhibitor of calcium release from sarcoplasmic reticulum, or ALLN, calpain-1 inhibitor, were performed in cultured cardiomyocytes stimulated during 24 hours with serum from animals infected with T. cruzi or subjected to severe sepsis, and dystrophin and calpain-1 expression were analyzed by Western blotting and immunofluorescence. Our results demonstrated loss of dystrophin associated with myofibers derangement and presence of cytoplasmic blebs as well increase of calpain-1 and NF-B expression. The dantrolene treatment in cultures stimulated with serum from animals infected with T. cruzi or subjected to severe sepsis recovey dystrophin expression and reduced calpain-1 levels. The ALLN treatment in cardiomyocytes stimulated with serum from animals infected with T. cruzi recovery dystrophin expression and preserved calpain-1 levels. In cultures stimulated with serum from animals subjected to severe sepsis, the ALLN treatment recovery dystrophin expression and decreased calpain-1 levels. Our results demonstrated that proinflammatory cytokines in serum from mice infected with T. cruzi or subjected to severe sepsis could induce an increase calcium influx with calpain activation, which could act in NF-B activation and dystrophin disruption. Possibly, this mechanism could be responsible to dystrophin proteolysis observed in experimentally-induced acute T. cruzi infection and experimental sepsis. More studies are needed to elucidate this mechanism, especially in relation to calcium channel blockers and inhibitors of pro-inflammatory cytokines and calpains, which may provide new routes for intervention to prevent cardiac damage in Chagas disease and sepsis.

L’objectif était d’évaluer l’efficacité de thérapies développées pour traiter la dystrophie musculaire de Duchenne (DMD, due à des mutations du gène de la dystrophine) dans la restauration de déficits cognitifs associés à ce syndrome. Deux pistes thérapeutiques visant à compenser les altérations cérébrales liées à la perte de dystrophine ont été explorées chez les souris mdx, modèle de DMD. Une approche pharmacologique basée sur la surexpression de l’utrophine, homologue de la dystrophine, n’améliore pas les déficits comportementaux des souris mdx. Par contre, une intervention génique basée sur l’épissage de l’exon muté conduit à la restauration d’une dystrophine endogène et une récupération d’altérations cérébrales comme l’agrégation des récepteurs GABAA et la plasticité synaptique hippocampique. Ceci suggère un rôle de la dystrophine dans la plasticité du cerveau adulte et l’applicabilité de cette approche de thérapie génique au traitement des altérations cognitives de la DMD. / Therapies have been developed to treat Duchenne muscular dystrophy (DMD, due to mutation in the dystrophin gene), but their ability to restore the cognitive deficits associated with this syndrome has not been yet studied. We explored two therapeutic approaches to compensate for the brain alterations resulting from the loss of dystrophin in the mdx mouse, a model of DMD. A pharmacological approach based on the overexpression of utrophin, a dystrophin homologue, does not alleviate the behavioural deficits in these mice. In contrast, a genetic intervention based on the splicing of the mutated exon leads to the restoration of endogenous dystrophin and a recovery of brain alterations such as the clustering of GABAA receptors and hippocampal synaptic plasticity in mdx mice. These results suggest a role for dystrophin in adult brain plasticity and indicate that this gene therapy approach is applicable to the treatment of cognitive impairments in DMD.