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Dystroglycan in cerebellar development and diseaseNguyen, Huy Tuan 01 December 2013 (has links)
Dystroglycanopathies are muscular dystrophies caused by mutations in genes involved the in O-linked glycosylation of alpha-dystroglycan. Severe forms exhibit brain and ocular developmental abnormalities in addition to muscular dystrophy. While cerebellar dysplasia is a common feature of dystroglycanopathy, its pathogenesis has not been thoroughly investigated. Here we evaluate the role of dystroglycan during cerebellar development. Brain-selective deletion of dystroglycan does not affect overall cerebellar growth, but causes malformations associated with glia limitans disruptions and granule cell heterotopia that recapitulate phenotypes found in dystroglycanopathy patients. Cerebellar pathology in these mice is not evident until birth even though dystroglycan is lost during the second week of embryogenesis. The severity and spatial distribution of glia limitans disruption, Bergmann glia disorganization, and granule cell heterotopia rapidly increase during postnatal development. Astrogliosis becomes prominent at these same sites by the time cerebellar development is complete. Interestingly, there is spatial heterogeneity in the glia limitans and granule neuron migration defects that spares the tips of lobules IV-V and VI. The full spectrum of developmental pathology is caused by loss of dystroglycan from Bergmann glia, as neither granule cell- nor Purkinje cell-specific deletion of dystroglycan results in similar pathology. These data illustrate the importance of dystroglycan function in radial/Bergmann glia, but not neurons, during cerebellar histogenesis. The spatial heterogeneity of pathology shows that the dependence on dystroglycan is not uniform.
Cognitive deficits are constant features of severe dystroglycanopathies, yet the precise molecular mechanism leading to neuronal dysfunction in these diseases is not known. Here, we show that dystroglycan interaction with dystrophin is required for the normal clustering of a subset of inhibitory synapses in Purkinje neurons. Using mouse models of dystroglycan mutants, we demonstrate that the number of gamma-aminobutyric acid receptor-containing synapses is significantly reduced in the absence of dystroglycan or portions of dystroglycan; a similar result is attained in dystrophin-deficient mice. Finally, we verify that the number of these receptors is retained when dystroglycan and dystrophin are preserved exclusively in Purkinje neurons. Our findings substantiate the notion that brain dystroglycan is important for neuronal function and suggest a molecular mechanism that may underline cognitive impairments in dystroglycanopathies.
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??-Dystroglycan is essential for the induction of Egr3, a transcription factor important in muscle spindle formationWilliams, Stacey 06 November 2014 (has links)
Muscle spindle fibers are specialized stretch receptors that allow the perception and coordination of limb movement. Differentiation of muscle spindles is initiated by signals derived from the in growing Ia sensory neurons during development. The sensory neuron secretes neuregulin which binds and signals through the ErbB receptors to initiate a signaling cascade. This cascade results in the expression of a specific repertoire of genes, one of which is the transcription factor Egr3, which is necessary in the development of muscle spindles.
Signaling occurs efficiently when the postsynaptic receptors are clustered into large aggregates in apposition to an innervating nerve. Using what is known about acetylcholine receptor clustering at neuromuscular junctions as a model, this study shows the importance of the basal lamina proteins agrin and laminin and their shared receptor ??-dystroglycan in aggregating ErbB receptors at sensory synapses. The study also shows that signaling through these receptors subsequently results in increased expression of Egr3, the transcription factor critical to muscle spindle fiber differentiation. Using an ??-dystroglycan silenced culture, it is shown that ??-dystroglycan is necessary to induce neuregulin, laminin and agrin induced Egr3. In these same myotube cultures there is also a reduced number of AChR-ErbB3 colocalized aggregates and this is not rescued with the addition of laminin. Taken together, these results suggest an essential role for basal lamina components and ??-dystroglycan, molecules that are crucial in acetylcholine receptor aggregation at neuromuscular junctions, in the induction of the transcription factor Egr3, a critical transcription factor involved in muscle spindle fiber differentiation.
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Estratégias de investigação de glicoproteínas de tecidos musculares de modelos animais distróficos / Strategies for investigation of glycoproteins extracted from muscle tissues of dystrophic animal modelsEugenio, Patrícia de Fátima Menegoci 09 May 2013 (has links)
A glicosilação é uma das modificações mais comuns ocorridas naturalmente nas cadeias polipeptídicas. As glicoproteínas exercem papéis essenciais para os seres vivos desde o ínicio vida e, por essa razão, qualquer mutação nos resíduos de açúcares a elas ligados causam diversos efeitos não desejados ao indivíduo. O padrão de glicosilação de proteínas é regido tanto por fatores genéticos quanto por fatores externos. Em relação aos defeitos de glicosilação hereditários, diversas mutações em genes específicos causam anormalidades na síntese de glicoproteínas. No grupo de doenças causadas por defeitos de glicosilação hereditários estão incluídas algumas distrofias musculares relacionadas a mutações em proteínas que são glicosiltransferases comprovadas ou putativas. O uso de modelos animais facilita o estudo dessas doenças neuromusculares e, por isso, o presente trabalho foi desenvolvido utilizando tecidos de camundongos controle (C57Black6) e LARGE. O camundongo LARGE possui características fenotípicas semelhantes às de humanos afetados pela distrofia muscular congênita tipo 1D. Diferentes estratégias de análise e de instrumentação foram empregadas para a obtenção de informações relacionadas tanto ao conjunto de glicoproteínas em geral quanto à alfa-distroglicana especificamente. A alfa-distroglicana mostra-se modificada em relação aos resíduos de açúcares nela ligados em animais com mutação no gene LARGE, resultando em diversos problemas de saúde. A técnica de eletroforese bidimensional, aliada à pré-purificação das glicoproteínas por colunas de lectinas e posterior identificação por espectrometria de massas, não garantiu a obtenção de resultados adequados para esta classe de estruturas. Portanto, a comparação glicoproteômica de tecidos musculares de animais controle e LARGE não foi bem sucedida por esta estratégia instrumental. Técnicas imunoanalíticas, em destaque o western blot, por sua vez, garantiram a visualização das diferenças de glicosilação da alfa-distroglicana, e experimentos de cromatografia de imunoafinidade iniciados neste trabalho mostraram o potencial da especificidade de interação anticorpo-antígeno no isolamento desta glicoproteína para estudos futuros de seus resíduos de oligossacarídeos. Finalmente, análises de espectrometria de massas dos resíduos de oligossacarídeos isolados das glicoproteínas foram realizadas. Os resultados obtidos indicaram a necessidade de otimização do preparo e purificação mais eficiente dessas amostras, mas alguns íons puderam ser relacionados a N- e O-glicanas. / Glycosylation is one of the most common modifications that occur naturally in the polypeptide chains. Glycoproteins play key roles in living organisms from the beginning of life and, therefore, any mutation in the sugar residues attached to them may cause many undesirable effects. The glycosylation pattern of proteins is regulated by both genetic and external factors. Regarding hereditary defects of glycosylation, different mutations in specific genes cause abnormalities in the synthesis of glycoproteins. In the group of diseases caused by defective glycosylation are included some hereditary muscular dystrophies, related to mutations in proteins that are proven or putative glycosyltransferase. The use of animal models facilitates the study of neuromuscular diseases and, therefore, this study was conducted using tissues from control mice (C57Black6) and LARGE. The LARGE mouse has phenotypic characteristics similar to those of humans affected by congenital muscular dystrophy type 1D. Different strategies for analysis and instrumentation were employed here to obtain information related both to the set of glycoproteins in general and specifically to alpha-dystroglycan. The alpha-dystroglycan is modified in relation to its linked sugar residues in animals with LARGE gene mutation, resulting in several health problems. Twodimensional electrophoresis technique, coupled with the pre-purification of glycoproteins by lectin column and subsequent identification by mass spectrometry, did not guarantee resultssuited for this class of structures. Therefore, the glycoproteomic comparison of muscle tissues from LARGE and control animals was not effective by this instrumental strategy. Immunoanalytical techniques, highlighting here the western blot, in turn, assured the visualization of the differences in glycosylation of alpha-dystroglycan, and immunoaffinity chromatography experiments undertaken in this work indicate the potential of the specific antibody-antigen interaction in isolation of this glycoprotein for the future study of their attached oligosaccharides. Finally, mass spectrometer analyses of the isolated oligosaccharides residues of the glycoproteins were performed. The results indicated the need for optimization of preparation and purification of these samples more efficiently, but some ions could be related to N-and O-glycans.
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Estratégias de investigação de glicoproteínas de tecidos musculares de modelos animais distróficos / Strategies for investigation of glycoproteins extracted from muscle tissues of dystrophic animal modelsPatrícia de Fátima Menegoci Eugenio 09 May 2013 (has links)
A glicosilação é uma das modificações mais comuns ocorridas naturalmente nas cadeias polipeptídicas. As glicoproteínas exercem papéis essenciais para os seres vivos desde o ínicio vida e, por essa razão, qualquer mutação nos resíduos de açúcares a elas ligados causam diversos efeitos não desejados ao indivíduo. O padrão de glicosilação de proteínas é regido tanto por fatores genéticos quanto por fatores externos. Em relação aos defeitos de glicosilação hereditários, diversas mutações em genes específicos causam anormalidades na síntese de glicoproteínas. No grupo de doenças causadas por defeitos de glicosilação hereditários estão incluídas algumas distrofias musculares relacionadas a mutações em proteínas que são glicosiltransferases comprovadas ou putativas. O uso de modelos animais facilita o estudo dessas doenças neuromusculares e, por isso, o presente trabalho foi desenvolvido utilizando tecidos de camundongos controle (C57Black6) e LARGE. O camundongo LARGE possui características fenotípicas semelhantes às de humanos afetados pela distrofia muscular congênita tipo 1D. Diferentes estratégias de análise e de instrumentação foram empregadas para a obtenção de informações relacionadas tanto ao conjunto de glicoproteínas em geral quanto à alfa-distroglicana especificamente. A alfa-distroglicana mostra-se modificada em relação aos resíduos de açúcares nela ligados em animais com mutação no gene LARGE, resultando em diversos problemas de saúde. A técnica de eletroforese bidimensional, aliada à pré-purificação das glicoproteínas por colunas de lectinas e posterior identificação por espectrometria de massas, não garantiu a obtenção de resultados adequados para esta classe de estruturas. Portanto, a comparação glicoproteômica de tecidos musculares de animais controle e LARGE não foi bem sucedida por esta estratégia instrumental. Técnicas imunoanalíticas, em destaque o western blot, por sua vez, garantiram a visualização das diferenças de glicosilação da alfa-distroglicana, e experimentos de cromatografia de imunoafinidade iniciados neste trabalho mostraram o potencial da especificidade de interação anticorpo-antígeno no isolamento desta glicoproteína para estudos futuros de seus resíduos de oligossacarídeos. Finalmente, análises de espectrometria de massas dos resíduos de oligossacarídeos isolados das glicoproteínas foram realizadas. Os resultados obtidos indicaram a necessidade de otimização do preparo e purificação mais eficiente dessas amostras, mas alguns íons puderam ser relacionados a N- e O-glicanas. / Glycosylation is one of the most common modifications that occur naturally in the polypeptide chains. Glycoproteins play key roles in living organisms from the beginning of life and, therefore, any mutation in the sugar residues attached to them may cause many undesirable effects. The glycosylation pattern of proteins is regulated by both genetic and external factors. Regarding hereditary defects of glycosylation, different mutations in specific genes cause abnormalities in the synthesis of glycoproteins. In the group of diseases caused by defective glycosylation are included some hereditary muscular dystrophies, related to mutations in proteins that are proven or putative glycosyltransferase. The use of animal models facilitates the study of neuromuscular diseases and, therefore, this study was conducted using tissues from control mice (C57Black6) and LARGE. The LARGE mouse has phenotypic characteristics similar to those of humans affected by congenital muscular dystrophy type 1D. Different strategies for analysis and instrumentation were employed here to obtain information related both to the set of glycoproteins in general and specifically to alpha-dystroglycan. The alpha-dystroglycan is modified in relation to its linked sugar residues in animals with LARGE gene mutation, resulting in several health problems. Twodimensional electrophoresis technique, coupled with the pre-purification of glycoproteins by lectin column and subsequent identification by mass spectrometry, did not guarantee resultssuited for this class of structures. Therefore, the glycoproteomic comparison of muscle tissues from LARGE and control animals was not effective by this instrumental strategy. Immunoanalytical techniques, highlighting here the western blot, in turn, assured the visualization of the differences in glycosylation of alpha-dystroglycan, and immunoaffinity chromatography experiments undertaken in this work indicate the potential of the specific antibody-antigen interaction in isolation of this glycoprotein for the future study of their attached oligosaccharides. Finally, mass spectrometer analyses of the isolated oligosaccharides residues of the glycoproteins were performed. The results indicated the need for optimization of preparation and purification of these samples more efficiently, but some ions could be related to N-and O-glycans.
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The role of viral strain in a congenital brain infectionPlume, Jeffrey Michael 01 July 2015 (has links)
Lymphocytic Choriomeningitis Virus (LCMV) is a common arenavirus and natural murine pathogen that causes congenital neurodevelopmental disease in humans. Exposure to the virus in utero often results in severe and permanent damage the fetal brain and eyes. While usually severe, symptoms vary from case to case. Little is known about the pathological mechanism of congenital LCMV disease. Animal models of congenital LCMV infections suggest that timing of infection during gestation may influence disease outcomes; however, time alone cannot explain all of the variation observed in humans. Another possibility is that individuals are infected with different strains of LCMV.
The LCMV genome is composed of four highly conserved genes, yet even single amino acid mutations can cause the virus to exhibit very different properties in animal models. However, the role of viral strain in the context of neurodevelopment remains relatively unexplored. Here, using a rat model of congenital LCMV infection, we demonstrate that three related strains of LCMV produce different patterns of infection and disease states.
Infection with the highly neurotropic E350 strain induces a disease comparable to that observed in many confirmed cases of human congenital LCMV infection. While most of these animals survive to adulthood, they suffer permanent motor and behavioral abnormalities. Postmortem analyses of infected brains suggest that this strain has a proclivity for infecting mitotically active regions of the brain, including the cerebellum, olfactory bulb, hippocampus and subventricular region. E350 is not known to induce immunosuppression and viral clearance is likely mediated by a robust T-cell response. Indeed, we find high numbers of inflammatory cells in the brains of E350 animals and elevated pro-inflammatory cytokines and chemokines. The immune response, though responsible for the clearance of the virus from the brain, is also implicated in severe and sometimes permanent brain damage.
The Clone 13 strain, a strain typically associated with lymphatic tissue, readily infects the brains of developing rats. Many of these animals do not live to adulthood. Those that survive exhibit extreme stunted growth, but relatively normal neurodevelopment and little discernable neurological disease. By adulthood, the brains of these animals are comparable in size and structure to controls, despite reduced body mass. The Clone 13 infects the same brain regions as the E350, but is not cleared and remained at high titers for the duration of the study. Chronic infection is likely a consequence of the immunosuppressive effects of Clone 13 on the host immune system.
The WE2.2 causes a severe disease with both neurological and systemic symptoms. These animals exhibit persistent seizure like discharges during peak infection and significant motor deficits. They all fail to thrive, losing weight shortly after infection and die invariably 9-11 days post-inoculation. The brains of WE2.2 animals exhibit widespread infection of neurons in the cerebellum, hippocampus, olfactory bulbs, and cortex. WE2.2 does not cause immunosuppression and high levels of inflammatory cells are observed in the brain. Cytokine and chemokine expression is complex, without discernible trends and variable by brain region.
Finally, we looked at alpha-dystroglycan (α-DG) expression in the brain and compared it with infectivity among the strains. Alpha-dystroglycan is recognized as the principle receptor for LCMV and due to mutations in the viral glycoprotein, certain strains are more dependent on α-DG for infection. Several associations between α-DG expression and viral infectivity were observed; however α-DG expression alone could not explain all the differences in infection patterns.
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Effects and regulation of dystroglycan glycosylation in cancerMiller, Michael Raymond 01 May 2015 (has links)
The interplay between cancer cells and the extracellular matrix (ECM) remains a critical regulator of both normal tissue organization and cancer cell invasion. Proteins that function as ECM receptors function to link the cell with the ECM. Abberations in either the structure of the ECM or the expression of ECM receptors leads to disrupted interaction and downstream signaling effects. Dystroglyan (DG) is an ECM receptor that is expressed in a variety of tissue types and functions to mediate sarcolemma stability, epithelial polarity, and is critical in the early formation of basement membranes. However, DG has primarily been studied in muscle where loss of its function is linked to a host of muscular dystrophies. In the epithelium, the role of DG remains enigmatic. While DG has repeatedly been shown to lose function during cancer development and progression, the mechanism and functional consequence of its loss are currently unknown.
In order to increase our understanding of DG in cancer development, we analyzed its expression and glycosylation, a functional requirement for DG, in a range of prostate cancer cell lines. Previous work has shown DG to be downregulated in prostate cancer, but the mechanism by which this occurs has remained largely unclear. We found that DG expression is maintained while its glycosylation was heterogeneous in the cell lines. Further investigation revealed that lines with hypoglycosylated DG strongly associated with the loss of expression of the glycosyltransferase LARGE2. Further this enzyme is frequently downregulated in human cancers and appears to serve as a required enzyme in DG glycosylation within prostate epithelium. This is the first work to demonstrate the functional requirement of LARGE2 for DG, and the only work to implicate loss-of-function of LARGE2 in cancer progression.
To determine whether loss of LARGE2 is found in other tumor types, we analyzed human clear cell renal cell carcinoma (ccRCC) samples by immunohistochemistry and via in silico analysis with the Cancer Genome Atlas (TCGA). Our work demonstrated a frequent and significant downregulation of LARGE2 expression and its association with DG hypoglycosylation. Additionally, we found the loss of LARGE2 strongly associated with increased mortality. Thus, we again demonstrated a functional requirement of LARGE2 but also found a clinical correlate with increased mortality.
Finally, we examined the functional outcome of DG hypoglycosylation or loss of expression in both a mouse model of prostate cancer and a variety of cell lines models. We found that while loss of DG expression does not increase prostate cancer growth or metastasis in one model of cancer, loss of its glycosylation does seem to mediate downstream metabolic changes within cells. The mechanism for this change remains unclear.
In summary, these studies have contributed to our understanding of DG glycosylation and function in both prostate and renal carcinoma. Additionally, we have shown a novel mechanism by which DG glycosylation is lost with downregulation of LARGE2 expression. Finally, while we were unable to demonstrate a clear mechanism by which signaling changes arose, we were able to demonstrate a strong correlation between DG hypoglycosylation and increased mortality in ccRCC. These insights could be used to improve treatment of multiple cancer types as our understanding of DG function continues to improve.
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Human natural killer-1 sulfotransferase (HNK-1ST)-induced sulfate transfer regulates laminin-binding glycans on α-dystroglycan / HNK-1STは硫酸基の転移によってα-ジストログリカン上のラミニン結合性糖鎖の発現を制御するNakagawa, Naoki 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18196号 / 人健博第13号 / 新制||人健||1(附属図書館) / 31054 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 齋藤 邦明, 教授 足立 壯一, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM
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Neurexophilin1 suppresses the proliferation of hematopoietic progenitor cellsKinzfogl, John M 16 March 2012 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) are membrane receptors which serve as mutual ligands in the neuronal system. Neurexophilins (NXPHs) bind NRXN1α. Both NRXN1α and DAG1 were expressed in primitive populations in human cord blood (huCB) and murine bone marrow (muBM), with high concentrations of NXPHs in huCB plasma. We evaluated effects of these molecules on huCB and muBM hematopoietic progenitor (HPC) and stem (HSC) cells. At both a single and population level in vitro, we found that NXPH1 is a potent inhibitor of HPC proliferation acting through NRXN1α, an effect antagonized by DAG1. Injection of recombinant NXPH1 in vivo resulted in myelo- and lymphosuppression, with absolute numbers and cycling status of functional and phenotypically defined HPCs dose- and time-dependently decreased, and absolute numbers and cycling status of phenotypically defined longer-term repopulation HSCs increased. Competitive transplants showed an initial decrease in engraftment of NXPH1-treated cells, with an intermediate stage increase in engraftment. The increase in HSCs is at least partially mediated by the mTOR pathway and is thought to be homeostatic in nature. These results demonstrate the presence and function of a regulated signaling axis in hematopoiesis centered on NRXN1α and its modulation by DAG1 and NXPH1.
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Determination of Genetic Interactions Required for Dystrophin-Dystroglycan Function and Regulation in a Drosophila Model of Muscular Dystrophy / Drosophila DGC function and regulationKucherenko, Mariya 29 October 2009 (has links)
No description available.
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The Laminins and their ReceptorsFerletta, Maria January 2002 (has links)
<p>Basement membranes are thin extracellular sheets that surround muscle, fat and peripheral nerve cells and underlay epithelial and endothelial cells. Laminins are one of the main protein families of these matrices. Integrins and dystroglycan are receptors for laminins, connecting cells to basement membranes. Each laminin consists of three different chains, (α, β, γ). Laminin-1 (α1β1γ1) was the first laminin to be found and is the most frequently studied. Despite this, it was unclear where its α1 chain was expressed. A restricted distribution of the α1 chain in the adult epithelial basement membranes was demonstrated in the present study. In contrast, dystroglycan was found to have a much broader distribution. Dystroglycan is an important receptor for α2-laminins in muscle, but binds also α1-laminins. The more ubiquitous α5-laminins were also shown to bind dystroglycan, but with distinctly lower affinity than α1- and α2- laminins. </p><p>The biological roles of different laminin isoforms have been investigated. Differences were found in the capacity of various tested laminins to promote epithelial cell adhesion. The α5-laminins were potent adhesive substrates, a property shown to be dependent on α3 and α6 integrins. Each receptor alone could promote efficient epithelial cell adhesion to α5-laminins. Yet, only α6 integrin and in particular the α6A cytoplasmic splice variant could be linked to laminin-mediated activation of a mitogen-activated protein kinase (MAP kinase) pathway. Attachment to either α1- or α5-laminins activated extracellular-signal regulated kinase (ERK) in cells expressing the integrin α6A variant, but not in cells expressing α6B. A new role for dystroglycan as a suppressor of this activation was demonstrated. Dystroglycan antibodies, or recombinant fragments with high affinity for dystroglycan, decreased ERK activation induced by integrin α6 antibodies. Integrin αvβ3 was identified as a novel co-receptor for α5-laminin trimers. Cell attachment to α5-laminins was found to facilitate growth factor induced cell proliferation. This proliferation could be blocked by antibodies against integrin αvβ3 or by an inhibitor of the MEK/ERK pathway. Therefore, integrin αvβ3 binding to α5-laminins could be of biological significance.</p>
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