Browsing by Subject "mdx"
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Item A biochemical and molecular analysis of functional differences between dystrophin and utrophin(2013-11) Belanto, Joseph JohnThe DMD gene encodes the protein dystrophin, a 427kD cytoplasmic protein responsible for linking the actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. Mutations in dystrophin that abolish its expression lead to Duchenne muscular dystrophy (DMD). Patients with DMD become wheelchair bound in their early teens and succumb to fatal cardiac and/or respiratory failure in their mid-twenties to early thirties. There is currently no effective or widely available treatment for DMD beyond ventilatory support and the use of corticosteroids. Many therapies for treating dystrophin deficiency aim at upregulating its autosomal homolog utrophin due to its structural similarity and ability to bind an almost identical repertoire of proteins that dystrophin binds. It was previously shown that utrophin cannot bind neuronal nitric oxide synthase (nNOS) even though dystrophin binds nNOS, establishing for the first time a functional difference between dystrophin and utrophin. Here, we show that transgenic overexpression of utrophin on the mdx mouse background (Fiona-mdx) is not sufficient to rescue the disorganized microtubule network of the mdx mouse. Thus, we have elucidated a second functional difference between dystrophin and utrophin. Additionally, Fiona-mdx mice lack full recovery of cage activity after mild exercise. Our results suggest that any deficiency in nNOS binding or microtubule lattice function caused by loss of dystrophin may not be restored by upregulation of utrophin. Previously, our lab reported that dystrophin directly binds to microtubules and organizes them beneath the sarcolemma. Using in vitro microtubule cosedimentation assays, we show that dystrophin binds to microtubules with strong affinity (KD=0.33µM). Through the use of various recombinant constructs tested via in vitro microtubule cosedimentation we show that spectrin-like repeats 20-22 of the dystrophin central rod are responsible for microtubule binding activity. However, we show that these repeats require flanking regions of dystrophin for proper binding activity, making microtubule binding context-dependent. Additionally, we show that recombinant utrophin does not bind microtubules in vitro, corroborating our in vivo findings of the disorganized subsarcolemmal microtubule lattice of the Fiona-mdx mouse. We also provide evidence showing that dystrophin functions as a molecular guidepost to organize microtubules into a rectilinear lattice.Item Skeletal Muscle Microtubule Organization and Stability is Regulated by the Dystrophin-Glycoprotein Complex and Cortical Actin(2020-08) Nelson, D'annaDuchenne muscular dystrophy (DMD) is a fatal X-linked myopathy caused by the loss of dystrophin in striated muscle. DMD is frequently studied in the mdx mouse which also lacks dystrophin. It has been observed by multiple independent research groups that mdx skeletal muscle presents with a disorganized cortical microtubule lattice that primarily lacks transverse microtubules as compared to the orthogonal microtubule lattice of wildtype mouse skeletal muscle. While transgenic expression of dystrophin in mdx skeletal muscle does restore microtubule organization, it is not understood how dystrophin regulates microtubule organization in vivo. This thesis implicates two regions of the dystrophin rod domain as regulators of microtubule organization and stability. Singular absence of dystrophin spectrin like repeats R4-15 or R20-24 does not impact basal microtubule organization. However, removal of both R4-15 and R20-23 from micro-dystrophin constructs results in a miniaturized dystrophin that is incapable of fully restoring microtubule organization when transgenically expressed in mdx muscle. In addition to the intermediate microtubule organization by micro-dystrophins lacking R4-15 and R20-23, we have characterized a novel microtubule pathology where transverse microtubules are lost upon eccentric contraction in the absence of either R4-15 or R20-24. Transverse microtubule loss is specific to eccentric contractions and occurs rapidly via a ROS mediated mechanism. Multiple sources of ROS appear to be involved including NOX2 but not nNOS. Moreover, loss of γ-cytoplasmic actin, β-cytoplasmic actin, or the dystrophin-glycoprotein complex (DGC) member α-dystrobrevin all cause a highly similar microtubule phenotype where transverse microtubules are lost post eccentric contraction. While both the intermediate microtubule organization and microtubule susceptibility to eccentric contraction exhibited by micro-dystrophin rescued muscle may have implications for micro-dystrophin gene therapy, the work presented in this thesis has also widened our understanding of skeletal muscle microtubule regulation to include cytoplasmic actins and DGC stability.