Browsing by Subject "Duchenne"

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    Biophysical, cellular, and animal models of dystrophin missense mutations
    (2014-12) Talsness, Dana
    The 427kDa protein dystrophin is expressed in skeletal muscle where it localizes to the costamere and physically links the interior of muscle fibers to the extracellular matrix. Mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a mild form known as Becker (BMD). Currently, there is no cure for DMD or BMD, but there are several therapies being investigated that target specific types of mutations found in the DMD gene. Nonsense mutations almost always lead to a complete lack of dystrophin protein, with stop codon read-through drugs being studied for personalized treatments. Out-of-frame deletions and insertions also cause nearly a complete lack of dystrophin, for which exon-skipping is currently being investigated. Missense mutations in dystrophin, however, cause a wide range of phenotypic severity in patients, the molecular and cellular consequences of such mutations are not well understood, and there are no therapies currently targeting this genotype. Here, we report on three separate model systems of missense mutations in dystrophin: an in vitro biochemical model, a myoblast cell culture model, and an in vivo animal model. Together, they provide evidence that different missense mutations cause variable degrees of thermal instability, which leads to proportionally decreased dystrophin expression, and subsequently causes dystrophic phenotypes. In addition, our initial studies of small molecule treatments show that it is possible to increase the levels of mutant dystrophin, and may lead to personalized therapeutics for patients with missense mutations.
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    Copolymer-based membrane stabilizers for Duchenne Muscular Dystrophy
    (2016-04) Houang, Evelyne
    The overarching objective of this work centers on a structure-function approach to investigate the mechanism of action of synthetic copolymer-based membrane stabilization in the context of Duchenne Muscular Dystrophy (DMD). The guiding theme is the investigation of mechanism of interaction of membrane stabilizing copolymers using cellular and whole animal physiology, chemical engineering, and supercomputational approaches. DMD is an X-linked recessive disease of marked striated muscle deterioration affecting 1 in 3500-5000 boys. DMD results from the lack of the cytoskeletal protein dystrophin, which is essential for maintaining the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle degeneration, along with clinically significant cardiomyopathy. There is no cure for DMD patients, or any effective treatment to halt, prevent or reverse DMD striated muscle deterioration. The primary pathophysiological defect in DMD is the marked susceptibility to contraction-induced membrane stress and the subsequent muscle damage and degeneration that occurs due to loss of muscle membrane barrier function. In this context, a unique therapeutic approach is the use of synthetic membrane stabilizers to prevent muscle damage by directly stabilizing the dystrophin-deficient muscle membrane. The triblock copolymer poloxamer 188 (P188) has numerous features that make it an attractive synthetic membrane stabilizer candidate for DMD treatment and has been demonstrated to target and stabilize damaged membranes in various pathophysiological contexts. The efficacy of P188 in protecting the dystrophic myocardium has been well established, but its effect on the dystrophic skeletal muscle has remained unclear. This work for the first time demonstrates that P188 stabilizes the dystrophic skeletal muscle membrane in vivo and protects it against the mechanical stress associated with lengthening contractions. This result validates P188 as a therapeutic strategy to directly target the hallmark of DMD: impaired membrane stability in all striated muscles. Very little is known on how P188 interacts with and stabilizes biological membranes. To fundamentally probe the mechanism of action of synthetic copolymers as membrane stabilizers, a structure-function approach was undertaken. The aim was to gain insight into the essential critical chemical parameters of copolymers in terms of membrane interacting properties. This work shows for the first time that copolymer mass, composition, architecture, and functional end group chemistries significantly define mechanism of action at the membrane. Based on these insights, an “anchor and chain” model is advanced whereby membrane interaction is critically dependent on end group hydrophobicity. Finally, leveraging the power of supercomputational approaches, Molecular Dynamics simulations were developed to further evaluate and understand copolymer-membrane interactions at atom level resolution. Using increases in surface tension applied to the lipid bilayer, an area-per- lipid dependence of adsorption vs. insertion was uncovered, supporting the hypothesis that copolymers insert into areas of decreased lipid density and then are “squeezed-out” once membrane integrity is restored. Collectively, these findings shed new light on block copolymer dynamic interaction with biological membranes.