Abstract
Loss of dystrophin protein due to mutations in the DMD gene causes Duchenne muscular dystrophy. Dystrophin loss also leads to the loss of the dystrophin glycoprotein complex (DGC) from the sarcolemma which contributes to the dystrophic phenotype. Tyrosine phosphorylation of dystroglycan has been identified as a possible signal to promote the proteasomal degradation of the DGC. In order to test the role of tyrosine phosphorylation of dystroglycan in the aetiology of DMD, we generated a knock-in mouse with a phenylalanine substitution at a key tyrosine phosphorylation site in dystroglycan, Y890. Dystroglycan knock-in mice (Dag1(Y890F/Y890F)) had no overt phenotype. In order to examine the consequence of blocking dystroglycan phosphorylation on the aetiology of dystrophin-deficient muscular dystrophy, the Y890F mice were crossed with mdx mice an established model of muscular dystrophy. Dag1(Y890F/Y890F)/mdx mice showed a significant improvement in several parameters of muscle pathophysiology associated with muscular dystrophy, including a reduction in centrally nucleated fibres, less Evans blue dye infiltration and lower serum creatine kinase levels. With the exception of dystrophin, other DGC components were restored to the sarcolemma including α-sarcoglycan, α-/β-dystroglycan and sarcospan. Furthermore, Dag1(Y890F/Y890F)/mdx showed a significant resistance to muscle damage and force loss following repeated eccentric contractions when compared with mdx mice. While the Y890F substitution may prevent dystroglycan from proteasomal degradation, an increase in sarcolemmal plectin appeared to confer protection on Dag1(Y890F/Y890F)/mdx mouse muscle. This new model confirms dystroglycan phosphorylation as an important pathway in the aetiology of DMD and provides novel targets for therapeutic intervention.
Highlights
In normal striated muscle dystrophin associates with a large group of proteins known as the dystrophin glycoprotein complex (DGC) [1]
This study demonstrates that preventing phosphorylation of a key tyrosine residue on murine dystroglycan -Y890, ameliorates many of the main pathological symptoms associated with dystrophin deficiency in the mdx mouse
Restoration of dystrophin or utrophin in mdx mice, by genetic, viral or chemical means is able to restore dystroglycan and other DGC components and effect a significant rescue of the dystrophic phenotype, a number of therapeutic strategies are predicated on the success of this approach
Summary
In normal striated muscle dystrophin associates with a large group of proteins known as the dystrophin glycoprotein complex (DGC) [1]. The Lisanti group further demonstrated that inhibition of the proteasome was able to restore other DGC components in both mdx mice that lack dystrophin and in explants of DMD patients [8, 9] From these studies it can be concluded that under normal circumstances binding of dystrophin to dystroglycan via the WW domain binding motif PPPY890 prevents tyrosine phosphorylation of -dystroglycan allowing the DGC to be maintained stably at the sarcolemma. It has been demonstrated that restoration of the DGC by Dp71 overexpression did not alleviate the dystrophic phenotype in mdx mice [10, 11] We surmise that this approach fails because whilst the dystrophin and utrophin binding site on dystroglycan is blocked by Dp71 and the complex is restored, Dp71 cannot bind to the actin cytoskeleton, so the link between extracellular matrix and cytoskeleton remains compromised. We have investigated whether preventing dystroglycan phosphorylation in mouse by a targeted gene knock-in of phenylalanine at tyrosine residue 890, which is predicted to block tyrosine phosphorylation, can restore dystroglycan function and reduce the dystrophic phenotype in mdx mice
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