Muscle microtubule defects implicated in the pathology of Duchenne muscular dystrophy can be induced or normalized in adult mouse muscles by changing the level of tubb6, a minor beta-tubulin isotype

Authors

• D Randazzo
• DM Talsness
• JJ Belanto
• KJ Zaal
• MD Tran
• A Kenea
• DL Sackett
• JM Ervasti
• E Ralston

Abstract

Duchenne muscular dystrophy (DMD) is a lethal progressive disease caused by the loss of dystrophin. Work with the mdx mouse model of DMD has implicated defects of microtubules in the pathology common to mdx and DMD. In mdx muscle, microtubules are disordered and abnormally dense, and alpha-tubulin post-translational detyrosination is increased. Analysis of DMD transcriptome revealed changes in several tubulin isotypes. We noticed that the most increased isotype (8.6-fold) is tubb6 (beta 6 class-V tubulin). Tubb6 is a minor, ubiquitous tubulin whose overexpression in proliferating cells impairs microtubule organization. We now report that tubb6 is involved in the microtubule defects and in the pathology of mdx. Our main tool is the cDNA injection and electroporation of the flexor digitorum brevis (FDB) muscle of the mouse footpad. Overexpressing GFP-tubb6 in adult wild-type FDB caused impressive microtubule disorganization and densification, assessed by confocal microscopy and quantitated by our directionality assessment software. Tubb6 overexpression also increased alpha-tubulin detyrosination and the number of centrally-nucleated fibers, a hallmark of muscle fiber regeneration in mdx. Conversely, shRNA-mediated knockdown of tubb6 from mdx fibers improved and normalized microtubule organization and density and decreased alpha-tubulin detyrosination. Overexpression of another tubulin isotype, tubb5, did not disturb microtubules in wild-type FDB and knockdown of tubb5 did not improve microtubules in mdx; furthermore tubb5 had no effect on alpha-tubulin detyrosination, indicating that the effects of tubb6 are specific. Thus, disregulation of tubb6 expression is an effector of dystrophin loss in mdx and a target for better understanding DMD pathology.

Scientific Focus Area: Cell Biology

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