Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice

Abstract

Duchenne muscular dystrophy (DMD) is a congenital X-linked myopathy caused by lack of dystrophin protein expression. In DMD, the expression of many dystrophin-associated proteins (DAPs) is reduced along the sarcolemmal membrane, but the same proteins remain concentrated at the neuromuscular junction where utrophin, a dystrophin homologue, is expressed [Matsumura, K., Ervasti, J. M., Ohlendieck, K., Kahl, K. D. & Campbell, K. (1992) Nature (London) 360, 588 -591]. This outcome has led to the concept that ectopic expression of a ''synaptic scaffold'' of DAPs and utrophin along myofibers might compensate for the molecular defects in DMD. Here we show that transgenic overexpression of the synaptic CT GalNAc transferase in the skeletal muscles of mdx animals (mdx͞CT) increases the expression of utrophin and many DAPs, including dystroglycans, sarcoglycans, and dystrobrevins, along myofibers. Protein expression of utrophin and DAPs was equal to or above that of wild-type mice. In addition, ␣-dystroglycan was glycosylated with the CT carbohydrate antigen in mdx͞CT but not in mdx muscles. mdx͞CT mice have little or no evidence of muscular dystrophy by several standard measures; Serum creatine kinase levels, percentage of centrally located myofiber nuclei, and variance in myofiber diameter in mdx͞CT muscles were dramatically reduced compared with mdx mice. These data suggest that ectopic expression of the CT GalNAc transferase creates a functional dystrophin-related complex along myofibers in the absence of dystrophin and should be considered as a target for therapeutic intervention in DMD. D uchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations or deletions in the dystrophin gene that abrogate dystrophin protein expression (1-3). Loss of dystrophin protein in DMD (4) or in the mdx mouse model for DMD (5) also leads to the reduced expression of a complex of membrane proteins that either bind to or associate with dystrophin. Among these are cytoplasmic membrane-associated proteins, including syntrophins and dystrobrevins, and transmembrane glycoproteins, including the dystroglycans and sarcoglycans (for review, see ref. 6). Through a series of intermolecular interactions, this complex of proteins ultimately links laminins in the extracellular matrix to the actin cytoskeleton. Mutations in genes encoding most of these proteins cause some form of muscular dystrophy. Mutations in dystrophin cause DMD (7) and Becker muscular dystrophy (8), mutations in sarcoglycans cause forms of limb-girdle muscular dystrophy (for review, see ref. 9), mutations in laminin ␣2 cause a form of congenital muscular dystrophy (10), and mutations in dystrobrevin (11), dystroglycan (12) and proteins that alter dystroglycan glycosylation (13, 14) cause muscular dystrophy.Although the loss of dystrophin reduces the expression of dystrophin-associated proteins along myofibers, most of these proteins are still highly concentrated at the neuromuscular junction (5). In addition, utrophin, a protein homologue of dystrophin ...

Postnatal overexpression of the CT GalNAc transferase inhibits muscular dystrophy in mdx mice without altering muscle growth or neuromuscular development: Evidence for a utrophin-independent mechanism

“…Here, we use adeno-associated virus (AAV) to introduce the Galgt2 transgene (AAV-Galgt2) at later developmental time points and show that the beneficial effects of Galgt2 in inhibiting muscular dystrophy can be divorced from its unwanted developmental effects on muscle growth and neuromuscular structure. Surprisingly, while embryonic Galgt2 transgene expression stimulates the overexpression of utrophin, synaptic laminins, and dystrophin-associated glycoproteins along transgenic myofibers [10,14], we show here that this does not occur with postnatal Galgt2 overexpression and that postnatal inhibition of muscular dystrophy by Galgt2 overexpression still occurs in mdx mice lacking utrophin. Thus, the therapeutic effects of Galgt2 overexpression are likely to occur via a novel molecular mechanism.…”

“…In our original study, we demonstrated that transgenic expression of the CT GalNAc transferase (Gal gt2) in mdx mouse muscles inhibited the development of muscular dystrophy for up to 6 months of age [14]. We have now followed significant numbers of these mice up to 18 months of age, with some to 24 months.…”

“…Transgenic mice that express the CT GalNAc transferase (Galgt2) specifically in skeletal muscles (via the skeletal α actin promoter [15]) were described by us previously [10,16], as were Galgt2 transgenic mdx mice [14]. Additional mdx mice were bred in our colony from animals purchased from The Jackson laboratory (Bar Harbor, ME).…”

“…Sections were either stained with hematoxylin and eosin or immunostained with various antibodies as previously described [10,14,17]. Quantitation of central nuclei, myofiber diameters, and neuromuscular diameters were all also done as previously described [10,14,17].…”

“…Transgenic overexpression of either utrophin [11][12][13] or Galgt2 [14] from embryonic time points onward in skeletal muscle can increase the expression of dystrophin-associated glycoproteins, including dystroglycan and sarcoglycans, along the myofiber membrane of mdx animals [11,12]. Overexpression of either utrophin [11][12][13] or Galgt2 [14] also inhibits the development of muscular dystrophy in the skeletal muscles of transgenic mdx mice. In addition to its effect on muscular dystrophy, embryonic overexpression of Galgt2 has a profound effect on neuromuscular development and skeletal muscle growth [10].…”