Department of Medicine, Department of Human Genetics, The University of Chicago, Chicago, Illinois, USA.
Address correspondence to: Elizabeth M. McNally, Department of Medicine, Department of Human Genetics, The University of Chicago, 5841 S. Maryland, MC6088, Chicago, Illinois 60637, USA. Phone: 773.702.2672; Fax: 773.702.2681; E-mail: firstname.lastname@example.org.
First published August 27, 2012 - More info
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Dystroglycan is a prominent cell surface protein that mediates attachment to the extracellular matrix. Although broadly expressed, glycosylated dystroglycan is critically important for muscle cell adherence to its surrounding matrix. A subgroup of muscular dystrophies, which often manifest in infancy, is associated with reduced glycosylation of dystroglycan. In this issue of the JCI, Beedle et al. used conditional gene targeting of Fktn, the gene responsible for Fukuyama congenital muscular dystrophy, to investigate a developmental requirement for glycosylation of dystroglycan.
The dystrophin glycoprotein complex is the major transmembrane linkage in muscle cells that allows the plasma membrane to attach to the surrounding matrix. Disruption of dystrophin, which occurs in Duchenne muscular dystrophy, renders the sarcolemma fragile and susceptible to contraction-induced damage because the intracellular connection to the membrane is disrupted. In a related class of genetic diseases, mutations that disrupt the post-translational modifications on the transmembrane protein dystroglycan also lead to muscular dystrophy (Figure 1). Dystroglycan is a heavily glycosylated protein that mediates muscle attachment by linking directly to matrix components with laminin globular domains including laminin-α2, agrin, and perlecan. The extracellular α-dystroglycan subunit undergoes extensive post-translational modification, including the unusual deposition of carbohydrate sugars onto serine/threonine residues known as O-linked glycosylation. Mutations in seven different genes that encode known or suspected glycosyltransferases lead to congenital muscular dystrophy (1). The congenital muscular dystrophies may be evident at birth, often presenting as floppy infant syndrome with reduced muscle tone (2). Progressive muscle degeneration is variably present in the congenital muscular dystrophies and may be accompanied by extramuscular features including eye and brain defects. Disruption of dystroglycan’s normal post-translational processing targets tissues and cells beyond muscle, and matrix binding to dystroglycan is an important feature in many different cell types, including the brain cells (3).
Shown is a schematic of dystroglycan and its position within the muscle cell membrane. Dystroglycan is composed of α and β subunits (αDG and βDG). βDG interacts directly with dystrophin, the protein product of the Duchenne muscular dystrophy locus. The sarcoglycan transmembrane complex stabilizes the interaction between αDG and βDG. Glycosylation of αDG is critical for mediating attachment to matrix components including laminin. Mutations in at least seven different genes are associated with reduced αDG glycosylation (listed in the red box), and protein products of these genes encode glycosylases resident to the secretory system. Decreased αDG glycosylation reduces attachment between the membrane and surrounding matrix. Beedle et al. (7) now report three different deletion strategies for FKTN, and show that FKTN loss is associated with dysregulated glycosylation of αDG and muscular dystrophy.
Fukuyama congenital muscular dystrophy (FCMD, OMIM 607440) is one of the most common autosomal recessive disorders in Japan (~1:10,000) and arises from a retrotransposon insertion in the 3′ UTR of the FKTN gene (4, 5) leading to missplicing of the mRNA and loss of function. FKTN encodes an enzyme resident in the secretory system that mediates O-linked glycosylation. FCMD is not only associated with congenital muscular dystrophy, but is also accompanied by micropolygyria, indicating the gene’s importance in brain formation. In the mouse, complete deletion of Fktn is not compatible with survival (6). In this issue, Beedle et al. describe targeted disruption of Fktn using three different approaches yielding a range of phenotypes (7). Deletion of the Fktn gene early in muscle development, using the Myf5 promoter to drive Cre-mediated recombination at murine E8, resulted in the most severe phenotype. This early muscle-specific deletion of Fktn resulted in reduced body mass, reduced grip strength, and histologic evidence of muscular dystrophy. Notably, the muscle fibers in the Myf5-Cre/Fktn mice were smaller, and the authors speculate that this reflects a delay in full maturation, reminiscent of what is thought to occur in human FCMD patients.
In contrast, deletion of Fktn later in muscle development using the muscle creatine kinase (MCK) promoter produced a less severe phenotype. The MCK promoter deleted the Fktn gene close to the time of birth, at E17. Although histological features of muscular dystrophy were present, there was no reduction in body mass or reduction in strength. The authors were careful to note technical caveats for each of the methods used for targeted deletion, namely that the degree of excision may differ. For example, mice carrying the Myf5-Cre used to delete early in development are also haploinsufficient for Myf5, an important muscle regulatory factor critical for muscle development and regeneration. Further, detecting Fktn deletion efficiency was technically challenging because of the lack of specific anti-Fktn antibodies. Even with these considerations, the data suggest a critical developmental window that is highly sensitive to Fktn level. As the major target of Fktn activity is the heavily glycosylated dystroglycan, these data reinforce the hypothesis that normal development requires interaction between dystroglycan and the matrix.
Whole-body deletion of Fktn was also induced in the mouse using tamoxifen-sensitive Cre expression beginning at 6 weeks of age. This resulted in typical muscular dystrophy features appearing approximately 6 weeks after gene deletion was induced, including an increase in centrally nucleated fibers. In the normal myofiber, nuclei are found at the periphery, and a centrally nucleated fiber is often an indication of recent regeneration (8). Mice with globally deleted Fktn had elevated serum creatine kinase (CK), consistent with muscle membrane disruption. Whole-body Fktn disruption produced histological features of muscular dystrophy including variably sized fibers and pockets of necrosis within the muscle. The authors speculated that aspects of muscle dysfunction in the whole-body-deleted Fktn mice may derive from loss of Fktn in peripheral nerve. Because these phenotypes mirror the clinical findings in FCMD patients, these mice provide a sound model for studying replacement and other therapies in muscle (Table 1).
Comparison of Fktn deletion strategies in mice as reported by Beedle et al.
The major target of Fktn activity is dystroglycan. Two subunits of dystroglycan, α and β (αDG and βDG), are produced from the same gene. βDG traverses the membrane and anchors the wholly extracellular αDG. Although the three models produced by Beedle and colleagues displayed differing phenotypic severity of muscle disease, all had similar biochemical profile in muscle in that glycosylation of αDG was markedly reduced. In each, βDG was readily detected and essentially unchanged by Fktn deletion. However, although the core unit of αDG was retained, its post-translational modifications were significantly depleted. There are two different O-mannosyl modifications on αDG, an O-mannosyl tetrasaccharide and the more recently described phosphodiester-linked branched structure (9, 10). Laminin binding by dystroglycan requires a phosphorylated O-mannosyl modification (9, 10), a type of modification that was previously described only in yeast. The αDG glycosylation defects that Beedle et al. describe in Fktn-deleted muscle were similar to those in muscle lacking Large (like-acetylglucosaminyltransferase), a bifunctional glycosyltransferase with both xylosyltransferase and glucuronyltransferase activities (11). The similar profile of αDG glycosylation in muscle deleted for either Fktn or Large is consistent with these two enzymes acting at the same step in αDG modification, and notably, mutations in Large are known to produce a similar spectrum of disease, as reflected by the phenotype of the myd/myd mouse (12, 13).
Although post-translational modification of αDG is known to be required for laminin binding, the precise residues and modifications sufficient to mediate the linkage have remained obscure. There are at least six genes that fall into this clinical and pathogenetic pathway. However, in many patients, the genetic defect that might explain muscle weakness and the hallmark finding of reduced αDG glycosylation is unknown. Most recently, the ISPD gene, encoding an isoprenoid synthetase domain, was the seventh gene implicated in the congenital muscular dystrophies (14). Based on homology to bacterially related genes, human ISPD may encode a new nucleotide sugar.
In addition, there may be redundancy among these enzymes, since overexpression or upregulation of compensatory enzymes may be corrective. Overexpression of Galgt2, the cytotoxic T cell (CT) GalNAc transferase that is normally enriched at the neuromuscular junction, protects dystrophin-deficient muscle against contraction-induced damage, and dystroglycan is thought to be the major target of this strategy (15). Mutations in fukutin-related protein (FKRP) also lead to a spectrum of muscle diseases that range from congenital to adult-onset (1).
Enzyme delivery is an effective means of treating some muscle diseases, but whether αDG can be adequately targeted by enzyme replacement has not yet been addressed. The retroposon insertion responsible for FCMD induces missplicing of FKTN, and this could be corrected by antisense oligonucleotide treatment, which restored fukutin activity and normal αDG glycosylation (5). Therefore, combinatorial approaches to partially restore FKTN expression and augment activity through other enzymes may prove feasible for enhancing muscle function in the congenital muscular dystrophies. Furthermore, the work of Beedle suggests that the most effective targeting strategy might require treatment early in muscle development.
E.M. McNally is supported by NIH grants HL61322, AR052646, and NS072027 and by the Doris Duke Charitable Foundation.
Conflict of interest: The author has declared that no conflict of interest exists.
Citation for this article:J Clin Invest. 2012;122(9):3046–3048. doi:10.1172/JCI65483.
See the related article beginning on page 3330.
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