Dystroglycan: important player in skeletal muscle and beyond

Neuromuscular Disorders 15 (2005) 207–217 www.elsevier.com/locate/nmd

Review

Dystroglycan: important player in skeletal muscle and beyond Ronald D. Cohn* Johns Hopkins Hospital, Children’s Center, McKusick-Nathans Institute of Genetic Medicine, 600 N Wolfe Street, Blalock 1008, Baltimore, MD 21287, USA Received 11 June 2004; received in revised form 2 August 2004; accepted 24 November 2004

Abstract Dystroglycan is a transmembrane protein that connects the extracellular matrix to the cytoskeleton. Given the ubiquitous tissue expression of dystroglycan, different functional roles in various organ systems have been characterized during the past decade. More recently, aberrant glycosylation of dystroglycan has been identified as a novel pathogenetic mechanism in several forms of congenital and late onset muscular dystrophy syndromes. The current review summarizes the recent scientific achievements as they relate to the function of dystroglycan under normal and pathophysiological conditions. q 2004 Elsevier B.V. All rights reserved. Keywords: Dystroglycan; Skeletal muscle; Muscular dystrophy; Neuronal migration disorders

1. New aspects of dystroglycan function in skeletal muscle Dystroglycan was originally isolated from skeletal muscle as an integral membrane component of the dystrophin–glycoprotein complex (DGC) [1]. In vertebrates dystroglycan is composed of alpha- and betasubunits encoded by a single gene and cleaved into two proteins by posttranslational processing [2]. At the sarcolemma, b-dystroglycan binds intracellularly to dystrophin, which binds to the actin cytoskeleton, and extracellularly to a-dystroglycan. a-Dystroglycan, a highly glycosylated peripheral membrane protein, completes the link from the cytoskeleton to the basal lamina by binding to extracellular matrix proteins containing LamG domains, such as laminin [3], neurexin [4], agrin [5–8], and perlecan [9] (Fig. 1). In addition to dystroglycan and dystrophin, the DGC in muscle cells contains the sarcoglycan complex composed of five sarcoglycan proteins (a,b,g,d,z) and sarcospan [1,10,11]. Via dystrophin, the sarcolemmal DGC interacts with a pair of syntrophins (a1 and b1) and a-dystrobrevin within the cytosol [12–16]. The C-terminal tail of b-dystroglycan also contains a PPXY motif that can * Tel.: C1 410 955 3071; fax: C1 410 614 9246. E-mail address: [email protected] 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.11.005

interact with dystrophin or caveolin-3 [17]. Recent evidence suggests that the ZZ domain of b-dystroglycan is essential for its physiological binding to dystrophin and utrophin [18]. Studies in animal models of muscular dystrophy have shown that a-dystroglycan is greatly reduced at the sarcolemma in dystrophin and sarcoglycan deficient mice [2,10,19]. These findings indicate that sarcolemmal expression of the sarcoglycans is a prerequisite for the membrane targeting and stabilization of a-dystroglycan. Though the exact function of the DGC is not entirely understood, it is thought to contribute to the structural stability of the muscle cell membrane during cycles of contraction and relaxation, thereby protecting the muscle from stress-induced membrane damage [20]. In humans, mutations in dystrophin cause Duchenne and Becker muscular dystrophy; mutations in sarcoglycans cause limb-girdle muscular dystrophy (LGMD 2C-F); and mutations in laminin a2 cause congenital muscular dystrophy [21–23]. Advances in technology have improved our understanding of dystroglycan function in skeletal muscle. Engineering of mice chimeric for dystroglycan expression in all tissues have been reported to cause muscular dystrophy [24]. Using the Cre-loxP system, mice with targeted disruption of the DAG1 gene in differentiated skeletal muscle revealed a role for dystroglycan in muscle regeneration [25]. Mice with skeletal muscle specific loss of dystroglycan (MCK-DG-null) showed muscular

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Fig. 1. The integral and peripheral components of the dystrophin glycoprotein complex and other membrane associated proteins and binding partners in skeletal muscle.

dystrophy. Interestingly, the severity of the dystrophic process was very mild and MCK-DG-null developed significant skeletal muscle hypertrophy, as opposed to the expected increase in adipose and fibrous tissue. The study demonstrated that maintenance of regenerating capacity by satellite cells expressing dystroglycan was likely to be responsible for the milder phenotype observed in this mouse model [25]. It has been shown that skeletal muscle seems capable of efficiently repairing itself during the early phase of the disease, and it is believed that the ongoing stimulus and activation of the repair mechanism eventually exhausts the satellite cell pool, subsequently leading to severe fibrosis and adipose tissue replacement [26,27]. The findings observed in MCK-DG-null mice suggest that dysfunction of the satellite cell population, resulting in impairment of the repair mechanism in skeletal muscle, represents a key mechanism in the pathogenesis of muscular dystrophy. Future efforts towards treatment should be aimed at identifying ways to maintain muscle regeneration capacity.

2. Aberrant glycosylation as a novel mechanism for neuromuscular and brain diseases There has been a recent boom in the identification of neuromuscular diseases caused by mutations in genes that affect carbohydrate metabolism or protein glycosylation. A number of these findings relate to putative and determined glycosyltransferase enzyme defects in the O-glycosylation of a-dystroglycan, which subsequently led to characterization of a novel disease entity called ‘dystroglycanopathies’ [28–31] (Table 1).

About 500 genes are known to be involved in glycosylation processes and roughly 50% of body proteins are glycosylated [32]. Congenital disorders of glycosylation (formerly carbohydrate deficient glycoprotein syndrome, CDG) were initially identified in 1980 by Jaeken et al., [33] following the observation of abnormal processing of arylsulfatase-A and thyroxin-binding globulin in patients with severe developmental delay and neurological deficits [33]. In contrast to dystroglycanopathies, which are caused by defects in the O-glycosylation pathway, most syndromes of congenital disorders of glycosylation are due to abnormalities in N-glycosylation [32]. The following paragraphs highlight the novel discoveries of dystroglycan function in skeletal muscle and the central nervous system as they relate to O-glycosylation disorders. 2.1. Dystroglycan glycosylation in skeletal muscle Dystroglycan undergoes N-linked and extensive O-linked glycosylation, and as a result a-dystroglycan migrates on SDS-PAGE as a broad band with an approximate molecular mass of 120–180 kDa, depending on tissue type (156 kDa in muscle, predicted molecular mass is w75 kDa) [2]. a-Dystroglycan contains a large mucin-like domain with a number of Serine or Threonine residues, which are potential sites for O-glycosylation [2] (Fig. 2). Dystroglycan also contains four potential N-linked glycosylation sites, three in a-dystroglycan and one in b-dystroglycan [2]. Deficiencies of a-dystroglycan have now been described in several forms of muscular dystrophies (see below) and these findings have been mainly based on loss of immunoreactivity with one or both of the two

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Table 1 Muscular dystrophies associated with aberrant glycosylation of a-dystroglycan Muscular dystrophy

Inheritance

Gene

Protein function

Phenotype

MDC1D/myd

AR

LARGE

Putative glycosyltransferase

FCMD

AR

Fukutin

Putative glycosyltransferase

MEB

AR

POMGnT1

O-mannose b-1,2-Nacetylglucosaminyltransferase

WWS

AR

POMT1

O-mannosyltransferase

MDC1C/LGMD2I

AR

FKRP

Putative glycosyltransferase

HIBM

AR

GNE

UDP-GlcNAc2-epimerase/Nacetylmannosamine kinase

Muscular dystrophy, eye abnormalities, abnormal neuronal migration and white matter changes Severe congenital muscular dystrophy, neuronal migration abnormalities associated with mental retardation, epilepsy and, frequently, eye abnormalities Muscular dystrophy, ocular abnormalities (congenital myopia, glaucoma and retinal hypoplasia), mental retardation and structural brain malformations (pachygyria, cerebellar hypoplasia and flat brain stem) Most severe disorder, congenital muscular dystrophy, ocular abnormalities, structural brain abnormalities (cobblestone lissencephaly, agenesis of the corpus callosum, cerebellar hypoplasia, hydrocephalus) Broad range of phenotype ranging from very severe to mild onset of skeletal muscle weakness. Often cardiomyopathy and respiratory failure. Morphological brain abnormalities recently observed Slowly progressive myopathic weakness and atrophy which usually manifests in the second or third decade with foot drop and weakness involving all limbs but typically spare the quadriceps muscle

Abbreviations: MDC1C/MDC1D, congenital muscular dystrophy type 1C/1D; myd, myodystrophy mouse mutant; FCMD, Fukuyama congenital muscular dystrophy; MEB, muscle eye brain disease; WWS, Walker Warburg syndrome; LGMD2I, limb girdle muscular dystrophy type 2I; HIBM, hereditary inclusion body myopathy; AR, autosomal recessive; FKRP, fukutin-related protein; POMT1, protein O-mannosyltransferase1; POMGnT1, protein O-mannose b-1,2Nacetylglucosaminyltransferase; GNE, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannoseamine kinase.

Fig. 2. Potential alterations of O-Mannosylglycosylation of a-dystroglycan in muscular dystrophy. Mutations in the genes POMT1, POMGnT1, fukutin, FKRP and LARGE cause abnormal glycosylation of a-dystroglycan and are associated with a broad clinical phenotype involving muscular dystrophy and, frequently, eye abnormalities and structural brain malformations. The only biochemical substrate has been characterized for POMGnT1. POMT1 is thought to initiate the biosynthesis of O-mannosyl glycan, whereas the substrate for the other putative glycosyltransferases has yet to be identified. Abbreviation: MDC 1C/MDC1D, congenital muscular dystrophy type 1C/1D; myd, myodystrophy mouse mutant; FCMD, Fukuyama congenital muscular dystrophy; MEB, muscle eye brain disease; WWS, Walker Warburg syndrome; LGMD2I, limb girdle muscular dystrophy type 2I; HIBM, hereditary inclusion body myopathy; AR, autosomal recessive; FKRP, fukutin-related protein; POMT1, protein O-mannosyltransferase1; ST3Gal, sialyltransferase; b4GalT, galactosyltransferase; POMGnT1, protein O-mannose b-1,2N-acetylglucosaminyltransferase. Key: Circle, monosaccharides; rectangle, mannose; diamond, N-acetylglucosamine; triangle, galactose.

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commercially available monoclonal antibodies, IIH6 and VIA4-1 [34–40]. The physiological significance of an intact glycosylation site of a-dystroglycan in skeletal muscle has recently been shown in several genetically engineered mouse models. Jayasinha et al. [41] introduced a point mutation at the normal site of proteolysis (serine 654 to Alanine, DGS654A), creating a mouse model in which dystroglycan cleavage was severely inhibited, and subsequently developed a muscular dystrophy. These data suggest that lack of proper processing of dystroglycan correlates with changes in glycosylation of a-dystroglycan, and that dystroglycan cleavage is essential for normal muscle function. Interestingly, the diaphragm of these mice seemed almost completely spared from any pathological alteration, suggesting that the diaphragm may have unique characteristics regarding its glycosylation pathways. In yet another animal model, Nguyen et al. [42] showed that transgenic overexpression of the synaptic CT GalNAc transferase in skeletal muscle ameliorated the muscular dystrophy phenotype in dystrophin deficient mdx mice. They demonstrated that overexpression of CT GalNAc transferase led to increased expression of utrophin and other members of the DGC [42]. Interestingly, overexpression of synaptic CT GalNAc transferase stimulated cleavage of dystroglycan in the DGS654A mutant which subsequently also prevented the development of a dystrophic phenotype in skeletal muscle [43]. In contrast, transgenic overexpression of dystroglycan alone did not alter the expression of the DGC, glycosylation or the dystrophic phenotype in mdx mice [44]. These data suggest that synaptic CT GalNAc transferase may be involved in the regulation of dystroglycan proteolysis and thereby alter a-dystroglycan in a way that could potentially be of therapeutic benefit for various forms of muscular dystrophy. 2.2. Dystroglycan and the central nervous system Basement membranes are essential for proper brain development. During development, meningeal cells cover the surface of the brain and secrete components of the extracellular matrix [45]. The formation of a continuous meningeal layer and accompanying basement membrane is necessary to separate folia in the cerebellum and give rise to the interhemispheric fissure in the cerebral cortex. Selective deletion of dystroglycan in mouse brain (astrocyte targeted) revealed striking abnormalities resembling those found in patients with congenital muscular dystrophy and associated brain malformations [46]. These mice showed discontinuous pial basement membranes surrounding the cerebral cortex and loss of the interhemispheric fissure [46]. Moreover, discontinuities were detected in the pial basement membrane of the cerebellum where adjacent folia are often fused to each other. Dystroglycan null mice also displayed disorganized cortical layering and misplaced neurons (heterotopia) in layer I, and had clusters of neurons and glia beyond the pial basement

membrane, demonstrating significantly perturbed migration of cortical neurons. It seems that impaired anchoring of radial glial processes and focal disruptions in the pial basement membrane are the primary cause for disrupted migration. Dystroglycan has been shown to be concentrated at synaptic sites in the hippocampus. Evaluation of longterm potentiation in dystroglycan null mice revealed severely blunted hippocampal long-term potentiation. These findings indicate that dystroglycan might have a role in synaptic transmission of learning and memory. Interestingly, similar findings of abnormal neuronal migration were observed by Michele et al. [39] in the myodystrophy (Largemyd) mutant mouse (see below). The authors demonstrated disruption of the basement membrane and abnormal neuronal migration in the cerebral cortex, cerebellum and hippocampus. Moreover, it was shown that posttranslational disruption of dystroglycan leading to hypoglycosylation of a-dystroglycan in the central nervous system directly abolished binding activity of dystroglycan for its ligands laminin, agrin and neurexin. Future studies need to further delineate the exact molecular and cellular mechanism of dystroglycan function in neuronal migration and possibly also synaptic transmission. O-linked glycosylation has been shown to be important for intracellular trafficking as well as for targeting proteins [47] and it will be interesting to investigate the role that a-dystroglycan plays in the developmental process of the central nervous system. Dystroglycan has also been shown to be expressed in the retina and the cochlea [48,49]. In the retina, distinct isoforms of a-dystroglycan are localized in apposition to the basal lamina in the inner limiting membrane and blood vessels as well as within the parenchyma of the retina along the Muller glia. These findings suggest that dystroglycan plays a role in organizing synapses and basement membrane assembly in the retina [48]. In the cochlea, dystroglycan has been shown to be present in the developing and adult stria vascularis, and to decline in expression with age when thickening of the basement membrane becomes a prominent pathological feature [49]. Experimental evidence suggests a functional role of dystroglycan in the homeostasis of the strial capillary basement membrane [49]. Further studies are needed to clarify the clinical implications of dystroglycan function in the ophthalmic and cochlear system in regard to patients with dystroglycanopathies (see below). 2.3. Congenital muscular dystrophy type 1D, the myodystrophy mouse mutant and LARGE The identification of altered glycosylation of a-dystroglycan due to a loss-of-function mutation of a putative glycosyltransferase named Large in the myodystrophy mouse model (Largemyd) was the first demonstration that abnormal glycosylation can cause muscular dystrophy [34, 39]. The Large gene encodes a putative glycosyltransferase with a transmembrane domain followed by a coiled-coil domain and two DxD-containing catalytic domains [31].

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The Largemyd mouse had previously been thought to represent an animal model of human facioscapulohumeral muscular dystrophy with elevated creatine kinase levels, sensorineural hearing loss, abnormal gait and posture and decreased reproductive fitness [50,51]. Several studies expanded the previously characterized phenotype and demonstrated neuronal migration defects, cardiac abnormalities and defects in retinal transmission (abnormal b-wave electroretinograms) [39,52]. Longman and colleagues [53] recently identified a heterozygous mutation G1525A (Glu509Lys) in exon 13 and a heterozygous 1 bp insertion, 1999insT in exon 15 in the Large gene in a 17-year-old girl. This novel entity was classified as MDC1D, congenital muscular dystrophy type 1D. The patient presented with congenital onset of weakness (around 5 months of age), profound mental retardation and an abnormal electroretinogram with significant alteration of the b-wave response. MRI of the brain revealed white matter changes and subtle structural abnormalities indicative of abnormal neuronal migration [53]. The skeletal muscle biopsy of this patient showed severe muscular dystrophy with reduced immunoreactivity for a-dystroglycan, and biochemical analysis revealed decreased molecular weight of a-dystroglycan as well as impairment of laminin-binding activity. Two very recently published manuscripts by the group of Kevin Campbell have shed significant insight into the functional role of LARGE and dystroglycan in skeletal muscle. Kanagawa et al. demonstrated that molecular recognition by LARGE represents a key mechanism in the biosynthetic pathway for a mature and functional dystroglycan [54]. The authors show that post-translational modification of a-dystroglycan by LARGE occurs within the mucin-like domain. Biochemical evidence revealed that interaction of LARGE with the N-terminal domain of a-dystroglycan represents an intracellular enzyme recognition motif which is required to initiate efficient glycosylation. These data indicate that disruption of the dystroglycan-laminin linkage, caused by absence of the critical glycosylation/LARGE recognition, represents an essential, common central mechanistic pathway ultimately leading to skeletal muscle cell necrosis and degeneration in muscular dystrophy [54]. Another study by Barresi et al. [55] showed that overexpression of LARGE in Largemyd mice induced synthesis of glycan-enriched a-dystroglycan accompanied by high affinity for extracellular ligands subsequently ameliorating the dystrophic pathology in these mice. The authors went on to demonstrate that LARGE overexpression in cell lines from patients with genetically distinct forms of dystroglycanopathies restored a-dystroglycan receptor function and organization of laminin on the cell surface [55]. These data emphasize that manipulation of endogenous LARGE expression and activity represents a promising future therapeutic target for various muscular dystrophy syndromes caused by abnormal glycosylation of a-dystroglycan.

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2.4. Fukuyama congenital muscular dystrophy and Fukutin Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder that is most often seen in Japanese populations [56]. In Japan, its incidence is roughly 1 per 10,000 births, a frequency equivalent for Duchenne muscular dystrophy in the worldwide population. FCMD is characterized by severe congenital muscular dystrophy, neuronal migration abnormalities associated with mental retardation and epilepsy, and frequently eye abnormalities. Most Japanese patients with the disease carry an ancestral 3kB retrotransposonal insertion in the 3 0 noncoding region of the FCMD gene [57], causing near absence of fukutin mRNA in lymphoblastic cells isolated from FCMD patients. Fukutin, the product of the FCMD gene, has sequence similarities to several putative glycosyltransferases and has an Asp-Xaa-Asp motif in its C-terminus. This motif is conserved in many families of glycosyltransferases and is essential for enzymatic activity [58]. The precise subcellular localization of fukutin in muscle cells is unclear since fukutin GFP or fukutin-myc constructs in COS and CHO cells co-localizes with Golgi-markers [59], while it is present in secretory-type granules in C2 muscle cells [57]. Hayashi et al. demonstrated that a-dystroglycan expression (using antibody VIA4-1) and laminin a2 expression were reduced in FCMD patient’s skeletal and cardiac muscle. Generation of chimeric mice for fukutin demonstrated muscular dystrophy with reduced survival rate, and a significant disorganization of the laminar structures of the cerebral and cerebellar cortices and the hippocampus [60]. These mice also exhibited defects in lens development and retinal detachment, as well as cortical neuronal overmigration and defects of the interhemispheric fissure similar to brain dystroglycan null mice. Biochemical analysis of these mice revealed decreased expression of a-dystroglycan and disrupted laminin ligand activity. Henion et al. [61] recently studied the spatial expression pattern of dystroglycan together with its putative modifying enzymes POMGnT1 and fukutin during the period of peak neuronal migration in the cerebellum. Their data suggest abnormal glycosylation of a-dystroglycan on glial scaffolds, neurons and their processes, and subsequent perturbation of a-dystroglycan ligands may prove to be a potential mechanism of neuronal migration defects in patients with FCMD [61]. A broad correlation between genotype and phenotype in FCMD patients has been recognized. It appears that patients who are homozygous for the initially described ancestral mutation have a rather milder phenotype, while disease severity (associated eye abnormalities such as retinal detachment and microphtalmos) increases in patients who are compound heterozygous for the ancestral mutation and a more severe loss-of-function mutation [57]. Interestingly, in contrast to mice which are not viable, homozygous null mutations in the FCMD gene have recently been characterized in two patients of Turkish origin, suggesting that

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human life is compatible with a homozygous null mutation [62,63]. These patients presented with a more severe, WWS-like phenotype than the general FCMD patient population and had a substantial depletion of a-dystroglycan as shown by immunofluorescence. This was the first case of a fukutin mutation found outside the Japanese population. 2.5. Muscle eye brain disease and POMGnT1 Muscle eye brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities (congenital myopia, glaucoma and retinal hypoplasia), mental retardation and structural brain malformations (pachygyria, cerebellar hypoplasia and flat brain stem) [64]. Most of the patients so far have been described in a genetically isolated population, the Finns [65]. Yoshida et al. [66] identified a mutation in the gene that encodes O-linked mannose b1,2-N-acetylglucosaminyltransferase (POMGnT1), a type II membrane protein similar to other Golgi glycosyltransferases. Skeletal muscle of patients with MEB showed reduced expression of a-dystroglycan and laminin a2. Moreover, hypoglycosylation of a-dystroglycan in skeletal muscle was associated with abolished ligand binding activity of laminin, agrin and neurexin [39]. Mutations of the POMGnT1 gene comprise the first biochemical evidence that congenital muscular dystrophies associated with loss of a-dystroglycan expression are indeed a defect of protein glycosylation, and enzyme activity of POMGnT1 has been found to be significantly reduced [66,67]. Mutations in the POMGnT1 gene have also been identified in patients outside of Finland [68], namely in Japan and Korea. Interestingly, data suggest a genotype phenotype correlation of disease severity. Patients with milder clinical cases often have a mutation located towards the 3 0 end of the POMGnT1 gene, while patients with a more severe phenotype tend to have mutations toward the 5 0 end of the gene [68]. Zhang et al. [69] used an enzymatic assay with commercially available reagents and demonstrated decreased POMGnT1 activity in skeletal muscle biopsies of four patients with POMGnT1 mutations. Thus, this method has the potential to be used as a screening test for patients with congenital muscular dystrophy and associated brain malformations. 2.6. Walker Warburg syndrome and POMT1 Walker Warburg syndrome (WWS) is the most severe amongst the disorders discussed here, with most patients dying by age of 3 years [64]. It is characterized by severe congenital muscular dystrophy, ocular abnormalities, and structural brain abnormalities (type II lissencephaly or cobblestone lissencephaly, agenesis of the corpus callosum, cerebellar hypoplasia, hydrocephalus and rarely encephalocele) [70]. The pathological changes in the central nervous

system are thought to be secondary to pial glial limitans defects which resemble the morphological findings observed in mice with a tissue specific deletion of dystroglycan in brain [46]. Interestingly, similar structural abnormalities of the brain can be observed in mice lacking focal adhesion kinase, b1 integrin and laminin g1 [45,71]. Belran-Valero de Bernabe et al. [63] have recently identified mutations in the gene encoding protein O-mannosyltransferase I (POMT1) in 6 out of 30 unrelated cases with WWS, suggesting genetic heterogeneity. POMT1 belongs to the family of protein mannosyltransferases that has three transmembrane segments in its C-terminal. As it has no apparent Asp-Xaa-Asp motif, POMT1 differs markedly from other glycosyltransferases and therefore might function outside of the Golgi apparatus. Profound depletion of a-dystroglycan in skeletal muscle and peripheral nerve has been described in patients with WWS [63,72,73]. Moreover, defective glycosylation of a-dystroglycan causing loss of laminin-binding activity has recently been demonstrated in one Japanese patient with WWS and a mutation in the POMT1 gene [74]. The genetic heterogeneity of WWS and its clinical overlap with severe forms of FCMD and MEB emphasizes the necessity to further identify and delineate the molecular etiology of this severe disease. 2.7. Congenital muscular dystrophy type 1C, limb girdle muscular dystrophy type 2I and the fukutin-related protein gene The fukutin-related protein gene (FKRP) was initially characterized based upon its sequence homology with fukutin [36]. FKRP, a type II transmembrane protein, is ubiquitously expressed in all tissues. It contains a DxD motif suggestive of a glycosyltransferase, and localization studies in vitro have shown a subcellular localization within the Golgi apparatus [59]. Interestingly, overexpression in Chinese hamster ovary cells revealed that FKRP directly affects dystroglycan processing, a phenomenon completely abolished in case of a mutated FKRP [59]. These in vitro data suggest that mutations in FKRP may inhibit its localization to the Golgi apparatus and therefore FKRP may no longer be able to modify dystroglycan processing. Clinically, mutations in the FKRP gene can be detected in a very broad patient population. The two distinct phenotypes can be categorized into congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD 2I). The main difference between these two disease entities is that patients in the MDC1C category generally present with severe muscle weakness early in life and usually do not achieve ambulation. In contrast, patients with LGMD 2I can present at any time from childhood to adolescence or even adulthood, and are often able to stay ambulant. Initial findings supported by clinical evidence and imaging studies suggested that both entities have no brain involvement [28]. However, recent

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reports of patients with FKRP mutations showed evidence of cerebellar abnormalities, lissencephaly, pachygyria and brain stem hypoplasia [63,75]. The latter abnormalities were detected in patients carrying the clinical diagnosis of MEB and WWS, respectively, underlining once more the heterogeneity of MEB and WWS. Another phenotypic feature observed in patients with LGMD2I was cardiomyopathy and respiratory failure [76]. Immunohistochemical studies of a-dystroglycan in skeletal muscle of MDC1C and LGMD2I muscle suggested a correlation of a-dystroglycan expression and disease severity [77,78]. Patients with MDC1C and a phenotype at the severe end of the disease spectrum showed profound depletion of a-dystroglycan expression. In contrast, patients with LGMD2I and a Duchenne-like phenotype had moderate reduction in a-dystroglycan staining, whereas LGMD2I patients with a milder phenotype had more variable and subtle alterations in a-dystroglycan labeling [77]. In a study of 22 patients with FKRP mutations by Mercuri et al. [78] four patients had MDC1C and 18 had LGMD2I. The patients with MDC1C were compound heterozygous for either one missense and one nonsense or two missense mutations, while patients with two nonsense mutations have not yet been described. The patients with LGMD2I phenotype shared a common mutation (C826A, Leu276Ileu) and the phenotypic severity correlated with the second allelic mutation. Another study of patients with LGMD2I by Poppe et al. [76] confirmed that the C826A change is a common mutation and suggested a carrier frequency of 1:400 in the United Kingdom population. Yet further evidence of clinical and molecular heterogeneity has come from reports of novel missense mutations in the FKRP gene, which causes marked phenotypic variability within the same family [79,80]. These observations suggest that MDC1C and LGMD2I are overlapping ends of the same entity, and that genetic modifiers and/or environmental factors play a role in modulating disease severity. Delineating the exact molecular and cellular mechanisms underlying this phenomenon may prove to be useful in developing future therapeutic strategies. 2.8. Hereditary inclusion body myopathy and GNE Hereditary inclusion body myopathy (HIBM) is an adult onset neuromuscular disorder associated with mutations in the gene UDP-N-acetylglucosamine-2-epimerase/N-acetylmannoseamine kinase (GNE) [81]. HIBM is characterized by slowly progressive myopathic process and atrophy, which usually manifests in the second or third decade with foot drop and weakness involving all limbs but typically sparing the quadriceps muscle. GNE is a bifunctional enzyme that catalyzes the first two steps in the biosynthesis of sialic acid [82]. Loss of GNE activity is thought to impair sialic acid production and interfere with proper sialylation of glycoconjugates. Huizing et al. [81] were the first to demonstrate hypoglycosylation of a-dystroglycan in four

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patients with HIBM and a compound heterozygous mutation in the GNE gene (one in the epimerase, one in the kinase domain). Future studies will need to address how alteration of sialic acid biology may be connected to a-dystroglycan glycosylation and whether abnormalities of glycosylation is indeed a pathomechanism for HIBM.

3. Functional role of dystroglycan in other organ systems When the gene encoding dystroglycan was cloned in 1992, expression-pattern analysis suggested roles for dystroglycan beyond skeletal muscle [2,83]. Indeed, it is now apparent that dystroglycan does play important roles outside of muscle, covering a broad range of biological phenomena. Disruption of the dystroglycan gene in mice results in embryonic lethality, and dystroglycan appears essential for the formation of the basement membrane (Reichert’s membrane) that separates the rodent embryo from the maternal circulation [84]. Moreover, studies in developing and adult tissues have shown that dystroglycan is essential for the assembly of basement membrane formation [85]. Coupled with an increasing understanding of dystroglycan function at the molecular level, we are finally beginning to probe the complexities of dystroglycan involvement in development, adhesion and disease entities. Despite the pivotal role of dystroglycan in the DGC, no primary mutations in dystroglycan have been identified in any human disease. The following paragraphs will highlight some of the multifunctional roles of dystroglycan in tissues others than skeletal muscle. 3.1. Dystroglycan and epithelial cells Several studies have demonstrated that dystroglycan has distinct roles in various organs containing epithelial cells. For example, dystroglycan has been implicated in branching morphogenesis of kidney, lung and salivary glands [86–88]. In kidney epithelial cells, the E3 fragment of laminin-1 perturbed branching morphogenesis in an integrin-dependent manner [87]. Immunohistochemical analysis of dystroglycan in adult kidney tissue has shown reduced glomerular expression in minimal change nephrosis [89]. Interestingly, dystroglycan expression was not altered in focal segmental glomerulosclerosis, indicating different pathogenetic mechanisms of foot process flattening in these glomerular diseases. In another study, dystroglycan expression has been demonstrated in the cell membrane and nucleus of human airway epithelial cells [90]. In vitro experiments causing mechanical injury to the airway epithelial cell suggested that a-dystroglycan is dynamically expressed during wound healing and serves as a lectin to bind laminin, implicating a potential significance for airway epithelial cell repair. One of the most consistent and clinical significant observations about dystroglycan in epithelial cells is that

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loss of dystroglycan can be associated with epithelial cancer progression [91]. Analysis of dystroglycan expression in breast and prostate tumors showed that loss of dystroglycan correlated with tumor progression [92,93]. Interestingly, a-dystroglycan expression was reduced or absent in cell lines which are most invasive, while b-dystroglycan expression is preserved in these cells, indicating continued expression of the dystroglycan gene [94,95]. These findings point to posttranslational modifications (such as altered glycosylation) that perturb a-dystroglycan binding to laminin in tumor cells.

laminin-2. It is assumed that binding of M. leprae– laminin-2 LG domain complexes to a-dystroglycan is dependent on a-dystroglycan glycosylation, since binding to a-dystroglycan was inhibited by periodic acid. In summary, pathogenetic bacteria are particularly adapted to exploit host cell functions, and it seems that M. leprae perturbs this matrix-cytoskeleton link of the peripheral nervous system by adhering and invading Schwann cells, which subsequently interferes with neural cell functions.

3.2. Dystroglycan and peripheral nerve

Two decades of research have characterized dystroglycan as a key player in skeletal muscle biology and various other organ systems. The recent characterization of mutations in glycosyltransferases that lead to alteration of dystroglycan processing in several clinically distinct forms of muscular dystrophy suggest a unique common pathogenetic pathway within this disease entity emphasizing that skeletal muscle cell integrity and function is dependent on glycosylation. Future studies will need to be aimed at identifying the direct and indirect biochemical pathways of how glycosyltransferases impact dystroglycan glycosylation. Moreover, further biochemical delineation of the O-linked mannose pathway will eventually lead to improved diagnostic tests and novel therapeutic strategies for this group of neuromuscular disorders. It will be interesting to see what role (if any) glycosylation of dystroglycan will play in physiology and disease of tissues other than skeletal muscle.

Though it has been known for years that dystroglycan is expressed in Schwann cells, its function in peripheral nerve has only recently been established. Targeted disruption of the DAG1 gene in peripheral nerve in mice revealed decreased nerve conduction velocity secondary to nodal changes, including reduced sodium channel density, abnormal myelin sheath folding and disorganization of the microvilli [96]. These structural alterations led to abnormal motor performance and altered pain responses, thereby demonstrating a unique role of dystroglycan for both myelination and nodal architecture [96]. The findings implicate a role for dystroglycan in the pathogenesis of Charcot-Marie-Tooth neuropathy type IV, leprous neuropathy, and other neuropathic phenotypes associated with muscular dystrophy.

3.4. Conclusion

3.3. Dystroglycan and infectious diseases The cellular receptor for lymphocytic choriomeningitis virus (LCMV) and the human pathogen Lassa fever virus (LFV) has been identified as a-dystroglycan [97,98]. Interestingly, soluble a-dystroglycan was able to inhibit target cells from LCMV infections [97]. Furthermore, a-dystroglycan knockout ES cells failed to become infected with LCMV, but in turn were readily infected when a-dystroglycan was reconstituted. These results suggest that a posttranslational modification, such as glycosylation, is likely to be critical in the pathogenesis of fatal human hemorrhagic fevers caused by arenaviruses and may represent an important target for therapeutic interventions. Another pathological role for dystroglycan has emerged from studies implicating laminin-2 as a target for Mycobacterium leprae (M. leprae) infection of Schwann cells [99]. M. leprae, the causative organism of leprosy, is an intracellular pathogen that invades the Schwann cell of the peripheral nervous system. During infection, M leprae causes significant damage to peripheral nerves, resulting in severe disabilities and deformities. Experiments have provided evidence that a-dystroglycan is the crucial Schwann cell receptor for M. leprae [100]. Interestingly, M. leprae binding to purified a-dystroglycan and Schwann cell a-dystroglycan was almost entirely dependent on

Acknowledgements I would like to thank Dr. Kevin Campbell and his laboratory for the magnificent support I received during my postdoctoral fellowship in the Campbell laboratory. I would also like to thank Rita Barresi, Will Parsons, Adel Gilbert and Sean Charbonneau for critical reading of the manuscript and helpful suggestions.

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