Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx,Largemyd and mdx/Largemyd

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Large^myd mouse and the recently generated and worst double mutant mdx/Large^myd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Large^myd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Large^myd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Large^myd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Large^myd showed similar values than both the mild mdx and worst mdx/Large^myd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.     

Mutation of Large,which encodes a putative glycosyltransferase,in an animal model of muscular dystrophy

The myodystrophy (myd) mutation arose spontaneously and has an autosomal recessive mode of inheritance. Homozygous mutant mice display a severe, progressive muscular dystrophy. Using a positional cloning approach, we identified the causative mutation in myd as a deletion within the Large gene, which encodes a putative glycosyltransferase with two predicted catalytic domains. By immunoblotting, the α-subunit of dystroglycan, a key muscle membrane protein, is abnormal in myd mice. This aberrant protein might represent altered glycosylation of the protein and contribute to the muscular dystrophy phenotype. Our results are discussed in the light of recent reports describing mutations in other glycosyltransferase genes in several forms of human muscular dystrophy.     

Molecular recognition by LARGE is essential for expression of functional dystroglycan

Reduced ligand binding activity of alpha-dystroglycan is associated with muscle and central nervous system pathogenesis in a growing number of muscular dystrophies. Posttranslational processing of alpha-dystroglycan is generally accepted to be critical for the expression of functional dystroglycan. Here we show that both the N-terminal domain and a portion of the mucin-like domain of alpha-dystroglycan are essential for high-affinity laminin-receptor function. Posttranslational modification of alpha-dystroglycan by glycosyltransferase, LARGE, occurs within the mucin-like domain, but the N-terminal domain interacts with LARGE, defining an intracellular enzyme-substrate recognition motif necessary to initiate functional glycosylation. Gene replacement in dystroglycan-deficient muscle demonstrates that the dystroglycan C-terminal domain is sufficient only for dystrophin-glycoprotein complex assembly, but to prevent muscle degeneration the expression of a functional dystroglycan through LARGE recognition and glycosylation is required. Therefore, molecular recognition of dystroglycan by LARGE is a key determinant in the biosynthetic pathway to produce mature and functional dystroglycan.     

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.     

Contribution of Dysferlin Deficiency to Skeletal Muscle Pathology in Asymptomatic and Severe Dystroglycanopathy Models: Generation of a New Model for Fukuyama Congenital Muscular Dystrophy

Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin ^sjl/sjl mice to the fukutin-knock-in fukutin ^Hp/− and Large-deficient Large ^myd/myd mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin ^Hp/− mice do not show a dystrophic phenotype; however, (dysferlin ^sjl/sjl: fukutin ^Hp/−) mice showed a deteriorated phenotype compared with (dysferlin ^sjl/sjl: fukutin ^Hp/+) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin ^Hp/− mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin ^Hp/− FCMD mouse model, and the (dysferlin ^sjl/sjl: fukutin ^Hp/−) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD.     

The role of defective glycosylation in congenital muscular dystrophy

The dystrophin glycoprotein complex (DGC) is an assembly of proteins spanning the sarcolemma of skeletal muscle cells. Defects in the DGC appear to play critical roles in several muscular dystrophies due to disruption of basement membrane organization. O-mannosyl oligosaccharides on α-dystroglycan, a major extracellular component of the DGC, are essential for normal binding of α-dystroglycan to ligands (such as laminin) in the extracellular matrix and subsequent signal transmission to actin in the cytoskeleton of the muscle cell. Muscle-Eye-Brain disease (MEB) and Walker-Warburg Syndrome (WWS) have mutations in genes encoding glycosyltransferases needed for O-mannosyl oligosaccharide synthesis. Myodystrophic myd mice and humans with Fukuyama Congenital Muscular Dystrophy (FCMD), congenital muscular dystrophy due to defective fukutin-related protein (FKRP) and MDC1D have mutations in putative glycosyltransferases. These human congenital muscular dystrophies and the myd mouse are associated with defective glycosylation of α-dystroglycan. It is expected other congenital muscular dystrophies will prove to have mutations in genes involved in glycosylation. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.     

Cell Specific Post-Translational Processing of Pikachurin, A Protein Involved in Retinal Synaptogenesis

Pikachurin is a recently identified, highly conserved, extracellular matrix-like protein. Murine pikachurin has 1,017 amino acids (∼110 kDa), can bind to α-dystroglycan, and has been found to localize mainly in the synaptic cleft of photoreceptor ribbon synapses. Its knockout selectively disrupts synaptogenesis between photoreceptor and bipolar cells. To further characterize this synaptic protein, we used an antibody raised against the N-terminal of murine pikachurin on Western blots of mammalian and amphibian retinas and found, unexpectedly, that a low weight ∼60-kDa band was the predominant signal for endogenous pikachurin. This band was predicted to be an N-terminal product of post-translational cleavage of pikachurin. A similar sized protein was also detected in human Y79 retinoblastoma cells, a cell line with characteristics of photoreceptor cells. In Y79 cells, endogenous pikachurin immunofluorescence was found on the cell surface of living cells. The expression of the N-fragment was not significantly affected by dystroglycan overexpression in spite of the biochemical evidence for pikachurin-α-dystroglycan binding. The presence of a corresponding endogenous C-fragment was not determined because of the lack of a suitable antibody. However, a protein of ∼65 kDa was detected in Y79 cells expressing recombinant pikachurin with a C-terminal tag. In contrast, in QBI-HEK 293A cells, whose endogenous pikachurin protein level is negligible, recombinant pikachurin did not appear to be cleaved. Instead pikachurin was found either intact or as dimers. Finally, whole and N- and C-fragments of recombinant pikachurin were present in the conditioned media of Y79 cells indicating the secretion of pikachurin. The site of cleavage, however, was not conclusively determined. Our data suggest the existence of post-translational cleavage of pikachurin protein as well as the extracellular localization of cleaved protein specifically by retinal cells. The functions of the pikachurin N- and C-fragments in the photoreceptor ribbon synapse are unknown.     

Protein O-mannosylation in animal development and physiology: From human disorders to Drosophila phenotypes

Protein O-mannosylation has a profound effect on the development and physiology of mammalian organisms. Mutations in genes affecting O-mannosyl glycan biosynthesis result in congenital muscular dystrophies. The main pathological mechanism triggered by O-mannosylation defects is a compromised interaction of cells with the extracellular matrix due to abnormal glycosylation of α-dystroglycan. Hypoglycosylation of α-dystroglycan impairs its ligand-binding activity and results in muscle degeneration and failure of neuronal migration. Recent experiments revealed the existence of compensatory mechanisms that could ameliorate defects of O-mannosylation. However, these mechanisms remain poorly understood. O-mannosylation and dystroglycan pathway genes show remarkable evolutionary conservation in a wide range of metazoans. Mutations and downregulation of these genes in zebrafish and Drosophila result in muscle defects and degeneration, also causing neurological phenotypes, which suggests that O-mannosylation has similar functions in mammals and lower animals. Thus, future studies in genetically tractable model organisms, such as zebrafish and Drosophila, should help to reveal molecular and genetic mechanisms of mammalian O-mannosylation and its role in the regulation of dystroglycan function.     

Disruption of perlecan binding and matrix assembly by post-translational or genetic disruption of dystroglycan function

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy.     

Dystroglycan: a possible mediator for reducing congenital muscular dystrophy?

Alpha-dystroglycan is a highly glycosylated peripheral protein forming a complex with the membrane-spanning beta-dystroglycan and establishing a connection between the extracellular matrix and the cytoskeleton. In skeletal muscle, as part of the larger dystrophin-glycoprotein complex, dystroglycan is believed to be essential for maintaining the structural and functional stability of muscle fibers. Recent work highlights the role of abnormal dystroglycan glycosylation at the basis of glycosyltransferase-deficient congenital muscular dystrophies. Notably, modulation of glycosyltransferase activity can restore alpha-dystroglycan receptor function in these disorders. Moreover, transgenic approaches favoring the interaction between dystroglycan and the extracellular matrix molecules also represent an innovative way to restore skeletal muscle structure. These pioneering approaches might comprise an important first step towards the design of gene-transfer-based strategies for the rescue of congenital muscular dystrophies involving dystroglycan.     

Recruitment of Dbl by Ezrin and Dystroglycan Drives Membrane Proximal Cdc42 Activation and Filopodia Formation

Dystroglycan is an essential laminin binding cell adhesion molecule which is also an adaptor for several SH2 domain-containing signalling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodial. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localisation of Cdc42 and dystroglycan at the tips of dynamic filopodia.     

Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of α-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual α-dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with Walker-Warburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This finding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modification of the α-dystroglycan protein. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Deficiency of alpha-dystroglycan in muscle-eye-brain disease

Alpha-dystroglycan is a component of the dystrophin-glycoprotein-complex, which is the major mechanism of attachment between the cytoskeleton and the extracellular matrix. Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities and lissencephaly. We recently found that MEB is caused by mutations in the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1) gene. POMGnT1 is a glycosylation enzyme that participates in the synthesis of O-mannosyl glycan, a modification that is rare in mammals but is known to be a laminin-binding ligand of alpha-dystroglycan. Here we report a selective deficiency of alpha-dystroglycan in MEB patients. This finding suggests that alpha-dystroglycan is a potential target of POMGnT1 and that altered glycosylation of alpha-dystroglycan may play a critical role in the pathomechanism of MEB and some forms of muscular dystrophy.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

The origin of dystrophin-glycoprotein complex(DGC)-related muscular dystrophies: the need for protection against an ancestral pathogen?

Because of its crucial role during the early stages of morphogenesis, no genetic defects associated to dystroglycan have been reported so far. Dystroglycan is an important member of the dystrophin-glycoprotein complex (DGC) and in several muscular dystrophies, depending on abnormalities of proteins belonging to or associated with the DGC, it is frequently observed a significant reduction of dystroglycan levels at the sarcolemma. Recently, it has been demonstrated that dystroglycan acts as a receptor for pathogens such as M. leprae and arenaviruses. It is well-known that mutated alleles causing diseases can be selected in order to confer an additional genetic advantage. Herein it is discussed the possibility that mutations leading to a certain number of muscular dystrophies might have been originally selected to indirectly gain a specific advantage: the absence or the lower levels of dystroglycan could have greatly reduced the risk of some ancestral lethal infections specifically directed against muscles.     

Novel POMGnT1 mutations cause muscle-eye-brain disease in Chinese patients

Muscle-eye-brain (MEB) disease is a congenital muscular dystrophy (CMD) phenotype characterized by hypotonia at birth, brain structural abnormalities and ocular malformations. To date, few MEB cases have been reported in China where clinical recognition and genetic confirmatory testing on a research basis are recent developments. Here, we report the clinical and molecular genetics of three MEB disease patients. The patients had different degrees of muscle, eye and brain symptoms, ranging from congenital hypotonia, early-onset severe myopia and mental retardation to mild weakness, independent walking and language problems. This confirmed the expanding phenotypic spectrum of MEB disease with varying degrees of hypotonia, myopia and cognitive impairment. Brain magnetic resonance imaging showed cerebellar cysts, hypoplasia and characteristic brainstem flattening and kinking. Four candidate genes (POMGnT1, FKRP, FKTN and POMT2) were screened, and six POMGnT1 mutations (four novel) were identified, including five missense and one splice site mutation. Pathogenicity of the two novel variants in one patient was confirmed by POMGnT1 enzyme activity assay, protein expression and subcellular localization of mutant POMGnT1 in HeLa cells. Transfected cells harboring this patient’s L440R mutant POMGnT1 showed POMGnT1 mislocalization to both the Golgi apparatus and endoplasmic reticulum. We have provided clinical, histological, enzymatic and genetic evidence of POMGnT1 involvement in three unrelated MEB disease patients in China. The identification of novel POMGnT1 mutations and an expanded phenotypic spectrum contributes to an improved understanding of POMGnT1 structure–function relationships, CMD pathophysiology and genotype–phenotype correlations, while underscoring the need to consider POMGnT1 in Chinese MEB disease patients.     

Human MR1 expression on the cell surface is acid sensitive, proteasome independent and increases after culturing at 26°C

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the transfer of GlcNAc to O-mannose of glycoproteins. Mutations in the POMGnT1 gene cause muscle–eye–brain disease (MEB). POMGnT1 is a typical type II membrane protein, which is localized in the Golgi apparatus. However, details of the catalytic and reaction mechanism of POMGnT1 are not understood. To develop a better understanding of POMGnT1, we examined the substrate specificity of POMGnT1 using a series of synthetic O-mannosyl peptides based on the human α-dystroglycan (α-DG) sequence as substrates. O-Mannosyl peptides consisting of three to 20 amino acids are recognized as substrates. Enzyme kinetics improved with increasing peptide length up to a length of 8 amino acids but the kinetics of peptides longer than 8 amino acids were similar to those of octapeptides. Our results also show that the amino acid sequence affects POMGnT1 activity. These data suggest that both length and amino acid sequence of mannosyl peptides are determinants of POMGnT1 substrate specificity.     

Congenital muscular dystrophies involving the O-mannose pathway

A number of forms of congenital muscular dystrophy (CMD) have been identified that involve defects in the glycosylation of dystroglycan with O-mannosyl-linked glycans. There are at least six genes that can affect this type of glycosylation, and defects in these genes give rise to disorders that have many aspects of muscle and brain pathology in common. Overexpression of one gene implicated in CMD, LARGE, was recently shown to increase dystroglycan glycosylation and restore its function in cells taken from CMD patients. Overexpression of Galgt2, a glycosyltransferase not implicated in CMD, also alters dystroglycan glycosylation and inhibits muscular dystrophy in a mouse model of Duchenne muscular dystrophy. These findings suggest that a common approach to therapy in muscular dystrophies may be to increase the glycosylation of dystroglycan with particular glycan structures.     

Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle–eye–brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of α-DG, and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^?/? muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1^?/? satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^?/? myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of α-DG is important for maintenance of the proliferative activity of satellite cells in vivo.