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1.  The glucuronyltransferase B4GAT1 is required for initiation of LARGE-mediated α-dystroglycan functional glycosylation 
eLife  2014;3:e03941.
Dystroglycan is a cell membrane receptor that organizes the basement membrane by binding ligands in the extracellular matrix. Proper glycosylation of the α-dystroglycan (α-DG) subunit is essential for these activities, and lack thereof results in neuromuscular disease. Currently, neither the glycan synthesis pathway nor the roles of many known or putative glycosyltransferases that are essential for this process are well understood. Here we show that FKRP, FKTN, TMEM5 and B4GAT1 (formerly known as B3GNT1) localize to the Golgi and contribute to the O-mannosyl post-phosphorylation modification of α-DG. Moreover, we assigned B4GAT1 a function as a xylose β1,4-glucuronyltransferase. Nuclear magnetic resonance studies confirmed that a glucuronic acid β1,4-xylose disaccharide synthesized by B4GAT1 acts as an acceptor primer that can be elongated by LARGE with the ligand-binding heteropolysaccharide. Our findings greatly broaden the understanding of α-DG glycosylation and provide mechanistic insight into why mutations in B4GAT1 disrupt dystroglycan function and cause disease.
eLife digest
Dystroglycan is a protein that is critical for the proper function of many tissues, especially muscles and brain. Dystroglycan helps to connect the structural network inside the cell with the matrix outside of the cell. The extracellular matrix fills the space between the cells to serve as a scaffold and hold cells together within a tissue. It is well established that the interaction of cells with their extracellular environments is important for structuring tissues, as well as for helping cells to specialize and migrate. These interactions also play a role in the progression of cancer.
As is the case for many proteins, dystroglycan must be modified with particular sugar molecules in order to work correctly. Enzymes called glycosyltransferases are responsible for sequentially assembling a complex array of sugar molecules on dystroglycan. This modification is essential for making dystroglycan ‘sticky’, so it can bind to the components of the extracellular matrix. If sugar molecules are added incorrectly, dystroglycan loses its ability to bind to these components. This causes congenital muscular dystrophies, a group of diseases that are characterized by a progressive loss of muscle function.
Willer et al. use a wide range of experimental techniques to investigate the types of sugar molecules added to dystroglycan, the overall structure of the resulting ‘sticky’ complex and the mechanism whereby it is built. This reveals that a glycosyltransferase known as B3GNT1 is one of the enzymes responsible for adding a sugar molecule to the complex. This enzyme was first described in the literature over a decade ago, and the name B3GNT1 was assigned, according to a code, to reflect the sugar molecule it was thought to transfer to proteins. However, Willer et al. (and independently, Praissman et al.) find that this enzyme actually attaches a different sugar modification to dystroglycan, and so should therefore be called B4GAT1 instead.
Willer et al. find that the sugar molecule added by the B4GAT1 enzyme acts as a platform for the assembly of a much larger sugar polymer that cells use to anchor themselves within a tissue. Some viruses–including Lassa virus, which causes severe fever and bleeding–also use the ‘sticky’ sugar modification of dystroglycan to bind to and invade cells, causing disease in humans. Understanding the structure of this complex, and how these sugar modifications are added to dystroglycan, could therefore help to develop treatments for a wide range of diseases like progressive muscle weakening and viral infections.
PMCID: PMC4227050  PMID: 25279699
glycosylation; B4GAT1; B3GNT1; LARGE; alpha-dystroglycan; basement membrane; mouse
2.  Illuminating regeneration: noninvasive imaging of disease progression in muscular dystrophy 
The Journal of Clinical Investigation  2013;123(5):1931-1934.
Muscular dystrophies are characterized by progressive muscle weakness and wasting. Among the key obstacles to the development of therapies is the absence of an assay to monitor disease progression in live animals. In this issue of the JCI, Maguire and colleagues use noninvasive bioluminescence imaging to monitor luciferase activity in mice expressing an inducible luciferase reporter gene in satellite cells. These cells proliferate in response to degeneration, therefore increasing the level of luciferase expression in dystrophic muscle.
PMCID: PMC3635748  PMID: 23619358
3.  Congenital disorder of glycosylation due to DPM1 mutations presenting with dystroglycanopathy-type congenital muscular dystrophy 
Molecular genetics and metabolism  2013;110(3):345-351.
Congenital disorders of glycosylation (CDG) are rare genetic defects mainly in the post-translational modification of proteins via attachment of carbohydrate chains. We describe an infant with the phenotype of a congenital muscular dystrophy, with borderline microcephaly, hypotonia, camptodactyly, severe motor delay, and elevated creatine kinase. Muscle biopsy showed muscular dystrophy and reduced α-dystroglycan immunostaining with glycoepitope-specific antibodies in a pattern diagnostic of dystroglycanopathy. Carbohydrate deficient transferrin testing showed a pattern pointing to a CDG type I. Sanger sequencing of DPM1 (dolichol-P-mannose synthase subunit 1) revealed a novel Gly>Val change c.455 G>T missense mutation resulting in p.Gly152Val) of unknown pathogenicity and deletion/duplication analysis revealed an intragenic deletion from exons 3 to 7 on the other allele. DPM1 activity in fibroblasts was reduced by 80%, while affinity for the substrate was not depressed, suggesting a decrease in the amount of active enzyme. Transfected cells expressing tagged versions of wild type and the p.Gly152Val mutant displayed reduced binding to DPM3, an essential, non-catalytic subunit of the DPM complex, suggesting a mechanism for pathogenicity. The present case is the first individual described with DPM1-CDG (CDG-Ie) to also have clinical and muscle biopsy findings consistent with dystroglycanopathy.
PMCID: PMC3800268  PMID: 23856421
congenital disorder of glycosylation; dystroglycanopathy; congenital muscular dystrophy; DPM1; DPM1-CDG; CDG-Ie; mutation
4.  Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies 
Neuromuscular Disorders  2011;22(1):34-42.
Loss of mobility influences the quality of life for patients with neuromuscular diseases. Common measures of mobility and chronic muscle damage are the six-minute walk test and serum creatine kinase. Despite extensive pre-clinical studies of therapeutic approaches, characterization of these measures is incomplete. To address this, a six-minute ambulation assay, serum creatine kinase, and myoglobinuria were investigated for the mdx mouse, a dystrophinopathy mouse model commonly used in pre-clinical studies. Mdx mice ambulated shorter distances than normal controls, a disparity accentuated after mild exercise. An asymmetric pathophysiology in mdx mice was unmasked with exercise, and peak measurements of serum creatine kinase and myoglobinuria were identified. Our data highlights the necessity to consider asymmetric pathology and timing of biomarkers when testing potential therapies for muscular dystrophy.
PMCID: PMC3264796  PMID: 22154712
mdx; Duchenne muscular dystrophy; Biomarkers; pre-clinical studies; ambulation
5.  Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy 
The Journal of Clinical Investigation  2012;122(9):3330-3342.
Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twenty-week-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue- and timing-specific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy.
PMCID: PMC3428090  PMID: 22922256
6.  Distinct Functions of Glial and Neuronal Dystroglycan in the Developing and Adult Mouse Brain 
Cobblestone (type II) lissencephaly and mental retardation are characteristic features of a subset of congenital muscular dystrophies that include Walker-Warburg Syndrome, Muscle-Eye-Brain disease, and Fukuyama-type congenital muscular dystrophy. Although the majority of clinical cases are genetically undefined, several causative genes have been identified that encode known or putative glycosyltransferases in the biosynthetic pathway of dystroglycan. Here we test the effects of brain-specific deletion of dystroglycan, and show distinct functions for neuronal and glial dystroglycan. Deletion of dystroglycan in the whole brain produced glial/neuronal heterotopia resembling the cerebral cortex malformation in cobblestone lissencephaly. In wild-type mice, dystroglycan stabilizes the basement membrane of the glia limitans, thereby supporting the cortical infrastructure necessary for neuronal migration. This function depends on extracellular dystroglycan interactions, since the cerebral cortex developed normally in transgenic mice that lack the dystroglycan intracellular domain. Also, forebrain histogenesis was preserved in mice with neuron-specific deletion of dystroglycan, but hippocampal long-term potentiation was blunted, as is also the case in the Largemyd mouse, in which dystroglycan glycosylation is disrupted. Our findings provide genetic evidence that neuronal dystroglycan plays a role in synaptic plasticity and that glial dystroglycan is involved in forebrain development. Differences in dystroglycan glycosylation in distinct cell types of the CNS may therefore contribute to the diversity of dystroglycan function in the CNS, as well as to the broad clinical spectrum of type II lissencephalies.
PMCID: PMC2979314  PMID: 20980614
7.  Genetic ablation of complement C3 attenuates muscle pathology in dysferlin-deficient mice  
The Journal of Clinical Investigation  2010;120(12):4366-4374.
Mutations in the dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood. Although muscle inflammation is widely recognized in dysferlinopathy and dysferlin is expressed in immune cells, the contribution of the immune system to the pathology of dysferlinopathy remains to be fully explored. Here, we show that the complement system plays an important role in muscle pathology in dysferlinopathy. Dysferlin deficiency led to increased expression of complement factors in muscle, while muscle-specific transgenic expression of dysferlin normalized the expression of complement factors and eliminated the dystrophic phenotype present in dysferlin-null mice. Furthermore, genetic disruption of the central component (C3) of the complement system ameliorated muscle pathology in dysferlin-deficient mice but had no significant beneficial effect in a genetically distinct model of muscular dystrophy, mdx mice. These results demonstrate that complement-mediated muscle injury is central to the pathogenesis of dysferlinopathy and suggest that targeting the complement system might serve as a therapeutic approach for this disease.
PMCID: PMC2993587  PMID: 21060153
8.  Visual Impairment in the Absence of Dystroglycan 
Ocular involvement in muscular dystrophy ranges from structural defects to abnormal electroretinograms. While the mechanisms underlying the abnormal retinal physiology in patients are not understood, it is thought that α-dystroglycan extracellular interactions are critical for normal visual function. Here we show that β-dystroglycan anchors dystrophin and the inward rectifying K+ channel Kir4.1 at glial endfeet and that disruption of dystrophin and potassium channel clustering in dystroglycan mutant mice is associated with an attenuation of the electroretinogram b-wave. Glial-specific inactivation of dystroglycan or deletion of the cytoplasmic domain of β-dystroglycan was sufficient to attenuate the electroretinogram b-wave. Unexpectedly, deletion of the β-dystroglycan cytoplasmic domain did not disrupt the laminar structure of the retina. In contrast to the role of α-dystroglycan extracellular interactions during early development of the central nervous system, β-dystroglycan intracellular interactions are important for visual function but not the laminar development of the retina.
PMCID: PMC2965532  PMID: 19846701
dystroglycan; electroretinogram; b-wave; vision; Muscle-Eye-Brain disease; congenital muscular dystrophy
9.  Skeletal muscle’s 3rd year anniversary 
Skeletal Muscle  2014;4:3.
PMCID: PMC3911964  PMID: 24456943
10.  Brain and Eye Malformations Resembling Walker–Warburg Syndrome Are Recapitulated in Mice by Dystroglycan Deletion in the Epiblast 
Walker–Warburg syndrome (WWS) is a severe congenital disease that is characterized by brain and eye malformations and lethality during the first year of life. Genetic mutations have been identified in a subset of WWS patients, but a majority of clinical cases have unknown etiologies. POMT1 and POMT2, two of the causative genes, form an active enzyme complex in the posttranslational biosynthetic pathway of dystroglycan. Deletion of either Pomt1 or the dystroglycan gene causes early embryonic lethality in mice. Here we report that mice with epiblast-specific loss of dystroglycan develop brain and eye defects that broadly resemble the clinical spectrum of the human disease, including aberrant neuron migration, hydrocephalus, and malformations of the anterior and posterior chambers of the eye. Breaches of basement membranes coincide with the pathology, revealing an important function for dystroglycan in the morphogenesis of the brain and eye. These findings demonstrate the central role of dystroglycan in WWS and suggest that novel defects in posttranslational processing or mutations of the dystroglycan gene itself may underlie cases in which no causative mutation has been found.
PMCID: PMC2714190  PMID: 18923033
Walker–Warburg syndrome; congenital muscular dystrophy; lissencephaly; hydrocephalus; microphthalmia; dystroglycan
11.  LARGE glycans on dystroglycan function as a tunable matrix scaffold to prevent dystrophy 
Nature  2013;503(7474):136-140.
The dense glycan coat that surrounds every cell is essential for cellular development and physiological function1, and it is becoming appreciated that its composition is highly dynamic. Post-translational addition of the polysaccharide repeating unit [-3-xylose-α1,3-glucuronic acid-β1-]n by like-acetylglucosaminyltransferase (LARGE) is required for the glycoprotein dystroglycan to function as a receptor for proteins in the extracellular matrix2,3. Reductions in the amount of [-3-xylose-α1,3-glucuronic acid-β1-]n (hereafter referred to as LARGE-glycan) on dystroglycan result in heterogeneous forms of muscular dystrophy4. However, neither patient nor mouse studies has revealed a clear correlation between glycosylation status and phenotype5,6. This disparity can be attributed to our lack of knowledge of the cellular function of the LARGE-glycan repeat. Here we show that coordinated upregulation of Large and dystroglycan in differentiating mouse muscle facilitates rapid extension of LARGE-glycan repeat chains. Using synthesized LARGE-glycan repeats we show a direct correlation between LARGE-glycan extension and its binding capacity for extracellular matrix ligands. Blocking Large upregulation during muscle regeneration results in the synthesis of dystroglycan with minimal LARGE-glycan repeats in association with a less compact basement membrane, immature neuromuscular junctions and dysfunctional muscle predisposed to dystrophy. This was consistent with the finding that patients with increased clinical severity of disease have fewer LARGE-glycan repeats. Our results reveal that the LARGE-glycan of dystroglycan serves as a tunable extracellular matrix protein scaffold, the extension of which is required for normal skeletal muscle function.
PMCID: PMC3891507  PMID: 24132234
12.  Binding of Lassa virus perturbs extracellular matrix-induced signal transduction via dystroglycan 
Cellular microbiology  2012;14(7):10.1111/j.1462-5822.2012.01784.x.
The arenavirus Lassa virus (LASV) causes a severe haemorrhagic fever with high mortality in man. The cellular receptor for LASV is dystroglycan (DG). DG is a ubiquitous receptor for extracellular matrix (ECM) proteins, which cooperates with β1 integrins to control cell–matrix interactions. Here, we investigated whether LASV binding to DG triggers signal transduction, mimicking the natural ligands. Engagement of DG by LASV resulted in the recruitment of the adaptor protein Grb2 and the protein kinase MEK1 by the cytoplasmic domain of DG without activating the MEK/ERK pathway, indicating assembly of an inactive signalling complex. LASV binding to cells however affected the activation of the MEK/ERK pathway via α6β1 integrins. The virus-induced perturbation of α6β1 integrin signalling critically depended on high-affinity LASV binding to DG and DG’s cytoplasmic domain, indicating that LASV–receptor binding perturbed signalling cross-talk between DG and β1 integrins.
PMCID: PMC3869547  PMID: 22405130
13.  Dysferlin and Muscle Membrane Repair 
Current opinion in cell biology  2007;19(4):409-416.
The ability to repair membrane damage is conserved across eukaryotic cells, and is necessary for the cells to survive a variety of physiological and pathological membrane disruptions. Membrane repair is mediated by rapid Ca2+-triggered exocytosis of various intracellular vesicles, such as lysosomes and enlargeosomes, which lead to the formation of a membrane patch that reseals the membrane lesion. Recent findings suggest a crucial role for dysferlin in this repair process in muscle, possibly as a Ca2+ sensor that triggers vesicle fusion. The importance of membrane repair is highlighted by the genetic disease, dysferlinopathy, in which the primary defect is the loss of Ca2+-regulated membrane repair due to dysferlin deficiency. Future research on dysferlin and its interacting partners will enhance the understanding of this important process, and provide novel avenues to potential therapies.
PMCID: PMC2144911  PMID: 17662592
C2 domains; cardiomyopathy; dysferlin; membrane fusion; membrane repair; muscular dystrophy
14.  SGK196 Is a Glycosylation-Specific O-Mannose Kinase Required for Dystroglycan Function 
Science (New York, N.Y.)  2013;341(6148):10.1126/science.1239951.
Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine-β3-N-acetylglucosamine-β4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain-containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. Here we found that glycosyltransferase-like domain containing 2 (GTDC2) is a protein O-linked mannose β 1,4-N-acetylglucosaminyltransferase whose product could be extended by β 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.
PMCID: PMC3848040  PMID: 23929950
15.  Dystroglycan on Radial Glia Endfeet Is Required For Pial Basement Membrane Integrity and Columnar Organization of the Developing Cerebral Cortex 
Interactions between the embryonic pial basement membrane (PBM) and radial glia (RG) are essential for morphogenesis of the cerebral cortex, as disrupted interactions cause cobblestone malformations. To elucidate the role of dystroglycan (DG) in PBM-RG interactions, we studied the expression of DG protein and Dag1 mRNA (which encodes DG protein) in developing cerebral cortex, and analyzed cortical phenotypes in Dag1 CNS conditional mutant mice. In normal embryonic cortex, Dag1 mRNA was expressed in the ventricular zone, which contains RG nuclei whereas DG protein was expressed at the cortical surface on RG endfeet. Breaches of PBM continuity appeared during early neurogenesis in Dag1 mutants. Diverse cellular elements streamed through the breaches to form leptomeningeal heterotopia that were confluent with the underlying residual cortical plate and contained variably truncated RG fibers, many types of cortical neurons, and radial and intermediate progenitor cells. Nevertheless, layer-specific molecular expression appeared normal in heterotopic neurons, and axons projected to appropriate targets. Dendrites, however, were excessively tortuous and lacked radial orientation. These findings indicate that DG is required on RG endfeet to maintain PBM integrity and suggest that cobblestone malformations involve disturbances of radial glia structure, progenitor distribution, and dendrite orientation in addition to neuronal “overmigration.”
PMCID: PMC3512206  PMID: 23147502
Dystroglycan; cerebral cortex; cobblestone malformation; lissencephaly; leptomeningeal heterotopia
16.  Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan 
Cellular microbiology  2012;15(5):689-700.
The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a hemorrhagic fever with high mortality in man. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.
PMCID: PMC3805106  PMID: 23279385
17.  MG53′s new identity 
Skeletal Muscle  2013;3:25.
Mitsugumin 53 (MG53) is a relatively newly identified tripartite motif-containing (TRIM) family muscle-specific E3 ubiquitin ligase that is expressed in skeletal muscle and the heart. It has been postulated to facilitate repair by targeting the site of an injury, and acting as a scaffold for assembly of a repair complex made up of dysferlin, annexin V, caveolin-3, and polymerase I and transcript release factor (PTRF). A recent letter published in Nature by Song et al. proposes an alternate function for MG53: as an E3 ligase that targets the insulin receptor and insulin receptor substrate 1 (IRS1) for degradation, therefore regulating muscle insulin signaling. This work is exciting, as it not only presents a novel role for MG53, but also suggests that muscle insulin signaling has a systemic influence on insulin resistance and the metabolic syndrome.
PMCID: PMC4177543  PMID: 24175977
Mitsugumin 53; MG53; Insulin receptor; Insulin signaling; E3 ubiquitin ligase; Metabolic syndrome; TRIM72
18.  Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury 
Journal of Clinical Investigation  2007;117(7):1805-1813.
Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of dysferlin in the heart remains to be determined. Here, we demonstrate that dysferlin is also involved in cardiomyocyte membrane repair and that dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in dysferlin-null mice and increases Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and dysferlin leads to early onset cardiomyopathy. Our results suggest that dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of dysferlin.
PMCID: PMC1904311  PMID: 17607357
19.  Glial scaffold required for cerebellar granule cell migration is dependent on dystroglycan function as a receptor for basement membrane proteins 
Cobblestone lissencephaly is a severe neuronal migration disorder associated with congenital muscular dystrophies (CMD) such as Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama-type CMD. In these severe forms of dystroglycanopathy, the muscular dystrophy and other tissue pathology is caused by mutations in genes involved in O-linked glycosylation of alpha-dystroglycan. While cerebellar dysplasia is a common feature of dystroglycanopathy, its pathogenesis has not been thoroughly investigated.
Here we evaluate the role of dystroglycan during cerebellar development. Brain-selective deletion of dystroglycan does not affect overall cerebellar growth, yet causes malformations associated with glia limitans disruptions and granule cell heterotopia that recapitulate phenotypes found in dystroglycanopathy patients. Cerebellar pathology in these mice is not evident until birth even though dystroglycan is lost during the second week of embryogenesis. The severity and spatial distribution of glia limitans disruption, Bergmann glia disorganization, and heterotopia exacerbate during postnatal development. Astrogliosis becomes prominent at these same sites by the time cerebellar development is complete. Interestingly, there is spatial heterogeneity in the glia limitans and granule neuron migration defects that spares the tips of lobules IV-V and VI.
The full spectrum of developmental pathology is caused by loss of dystroglycan from Bergmann glia, as neither granule cell- nor Purkinje cell-specific deletion of dystroglycan results in similar pathology. These data illustrate the importance of dystroglycan function in radial/Bergmann glia, not neurons, for normal cerebellar histogenesis. The spatial heterogeneity of pathology suggests that the dependence on dystroglycan is not uniform.
PMCID: PMC3893534  PMID: 24252195
Dystroglycan; Glia; Cerebellum; Development
20.  Dystroglycan Function Requires Xylosyl- and Glucuronyltransferase Activities of LARGE 
Science (New York, N.Y.)  2012;335(6064):93-96.
Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [–3-xylose–α1,3-glucuronic acid-β1–]. This modification allowed α-DG to bind laminin-G domain–containing ECM ligands.
PMCID: PMC3702376  PMID: 22223806
21.  Prevention of cardiomyopathy in mouse models lacking the smooth muscle sarcoglycan-sarcospan complex 
Cardiomyopathy is a multifactorial disease, and the dystrophin-glycoprotein complex has been implicated in the pathogenesis of both hereditary and acquired forms of the disease. Using mouse models of cardiomyopathy made by ablating genes for components of the sarcoglycan complex, we show that long-term treatment with verapamil, a calcium channel blocker with vasodilator properties, can alleviate the severe cardiomyopathic phenotype, restoring normal serum levels for cardiac troponin I and normal cardiac muscle morphology. Interruption of verapamil treatment leads again to vascular dysfunction and acute myocardial necrosis, indicating that predilection for cardiomyopathy is a continuing process. In contrast, verapamil did not prevent cardiac muscle pathology in dystrophin-deficient mdx mice, which neither show a disruption of the sarcoglycan complex in vascular smooth muscle nor vascular dysfunction. Hence, our data strongly suggest that pharmacological intervention with verapamil merits investigation as a potential therapeutic option not only for patients with sarcoglycan mutations, but also for patients with idiopathic cardiomyopathy associated with myocardial ischemia not related to atherosclerotic coronary artery disease.
PMCID: PMC199179  PMID: 11160141
22.  Membrane Targeting and Stabilization of Sarcospan Is Mediated by the Sarcoglycan Subcomplex  
The Journal of Cell Biology  1999;145(1):153-165.
The dystrophin–glycoprotein complex (DGC) is a multisubunit complex that spans the muscle plasma membrane and forms a link between the F-actin cytoskeleton and the extracellular matrix. The proteins of the DGC are structurally organized into distinct subcomplexes, and genetic mutations in many individual components are manifested as muscular dystrophy. We recently identified a unique tetraspan-like dystrophin-associated protein, which we have named sarcospan (SPN) for its multiple sarcolemma spanning domains (Crosbie, R.H., J. Heighway, D.P. Venzke, J.C. Lee, and K.P. Campbell. 1997. J. Biol. Chem. 272:31221–31224). To probe molecular associations of SPN within the DGC, we investigated SPN expression in normal muscle as a baseline for comparison to SPN's expression in animal models of muscular dystrophy. We show that, in addition to its sarcolemma localization, SPN is enriched at the myotendinous junction (MTJ) and neuromuscular junction (NMJ), where it is a component of both the dystrophin– and utrophin–glycoprotein complexes. We demonstrate that SPN is preferentially associated with the sarcoglycan (SG) subcomplex, and this interaction is critical for stable localization of SPN to the sarcolemma, NMJ, and MTJ. Our experiments indicate that assembly of the SG subcomplex is a prerequisite for targeting SPN to the sarcolemma. In addition, the SG– SPN subcomplex functions to stabilize α-dystroglycan to the muscle plasma membrane. Taken together, our data provide important information about assembly and function of the SG–SPN subcomplex.
PMCID: PMC2148225  PMID: 10189375
sarcospan; dystrophin; sarcoglycans; tetraspans; muscular dystrophy
23.  ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies 
Brain  2013;136(1):269-281.
Dystroglycanopathies are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe of the congenital muscular dystrophies, Walker–Warburg syndrome, to mild forms of adult-onset limb-girdle muscular dystrophy. Their hallmark is a reduction in the functional glycosylation of α-dystroglycan, which can be detected in muscle biopsies. An important part of this glycosylation is a unique O-mannosylation, essential for the interaction of α-dystroglycan with extracellular matrix proteins such as laminin-α2. Mutations in eight genes coding for proteins in the glycosylation pathway are responsible for ∼50% of dystroglycanopathy cases. Despite multiple efforts using traditional positional cloning, the causative genes for unsolved dystroglycanopathy cases have escaped discovery for several years. In a recent collaborative study, we discovered that loss-of-function recessive mutations in a novel gene, called isoprenoid synthase domain containing (ISPD), are a relatively common cause of Walker–Warburg syndrome. In this article, we report the involvement of the ISPD gene in milder dystroglycanopathy phenotypes ranging from congenital muscular dystrophy to limb-girdle muscular dystrophy and identified allelic ISPD variants in nine cases belonging to seven families. In two ambulant cases, there was evidence of structural brain involvement, whereas in seven, the clinical manifestation was restricted to a dystrophic skeletal muscle phenotype. Although the function of ISPD in mammals is not yet known, mutations in this gene clearly lead to a reduction in the functional glycosylation of α-dystroglycan, which not only causes the severe Walker–Warburg syndrome but is also a common cause of the milder forms of dystroglycanopathy.
PMCID: PMC3562076  PMID: 23288328
congenital muscular dystrophy; limb-girdle muscular dystrophy; dystroglycan; laminin; isoprenoid synthase
24.  Xylosyl- and glucuronyltransferase functions of LARGE in α-dystroglycan modification are conserved in LARGE2 
Glycobiology  2012;23(3):295-302.
LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.
PMCID: PMC3555503  PMID: 23125099
dystroglycan; glucuronyltransferase; LARGE; muscular dystrophy; xylosyltransferase
25.  Anti-Epileptic Drugs Delay Age-Related Loss of Spiral Ganglion Neurons via T-type Calcium Channel 
Hearing research  2011;278(1-2):106-112.
Loss of spiral ganglion neurons is a major cause of age-related hearing loss (presbycusis). Despite being the third most prevalent condition afflicting elderly persons, there are no known medications to prevent presbycusis. Because calcium signaling has long been implicated in age-related neuronal death, we investigated T-type calcium channels. This family is comprised of three members (Cav3.1, Cav3.2, and Cav3.3), based on their respective main pore-forming alpha subunits: α1G, α1H, and α1I. In the present study, we report a significant delay of age-related loss of cochlear function and preservation of spiral ganglion neurons in α1H null and heterozygous mice, clearly demonstrating an important role for Cav3.2 in age-related neuronal loss. Furthermore, we show that anticonvulsant drugs from a family of T-type calcium channel blockers can significantly preserve spiral ganglion neurons during aging. To our knowledge, this is the first report of drugs capable of diminishing age-related loss of spiral ganglion neurons.
PMCID: PMC3152691  PMID: 21640179
Presbycusis; Spiral ganglion neuron; Hair cell; Anti-epileptic drug; Aging

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