Jakob S. Satz

Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies

Muscle–eye–brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD) are congenital muscular dystrophies with associated, similar brain malformations. The FCMD gene, fukutin, shares some homology with fringe-like glycosyltransferases, and the MEB gene, POMGnT1, seems to be a new glycosyltransferase. Here we show, in both MEB and FCMD patients, that α-dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. We show that this post-translational biochemical and functional disruption of α-dystroglycan is recapitulated in the muscle and central nervous system of mutant myodystrophy (myd) mice. We demonstrate that myd mice have abnormal neuronal migration in cerebral cortex, cerebellum and hippocampus, and show disruption of the basal lamina. In addition, myd mice reveal that dystroglycan targets proteins to functional sites in brain through its interactions with extracellular matrix proteins. These results suggest that at least three distinct mammalian genes function within a convergent post-translational processing pathway during the biosynthesis of dystroglycan, and that abnormal dystroglycan–ligand interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities.

LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies

Several congenital muscular dystrophies caused by defects in known or putative glycosyltransferases are commonly associated with hypoglycosylation of α-dystroglycan (α-DG) and a marked reduction of its receptor function. We have investigated changes in the processing and function of α-DG resulting from genetic manipulation of LARGE, the putative glycosyltransferase mutated both in Largemyd mice and in humans with congenital muscular dystrophy 1D (MDC1D). Here we show that overexpression of LARGE ameliorates the dystrophic phenotype of Largemyd mice and induces the synthesis of glycan-enriched α-DG with high affinity for extracellular ligands. Notably, LARGE circumvents the α-DG glycosylation defect in cells from individuals with genetically distinct types of congenital muscular dystrophy. Gene transfer of LARGE into the cells of individuals with congenital muscular dystrophies restores α-DG receptor function, whereby glycan-enriched α-DG coordinates the organization of laminin on the cell surface. Our findings indicate that modulation of LARGE expression or activity is a viable therapeutic strategy for glycosyltransferase-deficient congenital muscular dystrophies.

A Dystroglycan Mutation Associated with Limb-Girdle Muscular Dystrophy

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

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 contribute to the diversity of dystroglycan function in the CNS, as well as to the broad clinical spectrum of type II lissencephalies.

Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection

α-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr317ProThr319ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.

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.

Glycomic Analyses of Mouse Models of Congenital Muscular Dystrophy

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1−/−). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with β1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical β1,2-elongation and β1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

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 Largemyd 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.

Evidence for a role of dystroglycan regulating the membrane architecture of astroglial endfeet

The dystrophin–dystroglycan complex (DDC) is a molecular array of proteins in muscle and brain cells. The central component of the DDC is dystroglycan, which comprises α‐ and β‐subunits. α‐Dystroglycan (α‐DG) binds to extracellular matrix components such as agrin, whereas β‐dystroglycan (β‐DG) is a membrane‐spanning protein linking α‐DG to the cytoskeleton and other intracellular components such as α‐syntrophin. In astrocytes, α‐syntrophin binds to the water channel protein aquaporin‐4 (AQP4). Recently, it has been shown that AQP4 expression is unaltered in agrin‐knockout mice, but that formation of orthogonal arrays of particles (OAPs), consisting of AQP4, is abnormal. As the brain‐selective deletion of the DG gene causes a disorganization of the astroglial endfeet, we investigated whether DG deletion has an impact on AQP4. Western blotting revealed reduced AQP4 in the parenchymal but not in the superficial compartment of the astrocyte‐conditioned DG‐knockout mouse brain. Accordingly, immunohistochemical stainings of AQP4 revealed a selective loss of AQP4 in perivascular but not in superficial astroglial endfeet. In both superficial and perivascular endfeet of the DG‐knockout brain, we observed a loss of OAPs. We conclude that in the absence of DG the majority of superficial AQP4 molecules did not form OAPs, and that expression of AQP4 in perivascular endfeet is compromised. However, the decreased number of perivascular AQP4 molecules obviously did form a few OAPs, even in the absence of DG.

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 CNS, β-dystroglycan intracellular interactions are important for visual function but not the laminar development of the retina.