Sarcoglycan complex: a muscular supporter of dystroglycan-dystrophin interplay?

In striated muscle, the cytoskeletal protein dystrophin, the protein product of the Duchenne muscular dystrophy gene, is associated with a number of sarcolemmal glycoproteins to form a large oligomeric complex, the dystrophin-glycoprotein complex (DGC). Over the last 10 years, four of these sarcolemmal glycoproteins, alpha-, beta-, gamma- and delta-sarcoglycans, have been shown to form a distinct subcomplex, the sarcoglycan complex, in the DGC. Furthermore, the genetic defects of alpha-, beta-, gamma- and delta-sarcoglycans have been identified as the causes of four distinct forms of muscular dystrophies, which are now collectively called sarcoglycanopathy. Current studies are beginning to focus on the biological functions of the sarcoglycan complex and the molecular mechanism by which its dysfunction leads to muscle cell degeneration.     

The Roles of the Dystrophin-Associated Glycoprotein Complex at the Synapse

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. A number of Duchenne patients also present with mental retardation. The dystrophin protein is part of the highly conserved dystrophin-associated glycoprotein complex (DGC) which accumulates at the neuromuscular junction (NMJ) and at a variety of synapses in the peripheral and central nervous systems. Many years of research into the roles of the DGC in muscle have revealed its structural function in stabilizing the sarcolemma. In addition, the DGC also acts as a scaffold for various signaling pathways. Here, we discuss recent advances in understanding DGC roles in the nervous system, gained from studies in both vertebrate and invertebrate model systems. From these studies, it has become clear that the DGC is important for the maturation of neurotransmitter receptor complexes and for the regulation of neurotransmitter release at the NMJ and central synapses. Furthermore, roles for the DGC have been established in consolidation of long-term spatial and recognition memory. The challenges ahead include the integration of the behavioral and mechanistic studies and the use of this information to identify therapeutic targets.     

Role of gelatinases in pathological and physiological processes involving the dystrophin–glycoprotein complex

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin–glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn²⁺-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.     

Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from <Emphasis Type="Italic">mdx</Emphasis> mouse muscle

The dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.     

Drosophila Dystrophin is required for integrity of the musculature

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. The highly conserved dystrophin gene encodes a number of protein isoforms. The Dystrophin protein is part of a large protein assembly, the Dystrophin glycoprotein complex, which stabilizes the muscle membrane during contraction and acts as a scaffold for signaling molecules. How the absence of Dystrophin results in the onset of muscular dystrophy remains unclear. Here, we have used transgenic RNA interference to examine the roles of the Drosophila Dystrophin isoforms in muscle. We previously reported that one of the Drosophila Dystrophin orthologs, the DLP2 isoform, is not required to maintain muscle integrity, but plays a role in neuromuscular homeostasis by regulating neurotransmitter release. In this report, we show that reduction of all Dystrophin isoform expression levels in the musculature does not apparently affect myogenesis or muscle attachment, but results in progressive muscle degeneration in larvae and adult flies. We find that a recently identified Dystrophin isoform, Dp117, is expressed in the musculature and is required for muscle integrity. Muscle fibers with reduced levels of Dp117 display disorganized actin-myosin filaments and the cellular hallmarks of necrosis. Our results indicate the existence of at least two possibly separate roles of dystrophin in muscle, maintaining synaptic homeostasis and preserving the structural stability of the muscle.     

The emerging family of dystrophin-related proteins

Duchenne and Becker muscular dystrophies are caused by mutations in the gene encoding dystrophin, a component of the subsarcolemmal cytoskeleton. Dystrophin-related proteins are identical or homologous to the cysteine-rich and C-terminal domains of dystrophin. This part of dystrophin binds to a membrane-spanning glycoprotein complex in muscle. At least five dystrophin-related proteins are encoded by the Duchenne muscular dystrophy locus. These proteins are found in many non-muscle tissues where dystrophin is not expressed and they are thought to be membrane-associated. Two other dystrophin-related proteins--utrophin and an 87 kDa postsynaptic protein--are encoded by separate loci and, like dystrophin, they are components of the neuromuscular junction.     

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 alpha-dystroglycan, a major extracellular component of the DGC, are essential for normal binding of alpha-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 alpha-dystroglycan. It is expected other congenital muscular dystrophies will prove to have mutations in genes involved in glycosylation.     

Gene expression profiling studies on Caenorhabditis elegans dystrophin mutants dys-1(cx-35) and dys-1(cx18)

The Caenorhabditis elegans genome contains a single dystrophin/utrophin orthologue, dys-1. Point mutations in this gene, dys-1(cx35) and dys-1(cx18), result in truncated proteins. Such mutants offer potentially valuable worm models of human Duchenne muscular dystrophy. We have used microarrays to examine genes expressed differentially between wild-type C. elegans and dys-1 mutants. We found 106 genes (115 probe sets) to be differentially expressed when the two mutants are compared to wild-type worms, 49 of which have been assigned to six functional categories. The main categories of regulated genes in C. elegans are genes encoding intracellular signalling, cell-cell communication, cell-surface, and extracellular matrix proteins; genes in these same categories have been shown by others to be differentially expressed in muscle biopsies of muscular dystrophy patients. The C. elegans model may serve as a convenient vehicle for future genetic and chemical screens to search for new drug targets.     

Animal Models for Muscular Dystrophy Show Different Patterns of Sarcolemmal Disruption

Genetic defects in a number of components of the dystrophin–glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy^2J/dy^2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin α2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.     

Cell and fiber-type distribution of dystrophin

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue. The dystrophin content of brain and spinal cord, however, is found not to correlate with any type of muscle cell, and it is suggested that neurons express dystrophin. The potential involvement of striated muscle fibers, the vasculature, and the nervous system in the etiology of Duchenne muscular dystrophy makes it likely that the disease is a complex disorder of combined pathogenesis. We also find that the dystrophic chicken does not represent an animal model for dystrophin deficiency.     

A mutation in CHN-1/CHIP suppresses muscle degeneration in Caenorhabditis elegans

Duchenne muscular dystrophy (DMD) is one of the most severe X-linked, inherited diseases of childhood, characterized by progressive muscle wasting and weakness as the consequence of mutations in the dystrophin gene. The protein encoded by dystrophin is a huge cytosolic protein that links the intracellular F-actin filaments to the members of the dystrophin-glycoprotein-complex (DGC). Dystrophin deficiency results in the absence or reduction of complex components that are degraded through an unknown pathway. We show here that muscle degeneration in a Caenorhabditis elegans DMD model is efficiently reduced by downregulation of chn-1, encoding the homologue of the human E3/E4 ubiquitylation enzyme CHIP. A deletion mutant of chn-1 delays the cell death of body-wall muscle cells and improves the motility of animals carrying mutations in dystrophin and MyoD. Elimination of chn-1 function in the musculature, but not in the nervous system, is sufficient for this effect, and can be phenocopied by proteasome inhibitor treatment. This suggests a critical role of CHIP/CHN-1-mediated ubiquitylation in the control of muscle wasting and degeneration and identifies a potential new drug target for the treatment of this disease.     

Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

Current knowledge of dystrophin and dystrophin-associated proteins in the retina

Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and beta-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains full-length dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). beta-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.     

Calmodulin-binding profiles for nebulin and dystrophin in human skeletal muscle

Nebulin and dystrophin are two high-molecular-mass skeletal muscle proteins that have both been associated with the defective gene in Duchenne muscular dystrophy, although the function of neither protein is known. Other high-molecular-mass, calmodulin-binding proteins have recently been implicated in regulating calcium release from skeletal muscle. Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin. Dystrophin was absent in a Duchenne muscle biopsy.     

Dynamic changes in the subcellular localization of Drosophila beta-sarcoglycan during the cell cycle

One of the proposed roles of sarcoglycan is to stabilize dystrophin glycoprotein complexes in muscle sarcolemma. Involvement in signal transduction has also been proposed and abnormalities in some sarcoglycan genes are known to be responsible for muscular dystrophy. While characterization of sarcoglycans in muscle has been performed, little is known about its functions in the non-muscle tissues in which mammalian sarcoglycans are expressed. Here, we investigated temporal and spatial expression patterns of Drosophila beta-sarcoglycan (dScgbeta) during development by immunohistochemistry. In addition to almost ubiquitous expression in various tissues and organs, as seen for its mammalian counterpart, anti-dScgbeta staining data of embryos, eye imaginal discs, and salivary glands demonstrated cytoplasmic localization during S phase in addition to plasma membrane staining. Furthermore we found that subcellular localization of dScgbeta dramatically changes during mitosis through possible association with tubulin. These observations point to a complex role of sarcoglycans in non-muscle tissues.     

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.     

Genomic integration of the full‐length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells

Plasmid vectors that express the full‐length human dystrophin coding sequence in human cells were developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co‐transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower‐copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single‐copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid‐mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.