γ-Sarcoglycan Deficiency Leads to Muscle Membrane Defects and Apoptosis Independent of Dystrophin

γ-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine γ-sarcoglycan gene was disrupted using homologous recombination. Mice lacking γ-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of γ-sarcoglycan produced secondary reduction of β- and δ-sarcoglycan with partial retention of α- and ε-sarcoglycan, suggesting that β-, γ-, and δ-sarcoglycan function as a unit. Importantly, mice lacking γ-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, β-dystroglycan and laminin were left intact, implying that the dystrophin–dystroglycan–laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking γ-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking γ-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.     

Dystroglycan glycosylation and muscular dystrophy

Dystroglycan is an integral member of the skeletal muscle dystrophin glycoprotein complex, which links dystrophin to proteins in the extracellular matrix. Recently, a group of human muscular dystrophy disorders have been demonstrated to result from defective glycosylation of the α-dystroglycan subunit. Genetic studies of these diseases have identified six genes that encode proteins required for the synthesis of essential carbohydrate structures on dystroglycan. Here we highlight their known or postulated functions. This glycosylation pathway appears to be highly specific (dystroglycan is the only substrate identified thus far) and to be highly conserved during evolution.     

Dystrophin and Utrophin Complexed with Different Associated Proteins in Cardiac Purkinje Fibres

Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin network and the extracellular matrix via the dystrophin-associated protein complex. In mature skeletal muscle, utrophin is a dystrophin-related protein localized mainly at the neuromuscular junction, with the same properties as dystrophin in terms of linking the protein complex. Utrophin could potentially overcome the absence of dystrophin in dystrophic skeletal muscles. In cardiac muscle, dystrophin and utrophin were both found to be present with a distinct subcellular distribution in Purkinje fibres, i.e. utrophin was limited to the cytoplasm, while dystrophin was located in the cytoplasmic membrane.In this study, we used this particular characteristic of cardiac Purkinje fibres and demonstrated that associated proteins of dystrophin and utrophin are different in this structure. We conclude, contrary to skeletal muscle, dystrophin-associated proteins do not form a complex in Purkinje fibres. In addition, we have indirect evidence of the presence of two different 400kDa dystrophins in Purkinje fibres.     

Mutations in B3GALNT2 Cause Congenital Muscular Dystrophy and Hypoglycosylation of α-Dystroglycan

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.     

High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.     

In vitro developmental expression of dystroglycan and laminin-alpha2 in human skeletal muscle

The alpha-subunit of dystroglycan, a member of the dystrophin associated protein complex, binds to extracellular laminin-alpha2, while its beta-subunit interacts with cytoskeletal dystrophin. The exact biological role of dystroglycan, especially during human skeletal muscle development, has not been fully explored. Here, we analysed the distribution and expression characteristics of both dystroglycan subunits and laminin-alpha2 in primary human skeletal muscle cells. During development, expression levels of all three proteins increased with differentiation. The proteins were relocated from the sarcoplasm to the sarcolemma. The size of alpha-dystroglycan decreased from 150-220 kDa at the proliferation stage to 100-120 kDa at the late developmental stage. Both alpha- and beta-dystroglycan were involved in forming a complex with their respective partners laminin-alpha2 and dystrophin/utrophin. Our data show that, during development, cells may employ tightly regulated post-translational species-specific modification to produce different isoforms of alpha-dystroglycan to participate in appropriate functions.     

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.     

Differential Membrane Localization and Intermolecular Associations of α-Dystrobrevin Isoforms in Skeletal Muscle

α-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length α-dystrobrevin-1 (84 kD), and COOH-terminal truncated α-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that α-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. α-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, α-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro–translated proteins, α-dystrobrevin-2 binds dystrophin, whereas α-dystrobrevin-1 binds both dystrophin and utrophin. α-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, α-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of α-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the α-dystrobrevins.     

Components of the Basal Lamina and Dystrophin�CDystroglycan Complex in the Neurointermediate Lobe of Rat Pituitary Gland: Different Localizations of ��-Dystroglycan, Dystrobrevins, ��1-Syntrophin, and Aquaporin-4

The so-called neurointermediate lobe is composed of the intermediate and neural lobes of the pituitary. The present immunohistochemical study investigated components of the basal lamina (laminin, agrin, and perlecan), the dystrophin–dystroglycan complex (dystrophin, β-dystroglycan, α1-dystrobrevin, β-dystrobrevin, utrophin, and α1-syntrophin), and the aquaporins (aquaporin-4 and -9). Glia markers (GFAP, S100, and glutamine synthetase) and components of connective tissue (collagen type I and fibronectin) were also labeled. In the neurohypophysis, immunostaining of basal lamina delineated meningeal invaginations. In these invaginations, vessels were seen to penetrate the organ without submerging into its parenchyma. On the parenchymal side of the invaginations, β-dystroglycan was detected, whereas utrophin was detected in the walls of vessels. Immunostaining of α1-dystrobrevin and α1-syntrophin did not delineate the vessels. The cells of the intermediate lobe were fully immunoreactive to α1-dystrobrevin and α1-syntrophin, whereas components of the basal lamina delineated the contours of the cells. GFAP-immunoreactive processes surrounded them. Aquaporin-4 localized at the periphery of the neurohypophysis, mainly adjacent to the intermediate lobe but not along the vessels. It colocalized only partially with GFAP and not at all with α1-syntrophin. Aquaporin-9 was not detected. These results emphasize the possibility that the components of the dystrophin–dystroglycan complex localize differently and raise the question about the roles of dystrobrevins, α1-syntrophin, and aquaporin-4 in the functions of the intermediate and neural lobes, respectively. (J Histochem Cytochem 58:463–479, 2010)     

Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts

Background

Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal Findings

We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with β-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions

We propose therefore, that Src-dependent phosphorylation of β-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.     

Loss of ��-Dystroglycan Laminin Binding in Epithelium-derived Cancers Is Caused by Silencing of LARGE

The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, β-dystroglycan is expressed, but α-dystroglycan is not detected. Here we report that α-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of α-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with the underlying basement membrane to maintain tissue architecture and function. During cancer progression, primitive cancer cells escape from this control by modifying the binding affinities of their cell membrane receptors. Several receptors have been described as important for this process. Of these, the integrins are the best studied (1). The receptor dystroglycan has been reported to be required for the development and maintenance of epithelial tissues (2, 3). A direct requirement for dystroglycan in epithelia is further demonstrated by the profound effect that loss of dystroglycan expression has on cell polarity and laminin binding in cultured mammary epithelial cells (4, 5). However, dystroglycan is not only important in the establishment and maintenance of epithelial structure. Associations have also been made between the loss of α-dystroglycan immunoreactivity and cancer progression in tumors of epithelial origin, including breast, colon, cervix, and prostate cancers (4, 6–9). The dystroglycan loss of function could thus serve as an effective means by which cancerous cells modify their adhesion to the extracellular matrix (ECM).²Dystroglycan is a ubiquitously expressed cell membrane protein that plays a key function in cellular integrity, linking the intracellular cytoskeleton to the extracellular matrix. The dystroglycan gene encodes a preprotein that is cleaved into two peptides (10). The C-terminal component, known as β-dystroglycan, is embedded within the cell membrane, whereas the N-terminal component, α-dystroglycan, is present within the extracellular periphery but remains associated with β-dystroglycan through non-covalent bonds. β-Dystroglycan binds to actin (11), dystrophin (11), utrophin (11), and Grb2 (12) through its C-terminal intracellular domain. α-Dystroglycan, on the other hand, binds to ECM proteins that contain laminin globular domains including laminins (13, 14), agrin (15), and perlecan (16), as well as to the transmembrane protein neurexin (17). α-Dystroglycan is extensively decorated by three different types of glycan modifications: mucin type O-glycosylation, O-mannosylation, and N-glycosylation. The state of α-dystroglycan glycosylation has been shown to be critical for the ability of the protein to bind to laminin globular domain-containing proteins of the ECM (18).Previous studies of epithelium-derived cancers (4, 9) demonstrated that the loss of immunoreactivity of α-dystroglycan antibodies correlates with tumor grade and poor prognosis. This reduced detection of α-dystroglycan, however, is based on a loss of α-dystroglycan reactivity to antibodies (known as IIH6 and VIA4-1) that recognize the laminin-binding glyco-epitope of α-dystroglycan, i.e. the protein is only functional when it is glycosylated in such a way (henceforth, referred to as functional glycosylation). However, in most of the cancer samples that have been studied to date, β-dystroglycan is expressed at normal levels at the cell membrane. Thus, the aforementioned cancer-associated loss of α-dystroglycan expression may reflect a failure in the post-translational processing of dystroglycan rather than in the synthesis of α-dystroglycan itself.A similar defect in dystroglycan has been reported in a group of congenital muscular dystrophies (19). This spectrum of human developmental syndromes involves the brain, eye, and skeletal muscle and shows a dramatic gradient of phenotypic severity that ranges from the most devastating in Walker-Warburg syndrome to the least severe in limb-girdle muscular dystrophy. Six distinct known and putative glycosyltransferases have been shown to underlie these syndromes: protein O-mannosyltransferase 1 (POMT1), protein O-mannosyltransferase 2 (POMT2), protein O-mannose β-1,2-acetylglucosaminyltransferase 1 (POMGnT1), like acetylglucosaminyltransferase (LARGE), Fukutin, and Fukutin-related protein (FKRP) (20–25). Indeed, all muscular dystrophy patients with mutations in any of these genes fail to express the functionally glycosylated α-dystroglycan epitope that is recognized by the IIH6 and VIA4-1 antibodies.To investigate the molecular mechanism responsible for the loss of α-dystroglycan in epithelium-derived cancers and its role in metastatic progression, we examined the expression and glycosylation status of α-dystroglycan in a group of breast, cervical, and lung cancer cell lines. Here we report that although α-dystroglycan is expressed in the metastatic cell lines MDA-MB-231, HeLa, H1299, and H2030, it is not functionally glycosylated. In screening these cell lines for expression of the six known α-dystroglycan-modifying proteins, we observed that only one, LARGE, was extensively down-regulated. We also report that the ectopic restoration of LARGE expression in these cell lines led not only to the production of a functional dystroglycan but also to the reversion of certain characteristics associated with invasiveness, namely cell attachment to ECM proteins and cell migration.     

Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protection of skeletal muscle from exercise injury

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.     

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.     

Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury.     

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.     

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

Background

The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice.

Methods and Findings

Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm² versus 12.8±0.9 N/cm²; PBS versus TAT).

Conclusions

These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.     

Muscle Structure Influences Utrophin Expression in mdx Mice

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx^4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin^−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx^4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin^−/−, mdx^4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.     

Maturation and maintenance of the neuromuscular synapse: genetic evidence for roles of the dystrophin--glycoprotein complex

The dystrophin-glycoprotein complex (DGC) links the cytoskeleton of muscle fibers to their extracellular matrix. Using knockout mice, we show that a cytoplasmic DGC component, alpha-dystrobrevin (alpha-DB), is dispensable for formation of the neuromuscular junction (NMJ) but required for maturation of its postsynaptic apparatus. We also analyzed double and triple mutants lacking other cytoskeletal DGC components (utrophin and dystrophin) and myotubes lacking a alpha-DB or a transmembrane DGC component (dystroglycan). Our results suggest that alpha-DB acts via its linkage to the DGC to enhance the stability of postsynaptic specializations following their DGC-independent formation; dystroglycan may play additional roles in assembling synaptic basal lamina. Together, these results demonstrate involvement of distinct protein complexes in the formation and maintenance of the synapse and implicate the DGC in the latter process.     

A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.     

Disruption of DAG1 in differentiated skeletal muscle reveals a role for dystroglycan in muscle regeneration

Striated muscle-specific disruption of the dystroglycan (DAG1) gene results in loss of the dystrophin-glycoprotein complex in differentiated muscle and a remarkably mild muscular dystrophy with hypertrophy and without tissue fibrosis. We find that satellite cells, expressing dystroglycan, support continued efficient regeneration of skeletal muscle along with transient expression of dystroglycan in regenerating muscle fibers. We demonstrate a similar phenomenon of reexpression of functional dystroglycan in regenerating muscle fibers in a mild form of human muscular dystrophy caused by disruption of posttranslational dystroglycan processing. Thus, maintenance of regenerative capacity by satellite cells expressing dystroglycan is likely responsible for mild disease progression in mice and possibly humans. Therefore, inadequate repair of skeletal muscle by satellite cells represents an important mechanism affecting the pathogenesis of muscular dystrophy.