Rachelle H. Crosbie-Watson

Sarcospan: a small protein with large potential for Duchenne muscular dystrophy.

Purification of the proteins associated with dystrophin, the gene product responsible for Duchenne muscular dystrophy, led to the discovery of the dystrophin-glycoprotein complex. Sarcospan, a 25-kDa transmembrane protein, was the last component to be identified and its function in skeletal muscle has been elusive. This review will focus on progress over the last decade revealing that sarcospan is an important regulator of muscle cell adhesion, strength, and regeneration. Investigations using several transgenic mouse models demonstrate that overexpression of sarcospan in the mouse model for Duchenne muscular dystrophy ameliorates pathology and restores muscle cell binding to laminin. Sarcospan improves cell surface expression of the dystrophin- and utrophin-glycoprotein complexes as well as α7β1 integrin, which are the three major laminin-binding complexes in muscle. Utrophin and α7β1 integrin compensate for the loss of dystrophin and the finding that sarcospan increases their abundance at the extra-synaptic sarcolemma supports the use of sarcospan as a therapeutic target. Newly discovered phenotypes in sarcospan-deficient mice, including a reduction in specific force output and increased drop in force in the diaphragm muscle, result from decreased utrophin and dystrophin expression and further reveal sarcospan’s role in determining abundance of these complexes. Dystrophin protein levels and the specific force output of the diaphragm muscle are further reduced upon genetic removal of α7 integrin (Itga7) in SSPN-deficient mice, demonstrating that interactions between integrin and sarcospan are critical for maintenance of the dystrophin-glycoprotein complex and force production of the diaphragm muscle. Sarcospan is a major regulator of Akt signaling pathways and sarcospan-deficiency significantly impairs muscle regeneration, a process that is dependent on Akt activation. Intriguingly, sarcospan regulates glycosylation of a specific subpopulation of α-dystroglycan, the laminin-binding receptor associated with dystrophin and utrophin, localized to the neuromuscular junction. Understanding the basic mechanisms responsible for assembly and trafficking of the dystrophin- and utrophin-glycoprotein complexes to the cell surface is lacking and recent studies suggest that sarcospan plays a role in these essential processes.

Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration.

Sarcospan signals through Akt to increase cell surface levels of utrophin and glycosylated α-dystroglycan and promote muscle repair after injury.

The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin– and utrophin–glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein‐replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein–protein interactions amongst the adhesion complexes and is an important potential therapeutic target. Here, we review current protein‐replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic.

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding

Background: Genetic alteration of muscle cell glycosylation in muscular dystrophy models has ameliorated disease. Results: A high throughput screen identified a small molecule, lobeline, which altered muscle cell glycosylation and improved laminin binding. Conclusion: Lobeline increased abundance of sarcolemmal glycoproteins and increased laminin binding in an N-glycan-dependent manner. Significance: A novel approach revealed an unexpected role for N-glycans in muscle cell function. 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.

Report on the Myomatrix Conference April 22-24, 2012, University of Nevada, Reno, Nevada, USA

The Myomatrix 2012 conference held April 22-24th, 2012 at the University of Nevada, Reno convened 73 international participants to discuss the dynamic relationship between muscle and its matrix in muscular dystrophy with a specific focus on congenital muscular dystrophy. Seven sessions over 2½ days defined three central themes: (1) the role of extracellular matrix proteins and compartments in development and specifically in congenital muscular dystrophy (CMD) (2) the role of extracellular matrix signaling and adhesion to membrane receptors and (3) the balance and interplay between inflammation and fibrosis as drivers of altered matrix stiffness, impaired regeneration and progressive dystrophy. This report highlights major conference findings and the translational roadmap as defined by conference attendees.

High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD

Abstract Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle weakness, ultimately leading to early mortality in affected teenagers and young adults. Previous work from our lab has shown that a small transmembrane protein called sarcospan (SSPN) can enhance the recruitment of adhesion complex proteins to the cell surface. When human SSPN is expressed at three-fold levels in mdx mice, this increase in adhesion complex abundance improves muscle membrane stability, preventing many of the histopathological changes associated with DMD. However, expressing higher levels of human SSPN (ten-fold transgenic expression) causes a severe degenerative muscle phenotype in wild-type mice. Since SSPN-mediated stabilization of the sarcolemma represents a promising therapeutic strategy in DMD, it is important to determine whether SSPN can be introduced at high levels without toxicity. Here, we show that mouse SSPN (mSSPN) can be overexpressed at 30-fold levels in wild-type mice with no deleterious effects. In mdx mice, mSSPN overexpression improves dystrophic pathology and sarcolemmal stability. We show that these mice exhibit increased resistance to eccentric contraction-induced damage and reduced fatigue following exercise. mSSPN overexpression improved pulmonary function and reduced dystrophic histopathology in the diaphragm. Together, these results demonstrate that SSPN overexpression is well tolerated in mdx mice and improves sarcolemma defects that underlie skeletal muscle and pulmonary dysfunction in DMD.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F

Background Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Methods Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Results Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd−/−) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. Conclusions Alternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865–71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes. Electronic supplementary material The online version of this article (doi:10.1186/s13395-017-0127-9) contains supplementary material, which is available to authorized users.BackgroundSarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models.MethodsStandard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections.ResultsNanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd−/−) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies.ConclusionsAlternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865–71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.

Benfotiamine reduces pathology and improves muscle function in mdx mice.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease which arises from mutations in the dystrophin gene (DMD) that result in the absence or severe reduction of the cytoskeletal protein dystrophin. In addition to the primary dystrophin defect, secondary processes such as inflammation, calcium influx, dysregulated autophagy and fibrosis exacerbate dystrophic pathology and thus increase disease progression. While therapies to restore dystrophin deficiency are being developed, strategies which target these secondary processes could be of benefit to patients. Benfotiamine is a lipid soluble precursor to thiamine that can reduce secondary processes such as inflammation and oxidative stress in diabetic patients. As such we tested it in the mdx mouse model of DMD and found that benfotiamine reduced multiple markers of dystrophic pathology and improved grip strength. In addition, members of the utrophin and dystrophin glycoprotein complexes were significantly increased at the sarcolemma which could improve cell adhesion. We also demonstrated that benfotiamine treatment lowered the expression of macrophage markers and pro-inflammatory cytokines suggesting that benfotiamine is reducing dystrophic pathology by acting on inflammatory processes.

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animals free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.