The Experts below are selected from a list of 480 Experts worldwide ranked by ideXlab platform
Kevin P. Campbell - One of the best experts on this subject based on the ideXlab platform.
-
Exome sequencing reveals independent SGCD deletions causing limb girdle muscular dystrophy in Boston terriers.
Skeletal muscle, 2017Co-Authors: Melissa L. Cox, Jacquelyn M. Evans, Alexander G. Davis, Ling T. Guo, Jennifer R. Levy, Alison N. Starr-moss, Elina Salmela, Marjo K. Hytönen, Hannes Lohi, Kevin P. CampbellAbstract:Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal myopathies that preferentially affect voluntary muscles of the shoulders and hips. LGMD has been clinically described in several breeds of dogs, but the responsible mutations are unknown. The clinical presentation in dogs is characterized by marked muscle weakness and atrophy in the shoulder and hips during puppyhood. Following clinical evaluation, the identification of the dystrophic histological phenotype on muscle histology, and demonstration of the absence of sarcoglycan-Sarcospan complex by immunostaining, whole exome sequencing was performed on five Boston terriers: one affected dog and its three family members and one unrelated affected dog. Within sarcoglycan-δ (SGCD), a two base pair deletion segregating with LGMD in the family was discovered, and a deletion encompassing exons 7 and 8 was found in the unrelated dog. Both mutations are predicted to cause an absence of SGCD protein, confirmed by immunohistochemistry. The mutations are private to each family. Here, we describe the first cases of canine LGMD characterized at the molecular level with the classification of LGMD2F.
-
Exome sequencing reveals independent SGCD deletions causing limb girdle muscular dystrophy in Boston terriers
Skeletal Muscle, 2017Co-Authors: Melissa L. Cox, Jacquelyn M. Evans, Alexander G. Davis, Ling T. Guo, Jennifer R. Levy, Alison N. Starr-moss, Elina Salmela, Marjo K. Hytönen, Hannes Lohi, Kevin P. CampbellAbstract:Background Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal myopathies that preferentially affect voluntary muscles of the shoulders and hips. LGMD has been clinically described in several breeds of dogs, but the responsible mutations are unknown. The clinical presentation in dogs is characterized by marked muscle weakness and atrophy in the shoulder and hips during puppyhood. Methods Following clinical evaluation, the identification of the dystrophic histological phenotype on muscle histology, and demonstration of the absence of sarcoglycan-Sarcospan complex by immunostaining, whole exome sequencing was performed on five Boston terriers: one affected dog and its three family members and one unrelated affected dog. Results Within sarcoglycan-δ ( SGCD ), a two base pair deletion segregating with LGMD in the family was discovered, and a deletion encompassing exons 7 and 8 was found in the unrelated dog. Both mutations are predicted to cause an absence of SGCD protein, confirmed by immunohistochemistry. The mutations are private to each family. Conclusions Here, we describe the first cases of canine LGMD characterized at the molecular level with the classification of LGMD2F.
-
Membrane Targeting and Stabilization of Sarcospan Is Mediated by the
2013Co-Authors: Sarcoglycan Subcomplex, Jane C. Lee, Mark R. Grady, Jeffery S. Chamberlain, Joshua R. Sanes, Kevin P. CampbellAbstract:Abstract. The dystrophin–glycoprotein complex (DGC) is a multisubunit complex that spans the muscle plasma membrane and forms a link between the F-actin cytoskeleton and the extracellular matrix. The proteins of the DGC are structurally organized into distinct subcomplexes, and genetic mutations in many individual components are manifested as muscular dystrophy. We recently identified a unique tetraspan-like dystrophinassociated protein, which we have named Sarcospan (SPN) for its multiple sarcolemma spanning domain
-
Sarcoglycan complex: implications for metabolic defects in muscular dystrophies.
The Journal of biological chemistry, 2009Co-Authors: Séverine Groh, Haihong Zong, Matthew M. Goddeeris, Connie S. Lebakken, David Venzke, Jeffrey E. Pessin, Kevin P. CampbellAbstract:The sarcoglycans are known as an integral subcomplex of the dystrophin glycoprotein complex, the function of which is best characterized in skeletal muscle in relation to muscular dystrophies. Here we demonstrate that the white adipocytes, which share a common precursor with the myocytes, express a cell-specific sarcoglycan complex containing beta-, delta-, and epsilon-sarcoglycan. In addition, the adipose sarcoglycan complex associates with Sarcospan and laminin binding dystroglycan. Using multiple sarcoglycan null mouse models, we show that loss of alpha-sarcoglycan has no consequence on the expression of the adipocyte sarcoglycan complex. However, loss of beta- or delta-sarcoglycan leads to a concomitant loss of the sarcoglycan complex as well as Sarcospan and a dramatic reduction in dystroglycan in adipocytes. We further demonstrate that beta-sarcoglycan null mice, which lack the sarcoglycan complex in adipose tissue and skeletal muscle, are glucose-intolerant and exhibit whole body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles. Thus, our data demonstrate a novel function of the sarcoglycan complex in whole body glucose homeostasis and skeletal muscle metabolism, suggesting that the impairment of the skeletal muscle metabolism influences the pathogenesis of muscular dystrophy.
-
A common disease-associated missense mutation in alpha-sarcoglycan fails to cause muscular dystrophy in mice
Human molecular genetics, 2008Co-Authors: Kazuhiro Kobuke, Steven A. Moore, F. Piccolo, Keith W. Garringer, Eileen M. Sweezer, Baoli Yang, Kevin P. CampbellAbstract:Limb-girdle muscular dystrophy type 2D (LGMD2D) is caused by autosomal recessive mutations in the alpha-sarcoglycan gene. An R77C substitution is the most prevalent cause of the disease, leading to disruption of the sarcoglycan-Sarcospan complex. To model this common mutation, we generated knock-in mice with an H77C substitution in alpha-sarcoglycan. The floxed neomycin (Neo)-cassette retained at the targeted H77C alpha-sarcoglycan locus caused a loss of alpha-sarcoglycan expression, resulting in muscular dystrophy in homozygotes, whereas Cre-mediated deletion of the floxed Neo-cassette led to recovered H77C alpha-sarcoglycan expression. Contrary to expectations, mice homozygous for the H77C-encoding allele expressed both this mutant alpha-sarcoglycan and the other components of the sarcoglycan-Sarcospan complex in striated muscle, and did not develop muscular dystrophy. Accordingly, conditional rescued expression of the H77C protein in striated muscle of the alpha-sarcoglycan-deficient mice prevented the disease. Adding to the case that the behavior of mutant alpha-sarcoglycan is different between humans and mice, mutant human R77C alpha-sarcoglycan restored the expression of the sarcoglycan-Sarcospan complex when introduced by adenoviral vector into the skeletal muscle of previously created alpha-sarcoglycan null mice. These findings indicate that the alpha-sarcoglycan with the most frequent missense mutation in LGMD2D is correctly processed, is transported to the sarcolemma, and is fully functional in mouse muscle. Our study presents an unexpected difference in the behavior of a missense-mutated protein in mice versus human patients, and emphasizes the need to understand species-specific protein quality control systems.
Rachelle H. Crosbie - One of the best experts on this subject based on the ideXlab platform.
-
Loss of Sarcospan exacerbates pathology in mdx mice, but does not affect utrophin amelioration of disease.
Human molecular genetics, 2021Co-Authors: Elizabeth M. Gibbs, Jamie L. Marshall, Katherine G Hammond, Jackie L Mccourt, Kara M Shin, Rachelle H. CrosbieAbstract:The dystrophin-glycoprotein complex (DGC) is a membrane adhesion complex that provides structural stability at the sarcolemma by linking the myocyte's internal cytoskeleton and external extracellular matrix. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to the loss of the DGC at the sarcolemma, resulting in sarcolemmal instability and progressive muscle damage. Utrophin, an autosomal homologue of dystrophin, is upregulated in dystrophic muscle and partially compensates for loss of dystrophin in muscle from patients with DMD. Here, we examine the interaction between utrophin and Sarcospan (SSPN), a small transmembrane protein that is a core component of both the utrophin- and dystrophin-glycoprotein complexes (UGC and DGC). We show that additional loss of SSPN causes an earlier onset of disease in dystrophin-deficient mdx mice by reducing expression of the UGC at the sarcolemma. In order to further evaluate the role of SSPN in maintaining therapeutic levels of utrophin at the sarcolemma, we tested the effect of utrophin transgenic overexpression in mdx mice lacking SSPN (mdx:SSPN-/-:Utr-Tg). We found that overexpression of utrophin restored SSPN to the sarcolemma in mdx muscle, but that ablation of SSPN in mdx muscle reduced utrophin at the membrane. Nevertheless, utrophin overexpression reduced central nucleation and improved grip strength in both lines. These findings demonstrate that high levels of utrophin transgenic overexpression ameliorate the mdx phenotype independently of SSPN expression, but that loss of SSPN may impair utrophin-based mechanisms that rely on lower levels of utrophin protein.
-
Development of a high-throughput screen to identify small molecule enhancers of Sarcospan for the treatment of Duchenne muscular dystrophy
Skeletal muscle, 2019Co-Authors: Cynthia Shu, Judd R Collado, Ariana N. Kaxon-rupp, Robert Damoiseaux, Rachelle H. CrosbieAbstract:Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein Sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.
-
JCB: REPORT Sarcospan reduces dystrophic pathology: stabilization of the utrophin – glycoprotein complex
2013Co-Authors: Angela K Peter, Jamie L. Marshall, Rachelle H. CrosbieAbstract:Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin – glycoprotein complex (DGC) from the sarcolemma. We show that Sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophindeficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin – glycoprotein complex (UGC) at the extrasynaptic membrane to compensate fo
-
Altered calcium pump and secondary deficiency of γ-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from δ-sarcoglycan knockout mice
Cell calcium, 2010Co-Authors: Alhondra Solares-pérez, Rachelle H. Crosbie, Francisco J. Estrada, Rocío Álvarez, Jesús Vega-moreno, Joel Medina-monares, Alicia Ortega, Ramón Mauricio Coral-vázquezAbstract:Abstract Sarcoglycans (SGs) and Sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and δ-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of δ-SG isoforms in TT and SR results in a secondary deficiency of γ-SG and μSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the δ-SG isoforms may stabilize the expression of γ-SG and μSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle.
-
Sarcospan reduces dystrophic pathology: stabilization of the utrophin–glycoprotein complex
The Journal of cell biology, 2008Co-Authors: Angela K Peter, Jamie L. Marshall, Rachelle H. CrosbieAbstract:Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin–glycoprotein complex (DGC) from the sarcolemma. We show that Sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophin-deficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin–glycoprotein complex (UGC) at the extrasynaptic membrane to compensate for the loss of dystrophin. Utrophin is normally restricted to the neuromuscular junction, where it replaces dystrophin to form a functionally analogous complex. SSPN directly interacts with the UGC and functions to stabilize utrophin protein without increasing utrophin transcription. These findings reveal the importance of protein stability in the prevention of muscular dystrophy and may impact the future design of therapeutics for muscular dystrophies.
Rachelle H Crosbie-watson - One of the best experts on this subject based on the ideXlab platform.
-
Nanospan, an alternatively spliced isoform of Sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.
Skeletal muscle, 2017Co-Authors: Angela K Peter, Gaynor Miller, Ramón Mauricio Coral-vázquez, Emily L. Wang, Jim Heighway, Joana Capote, Marino Difranco, Alhondra Solares-pérez, Julio L. Vergara, Rachelle H Crosbie-watsonAbstract: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. 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. 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. 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.
-
Nanospan, an alternatively spliced isoform of Sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F
Skeletal Muscle, 2017Co-Authors: Angela K Peter, Gaynor Miller, Ramon Coral-vazquez, Emily L. Wang, Jim Heighway, Joana Capote, Marino Difranco, Alhondra Solares-pérez, Julio Vergara, Rachelle H Crosbie-watsonAbstract: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.
-
High levels of Sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.
Human molecular genetics, 2016Co-Authors: Elizabeth M. Gibbs, Jamie L. Marshall, Thien M. Nguyen, Grace Hong, Jessica S. Lam, Melissa J. Spencer, Rachelle H Crosbie-watsonAbstract: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.
-
Abstract 79: Sarcospan Has a Protective Effect During Development of Cardiac Disease
Circulation Research, 2016Co-Authors: Michelle S. Parvatiyar, Reginald T. Nguyen, Maria C. Jordan, Kenneth P. Roos, Rachelle H Crosbie-watsonAbstract:Sarcospan (SSPN) has an important role in stabilizing sarcolemmal dystrophin- and utrophin-glycoprotein adhesion complexes at the cell membrane. Loss of cell adhesion leads to contraction-induced m...
-
Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin
Human molecular genetics, 2014Co-Authors: Jamie L. Marshall, Eric Chou, Joy A. Lee, Johan Holmberg, Dean J. Burkin, Rachelle H Crosbie-watsonAbstract:Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of Sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying Sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that Sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of Sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin.
Jamie L. Marshall - One of the best experts on this subject based on the ideXlab platform.
-
Loss of Sarcospan exacerbates pathology in mdx mice, but does not affect utrophin amelioration of disease.
Human molecular genetics, 2021Co-Authors: Elizabeth M. Gibbs, Jamie L. Marshall, Katherine G Hammond, Jackie L Mccourt, Kara M Shin, Rachelle H. CrosbieAbstract:The dystrophin-glycoprotein complex (DGC) is a membrane adhesion complex that provides structural stability at the sarcolemma by linking the myocyte's internal cytoskeleton and external extracellular matrix. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to the loss of the DGC at the sarcolemma, resulting in sarcolemmal instability and progressive muscle damage. Utrophin, an autosomal homologue of dystrophin, is upregulated in dystrophic muscle and partially compensates for loss of dystrophin in muscle from patients with DMD. Here, we examine the interaction between utrophin and Sarcospan (SSPN), a small transmembrane protein that is a core component of both the utrophin- and dystrophin-glycoprotein complexes (UGC and DGC). We show that additional loss of SSPN causes an earlier onset of disease in dystrophin-deficient mdx mice by reducing expression of the UGC at the sarcolemma. In order to further evaluate the role of SSPN in maintaining therapeutic levels of utrophin at the sarcolemma, we tested the effect of utrophin transgenic overexpression in mdx mice lacking SSPN (mdx:SSPN-/-:Utr-Tg). We found that overexpression of utrophin restored SSPN to the sarcolemma in mdx muscle, but that ablation of SSPN in mdx muscle reduced utrophin at the membrane. Nevertheless, utrophin overexpression reduced central nucleation and improved grip strength in both lines. These findings demonstrate that high levels of utrophin transgenic overexpression ameliorate the mdx phenotype independently of SSPN expression, but that loss of SSPN may impair utrophin-based mechanisms that rely on lower levels of utrophin protein.
-
stabilization of the cardiac sarcolemma by Sarcospan rescues dmd associated cardiomyopathy
JCI insight, 2019Co-Authors: Michelle S. Parvatiyar, Jamie L. Marshall, Alexandra J Brownstein, Rosemeire M Kanashirotakeuchi, Judd R Collado, Karissa Dieseldorff M Jones, Jay Gopal, Katherine G Hammond, Abel Ferrel, Aaron M BeedleAbstract:In the current preclinical study, we demonstrate the therapeutic potential of Sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN-Tg mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography revealed that SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN-Tg mdx mice by hemodynamic pressure-volume methods highlighted enhanced systolic performance compared with mdx controls. SSPN restored cardiac sarcolemma stability, the primary defect in DMD disease; reduced fibrotic response; and improved contractile function. We demonstrate that SSPN ameliorated more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and β1D integrin, which mitigate disease severity, and partially restored responsiveness to β-adrenergic stimulation. Overall, our current and previous findings suggest that SSPN overexpression in DMD mouse models positively affects skeletal, pulmonary, and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.
-
Stabilization of the cardiac sarcolemma by Sarcospan rescues DMD-associated cardiomyopathy.
JCI insight, 2019Co-Authors: Michelle S. Parvatiyar, Jamie L. Marshall, Alexandra J Brownstein, Judd R Collado, Jay Gopal, Katherine G Hammond, Abel Ferrel, Rosemeire M Kanashiro-takeuchi, Karissa M Dieseldorff Jones, Aaron M BeedleAbstract:In the current preclinical study, we demonstrate the therapeutic potential of Sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and β1D integrin that mitigate disease severity and partially restores responsiveness to β-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.
-
High levels of Sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.
Human molecular genetics, 2016Co-Authors: Elizabeth M. Gibbs, Jamie L. Marshall, Thien M. Nguyen, Grace Hong, Jessica S. Lam, Melissa J. Spencer, Rachelle H Crosbie-watsonAbstract: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.
-
Sarcospan regulates cardiac isoproterenol response and prevents duchenne muscular dystrophy associated cardiomyopathy
Journal of the American Heart Association, 2015Co-Authors: Michelle S. Parvatiyar, Jamie L. Marshall, Reginald T. Nguyen, Maria C. Jordan, Kenneth P. Roos, Vanitra Richardson, Rachelle H CrosbiewatsonAbstract:BackgroundDuchenne muscular dystrophy is a fatal cardiac and skeletal muscle disease resulting from mutations in the dystrophin gene. We have previously demonstrated that a dystrophin‐associated pr...
Angela K Peter - One of the best experts on this subject based on the ideXlab platform.
-
Nanospan, an alternatively spliced isoform of Sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.
Skeletal muscle, 2017Co-Authors: Angela K Peter, Gaynor Miller, Ramón Mauricio Coral-vázquez, Emily L. Wang, Jim Heighway, Joana Capote, Marino Difranco, Alhondra Solares-pérez, Julio L. Vergara, Rachelle H Crosbie-watsonAbstract: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. 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. 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. 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.
-
Nanospan, an alternatively spliced isoform of Sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F
Skeletal Muscle, 2017Co-Authors: Angela K Peter, Gaynor Miller, Ramon Coral-vazquez, Emily L. Wang, Jim Heighway, Joana Capote, Marino Difranco, Alhondra Solares-pérez, Julio Vergara, Rachelle H Crosbie-watsonAbstract: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.
-
JCB: REPORT Sarcospan reduces dystrophic pathology: stabilization of the utrophin – glycoprotein complex
2013Co-Authors: Angela K Peter, Jamie L. Marshall, Rachelle H. CrosbieAbstract:Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin – glycoprotein complex (DGC) from the sarcolemma. We show that Sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophindeficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin – glycoprotein complex (UGC) at the extrasynaptic membrane to compensate fo
-
Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration
The Journal of cell biology, 2012Co-Authors: Jamie L. Marshall, Angela K Peter, Eric Chou, Joy A. Lee, Johan Holmberg, Amber C. Ocampo, Paul T. Martin, Rachelle H Crosbie-watsonAbstract:Utrophin is normally confined to the neuromuscular junction (NMJ) in adult muscle and partially compensates for the loss of dystrophin in mdx mice. We show that Akt signaling and utrophin levels were diminished in Sarcospan (SSPN)-deficient muscle. By creating several transgenic and knockout mice, we demonstrate that SSPN regulates Akt signaling to control utrophin expression. SSPN determined α-dystroglycan (α-DG) glycosylation by affecting levels of the NMJ-specific glycosyltransferase Galgt2. After cardiotoxin (CTX) injury, regenerating myofibers express utrophin and Galgt2-modified α-DG around the sarcolemma. SSPN-null mice displayed delayed differentiation after CTX injury caused by loss of utrophin and Akt signaling. Treatment of SSPN-null mice with viral Akt increased utrophin and restored muscle repair after injury, revealing an important role for the SSPN-Akt-utrophin signaling axis in regeneration. SSPN improved cell surface expression of utrophin by increasing transportation of utrophin and DG from endoplasmic reticulum/Golgi membranes. Our experiments reveal functions of utrophin in regeneration and new pathways that regulate utrophin expression at the cell surface.
-
Sarcospan reduces dystrophic pathology: stabilization of the utrophin–glycoprotein complex
The Journal of cell biology, 2008Co-Authors: Angela K Peter, Jamie L. Marshall, Rachelle H. CrosbieAbstract:Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin–glycoprotein complex (DGC) from the sarcolemma. We show that Sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophin-deficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin–glycoprotein complex (UGC) at the extrasynaptic membrane to compensate for the loss of dystrophin. Utrophin is normally restricted to the neuromuscular junction, where it replaces dystrophin to form a functionally analogous complex. SSPN directly interacts with the UGC and functions to stabilize utrophin protein without increasing utrophin transcription. These findings reveal the importance of protein stability in the prevention of muscular dystrophy and may impact the future design of therapeutics for muscular dystrophies.
