Disrupted mechanical stability of the dystrophin-glycoprotein complex causes severe muscular dystrophy in sarcospan transgenic mice

Abstract

The dystrophin-glycoprotein complex spans the muscle plasma membrane and provides a mechanical linkage between laminin in the extracellular matrix and actin in the intracellular cytoskeleton. Within the dystrophin-glycoprotein complex, the sarcoglycans and sarcospan constitute a subcomplex of transmembrane proteins that stabilize α-dystroglycan, a receptor for laminin and other components of the extracellular matrix. In order to elucidate the function of sarcospan, we generated transgenic mice that overexpress sarcospan in skeletal muscle. Sarcospan transgenic mice with moderate (tenfold) levels of sarcospan overexpression exhibit a severe phenotype that is similar to mouse models of laminin-deficient congenital muscular dystrophy (MD). Sarcospan transgenic mice display severe kyphosis and die prematurely between 6 and 10 weeks of age. Histological analysis reveals that sarcospan expression causes muscle pathology marked by increased muscle fiber degeneration and/or regeneration. Sarcospan transgenic muscle does not display sarcolemma damage, which is distinct from dystrophin- and sarcoglycan-deficient muscular dystrophies. We show that sarcospan clusters the sarcoglycans into insoluble protein aggregates and causes destabilization of α-dystroglycan. Evidence is provided to demonstrate abnormal extracellular matrix assembly, which represents a probable pathological mechanism for the severe and lethal dystrophic phenotype. Taken together, these data suggest that sarcospan plays an important mechanical role in stabilizing the dystrophin-glycoprotein complex.

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

“…S1C). Previous work from our lab has shown that ten-fold transgenic expression of human SSPN in wild-type mice leads to a severe muscle phenotype that includes muscle degeneration, kyphosis and death at 6-10 weeks of age (27). By contrast, wild-type mice overexpressing mSSPN are healthy, breed well, exhibit no signs of muscle pathology, and appear to have a normal lifespan (the oldest transgenic carriers in our colony are currently over 2.5 years of age).…”

“…SSPN-null mice were a generous gift from Dr. Kevin P. Campbell (University of Iowa Carver College of Medicine, Iowa City, IA) (37). Transgenic constructs were designed with the human skeletal actin promoter and VP1 intron upstream of the full-length mouse SSPN cDNA, as described previously (27,43). Transgenic mice were generated by microinjection of transgenic constructs into the pronucleus of fertilized single-cell embryos (C57BL/6J background, Transgenic Mouse Facility, University of California, Irvine), and four viable lines were recovered (Lines 28, 23, 32 and 15).…”

“…At levels of approximately 0.5 or 1.5 fold expression, hSSPN was not able to ameliorate mdx pathology, suggesting that a minimum three-fold level overexpression of SSPN is necessary for rescue (19). However, expressing higher levels of hSSPN in wild-type mice (ten-fold transgenic expression) caused a severe degenerative muscle phenotype that led to premature death (27). Biochemical evidence suggests that human-specific and mouse-specific SSPN proteins in hSSPN-Tg muscle form insoluble aggregates of SG-SSPN complexes.…”

The Dystrophin Complex: Structure, Function, and Implications for Therapy

“…An appropriate level of SSPN in the cell is also essential for the integrity of DGC. When overexpressed in mice by 10 fold, SSPN clusters the sarcoglycans into insoluble protein aggregates and causes the destabilization of α-dystroglycan (123). Microspan is an alternatively spliced product of the SSPN gene.…”

Caveolae – mechanosensitive membrane invaginations linked to actin filaments

“…Notably, near-complete loss of α-dystroglycan expression has been reported in a patient with limb girdle muscular dystrophy (LGMD) who carried a dominant-negative mutation in Cav3, again suggesting that there is a functional relationship (Herrmann et al, 2000). A transgenic mouse overexpressing sarcospan, a protein component of the DGC, induces elevated levels of many DGC proteins and also Cav3 (Peter et al, 2007). Dystrophin localizes to caveolae-enriched areas in smooth muscle cells (North et al, 1993) and can be detected in Cav3 immunoprecipitates (Doyle et al, 2000).…”