Rachelle H. Crosbie

Sarcospan, the 25-kDa Transmembrane Component of the Dystrophin-Glycoprotein Complex

The dystrophin-glycoprotein complex is a multisubunit protein complex that spans the sarcolemma and forms a link between the subsarcolemmal cytoskeleton and the extracellular matrix. Primary mutations in the genes encoding the proteins of this complex are associated with several forms of muscular dystrophy. Here we report the cloning and characterization of sarcospan, a unique 25-kDa member of this complex. Topology algorithms predict that sarcospan contains four transmembrane spanning helices with both N- and C-terminal domains located intracellularly. Phylogenetic analysis reveals that sarcospan’s arrangement in the membrane as well as its primary sequence are similar to that of the tetraspan superfamily of proteins. Sarcospan co-localizes and co-purifies with the dystrophin-glycoprotein complex, demonstrating that it is an integral component of the complex. We also show that sarcospan expression is dramatically reduced in muscle from patients with Duchenne muscular dystrophy. This suggests that localization of sarcospan to the membrane is dependent on proper dystrophin expression. The gene encoding sarcospan maps to human chromosome 12p11.2, which falls within the genetic locus for congenital fibrosis of the extraocular muscle, an autosomal dominant muscular dystrophy.

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.

Biosynthesis of dystroglycan: processing of a precursor propeptide

Dystroglycan is a cytoskeleton‐linked extracellular matrix receptor expressed in many cell types. Dystroglycan is composed of α‐ and β‐subunits which are encoded by a single mRNA. Using a heterologous mammalian expression system, we provide the first biochemical evidence of the α/β‐dystroglycan precursor propeptide prior to enzymatic cleavage. This 160 kDa dystroglycan propeptide is processed into α‐ and β‐dystroglycan (120 kDa and 43 kDa, respectively). We also demonstrate that the precursor propeptide is glycosylated and that blockade of asparagine‐linked (N‐linked) glycosylation did not prevent the cleavage of the dystroglycan precursor peptide. However, inhibition of N‐linked glycosylation results in aberrant trafficking of the α‐ and β‐dystroglycan subunits to the plasma membrane. Thus, dystroglycan is synthesized as a precursor propeptide that is post‐translationally cleaved and differentially glycosylated to yield α‐ and β‐dystroglycan.

Structural connectivity in actin: effect of C-terminal modifications on the properties of actin

In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.

Loss of sarcolemma nNOS in sarcoglycan-deficient muscle

nNOS, anchored to the sarcolemma through its interactions with the dystrophin–glycoprotein complex, is dramatically reduced in dystrophindeficient mdx mice and Duchenne muscular dystrophy patients. Recent evidence suggests that loss of nNOS in dystrophin‐deficient muscle may contribute significantly to the progression of muscle pathology through a variety of mechanisms. To investigate whether nNOS plays a role in other forms of muscular dystrophy, we analyzed protein expression of nNOS in several sarcoglycan‐deficient animal models of muscular dystrophy as well as patients with primary mutations in the sarcoglycan genes. Primary mutations in α‐, β‐, δ‐, and γ‐sarcoglycan result in autosomal recessive limb girdle muscular dystrophy (AR‐LGMD). We report that loss of the sarcoglycan–sarcospan complex in muscle causes a dramatic reduction in the levels of nNOS expression at the membrane, even in the presence of normal dystrophin and syntrophin expression. Furthermore, we show that expression of three out of four sarcoglycans is not sufficient to maintain nNOS at the sarcolemma. Our data suggest that loss of nNOS may contribute to muscle pathology in AR‐LGMD with primary mutations in the sarcoglycans.—Crosbie, R. H., Barresi, R., Campbell, K. P. Loss of sarcolemma nNOS in sarcoglycandeficient muscle. FASEB J. 16, 1786–1791 (2002)

NO vascular control in Duchenne muscular dystrophy

A new investigation into Duchenne muscular dystrophy (DMD) pathogenesis suggests that at least part of the muscle degeneration observed in DMD patients may result from the reduced production of muscle membrane-associated neuronal nitric oxide synthase. This reduction may lead to impaired regulation of the vasoconstrictor response and eventual muscle damage.

Characterization of aquaporin-4 in muscle and muscular dystrophy

Aquaporins are a growing family of transmembrane proteins that transport water and, in some cases, glycerol and urea across cellular membranes. Aquaporin‐4 (AQP4) is enriched at the sarcolemma of skeletal muscle and may play a role in accommodating the rapid changes in cell volume and hydrostatic forces that occur during contraction in order to prevent damage to the sarcolemma. Recent evidence has shown that AQP4 is absent in dystrophindeficient mdx mice, suggesting that AQP4 associates with dystrophin and has a role in the dystrophic process. To examine the relationship between aquaporins and muscle disease, and between aquaporins and dystrophin, we have investigated aquaporin expression in various mouse models of muscular dystrophy and cardiomyopathy before and after the onset of pathology. We find that AQP4 is expressed in prenecrotic mdx muscle despite the absence of dystrophin and that AQP4 is lost after the onset of muscle degeneration. Analysis of various dystrophin transgenic mice reveals that AQP4 is lost even when the dystrophin‐glycoprotein complex is present, suggesting that loss of AQP4 is not directly resulting from loss of the DGC. AQP4 was also lost in muscular dystrophies caused by primary mutations in the sarcoglycan genes. Taken together, our data demonstrate that AQP4 loss in skeletal muscle correlates with muscular dystrophy and is a common feature of pathogenesis.—Crosbie, R. H., Dovico, S. A., Flanagan, J. D., Chamberlain, J. S., Ownby, C. L., Campbell, K. P. Characterization of aquaporin‐4 in muscle and muscular dystrophy. FASEB J. 16, 943–949 (2002)

Myogenic Akt signaling upregulates the utrophin–glycoprotein complex and promotes sarcolemma stability in muscular dystrophy

Duchenne muscular dystrophy is caused by dystrophin mutations that lead to structural instability of the sarcolemma membrane, myofiber degeneration/regeneration and progressive muscle wasting. Here we show that myogenic Akt signaling in mouse models of dystrophy promotes increased expression of utrophin, which replaces the function of dystrophin thereby preventing sarcolemma damage and muscle wasting. In contrast to previous suggestions that increased Akt in dystrophy was a secondary consequence of pathology, our findings demonstrate a pivotal role for this signaling pathway such that modulation of Akt can significantly affect disease outcome by amplification of existing, physiological compensatory mechanisms.

Myogenic Akt signaling attenuates muscular degeneration, promotes myofiber regeneration and improves muscle function in dystrophin-deficient mdx mice

Duchenne muscular dystrophy, the most common form of childhood muscular dystrophy, is caused by X-linked inherited mutations in the dystrophin gene. Dystrophin deficiencies result in the loss of the dystrophin-glycoprotein complex at the plasma membrane, which leads to structural instability and muscle degeneration. Previously, we induced muscle-specific overexpression of Akt, a regulator of cellular metabolism and survival, in mdx mice at pre-necrotic (<3.5 weeks) ages and demonstrated upregulation of the utrophin-glycoprotein complex and protection against contractile-induced stress. Here, we found that delaying exogenous Akt treatment of mdx mice after the onset of peak pathology (>6 weeks) similarly increased the abundance of compensatory adhesion complexes at the extrasynaptic sarcolemma. Akt introduction after onset of pathology reverses the mdx histopathological measures, including decreases in blood serum albumin infiltration. Akt also improves muscle function in mdx mice as demonstrated through in vivo grip strength tests and in vitro contraction measurements of the extensor digitorum longus muscle. To further explore the significance of Akt in myofiber regeneration, we injured wild-type muscle with cardiotoxin and found that Akt induced a faster regenerative response relative to controls at equivalent time points. We demonstrate that Akt signaling pathways counteract mdx pathogenesis by enhancing endogenous compensatory mechanisms. These findings provide a rationale for investigating the therapeutic activation of the Akt pathway to counteract muscle wasting.

Sarcospan reduces dystrophic pathology: stabilization of the utrophin–glycoprotein complex

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.