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Cell Membrane Integrity and Muscular Dystrophy
Summary: Kevin Campbell is interested in understanding the molecular pathogenesis of various forms of muscular dystrophy, and developing therapeutic strategies to treat muscular dystrophy.
Muscular dystrophies are a group of genetic diseases that primarily affect skeletal muscle and are characterized by progressive muscle weakness. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that lead to the complete absence of dystrophin in skeletal muscle. Research in my laboratory on the function of dystrophin led to the identification and purification of the skeletal muscle dystrophin-glycoprotein complex (DGC), which provides an essential structural link between the actin cytoskeleton and the extracellular matrix. Defects in genes encoding a number of components of this complex lead to distinct forms of muscular dystrophy. Current projects in my laboratory are aimed at determining the function of dystroglycan, understanding the molecular pathogenesis of muscular dystrophy, and developing therapeutic strategies to treat muscular dystrophy.
Dystrophin-Glycoprotein Complex
The DGC is a large oligomeric protein complex found within the sarcolemma of skeletal muscle. Biochemical and structural characterization of the DGC indicates that it consists of dystrophin, a large, rod-shaped cytoskeletal protein that binds F-actin; α-dystroglycan, which binds extracellular matrix proteins possessing laminin globular (LG) domains (i.e., laminin-2, agrin, and perlecan); β-dystroglycan, which binds the cysteine-rich region of dystrophin; the syntrophins and dystrobrevin, intracellular proteins that bind to the carboxyl terminus of dystrophin; and the sarcoglycan-sarcospan complex. Based on interactions of the DGC with the extracellular matrix and the cytoskeleton, and the consequences of loss of function in protein components of the DGC, we have proposed that at least one function of the DGC is to provide mechanical reinforcement of the sarcolemma and to maintain membrane integrity during cycles of contraction and relaxation. The absence of dystrophin would disrupt these interactions, rendering the sarcolemma susceptible to damage incurred during muscle contraction and thus leading to muscle cell necrosis and progressive muscle weakness in patients with DMD.
Dystroglycan and Muscular Dystrophy
A major emphasis of my laboratory is determining the cellular function of dystroglycan. α-Dystroglycan binds LG-domain–containing extracellular matrix proteins with high affinity, while β-dystroglycan anchors dystrophin to the sarcolemma. Thus, through its interactions with LG-domain proteins and dystrophin, dystroglycan acts as a transmembrane link between the extracellular matrix and the cytoskeleton.
Recent research in my lab has produced further insights into the role of dystroglycan in the pathogenesis of Fukuyama congenital muscular dystrophy, muscle-eye-brain disease, and Walker-Warburg syndrome, all of which are congenital muscular dystrophies with associated developmental brain defects. We have shown that dystroglycan is abnormally glycosylated in patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy. This abnormal glycosylation of dystroglycan disrupts its normal binding activity toward each of its major extracellular matrix ligands in muscle and brain, namely laminin, neurexin, and agrin. Our findings demonstrate that disruption of the post-translational processing of dystroglycan, which results in a loss of the functional link between the cytoskeleton and the extracellular matrix, leads to severe muscular dystrophy with associated central nervous system (CNS) malformations.
In support of this conclusion, we also have identified a convergent phenotype in the spontaneously arising myodystrophy (myd) mouse. We demonstrated that the causative mutation, which is in the myodystrophy gene, LARGE, results in abnormal dystroglycan glycoslyation in muscle and brain, and a loss of ligand-binding activity toward laminin, neurexin, and agrin. We found that these defects are concomitant with significant levels of abnormal neuronal migration in the cerebral cortex, cerebellum, and hippocampus.
Related secondary α-dystroglycanopathies, limb girdle muscular dystrophy 2I and congenital muscular dystrophy 1C, are caused by mutations in the gene encoding fukutin-related protein (FKRP). Like Fukuyama, muscle-eye-brain, Walker-Warburg, and myd muscular dystrophies, FKRP patients exhibit defects in α-dystroglycan post-translational glycosylation; however, no FKRP enzymatic activity has been discovered. Recent work from my laboratory offers new insight into the role of FKRP in muscle, as our findings demonstrate that FKRP associates with the DGC complex at the muscle cell surface. Furthermore, FKRP localization is disrupted in mouse models with dystroglycan deficiency. These findings implicate FKRP for a nontraditional, perhaps nonenzymatic, role in α-dystroglycan post-translational regulation and suggest that FKRP may act downstream of other known processing components to regulate the functional stability of α-dystroglycan at the membrane.
Tissue-Specific Inactivation of Dystroglycan
Targeted inactivation of the dystroglycan gene in the mouse demonstrated that dystroglycan is required for embryonic development. Therefore, a floxed dystroglycan mouse—in which dystroglycan could be selectively removed from specific tissues of interest—was created. Using this mouse model, we found that brain-selective deletion is sufficient to cause congenital muscular dystrophy–like brain malformations, including disarray of cerebral cortical layering, fusion of cerebral hemispheres, and discontinuities in the surface basal lamina of the pia (glia limitans). Furthermore, mutant mice have severely blunted hippocampal long-term potentiation, indicating that dystroglycan on the postsynaptic membrane may play a novel role in learning and memory. Our work places dystroglycan at center stage of the pathogenetic pathway for the CNS defects of congenital muscular dystrophy and allows us to conclude that the functional disruption of dystroglycan likely underlies the pathogenesis of muscular dystrophy and developmental brain abnormalities.
We have also studied the function of dystroglycan in differentiated skeletal muscle, using the muscle creatine kinase promoter to eliminate its expression only in muscle fibers. Although the resulting mice developed muscular dystrophy when aged, they displayed a remarkable ability to regenerate muscle compared to other DGC-associated dystrophic mouse models. Eliminating dystroglycan expression in muscle fibers and satellite cells resulted in a much more severe phenotype. This suggests that dystroglycan may play an important role in satellite cell survival or function. Establishing an understanding of the dystroglycan-processing pathway and its developmental regulation may shed light on how dystroglycan modulates satellite cell function, and how dystroglycan processing might be targeted therapeutically to increase functional dystroglycan expression, promote muscle regeneration, and ameliorate the dystrophic phenotype.
Post-translational Processing of Dystroglycan
We are also studying post-translational processing of dystroglycan by the novel glycosyltransferase LARGE. My laboratory found that the amino-terminal domain of α-dystroglycan is essential for recognition by LARGE. This enzyme-substrate recognition motif is critical for the post-translational modification of dystroglycan and is necessary for the functional maturation of the protein into a receptor for extracellular matrix components. We revealed the significance of this unique pathway in vivo, as gene transfer of LARGE into muscle-specific gene-knockout mice rescued domain-specific functions of dystroglycan in the whole animal. Thus, molecular recognition of dystroglycan by LARGE in the biosynthetic pathway is essential to the production of functional dystroglycan.
Therapeutic Studies
We have explored the potential of LARGE overexpression as a treatment for glycosyltransferase-deficient muscular dystrophies. We showed that the enzyme LARGE modifies the sugar moieties of α-dystroglycan and prevents muscular dystrophy in myd mice. Importantly, we demonstrated that high levels of LARGE restore the function of α-dystroglycan and modulate its glycosylation in myoblasts and fibroblasts from patients afflicted with Fukuyama congenital muscular dystrophy, muscle-eye-brain disease, and Walker-Warburg syndrome. We also discovered that LARGE-dependent glycosylation of α-dystroglycan is required for dystroglycan function, and that induction of LARGE restores the function of α-dystroglycan, regardless of the type of glycosyltransferase that is mutated in patients. This work indicates that LARGE has a broad therapeutic potential.
Membrane Repair in Skeletal Muscle
In addition to the DGC, muscle cells utilize other mechanisms to maintain sarcolemmal integrity. Plasma membrane repair is a basic cellular mechanism for restoring the integrity of the cell membrane following physical injuries. To repair a disrupted membrane, new membrane is added to the plasmalemma at or near the membrane disruption site. We have shown that this additional membrane comes from an intracellular source and is targeted to the disruption site in the form of vesicles. Our recent data have demonstrated that the protein dysferlin is enriched at these membrane disruption sites in skeletal muscle. (Dysferlin has been identified as the gene mutated in two forms of dystrophy—limb girdle muscular dystrophy type 2B [LGMD2B] and Miyoshi myopathy.) Membrane injury causes a transient loss or reduction of sarcolemma markers at the disruption site, and the enrichment of protein markers involved in resealing of the disruption. Enrichment of dysferlin at membrane disruption sites suggests that dysferlin is a component of vesicles that fuse at sites of membrane damage.
Ultrastructural studies have shown that vesicles accumulate under sites of membrane disruption in muscle from patients with dysferlin mutations (LGMD2B), as well as in dysferlin-deficient mice. This suggests that dysferlin deficiency impairs the ability of vesicles to repair membrane disruptions. Similar vesicle accumulations under the plasma membrane have been reported in the spermatocytes of Caenorhabditis elegans with mutated fer-1, a dysferlin homolog. These data further suggest that it is not the targeting, but the fusion, of the vesicles that is compromised in the absence of functional dysferlin, indicating that dysferlin plays a role in membrane fusion.
Dysferlin and Membrane Repair in Muscular Dystrophy and Cardiomyopathy
A direct assessment of membrane repair in dysferlin-deficient muscle was performed using a membrane repair assay. Although laser-induced disruption of the sarcolemma of wild-type skeletal muscle fibers was efficiently repaired in a calcium-dependent manner, the repair of similar membrane disruption in dysferlin-null muscle was significantly delayed.
A modified membrane repair assay was performed in dysferlin-null cardiac muscle cells. Similar to dysferlin-null skeletal muscle cells, the dysferlin-null cardiac muscle cells failed to repair laser-induced membrane damage. This result suggests that dysferlin is also a critical component of membrane repair in cardiac muscle cells. Dysferlin-deficient mice showed hallmarks of cardiomyopathy (e.g., elevated serum cardiac troponin T levels, increased necrosis and fibrosis) during aging. Exhaustive uphill exercise exacerbated the cardiomyopathy. Moreover, dysferlin deficiency resulted in an early-onset cardiomyopathy in dystrophin-mutant mice. These data demonstrate that dysferlin-mediated membrane is physiologically important for the heart muscle.
Recent findings have led to the proposal of a model describing dysferlin-mediated membrane repair in which membrane disruption causes an influx of extracellular calcium, transiently creating a zone near the injured membrane in which the calcium concentration is quite high. Dysferlin-carrying vesicles targeted to disruption sites then fuse with each other and the plasma membrane in the presence of these localized high levels of calcium ions. Dysferlin is suggested to play a primary role in the fusion step of this repair process, with its presence on the vesicles facilitating vesicle docking and fusion with the plasma membrane. It may do so by interacting with other dysferlin molecules, annexins, or some unknown protein-binding partner(s) integral to or associated with the plasma membrane. Fusion of the vesicles then creates a membrane patch across the disrupted membrane, thereby resealing it. According to this model, the loss of dysferlin in LGMD2B and Miyoshi myopathy patients leads to inefficient resealing of membrane disruptions, such that damaged muscle fibers may undergo necrosis.
We anticipate that our ongoing investigations will lead to a better understanding of how dysferlin-dependent membrane repair protects muscle cells from damage induced by contraction, and this may shed light on how dysferlin might be targeted therapeutically to promote muscle repair and improve the dystrophic phenotype.
Last updated: September 19, 2007
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