Myotilin is mutated in limb girdle muscular dystrophy 1A.
(65/1324)
We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin. (+info)
Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3.
(66/1324)
Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C. (+info)
Expression of gamma -sarcoglycan in smooth muscle and its interaction with the smooth muscle sarcoglycan-sarcospan complex.
(67/1324)
The sarcoglycan complex in striated muscle is a heterotetrameric unit integrally associated with sarcospan in the dystrophin-glycoprotein complex. The sarcoglycans, alpha, beta, gamma, and delta, are mutually dependent with regard to their localization at the sarcolemma, and mutations in any of the sarcoglycan genes lead to limb-girdle muscular dystrophies type 2C-2F. In smooth muscle beta- and delta-sarcoglycans are associated with epsilon-sarcoglycan, a glycoprotein homologous to alpha-sarcoglycan. Here, we demonstrate that gamma-sarcoglycan is also a component of the sarcoglycan complex in the smooth muscle. First, we show the presence of gamma-sarcoglycan in a number of smooth muscle-containing organs, and we verify the existence of identical transcripts in skeletal and smooth muscle. The specificity of the expression of gamma-sarcoglycan in smooth muscle was confirmed by analysis of smooth muscle cells in culture. Next, we provide evidence for the association of gamma-sarcoglycan with the sarcoglycan-sarcospan complex by biochemical analysis and comparison among animal models for muscular dystrophy. Moreover, we find disruption of the sarcoglycan complex in the vascular smooth muscle of a patient with gamma-sarcoglycanopathy. Taken together, our results prove that the sarcoglycan complex in vascular and visceral smooth muscle consists of epsilon-, beta-, gamma-, and delta-sarcoglycans and is associated with sarcospan. (+info)
Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain.
(68/1324)
Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex. (+info)
Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA.
(69/1324)
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of the disease is caused by short (GCG)(8-13) expansions in the PABP2 gene. This gene encodes the poly(A) binding protein 2 (PABP2), an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we report that PABP2 is detected in filamentous nuclear inclusions, which are the pathological hallmark of OPMD. Using both immunoelectron microscopy and fluorescence confocal microscopy, the OPMD-specific nuclear inclusions appeared decorated by anti-PABP2 antibodies. In addition, the inclusions were labeled with antibodies directed against ubiquitin and the subunits of the proteasome and contained a form of PABP2 that was more resistant to salt extraction than the protein dispersed in the nucleoplasm. This suggests that the polyalanine expansions in PABP2 induce a misfolding and aggregation of the protein into insoluble inclusions, similarly to events in neurodegenerative diseases caused by CAG/polyglutamine expansions. No significant differences were observed in the steady-state poly(A) tail length in OPMD and normal myoblasts. However, the nuclear inclusions were shown to sequester poly(A) RNA. This raises the possibility that in OPMD the polyalanine expansions in the PABP2 protein may interfere with the cellular traffic of poly(A) RNA. (+info)
Dissociation of the dystroglycan complex in caveolin-3-deficient limb girdle muscular dystrophy.
(70/1324)
Limb girdle muscular dystrophy is a group of clinically and genetically heterogeneous disorders inherited in an autosomal recessive or dominant mode. Caveolin-3, the muscle-specific member of the caveolin gene family, is implicated in the pathogenesis of autosomal dominant limb girdle muscular dystrophy 1C. Here we report on a 4-year-old girl presenting with myalgia and muscle cramps due to a caveolin-3 deficiency in her dystrophic skeletal muscle as a result of a heterozygous 136G-->A substitution in the caveolin-3 gene. The novel sporadic missense mutation in the caveolin signature sequence of the caveolin-3 gene changes an alanine to a threonine (A46T) and prevents the localization of caveolin-3 to the plasma membrane in a dominant negative fashion. Caveolin-3 has been suggested to interact with the dystrophin-glycoprotein complex, which in striated muscle fibers links the cytoskeleton to the extracellular matrix and with neuronal nitric oxide synthase. Similar to dystrophin-deficient Duchenne muscular dystrophy, a secondary decrease in neuronal nitric oxide synthase and alpha-dystroglycan expression was detected in the caveolin-3-deficient patient. These results implicate an important function of the caveolin signature sequence and common mechanisms in the pathogenesis of dystrophin-glycoprotein complex-associated muscular dystrophies with caveolin-3-deficient limb girdle muscular dystrophy. (+info)
Animal models for muscular dystrophy: valuable tools for the development of therapies.
(71/1324)
Since the identification of dystrophin as the causative factor in Duchenne muscular dystrophy, an increasing amount of information on the molecular basis of muscular dystrophies has facilitated the division of these heterogeneous disorders into distinct groups. As more light is being shed on the genes and proteins involved in muscular dystrophy, diagnosis of patients has improved enormously. In addition to naturally occurring animal models, a number of genetically engineered murine models for muscular dystrophy have been generated. These animal models have provided valuable clues to the understanding of the pathogenesis of these disorders. Furthermore, as therapeutic approaches are being developed, mutant animals represent good models in which they can be tested. The present review focuses on the recent advancements of gene transfer-based strategies, with a special emphasis on animal models for Duchenne and limb-girdle muscular dystrophies. (+info)
Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography.
(72/1324)
In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1-2 kb, digested in fragments of 90-600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA(Leu(UUR)) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA(Leu(UUR)) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes. (+info)