Characterization of the intermolecular associations of the dystrophin-associated glycoprotein complex in retinal Muller glial cells.
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The abnormal retinal neurotransmission observed in Duchenne muscular dystrophy patients has been attributed to altered expression of C-terminal products of the dystrophin gene in this tissue. Muller glial cells from rat retina express dystrophin protein Dp71, utrophin and the members of the dystrophin-associated glycoprotein complex (DGC), namely beta-dystroglycan, delta- and gamma-sarcoglycans and alpha1-syntrophin. The DGC could function in muscle as a link between the cystoskeleton and the extracellular matrix, as well as a signaling complex. However, other than in muscle the composition and intermolecular associations among members of the DGC are still unknown. Here we demonstrate that Dp71 and/or utrophin from rat retinal Muller glial cells form a complex with beta-dystroglycan, delta-sarcoglycan and alpha1-syntrophin. We also show that beta-dystroglycan is associated with alpha-dystrobrevin-1 and PSD-93 and that anti-PSD antibodies coimmunoprecipitated alpha-syntrophin with PSD-93. By overlay experiments we also found that Dp71and/or utrophin and alpha-dystroglycan from Muller cells could bind to actin and laminin, respectively. These results indicate that the DGC could have both structural and signaling functions in retina. On the basis of our accumulated evidence, we propose a hypothetical model for the molecular organization of the dystrophin-associated glycoprotein complex in retinal Muller glial cells, which would be helpful for understanding its function in the central nervous system. (+info)
Dystrophin associates with caveolae of rat cardiac myocytes: relationship to dystroglycan.
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The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan. Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan. Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies. In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan. Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies. To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes. Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped. Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae. These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex. (+info)
Dissociation of sarcoglycans and the dystrophin carboxyl terminus from the sarcolemma in enteroviral cardiomyopathy.
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Enteroviral infection can cause an acquired form of dilated cardiomyopathy. We recently reported that dystrophin is cleaved, functionally impaired, and morphologically disrupted in vitro as well as in vivo during infection with coxsackievirus B3. Genetic dystrophin truncations lead to a marked decrease in dystrophin-associated glycoproteins, whereas expression of only the naturally occurring dystrophin carboxyl terminus, Dp-71, restores the sarcolemmal association of the dystrophin-associated glycoproteins. We sought to determine whether acute cleavage of dystrophin leads to a dissociation of the carboxyl-terminal dystrophin fragment and of the sarcoglycans from the sarcolemma during coxsackievirus B3 infection. We found that in cultured cardiac myocytes and murine hearts infected with coxsackievirus B3, the sarcolemmal localization of the dystrophin carboxyl terminus is lost. The dystrophin-associated glycoproteins alpha-, beta-, gamma-, and delta-sarcoglycan and beta-dystroglycan were markedly decreased in the membrane fraction of infected cells in culture, and the typical sarcolemmal localization for each of these proteins was lost in coxsackievirus-B3-infected cardiomyocytes in vivo. Furthermore, sucrose gradient ultracentrifugation demonstrated that delta-sarcoglycan was physically dissociated from dystrophin within the membrane fraction. In vivo, the sarcolemmal integrity was functionally impaired with Evans blue dye uptake even though there was no generalized disruption of the sarcolemma of infected myocytes evidenced by intact wheat germ agglutinin staining. In analogy to hereditary sarcoglycanopathies, this disintegration of the sarcoglycan complex may, in addition to the dystrophin cleavage, play an important role in the pathogenesis of enterovirus-induced cardiomyopathy. These results imply a potential role for disruption of the sarcoglycans in an acquired form of heart failure. (+info)
Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin.
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The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated alpha-syntrophin null mice by targeted gene disruption to test the function of this association. The alpha-Syn(-/)- mice show no evidence of myopathy, despite reduced levels of alpha-dystrobrevin-2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the alpha-Syn(-/)- mice, even where other syntrophin isoforms are present. alpha-Syn(-/)- neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, alpha-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse. (+info)
Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain.
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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)
Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans.
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BACKGROUND: Dystrophin is the product of the gene that is mutated in Duchenne muscular dystrophy (DMD), a progressive neuromuscular disease for which no treatment is available. Mice carrying a mutation in the gene for dystrophin (mdx mice) display only a mild phenotype, but it is aggravated when combined with a mutation in the MyoD gene. The nematode worm Caenorhabditis elegans has a dystrophin homologue (dys-1), but null mutations in dys-1 do not result in muscle degeneration. RESULTS: We generated worms carrying both the dys-1 null mutation cx18, and a weak mutation, cc561ts, of the C. elegans MyoD homologue hlh-1. The double mutants displayed a time-dependent impairment of locomotion and egg laying, a phenotype not seen in the single mutants, and extensive muscle degeneration. This result allowed us to look for genes that, when misexpressed, could suppress the dys-1; hlh-1 phenotype. When overexpressed, the dyc-1 gene - whose loss-of-function phenotype resembles that of dys-1 - partially suppressed the dys-1; hlh-1 phenotype. The dyc-1 gene encodes a novel protein sharing similarities with the mammalian neural nitric oxide synthase (nNOS)-binding protein CAPON, and is expressed in the muscles of the worm. CONCLUSIONS: As a C. elegans model for dystrophin-dependent myopathy, the dys-1; hlh-1 worms should permit the identification of genes, and ultimately drugs, that would reverse the muscle degeneration in this model. (+info)
Immune responses to dystropin: implications for gene therapy of Duchenne muscular dystrophy.
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Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse full-length or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be tolerant due to the presence of rare 'revertant' dystrophin-positive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of 'revertant' dystrophin-positive muscle fibres. (+info)
Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice.
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Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein. (+info)