Dystrophin Molecule Research Articles

Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse

Duchenne muscular dystrophy (DMD) is caused by the absence of full-length dystrophin molecules in skeletal muscle fibers. In normal muscle, dystrophin is found along the length of the sarcolemma where it links the intracellular actin cytoskeleton to the extracellular matrix, via the dystrophin-associated protein (DAP) complex. Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified and shown to be expressed in a variety of tissues, including skeletal muscle. However, in contrast to the localization of dystrophin in extrajunctional regions of muscle fibers, utrophin preferentially accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibers. Since it has recently been suggested that the upregulation of utrophin might functionally compensate for the lack of dystrophin in DMD, considerable interest is now directed toward the elucidation of the various regulatory mechanisms presiding over expression of utrophin in normal and dystrophic skeletal muscle fibers. In this review, we discuss some of the most recent data relevant to our understanding of the impact of myogenic differentiation and innervation on the expression and localization of utrophin in skeletal muscle fibers.

The role of the cytoskeleton in heart failure.

The cytoskeleton of cardiac myocytes consists of actin, the intermediate filament desmin and of alpha- and beta-tubulin that form the microtubules by polymerization. Vinculin, talin, dystrophin and spectrin represent a separate group of membrane-associated proteins. In numerous experimental studies, the role of cytoskeletal alterations especially of microtubules and desmin, in cardiac hypertrophy and failure (CHF) has been described. Microtubules were found to be accumulated thereby posing an increased load on myocytes which impedes sarcomere motion and promotes cardiac dysfunction. Other groups were unable to confirm microtubular densification. The possibility exists that these changes are species, load and chamber dependent. Recently, damage of the dystrophin molecule and MLP (muscle LIM protein) were identified as possible causes of CHF. Our own studies in human hearts with chronic CHF due to dilated cardiomyopathy (DCM) showed that a morphological basis of reduced contractile function exists: the cytoskeletal and membrane-associated proteins are disorganized and increased in amount confirming experimental reports. In contrast, the contractile myofilaments and the proteins of the sarcomeric skeleton including titin, alpha-actinin, and myomesin are significantly decreased. These changes can be assumed to occur in stages and are here presented as a testable hypothesis: (1) The early and reversible stage as present in animal experiments is characterized by accumulation of cytoskeletal proteins to counteract an increased strain without loss of contractile material. (2) Further accumulation of microtubules and desmin to compensate for the increasing loss of myofilaments and titin represents the late clinical and irreversible state. We suggest, based on a structural basis for heart failure, an integrative view which closes the gap between changes within cardiac myocytes and the involvement of the extracellular matrix, including the development of fibrosis. These factors contribute significantly to structural ventricular remodeling and dilatation finally resulting in reduced cardiac function.

Dystrophin and the cardiomyocyte membrane cytoskeleton in the healthy and failing heart.

The cardiomyocyte membrane cytoskeleton consists of the costameric proteins that mediate force transduction from the cell to the extracellular matrix, and a sub-membrane network composed of dystrophin and associated proteins. Studies of the precise cellular distribution of dystrophin and of the consequences of genetic mutations leading to abnormal expression of the dystrophin molecule, as occurs in Duchenne and Becker's muscular dystrophies, highlight potential functional roles of this sub-membrane protein complex in cardiomyocytes. Detailed investigation of dystrophin distribution using the complementary cell imaging techniques of immunoconfocal microscopy and freeze-fracture cytochemistry at the electron-microscopical level show that, in contrast to rat cardiomyocytes, the dystrophin network in human cardiomyocytes is locally enriched at costameres. Thus located, the dystrophin network appears to have a mechanical role, involving stabilization of the peripheral plasma membrane during the repetitive distortion associated with cardiac contraction and, in the human myocyte, contributing to lateral force-transduction. Evidence from animal models of muscular dystrophy and from investigation of the interactions of the sub-membrane cytoskeleton with other membrane-associated proteins including ion channels, receptors and enzymes, further suggests a role for dystrophin in organization and regulation of membrane domains. The relative preservation of the membrane cytoskeleton in non-dystrophic dilated cardiomyopathy and in ischemic cardiomyopathy, conditions in which the myocyte contractile apparatus and internal desmin-based cytoskeleton are commonly disrupted, emphasizes the vital role of the membrane cytoskeleton in cell survival. Continued cardiomyocyte survival despite loss of contractile protein organization has implications in the potential for reversibility of left ventricular remodeling that can be achieved in the clinical setting.

Molecular mechanisms and putative signalling events controlling utrophin expression in mammalian skeletal muscle fibres

The absence of full-length dystrophin molecules in skeletal muscle fibres results in the most severe form of muscular dystrophy, the Duchenne form (DMD). Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified. Although utrophin is expressed along the sarcolemma in developing, regenerating and DMD muscles, it nonetheless accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibres. Due to the high degree of sequence identity between dystrophin and utrophin, it has been previously suggested that utrophin could in fact functionally compensate for the lack of dystrophin. Recent studies using transgenic mouse model systems have directly tested this hypothesis and revealed that upregulation of utrophin throughout dystrophic muscle fibres represents indeed, a viable approach for the treatment of DMD. Current studies are therefore focusing on the elucidation of the various regulatory mechanisms presiding over expression of utrophin in muscle fibres in attempts to ultimately identify small molecules which could systematically increase utrophin levels in extrasynaptic compartments of dystrophic muscle fibres. This review presents some of the recent data relevant for our understanding of the transcriptional regulatory mechanisms involved in maintaining expression of utrophin at the neuromuscular junction. In addition, the contribution of specific cues originating from motoneurons and the putative involvement of signalling events are also discussed.

Dystrophin-Glycoprotein Complex Is Monomeric and Stabilizes Actin Filaments in Vitro through a Lateral Association

The native molecular weight of the dystrophin-glycoprotein complex and its effect on actin depolymerization and polymerization were examined. First, we determined that the native molecular weight of purified dystrophin-glycoprotein complex is only large enough (Mr 1,200,000) to contain one copy of each protein in the complex, including dystrophin. Using different approaches, we also demonstrated that dystrophin-glycoprotein complex significantly protected a fraction of actin filaments from disassembly, while individual recombinant actin binding fragments of dystrophin or calpain-digested dystrophin-glycoprotein complex had no effect on F-actin depolymerization. The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization saturated at a dystrophin:actin molar ratio of 0.04, corresponding to 1 dystrophin/25 actin monomers, which is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin previously measured at equilibrium. However, dystrophin-glycoprotein complex did not bind G-actin or alter the kinetics or extent of actin polymerization. This excluded the possibility that dystrophin-glycoprotein complex inhibited actin depolymerization by capping the ends of actin filaments. It therefore appears that actin binding domains separated on the dystrophin molecule from each other by almost 1,200 amino acids act in concert to protect F-actin from depolymerization. Our data suggest that dystrophin stabilizes F-actin in vitro by binding alongside an actin filament and bridging actin monomers in a manner analogous to the actin side binding protein tropomyosin. It is noteworthy that we did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin. This indicates that dystrophin-glycoprotein complex and tropomyosin may simultaneously bind the same actin filament and identifies another feature that distinguishes dystrophin from the other proteins in the actin-cross-linking superfamily.

Phosphorylation of the carboxyl-terminal region of dystrophin.

Dystrophin is a protein product of the gene responsible for Duchenne and Becker muscular dystrophy. The protein is localized to the inner surface of sarcolemma and is associated with a group of membrane (glyco)proteins. Dystrophin links cytoskeletal actins via the dystrophin-associated protein complex to extracellular matrix protein, laminin. This structural organization implicates the role of dystrophin in stabilizing the sarcolemma of muscle fibers. Precisely how dystrophin functions is far from clear. The presence of an array of isoforms of the C-terminal region of dystrophin suggests that dystrophin may have functions other than structural. In agreement, many potential phosphorylation sites are found in the C-terminal region of dystrophin, and the C-terminal region of dystrophin is phosphorylated both in vitro and in vivo by many protein kinases, including MAP kinase, p34cdc2 kinase, CaM kinase, and casein kinase, and is dephosphorylated by calcineurin. The C-terminal domain of dystrophin is also a substrate for hierarchical phosphorylation by casein kinase-2 and GSK-3. These observations, in accordance with the finding that the cysteine-rich region binds to Ca2+, Zn2+, and calmodulin, suggest an active involvement of dystrophin in transducing signals across muscle sarcolemma. Phosphorylation-dephosphorylation of the C-terminal region of dystrophin may play a role in regulating dystrophin-protein interactions and (or) transducing signal from the extracellular matrix via the dystrophin molecule to the cytoskeleton.

Modulation of pre-mRNA splicing in the Duchenne muscular dystrophy gene.

Duchenne & Becker muscular dystrophies (DMDIBMD) are allelic disorders caused by mutations in a 2.5Mb gene on the Xchromosome( 1). Over two-thirds of mutations are deletions(2). causing a shift in the reading frame or an in-frame loss of part of the dystrophin molecule associated with DMD or BMD forms of the disease respectively(3). The remaining disease-causing mutations are probably due to point mutations, or defects in transcriptional regulation(4). While muscle fibres of DMD patients and animal models of the disease are generally characterised by a lack of detectable dystrophin at the sarcolemma(5), rare dystrophin-positive “revertant” myofibres are encountered in muscle biopsies(6,7). Proposed mechanisms include reinitiation of translation at downstream methionine codons, frame-restoring mutations, and exon skipping to frame-restoring exons in a proportion of dystrophin mRNAs(8). Evidence of exon skipping has been found by reverse transcriptase-mediated PCR (RT-PCR) analysis of patients’ muscle tissue(9,lO) but it is unclear whether these transcripts can improve muscle function. It is clear from BMD patients, however, that in-frame gene products are normally at least semi-functional and lead to milder muscle pathology(1 l), so it has been proposed that the severe muscle disease experienced by DMD patients may be alleviated by increasing the proportion of in-frame transcripts arising in their myofibre nuclei( 12). This study aims to induce exon-skipping across DMD mutations during pre-mRNA splicing in order to force production of framerestored mRNA transcripts. Initial work seeks to analyse revertant fibres in the mdr mouse model by RT-PCR of muscle transcripts, followed by PCR-mediated construction of a recombinant premRNA template in order to study splicing using in vitro transcription assays( 13). Transcription of the template is driven by a “7 promoter and incorporates exon 23 (mutated in the mdx mouse) and flanking intron sequences within the context of a partial P-globin genome. The 5’ region of mouse dystrophin intron 23 has now been cloned and sequenced using the vectorette PCR technique for inclusion in the template DNA. Sequence-specific antisense 2’-O-methyl oligoribonucleotides targeted to splicing signals will be added to these assays to try to induce exonskipping at high frequency(l4). Parallel studies will focus on the effective delivery of oligonucleotides to the nuclei of cultured muscle cells from normal and dystrophic animals/patients. Methods to be tested include various liposomelnucleic acid formulations( 15, 16) and oligonucleotides tagged with nucleartargeting peptides(l7). Ultimately, these studies will be drawn together in order to induce exon skipping by the introduction of antisense oligonucleotides into cultured cells. Carefully-controlled analyses will involve RT-PCR of total and poly(A)+ RNA from in vitro assays, cultured cells and tissues and Northern blotting( 19) as well as immunocytochemistry using exon-specific monoclonal antibodies(20). Direct sequencing of PCR products will be performed to examine the precise nature of mRNAs in both experimental and control preparations(2 1). REFERENCES:

Dystrophin characterization in BMD patients: correlation of abnormal protein with clinical phenotype

We have investigated protein expression and genotype in 59 Becker muscular dystrophy (BMD) patients. The aim was to identify possible causes of the marked variability in phenotype in patients with similar deletions/mutations. The patients were examined neurologically and functionally and underwent Manual Muscle Testing. Dystrophin expression was analysed by immunohistochemistry and western blot using antibodies against six different segments of the protein. DNA mutations were investigated by PCR amplification of 30 exons. Based on dystrophin expression at the sarcolemma, two groups of patients were identified: group A (29 patients) with the classic patchy distribution of dystrophin and group B (30 patients) with absence or reduction of one or more dystrophin portions and variable, although mostly normal, expression of the other portions of the protein. Dystrophin molecular weight was normal or slightly reduced in group A and was variably reduced, generally conspicuously so, in group B. The quantity of dystrophin expressed varied markedly in both groups. The pattern of immunohistochemical staining in group B patients correlated with milder clinical phenotype, suggesting that small dystrophin molecules lacking a portion in the N-terminus or in the rod domain, are more functional than proteins with normal or slightly reduced molecular weight that display the BMD-typical patchy distribution at the sarcolemma.

Serum Antibodies to the Deleted Dystrophin Sequence after Cardiac Transplantation in a Patient with Becker's Muscular Dystrophy

To the Editor: Becker's muscular dystrophy and Duchenne's muscular dystrophy are allelic disorders caused by mutations of the dystrophin gene at Xp21.1 The 400-kd dystrophin is expressed most abundantly in the plasma membranes of skeletal and cardiac muscle fibers but is absent in patients with severe Duchenne's muscular dystrophy and in mdx mice. The expression of a truncated, “semifunctional” dystrophin molecule is usually associated with Becker's muscular dystrophy, which is milder than Duchenne's muscular dystrophy.2 It is not clear whether the full-length dystrophin molecule induces a specific immune response against the deleted sequence in a patient with Becker's muscular dystrophy. . . .

Expression of Deletion-Containing Dystrophins in mdx Muscle: Implications for Gene Therapy and Dystrophin Function

The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.

Sarcolemmal expression of dystrophin C-terminus but reduced expression of 6q-dystrophin-related protein in two DMD patients with large deletions of the dystrophin gene

Partial deletions of the dystrophin gene are the predominant genetic lesions in Duchenne (DMD) and Becker (BMD) muscular dystrophies. According to the reading frame hypothesis [1], any deletion disrupting the translational reading frame of the mRNA cannot result in expression of the dystrophin molecule and should lead to severe phenotypes of DMD. In contrast, deletions which maintain the reading frame across the deleted exons may give rise to truncated, semifunctional proteins and milder courses of the disease (i.e. BMD). Among the notable exceptions of this hypothesis are very large “in-frame” deletions by which functionally indispensable domains of the dystrophin molecule have been removed.Here, we report on two DMD patients with large intragenic in-frame deletions. Grossly truncated, but stable dystrophin molecules with preserved C-terminal domains were detected at the sarcolemma on cryosections in both patients. However, dystrophin organization on single-teased muscle fibers revealed disarrangement of the costameric pattern, if compared to normal skeletal muscle fibers. Compared to dystrophin-deficient DMD muscle, expression of chromosome-6-encoded dystrophin-related protein (DRP) was greatly diminished in skeletal muscle of both patients. We show, that loss of more than 50% of dystrophin seems to be deleterious for the protein's function and therefore, the extent of the deletions may have an impact on construction of dystrophin mini genes. Moreover, these findings shed new light on the functional significance of the C-terminal domain of dystrophin. They also suggest a negative correlation between sarcolemma) expression of the dystrophin C-terminus and DRP expression at the sarcolemma.

Mammalian alpha 1- and beta 1-syntrophin bind to the alternative splice-prone region of the dystrophin COOH terminus.

The carboxy-terminal region of dystrophin has been suggested to be crucially important for its function to prevent muscle degeneration. We have previously shown that this region is the locus that interacts with the sarcolemmal glycoprotein complex, which mediates membrane anchoring of dystrophin, as well as with the cytoplasmic peripheral membrane protein, A0 and beta 1-syntrophin (Suzuki, A., M. Yoshida, K. Hayashi, Y. Mizuno, Y. Hagiwara, and E. Ozawa. 1994. Eur. J. Biochem. 220:283-292). In this work, by using the overlay assay technique developed previously, we further analyzed the dystrophin-syntrophin/A0 interaction. Two forms of mammalian syntrophin, alpha 1- and beta 1-syntrophin, were found to bind to very close but discrete regions on the dystrophin molecule. Their binding sites are located at the vicinity of the glycoprotein-binding site, and correspond to the amino acid residues encoded by exons 73-74 which are alternatively spliced out in some isoforms. This suggests that the function of syntrophin is tightly linked to the functional diversity among dystrophin isoforms. Pathologically, it is important that the binding site for alpha 1-syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region whose deletion was suggested to result in a severe phenotype. In addition, A0, a minor component of dystrophin-associated proteins with a molecular mass of 94 kD which is immunochemically related to syntrophin, binds to the same site as beta 1-syntrophin. Finally, based on our accumulated evidence, we propose a revised model of the domain organization of dystrophin from the view point of protein-protein interactions.

Molecular Shape of Dystrophin with Special References to Type VI Collagen.

We have reported a dumbbell-shaped structure of dystrophin molecule (Murayama et al., 1990; Sato et al., 1992). However its small population has raised a possibility that the dumbbell structure was due to a contaminant protein, such as type VI collagen of aorta origin. In the present study, type VI collagen was purified from bacterial collagenase-treated aorta of the rabbit. Its rotary shadowed images were very similar to those present in the dystrophin preparations. However, the antiserum to type VI collagen raised in a mouse did not react with dystrophin samples. A tentative explanation for the small population of the dumbbell structure is described.

Expression of the 43 kDa dystrophin-associated glycoprotein in human neuromuscular disease

The expression of the 43 kDa dystrophin-associated glycoprotein (43DAG) has been studied using immunohistochemical labelling with a monoclonal antibody, MANDAG-1, and compared with immunolabelling for dystrophin and the dystrophin-related protein, utrophin, in normal muscle and in muscle from 50 patients with neuromuscular disease. 43DAG and dystrophin were expressed in vascular smooth muscle and at the sarcolemma of normal muscle fibres, with increased labelling at neuromuscular and myotendinous junctions. 43DAG expression was reduced in Duchenne and Becker dystrophies with patchy labelling, more intense around presumptive satellite cells. In Duchenne dystrophy, there was increased 43DAG expression in “revertant” fibres. In Becker dystrophy, 43DAG expression was more extensive around individual fibres, showed more interfibre variation and was more closely related to the intensity of immunolabelling for both dystrophin and utrophin than in Duchenne dystrophy. In other neuromuscular diseases, including congenital muscular dystrophy, no abnormalities of 43DAG expression were identified. The results suggest that in the absence of dystrophin, 43DAG is synthesized but is not stabilized in the sarcolemma. Stability is greater in Becker dystrophy but a normal dystrophin molecule appears to be required for the complete and stable membrane integration of 43DAG. Utrophin may confer some additional stability to the membrane integration of 43DAG but this is incomplete where dystrophin is absent or abnormal.

Multiplicity of abnormal dystrophin in Becker muscular dystrophy: A Becker muscular dystrophy gene frequently produced two smaller sizes of dystrophin

Dystrophin is a muscle cytoskeletal protein with a molecular mass (MM) of ∼ 420 kDa and an isoelectric point (p I) of ∼ 5.5, which is abnormal in size and/or abundance in Becker muscular dystrophy (BMD). We investigated the abnormality of dystrophin molecule in muscles biopsied from 23 BMD patients using the two-dimensional gel electrophoresis (TDGE). We found 7 protein spots which reacted specifically with the monoclonal anti-dystrophin antibody (mAb) A1C raised against N-terminal domain of the normal dystrophin. These spots were focused on the two-dimensional gel at the same position as the normal dystrophin (#1), at the position with MM ∼ 480 kDa pI ∼ 5.35 (#2), the position with MM ∼ 400−330 kDa pI ∼ 5.51−5.47 (#3), the position with MM ∼ 300 kDa pI ∼ 5.4 (#4), the position with MM ∼ 235−250 kDa pI ∼ 5.53−5.5 (#5), the position with MM ∼ 165 kDa pI ∼ 6.0 (#6), and the position with MM ∼ 160 kDa pI ∼ 5.75 (#7). These spots were classified into five patterns in individuals, that is, #1 alone in 3 patients, #3 alone in 1, the combination of # 3 and 5 in 17, the combination of # 1, 3 and 5 in 1 and the combination of # 1, 2, 4, 6 and 7 in 1. The combination of # 3 and 5 was observed in 17 of 23 patients (75%). In addition, both of # 3 and 5 reacted not only with the mAbs, A1C, Dys 3 and 5E2, which recognize the N-terminal domain of the normal dystrophin, but also with the mAbs, 4C5 and Dys 2, which recognize the C-terminal domain. Thus, each of # 3 and 5 preserved both N- and C-terminal domains of the normal dystrophin in spite of significant differences in MM and p I. Our observations conclude that the #5 is not a proteolytic fragment of the “full-length” dystrophin (#3), and suggest that some exons encoding triple helical segments at the central portion of dystrophin are spliced out to produce two abnormal dystrophins from a single mutated gene in the majority of BMD.

Abnormal dystrophin expression in patients with limb girdle syndromes.

Clinical differential diagnosis between Becker muscular dystrophy (BMD) and limb gridle muscular dystrophy (LGMD) may be difficult because the BMD clinical phenotype tends to overlap with other limb girdle syndromes, especially with LGMD. Therefore we studied the expression of dystrophin, the protein product of the Becker and Duchenne muscular dystrophy gene, in muscle biopsy specimens of 30 patients (18 males, of whom 15 represented spradic cases, and 12 females) diagnosed as having LGMD according to traditional clinical, electrophysiological and histological criteria. For dystrophin analysis, six different monoclonal antibodies directed against different epitopes of the dystrophin molecule were used. Immunocytochemically, five of the 30 LGMD patients (17%) showed abnormal dystrophin staining patterns diagnostic of BMD. Western blotting in these five patients, all sporadic cases, showed dystrophin of reduced size and/or abundance. Analysis of blood or muscle DNA using multiplex polymerase chain reaction revealed deletions in the dystrophin gene in three of the five. Thus, 5 of 15 (33%) sporadic male patients previously thought to have LGMD were identified as having BMD.

Size and localization of dystrophin molecule: immunoelectron microscopic and freeze etching studies of muscle plasma membranes of murine skeletal myofibers.

The ultrastructure and mode of existence of the dystrophin molecule and its relations to actin filaments were examined in murine skeletal myofibers. Electron microscopy of freeze-etched replicas of gold-labelled dystrophin molecules in quick-freeze, deep-etch, rotary-shadow preparations revealed rod-like structures 108.2 +/- 16.3 nm long and 3.1 +/- 1.5 nm thick. Some dystrophin molecules appeared to link their ends to form anastomosing networks; others were separate from each other. The dystrophin molecules were parallel or nearly parallel to the inner surface of the muscle plasma membrane. Double immuno-labelling transmission electron microscopy using N- and C-terminal dystrophin antibodies showed that the group mean distances of the N- and C-terminal signals from the muscle plasma membrane were 52.7 +/- 8.1 nm and 45.9 +/- 11.3 nm, respectively, which were not significantly different. Histograms of the distribution of the N- and C-terminal distances from the muscle plasma membrane had similar patterns with peaks 10-20 nm from the membrane. This was consistent with the findings of the mode of existence of dystrophin molecules seen in freeze-etched replicas. Finally, the dystrophin molecules were linked with the most peripheral sarcoplasmic actin like filaments, end to side as well as end to end.

Immunohistological evidence for second or somatic mutations as the underlying cause of dystrophin expression by isolated fibres in Xp21 muscular dystrophy of Duchenne-type severity

Using five monoclonal antibodies against different parts of the dystrophin molecule, we have studied the dystrophin composition of 17 dystrophin-positive fibres in a muscle biopsy from a boy with Xp21 muscular dystrophy of Duchenne-type severity. The fibres showed five distinct, reproducible, immunoreactive dystrophin profiles. All the profiles included both the N-terminal and the C-terminal domains, but between these domains, different fibres were negative for different antibodies, suggesting the somatic loss of certain exons. We interpret this as the first in situ evidence of an individual having different patterns of missing exons leading to restoration of the reading frame in various ways in the original germline frame-shifting deletion of exons 35–43. It follows that various somatic mutations had taken place in different fibres.

A quantitative ELISA for dystrophin

A novel approach to the quantitation of the muscular dystrophy protein, dystrophin, in muscle extracts is described. The two-site ELISA uses two monoclonal antibodies against dystrophin epitopes which lie close together in the rod domain of the dystrophin molecule in order to minimize the effects of dystrophin degradation. Dystrophin is assayed in its native form by extracting with non-ionic detergents and avoiding the use of SDS.

The distribution of dystrophin in the murine central nervous system: An immunocytochemical study

A mild non-progressive cognitive defect is a feature of the fatal X-linked disease, Duchenne muscular dystrophy. Recent studies have identified the genetic defect and the resulting loss of the protein dystrophin, and shown that dystrophin messenger RNA and protein are present in normal brain tissue. We have performed western immunoblotting and fluorescence immunocytochemistry using a sensitive antibody made against a large fragment of the dystrophin molecule to study the regional, cellular and subcellular distribution of dystrophin in the mammalian brain. The brains of B 10 (control) and mdx (dystrophin deficient null mutant) mouse brain were compared on a point-by-point basis to verify that only dystrophin and not autosomal dystrophin related protein or cross-reacting proteins were being identified. In addition three murine neurologic mutants, nervous, lurcher, and weaver, were studied to refine the localization of dystrophin. In western immunoblots, dystrophin is present in all regions of the brain and in greatest abundance in the cerebellum. Dystrophin, as demonstrated in immunofluorescence, is present in neurons, but not in glia or myelin, and forms punctate foci associated with the plasma membrane of perikarya and dendrites, but not axons. While dystrophin is abundant in cerebral cortical neurons and cerebellar Purkinje cells, it is absent from most subcortical neurons, the granule cells of fascia dentata, and cerebellar neurons other than Purkinje cells. The absence of dystrophin in the cerebellum of the Purkinje cell deficient mutants nervous and lurcher, and its presence in the granule cell deficient mutant weaver indicate that dystrophin is a component of Purkinje cells rather than closely apposed afferents to those cells. The distribution and localization of dystrophin suggests a role in organizing the plasma membrane, possibly as an anchor of the postsynaptic apparatus, a possible basis for the cognitive defect in Duchenne dystrophy.