Association of dystrophin and an integral membrane glycoprotein

Duchenne muscular dystrophy (DMD) is caused by a defective gene found on the X-chromosome. Dystrophin is encoded by the DMD gene and represents about 0.002% of total muscle protein. Immunochemical studies have shown that dystrophin is localized to the sarcolemma in normal muscle but is absent in muscle from DMD patients. Many features of the predicted primary structure of dystrophin are shared with membrane cytoskeletal proteins, but the precise function of dystrophin in muscle is unknown. Here we report the first isolation of dystrophin from digitonin-solubilized skeletal muscle membranes using wheat germ agglutinin (WGA)-Sepharose. We find that dystrophin is not a glycoprotein but binds to WGA-Sepharose because of its tight association with a WGA-binding glycoprotein. The association of dystrophin with this glycoprotein is disrupted by agents that dissociate cytoskeletal proteins from membranes. We conclude that dystrophin is linked to an integral membrane glycoprotein in the sarcolemma. Our results indicate that the function of dystrophin could be to link this glycoprotein to the underlying cytoskeleton and thus help either to preserve membrane stability or to keep the glycoprotein non-uniformly distributed in the sarcolemma.     

Expression of recombinant dystrophin and its localization to the cell membrane.

Duchenne's muscular dystrophy (DMD) is an X-linked progressive myopathy caused by a defect in the DMD gene locus. The gene corresponding to the DMD locus produces a 14-kilobase (kb) messenger RNA that codes for a large cytoskeletal membrane protein, dystrophin. DMD and Becker's muscular dystrophy are the consequences of dystrophin mutations. The exact biological function of dystrophin remains unknown but it has been demonstrated that it is localized to the cytoplasmic face of the cell membrane and has direct interaction with several other membrane proteins. We report here the synthesis of a 14-kb full-length complementary DNA for the mouse muscle dystrophin mRNA and the expression of this cDNA in COS cells. The recombinant dystrophin is indistinguishable from mouse muscle dystrophin by western blot analysis with anti-dystrophin antibodies and was shown by an immunofluorescent technique to be localized in the cell membrane. Our successful construction of a functional full-length cDNA opens opportunities for the study of structure and function of dystrophin and provides an opportunity to initiate gene therapy studies.     

Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs

Duchenne's muscular dystrophy (DMD), which affects one in 3,500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death. One approach to treatment would be to introduce the normal dystrophin gene into diseased muscle cells. When pure plasmid DNA is injected into rodent skeletal or cardiac muscle, the cells express reporter genes. We now show that a 12-kilobase full-length human dystrophin complementary DNA gene and a 6.3-kilobase Becker-like gene can be expressed in cultured cells and in vivo. When the human dystrophin expression plasmids are injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins are present in the cytoplasm and sarcolemma of approximately 1% of the myofibres. Myofibres expressing human dystrophin contain an increased proportion of peripheral nuclei. The results indicate that transfer of the dystrophin gene into the myofibres of DMD patients could be beneficial, but a larger number of genetically modified myofibres will be necessary for clinical efficacy.     

Immunoelectron microscopic localization of dystrophin in myofibres

Duchenne muscular dystrophy, a common X-linked recessive human disease, has recently been shown to be caused by the deficiency of a large, low abundance protein called 'dystrophin'. Biochemical techniques have shown dystrophin to be membrane-associated in skeletal muscle, with enrichment of dystrophin in the t-tubules of 'triads'. Other studies using immunohistochemistry on thick (10 micron) sections have shown dystrophin to be located at the periphery of muscle fibres, possibly at the plasma membrane. These results have been interpreted as being either consistent and complementary, or contradictory. To localize dystrophin more precisely relative to these membrane systems we have employed highly sensitive and spatially accurate immuno-gold electron microscopy of ultra-thin (70-100 nm) cryosections. The major distribution of dystrophin was on the cytoplasmic face of the plasma membrane of muscle fibres, and possibly on the contiguous t-tubule membranes. The presented data, taken together with recently accumulated information regarding the primary structure of dystrophin, suggests that dystrophin is a component of the membrane cytoskeleton in myogenic cells. Thus, myofibre necrosis in patients affected with Duchenne muscular dystrophy is likely the result of plasma membrane instability.     

Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons

Moderate non-progressive cognitive impairment is a consistent feature of Duchenne muscular dystrophy (DMD), although no central nervous system (CNS) abnormality has been identified. Recent studies have elucidated the molecular defect in DMD, including the absence of the protein dystrophin in affected individuals. Normal brain tissue contains dystrophin messenger RNA and dystrophin is present in low abundance in the brain and seems to be regulated in this tissue, at least in part, by a promoter that differs from that in muscle. Until now, antibodies and immunocytochemical methods used to demonstrate dystrophin at the plasma membrane of mouse and human muscle have proven inadequate to localize precisely dystrophin in the mammalian CNS. We have now made an antibody (anti 6-10) which is much more sensitive than those previously available to immunolabel dystrophin in the CNS. Using this antibody, we found that in the mouse, dystrophin is particularly abundant in the neurons of the cerebral and cerebellar cortices, and that it is localized at postsynaptic membrane specializations. Dystrophin may have a different role in neurons than in muscle, and an alteration at the synaptic level may be the basis of the cognitive impairment in DMD.     

Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse

Human X-linked Duchenne and Becker muscular dystrophies are due to defects in dystrophin, the product of an exceptionally large gene. Although dystrophin has been characterized as a spectrin-like submembranous cytoskeletal protein, there is no experimental evidence for its function in the structural maintenance of muscle. Current hypotheses attribute necrosis of dystrophin-less fibres in situ to mechanical weakening of the outer membrane, to an excessive influx of Ca2+ ions, or to a combination of these two mechanism, possibly mediated by stretch-sensitive ion channels. Using hypo-osmotic shock to determine stress resistance and a mouse model (mdx) for the human disease, we show that functional dystrophin contributes to the stability of both cultured myotubes and isolated mature muscle fibres.     

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.     

Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle.

Dystrophin is associated with a complex of muscle membrane (sarcolemmal) glycoproteins that provide a linkage to the extracellular matrix protein, laminin. The absence of dystrophin leads to a dramatic reduction of the dystrophin-associated proteins (156DAG, 59DAP, 50DAG, 43DAG and 35DAG) in the sarcolemma of patients with Duchenne muscular dystrophy and mdx mice. Here we demonstrate that dystrophin-related protein (DRP, utrophin), an autosomal homologue of dystrophin, is associated with an identical or antigenically similar complex of sarcolemmal proteins and that DRP and the dystrophin/DRP-associated proteins colocalize to the neuromuscular junction in Duchenne muscular dystrophy and mdx muscle. The DRP and dystrophin/DRP-associated proteins are found throughout the sarcolemma in small-calibre skeletal muscles and cardiac muscle of adult mdx mice. Because these muscles show minimal pathological changes, our results could provide a basis for the upregulation of DRP as a potential therapeutic approach.     

PTC124 targets genetic disorders caused by nonsense mutations

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.     

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.     

The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle

Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are allelic X-linked muscle disorders in man. The gene responsible for the disease has been cloned from knowledge of its map location at band Xp21 on the short arm of the X chromosome. The product of the DMD gene, a protein of relative molecular mass 400,000 (Mr 400K) recently named dystrophin, has been reported to co-purify with triads of mouse and rabbit skeletal muscle when assayed using polyclonal antibodies raised against fusion proteins encoded by regions of mouse DMD complementary DNA. Here we show that antibodies directed against synthetic peptides and fusion proteins derived from the N-terminal region of human DMD cDNA strongly react with an antigen present in skeletal muscle sarcolemma on cryostat sections of normal human muscle biopsies. This immunoreactivity is reduced or absent in muscle fibres from DMD patients but appears normal in muscle fibres from patients with other myopathic diseases. The same antibodies specifically react with a 400K protein in sodium dodecyl sulphate (SDS) extracts of normal human muscle subjected to Western blot analysis. We conclude that the product of the DMD gene is associated with the sarcolemma rather than with the triads and speculate that it strengthens the sarcolemma by anchoring elements of the internal cytoskeleton to the surface membrane.     

Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts

An important corollary to the recent advances in our understanding of the primary cause of Duchenne muscular dystrophy, is the validation of genuine genetic homologues as animal models of the disease in which potential therapies can be tested. The persistent skeletal muscle necrosis that characterizes human Duchenne muscular dystrophy is also seen in the mdx mouse and is, in both, a consequence of a deficiency of dystrophin, probably within the muscle fibres themselves. As injected muscle precursor cells of one genotype can fuse with host muscle fibres of a different genotype and express the donor genes, we decided to test grafts of normal muscle precursor cells to see if they could induce synthesis of dystrophin in innately dystrophin-deficient mdx muscle fibres. We show that injected normal muscle precursor cells can fuse with pre-existing or regenerating mdx muscle fibres to render many of these fibres dystrophin-positive and so to partially or wholly rescue them from their biochemical defect.