Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

Duchenne muscular dystrophy and the neuromuscular junction: The utrophin link

Although the precise function of utrophin at the postsynaptic membrane of the neuromuscular junction still remains unclear, despite recent genetic ‘knockout’ experiments^(1,2), a separate study in a transgenic mouse model system for Duchenne muscular dystrophy (DMD) has nonetheless shown that overexpression of utrophin into extrasynaptic regions of muscle fibers can functionally compensate for the lack of dystrophin and alleviate the muscle pathology⁽³⁾. In this context, the next step is to identify the mechanisms presiding over expression of utrophin at the neuromuscular synapse in attempts to induce its expression throughout DMD muscle fibers. In fact, additional studies have shown that an important DNA element contained with the utrophin promoter may confer synapse-specific expression to the utrophin gene^(4,5). Identification of the events culminating in the transaction of the utrophin gene within synaptic myonuclei should provide important cues for the development of an effective therapeutic strategy for DMD.     

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.     

Structure of neuromuscular junctions and differentiation of striated muscle fibers in mdx mice after bone-marrow stem cell therapy

Mdx mice are an experimental model of Duchenne muscular dystrophy caused by mutations in the dystrophin gene. Repeated cycles of muscle degeneration-regeneration are common for mdx mice. Disrupted neuromuscular junctions also characterize mdx mice. The structure of mdx mice neuromuscular junctions and the differentiation of striated muscle fibers were investigated 4, 8, 16, and 24 weeks after transplantation of C57BL/6 Lin(−) bone-marrow stem cells. We found that the death of striated muscle fibers decreased 4 weeks after the transplantation of bone-marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began in 8 weeks and dystrophin synthesis increased in 16–24 weeks after the bone-marrow stem cells transplantation. On the longitudinal sections of quadriceps muscle of mdx mice 4 weeks after transplantation, we observed a reduced quantity of acetylcholine receptor clusters and an increase in their area in neuromuscular junctions. Sixteen weeks after the transplantation, the total area of neuromuscular junctions increased due to an enlarged number of acethylcholine receptors and their extended area. The single intramuscular transplantation of C57BL/6 Lin(−) bone-marrow stem cells induces the differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.     

Subtle Neuromuscular Defects in Utrophin-deficient Mice

Utrophin is a large cytoskeletal protein that is homologous to dystrophin, the protein mutated in Duchenne and Becker muscular dystrophy. In skeletal muscle, dystrophin is broadly distributed along the sarcolemma whereas utrophin is concentrated at the neuromuscular junction. This differential localization, along with studies on cultured cells, led to the suggestion that utrophin is required for synaptic differentiation. In addition, utrophin is present in numerous nonmuscle cells, suggesting that it may have a more generalized role in the maintenance of cellular integrity. To test these hypotheses we generated and characterized utrophin-deficient mutant mice. These mutant mice were normal in appearance and behavior and showed no obvious defects in muscle or nonmuscle tissue. Detailed analysis, however, revealed that the density of acetylcholine receptors and the number of junctional folds were reduced at the neuromuscular junctions in utrophin-deficient skeletal muscle. Despite these subtle derangements, the overall structure of the mutant synapse was qualitatively normal, and the specialized characteristics of the dystrophin-associated protein complex were preserved at the mutant neuromuscular junction. These results point to a predominant role for other molecules in the differentiation and maintenance of the postsynaptic membrane.     

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.     

Presence of dystrophine-like protein at the neuromuscular junction in Duchenne muscular dystrophy and in "mdx" mutant mice

We have studied by indirect immunofluorescence, using three different polyclonal antidystrophin antibodies raised against fusion proteins, the neuromuscular junctions (NMJs) in muscle biopsies from Duchenne muscular dystrophy (DMD) patients, from human controls and mutant "mdx" mice and normal mice. In controls the periphery of all muscle fibres was strongly labelled by the three dystrophin antibodies and there was a high concentration of labelling at the NMJs (where it was co-localized with acetylcholine receptor labelled by the alpha-bungarotoxin). In DMD and in "mdx" mice the NMJs were equally labelled whereas there was an absence of reaction at the periphery of all (DMD) or most ("mdx" mice) muscle fibers. These findings suggest that a dystrophin-like protein, which was identified by the antibodies we have used, is present at the NMJs in the Duchenne's myopathy and "mdx" mice.     

Absence of utrophin in intercalated discs of human cardiac muscle

Utrophin is the autosomal homologue of dystrophin. In normal skeletal muscle it is localised only to neuromuscular and myotendinous junctions, nerves and vascular tissue. In Xp21 muscular dystrophies, utrophin is also detected on the sarcolemma of skeletal and cardiac muscle, while dystrophin is absent or reduced. In normal cardiac muscle, some reports have demonstrated utrophin at intercalated discs and T-tubules. We have re-examined the distribution of utrophin in normal human cardiac muscle using a panel of eight monoclonal antibodies against different epitopes in N- and C-terminal domains. In contrast to previous studies, utrophin was not detected at the intercalated discs or T-tubules, although labelling of blood vessels was strong. We conclude that the primary location of utrophin in normal heart is in the vascular system. In addition, our results show that the utrophin on cardiac blood vessels is full length, similar to that of skeletal muscle blood vessels.     

Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin

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.     

Utrophin upregulation for treating Duchenne or Becker muscular dystrophy: how close are we?

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder for which there is currently no effective treatment. This disorder is caused by mutations or deletions in the gene encoding dystrophin that prevent expression of dystrophin at the sarcolemma. A promising pharmacological treatment for DMD aims to increase levels of utrophin, a homolog of dystrophin, in muscle fibers of affected patients to compensate for the absence of dystrophin. Here, we review recent developments in our understanding of the regulatory pathways that govern utrophin expression, and highlight studies that have used activators of these pathways to alleviate the dystrophic symptoms in DMD animal models. The results of these preclinical studies are promising and bring us closer to implementing appropriate utrophin-based drug therapies for DMD patients.