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.     

Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin-binding domains of utrophin and dystrophin

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms.     

Utrophin, a way to cure Duchenne muscle dystrophy

Duchenne muscle dystrophy results from the absence of dystrophin, a cytoskeletal protein of the muscle fibre. Dystrophin plays an essential role in the integrity of the membrane-associated protein complexes connected to the extracellular matrix. On chromosome 6 is located the gene of a protein presenting 80 % homology with dystrophin : utrophin, which is expressed at the neuromuscular junction. The review examines if utrophin can replace dystrophin and correct the structural and functional characteristics of the myopathy, and how the improvements can be quantitatively expressed. In transgenic mice, deficient in dystrophin, but overexpressing large quantities of utrophin, the latter is found on structures where dystrophin is normally located, histological signs of necrosis disappear and the recovery of functional disorders, specially affecting the mechanical properties of the muscle fibres, can be complete. The review examines also several ways of obtaining overexpression of utrophin in adult mdx mice, such as conditioned expression of the utrophin transgene (using a tetracycline-sensitive transactivator), transfection with viral vectors containing the utrophin cDNA (complete or truncated), actions on factor(s) controlling utrophin expression at the neuromuscular junction (heregulin, 4 N-acetylgalactosamine), and pharmacological ways of inducing expression (NO, arginine). Though partial improvements of the myopathy status have been obtained by these various approaches, they remain limited by their localized action and/or by the moderate level of utrophin expression obtained. Further researchs to overcome these limitations are urgently needed in order to transform the very promising effect of utrophin overexpression into a real treatment of Duchenne myopathy.     

Pharmacological treatments for Duchenne and Becker dystrophies

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disease affecting 1 boy out of 3500. DMD is due to the lack of a submembranous cytoskeletal protein named dystrophin, leading to the progressive degeneration of skeletal, cardiac and smooth muscle tissue. A milder form of the disease, Becker muscular dystrophy (BMD), is characterised by the presence of a semi-functional truncated dystrophin, or the full-length dystrophin at reduced level. Three different therapeutic approaches are currently under study, gene therapy, cellular therapy and pharmacological therapy. One of the chosen strategies consists of the overexpression of utrophin, a protein 80% homologous with dystrophin, and able to perform similar functions. In this review, we shall consider studies of pharmacological therapy, the aims of which can be classified in three categories: reversal of dystrophic phenotype, dystrophin expression, utrophin overexpression.     

Dystrophin and Utrophin Complexed with Different Associated Proteins in Cardiac Purkinje Fibres

Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin network and the extracellular matrix via the dystrophin-associated protein complex. In mature skeletal muscle, utrophin is a dystrophin-related protein localized mainly at the neuromuscular junction, with the same properties as dystrophin in terms of linking the protein complex. Utrophin could potentially overcome the absence of dystrophin in dystrophic skeletal muscles. In cardiac muscle, dystrophin and utrophin were both found to be present with a distinct subcellular distribution in Purkinje fibres, i.e. utrophin was limited to the cytoplasm, while dystrophin was located in the cytoplasmic membrane.In this study, we used this particular characteristic of cardiac Purkinje fibres and demonstrated that associated proteins of dystrophin and utrophin are different in this structure. We conclude, contrary to skeletal muscle, dystrophin-associated proteins do not form a complex in Purkinje fibres. In addition, we have indirect evidence of the presence of two different 400kDa dystrophins in Purkinje fibres.     

The NO way to increase muscular utrophin expression?

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder that results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to the search for a treatment is to compensate for dystrophin loss by utrophin, another cytoskeletal protein. During development, in normal as in dystrophic embryos, utrophin is found at the membrane surface of immature skeletal fibres and is progressively replaced by dystrophin. Thus, it is possible to consider utrophin as a 'foetal homologue' of dystrophin. In a previous work, we studied the effect of L-arginine, the substrate of nitric oxide synthetase (NOS), on utrophin expression at the muscle membrane. Using a novel antibody, we confirm here that the immunocytochemical staining was indeed due to an increase in utrophin at the sarcolemma. The result is observed not only on mdx (an animal model of DMD) myotubes in culture but also in mdx mice treated with L-arginine. In addition, we show here the utrophin increase in muscle extracts of mdx mice treated with L-arginine, after electrophoretic separation and western-blotting using this novel antibody, and thus extending the electrophoretic results previously obtained on myotube cultures to muscles of treated 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.     

Dystrophin and utrophin bind actin through distinct modes of contact

This study was designed to define the molecular epitopes of dystrophin-actin interaction and to directly compare the actin binding properties of dystrophin and utrophin. According to our data, dystrophin and utrophin both bound alongside actin filaments with submicromolar affinities. However, the molecular epitopes involved in actin binding differed between the two proteins. In utrophin, the amino-terminal domain and an adjacent string of the first 10 spectrin-like repeats more fully recapitulated the activities measured for full-length protein. The homologous region of dystrophin bound actin with low affinity and near 1:1 stoichiometry as previously measured for the isolated amino-terminal, tandem (CH) domain. In contrast, a dystrophin construct including a cluster of basic spectrin-like repeats and spanning from the amino terminus through repeat 17, bound actin with properties most similar to full-length dystrophin. Dystrophin and utrophin both stabilized preformed actin filaments from forced depolymerization with similar efficacies but did not appear to compete for binding sites on actin. We also found that dystrophin binding to F-actin was markedly sensitive to increasing ionic strength, although utrophin binding was unaffected. Although dystrophin and utrophin are functionally homologous actin-binding proteins, these results indicate that their respective modes of contact with actin filaments are markedly different. Finally, we reassessed the abundance of dystrophin in striated muscle using full-length protein as the standard and measured greater than 10-fold higher values than previously reported.     

Immunogold confirmation that utrophin is localized to the normal position of dystrophin in dystrophin-negative transgenic mouse muscle

It has been shown previously that when utrophin is highly expressed in mice which lack dystrophin, the muscle pathology is prevented. Immunohistochemical evidence strongly suggests that utrophin in these transgenic mice occupies the position normally filled by dystrophin, although it is not possible to establish this firmly at the level of the light microscope. Using the higher resolution provided by the electron microscope, we demonstrate here by immunogold labelling with both monoclonal and polyclonal antibodies that utrophin, in both its truncated and full-length forms, is indeed specifically located in the subcellular position usually occupied by dystrophin in normal muscle. Moreover, when double-labelling of utrophin and beta-dystroglycan was carried out, colocalisation of the two labels was often seen, indicating an association of the two proteins. Furthermore, when both utrophin and dystrophin were labelled in a transgenic line in which both were simultaneously expressed, the sites of both proteins were in the same zone in relation to the plasma membrane. When both proteins were present, the density of labelling of each was reduced compared with when they are expressed individually, suggesting that there is a finite number of binding sites. These results constitute further support for the view that utrophin might be therapeutically substituted for dystrophin in dystrophic muscle.     

Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

Utrophin is normally confined to the neuromuscular junction (NMJ) in adult muscle and partially compensates for the loss of dystrophin in mdx mice. We show that Akt signaling and utrophin levels were diminished in sarcospan (SSPN)-deficient muscle. By creating several transgenic and knockout mice, we demonstrate that SSPN regulates Akt signaling to control utrophin expression. SSPN determined α-dystroglycan (α-DG) glycosylation by affecting levels of the NMJ-specific glycosyltransferase Galgt2. After cardiotoxin (CTX) injury, regenerating myofibers express utrophin and Galgt2-modified α-DG around the sarcolemma. SSPN-null mice displayed delayed differentiation after CTX injury caused by loss of utrophin and Akt signaling. Treatment of SSPN-null mice with viral Akt increased utrophin and restored muscle repair after injury, revealing an important role for the SSPN-Akt-utrophin signaling axis in regeneration. SSPN improved cell surface expression of utrophin by increasing transportation of utrophin and DG from endoplasmic reticulum/Golgi membranes. Our experiments reveal functions of utrophin in regeneration and new pathways that regulate utrophin expression at the cell surface.     

Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.     

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.     

Fetal muscle-derived cells can repair dystrophic muscles in mdx mice

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.     

Impacts of dystrophin and utrophin domains on actin structural dynamics: implications for therapeutic design

We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics-restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a "mini-dystrophin," lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a "micro-utrophin," with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin-protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.