Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.     

Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p < 0.0001), and by 50% in skeletal muscle (p < 0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labelling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.     

The membrane-cytoskeleton interface: the role of dystrophin and utrophin

Recent studies with transgenic animals have considerably advanced our knowledge of the roles of dystrophin and utrophin in both muscle and non-muscle tissues. Rigorous analyses of the roles of the various mdx mutations in mice, as well as the use of artificial transgenes in an mdx background, are beginning to define the functional importance of various regions of the dystrophin protein in normal muscle. Furthermore, recent biochemical analyses have revealed new insights into the role and organization of dystrophin at the membrane-cytoskeleton interface. Transgenic approaches have also revealed surprising and encouraging results with respect to utrophin. Against expectations, the long-awaited utrophin knockout mice have a remarkably mild phenotype with only subtle changes in neuromuscular junction architecture. On the other hand, mdx mice transgenic for a mini-utrophin construct showed rescue of the muscular dystrophy phenotype, clearly an encouraging finding with obvious therapeutic possibilities. These and other recent findings are discussed in the context of the structure and function of dystrophin and utrophin at the membrane-cytoskeleton interface.     

Does utrophin expression in muscles of mdx mice during postnatal development functionally compensate for dystrophin deficiency?

We correlated utrophin expression with the physiopathological course in mdx mice. Evolution of the pathology was assessed by monitoring expression of developmental MHC in mdx mice versus control. Utrophin expression is detected by dystrophin/utrophin cross-reacting antibodies and can only be evaluated in mdx mouse muscles (in absence of dystrophin). This protein was expressed at the periphery of all myotubes and myofibers during the first postnatal week. It began declining in fast muscles before the third week and disappeared from the soleus between the 3rd and the 4th week. The decrease was concomitant with a sudden degenerative/regenerative process affecting slow muscle earlier and more massively than fast muscles. The pathological process became stable in all muscle types (except the diaphragm), with greater utrophin expression in the soleus. These results in mdx mice along with observed utrophin expression in severely affected DMD patients suggest that overexpression of utrophin is not enough to explain the stability of regenerated fibers in mdx mice.     

Utrophin: a potential replacement for dystrophin?

This paper reviews the evidence that utrophin, the autosomally encoded protein related to dystrophin, may be capable of performing the same cellular functions as dystrophin. If this is the case, it may be possible to modify the regulation of utrophin expression as an alternative route to dystrophin gene therapy for sufferers of DMD and/or BMD.     

Development of muscle pathology in canine X-linked muscular dystrophy. I. Delayed postnatal maturation of affected and normal muscle as revealed by myosin isoform analysis and utrophin expression

Canine X-linked muscular dystrophy (CXMD) is genetically homologous to Duchenne muscular dystrophy and shares the severe myopathy and lethal clinical development of the human disease. We used immunohistochemistry to characterize the time course of postnatal expression of adult fast, adult slow and developmental myosin in the muscle of CXMD dogs, carriers and healthy controls. We also characterized the expression of utrophin and dystrophin. This detailed immunolocalization study confirmed that postnatal muscle maturation is delayed in normal dogs compared to other animals and humans, and is only achieved at around 60 days. In CXMD dogs major derangement of myosin expression became evident from about 15 days; there was a selective loss of fibers expressing fast myosin and persistence of developmental fibers compared to controls. In carriers, the proportion of dystrophin-deficient fibers, which mainly expressed fast myosin, decreased with age. In controls and carriers utrophin was absent from muscle fiber surfaces in 2-day-old animals but present between 15 and 30 days, to mostly disappear by 60 days. In dystrophic animals, sarcolemmal expression of utrophin was more marked and persistent. That immature neonatal muscle from control dogs normally contains sarcolemmal utrophin may have implications for the success of utrophin up-regulation therapy to correct the dystrophic phenotype. The data of this study provide important baseline information for further studies on the development and progression of pathological changes in the muscle of CXMD dogs.     

Premorbid psychosocial behavior in demented patients

Formation of the skeletal neuromuscular junction is a multi-step process that requires communication between the nerve and muscle. Studies in many laboratories have led to identification of factors that seem likely to mediate these interactions. 'Knock-out' mice have now been generated with mutations in several genes that encode candidate transsynaptic messengers and components of their effector mechanisms. Using these mice, it is possible to test hypotheses about the control of synaptogenesis. Here, we review our studies on neuromuscular development in mutant mice lacking agrin alpha CGRP, rapsyn, MuSK, dystrophin, dystrobrevin, utrophin, laminin alpha 5, laminin beta 2, collagen alpha 3 (IV), the acetylcholine receptor epsilon subunit, the collagenous tail of acetylcholinesterase, fibroblast growth factor-5, the neural cell adhesion molecule, and tenascin-C.     

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.     

Alkaline phosphatase (ALP) activity in rheumatoid arthritis (RA): its clinical significance and synthesis of ALP in RA synovium

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.     

Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.     

Dystrophin, utrophin and beta-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

The precise localization and semiquantitative correlation of dystrophin, utrophin and beta-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of beta-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of beta-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of beta-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to beta-dystroglycan.     

Nitric oxide synthase in skeletal muscle fibers: a signaling component of the dystrophin-glycoprotein complex

The present review deals with the anatomical distribution, physiological importance, and pathological implications of the neuronal-type nitric oxide synthase (nNOS) in skeletal muscle. Throughout the body, nNOS is located beneath the sarcolemma of skeletal muscle fibers. In rodents, nNOS is enriched in type IIb muscle fibers, but is more homogenously distributed among type II and type I fibers in humans and subhuman primates. It is accumulated on the postsynaptic membrane at the neuromuscular junction. An increased concentration of nNOS is noted at the sarcolemma of muscle spindle fibers, in particular nuclear bag fibers, which belong to type I fibers. The association of nNOS with the sarcolemma is mediated by the dystrophin-glycoprotein complex. Specifically, nNOS is linked to alpha 1-syntrophin through PDZ domain interactions. Possibly, it also directly binds to dystrophin. The activity and expression of nNOS are regulated by both myogenic and neurogenic factors. Besides acetylcholine, glutamate has also been shown to stimulate nNOS, probably acting through N-methyl-D-aspartate receptors, which are colocalized with nNOS at the junctional sarcolemma. Functional studies have implicated nitric oxide as a modulator of skeletal muscle contractility, mitochondrial respiration, carbohydrate metabolism, and neuromuscular transmission. A clinically relevant aspect of nNOS is its absence from the skeletal muscle sarcolemma of patients with Duchenne muscular dystrophy (DMD). A concept is presented which suggests that, as a consequence of the disruption of the dystrophin-glyoprotein complex in DMD, nNOS fails to become attached to the sarcolemma and is subject to downregulation in the cytosol.     

Formation of the neuromuscular junction: molecules and mechanisms

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron-neuron synapses in the peripheral as well as the central nervous system.     

Targeting acetylcholinesterase molecules to the neuromuscular synapse

The functional integrity of the neuromuscular synapse requires that sufficient numbers of acetylcholinesterase (AChE) molecules be localized on the specialized extracellular matrix between the nerve terminal and the post-synaptic membrane. Multiple interrelated levels of regulation are necessary to accomplish this complex task including the spatial and temporal restriction of AChE mRNA expression within the muscle fiber, local translation and assembly of AChE polypeptides, and focused accumulation of AChE molecules on the extracellular matrix. This is accomplished in part through the organization of other extracellular matrix molecules into a complex which further associates with acetylcholine receptors and their accompanying molecules. Finally, the mature neuromuscular junction contains molecules which can act as receptors for the attachment of AChE which in turn may allow for the turnover of this enzyme at the synapse. This brief review will focus mainly on contributions from our laboratory towards understanding the mechanisms involved in organizing AChE molecules at the neuromuscular synapse.     

Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes

We have identified isoforms of dystrophin and utrophin, a dystrophin homologue, expressed in astrocytes and examined their expression patterns during dibutyryl-cAMP (dBcAMP)-induced morphological differentiation of astrocytes. Immunoblot and immunocytochemical analyses showed that full-length-type dystrophin (427 kDa), utrophin (395 kDa), and Dp71 (75 kDa), a small-type dystrophin isoform, were coexpressed in cultured nondifferentiated rat brain astrocytes and were found to be located in the cell membrane. During morphological differentiation of the astrocytes induced by 1 mM dBcAMP, the amount of Dp71 markedly increased, whereas that of dystrophin and utrophin decreased. Northern blot analyses revealed that dBcAMP regulates the mRNA levels of Dp71 and dystrophin but not that of utrophin. dBcAMP slightly increased the amount of the beta-dystroglycan responsible for anchoring dystrophin isoforms and utrophin to the cell membrane. Immunocytochemical analyses showed that most utrophin was observed in the cytoplasmic area during astrocyte differentiation, whereas Dp71 was found along the cell membrane of the differentiated astrocytes. These findings suggest that most of the dystrophin/utrophin-dystroglycan complex on cell membrane in cultured astrocytes was replaced by the Dp71-dystroglycan complex during morphological differentiation. The cell biological roles of Dp71 are discussed.     

Consequences of the combined deficiency in dystrophin and utrophin on the mechanical properties and myosin composition of some limb and respiratory muscles of the mouse

The mechanical properties and the myosin isoform composition were studied in three isolated muscles (EDL, soleus, diaphragm) of mutant mice lacking both dystrophin and utrophin (dko). They were compared with the corresponding muscles of the normal and the dystrophin-deficient (mdx) and the utrophin-deficient (uko) mice. In comparison with mdx muscles, dko muscles show a significant reduction of the normalized isometric force, confirmed by the reduced muscular activity of the whole animal. Kinetics parameters (twitch time-to-peak and half-relaxation time) were slightly reduced, and the maximal speed of shortening of soleus, Vmax, was reduced by 30%. The maximal power output (muW/mm3) was reduced by 50% in dko soleus. In the three muscles studied, the relative myosin heavy chains (MHC) composition showed a shift towards slower isoforms. dko EDL presented a dramatic decrease of the resistance ot tetanic contraction with forced lengthenings (eccentric contractions), while muscle lacking only utrophin (uko mutants) display a normal resistance to this exacting mechanical challenge. These experiments suggest that lack of both dystrophin and utrophin is very detrimental to the mice and that mechanical properties of the muscles may explain the overall phenotype. Moreover these results bring some support to the idea that the expression of utrophin in mdx muscle compensates, to some extent, for the lack of dystrophin.     

No health risk?

Female carriers of Duchenne muscular dystrophy (DMD) may demonstrate elevated serum creatine kinase (CK) and reduction of muscle dystrophin in all muscle types. We hypothesized that decreased dystrophin in uterine or pelvic girdle musculature might affect the obstetrical performance of females heterozygous for a dystrophin mutation. We reviewed the outcome of 34 deliveries resulting in 35 children from 13 women who were mothers of males attending a muscular dystrophy clinic. Obstetrical performance was examined retrospectively by chart review and patient contact. Of 35 children, 6 (17%) were delivered in the breech position, which is a fivefold increase above the national standards for term pregnancies. Of the six infants with breech presentation, two were males affected with DMD, one was a female heterozygote, one was a male who died perinatally, and the carrier status of the other two females is unknown. Most DMD affected males (12/14) were delivered in the vertex position. Thus, it is likely that maternal, rather than fetal, muscle weakness was the significant factor in determination of fetal position at term. We speculate that subtle changes in uterine or pelvic girdle muscle tone may contribute to a higher rate of fetal breech position in carriers of the DMD gene.     

M-cadherin distribution in the mouse adult neuromuscular system suggests a role in muscle innervation

M-cadherin belongs to the Ca(2+)-dependent cadherin family of cell adhesion molecules and was first isolated from a mouse muscle cell line cDNA library. It is specifically expressed in muscle tissue during development and is supposed to play an important role in secondary myogenesis. In the present study the expression of M-cadherin mRNA and protein and its localization were investigated in adult mouse skeletal muscle and peripheral nerve. The mRNA was abundant in embryonic legs from embryonic day (E)14 to E18. It remained expressed in new-born and adult muscles. In the adult muscle M-cadherin immunoreactivity was only detected at the neuromuscular junction, associated with perijunctional mononucleated cells and on intramuscular nerves. Peripheral nerves were also M-cadherin-positive. The molecule was found at the surface of myelinated nerve fibres where it was concentrated at the node of Ranvier. When a nerve was crushed and allowed to regenerate, M-cadherin was over-expressed at the site of nerve injury and in the distal stump. M-cadherin was also upregulated on the sarcolemma of denervated muscle fibres. Taken together, these observations point toward a much wider tissue distribution of M-cadherin than previously thought. M-cadherin might be involved not only in specific steps of myogenesis but also in some aspects of synaptogenesis, axon/Schwann cell interactions and node of Ranvier structural maintenance.     

Polymeric conjugates of Gd(3+)-diethylenetriaminepentaacetic acid and dextran. 2. Influence of spacer arm length and conjugate molecular mass on the paramagnetic properties and some biological parameters

Myoblast transplantation and gene therapy are two promising therapeutical approaches for the treatment of Duchenne Muscular Dystrophy (DMD). So far, both strategies have met many hurdles, mainly because of immune reactions. In this study, we investigated a third and novel strategy based on the combination of these two basic ones, i.e., transplantation of genetically modified myoblasts. We first derived a primary culture from a muscle biopsy of a young DMD patient (3 years old). Adenoviral-mediated dystrophin gene transfer into these DMD cultures and expression of the dystrophin transgene were achieved in vitro. The transduced cultures were then transplanted the same day in immunodeficient SCID mouse muscles. Three weeks following the graft, many human dystrophin-positive fibers were observed throughout sections of the injected muscles. However, many fibers expressed human MHC antigens without expressing human dystrophin due to the low percentage of infected primary muscle cells in vitro (even when a high MOI [400] was used) and to a reduction and even to a complete loss of transgene copy number during myoblast replication. From our results, we conclude that, although not at a high proportion, (1) DMD primary myoblast cultures are infectable by adenoviruses; (2) they can be efficiently transplanted back in a muscle, leading to normal fusion of infected myoblasts with the host fibers; and (3) they can correct the dystrophin deficiency in the host fibers by the expression of a mini-dystrophin transgene.