ANÁLISIS ESTADÍSTICO DE RESULTADOS DEL EXPERIMENTO 1

Utrophin appears to localise to the extrajunctional

sarcolemma of dystrophin deficient muscle in both mouse and man (i.e. tissue from DMD patients and m dx mice), in addition

to retaining high levels of expression in the postjunctional sarcolemma [Fardeau et al., 1990, Khurana et al., 1991, Love e t al., 1991, Nguyen thi Man et al., 1991, Takemitsu et al., 1991, Tanaka et al., 1989, 1991, Voit et al., 1991a, Bewick et al.,

1992, Blake et al., 1992, Cox et al., 1993b, Karpati et al.,

1993a, Takemitsu et al., 1993, Mizuno et al., 1993, Zhao et al., 1993]. In BMD the strength of expression in the

extrajunctional sarcolemma is variable, but in both DMD and BMD expression was markedly increased, 17 and 15 fold

respectively [Karpati et al., 1993a]. Utrophin also accumulates in the extrajunctional sarcolemma of regenerating muscle fibres, in both dystrophin competent and deficient tissue, (characterised using desmin and neonatal myosin antibodies) [Helliwell et al., 1992b, Karpati et al., 1993a].

Studies of human dystrophin deficient muscle have been carried out on asymptomatic BMD patients. The dystrophin in these patients was demonstrated to be of normal molecular weight but reduced in amount. Immunocytochemical staining of muscle biopsies revealed that dystrophin was detected more weakly than in normal controls and absent in clustered

regenerating fibres [Tachi et al., 1992, 1993]. In the cells that stained weakly for dystrophin, utrophin was detected at the non-junctional sarcolemma with a non-uniform distribution, but, in the dystrophin deficient regenerating fibres, utrophin was detected more strongly. Over expression of utrophin in the regenerating fibres may contribute to the lack of

progressive muscle weakness or atrophy shown by these patients [Tachi et al., 1993].

Utrophin has been detected in muscle from patients with a wide range of muscular disorders apart from DMD/BMD, i.e. limb-girdle dystrophy, myotonic dystrophy, congenital

muscular dystrophy, polymyositis and dermatomyositis, in the latter two diseases utrophin expression is increased 4 and 10 fold respectively [Helliwell et al., 1992b, Karpati et al.,

1993a] (It must be noted that Karpati et al. did not detect extrajunctional sarcolemmal expression of utrophin in

biopsies from patients with limb-girdle dystrophy which may be due to the sensitivity of the detection system). It should

also be noted that, In a wide range of muscle disorders,

Helliwell et al. [1992b] came to the conclusion that there was no proportional correlation between the levels of expression of DMD and DMDL (UTRN) mRNA and protein. Interestingly,

utrophin can occasionally be detected at the periphery of

larger morphologically normal fibres that are close to a site of damage in diseased muscle. This could be due to previous

episodes of regeneration or it may be that utrophin is

expressed in response to stress [Helliwell at a/., 1992b]. In addition, more recent work has demonstrated that in intrafusal muscle fibres from DMD patients, where no necrosis or

regeneration is ever observed, utrophin is still detected in the extrajunctional sarcolemma [Karpati at a/., 1993a]. However, no extrajunctional sarcolemmal utrophin was detected in other diseases where regenerating fibres were not present, for

example, M^Ardle's disease, congenital myopathies and

facioscapulohumeral dystrophy. It has been suggested that the presence of utrophin in the extrajunctional sarcolemma of regenerating muscle may occur because a foetal pattern of expression has been reactivated [Khurana at a!., 1991,

Takemitsu at a/., 1991, Helliwell at a!., 1992b] or that it is due to a non-specific stress reaction [Karpati at a!., 1993a]. The argument for a non-specific reaction is based on studies of other proteins, for example, the class 1 major

histocompatibility complex protein products are not expressed at the sarcolemma of normal muscle fibres but can be detected strongly throughout the sarcolemma in muscle from

polymyositis and dermatomyositis patients and in some cases of DMD [Karpati at a/., 1988b] and a similar pattern has been observed for the heat-shock protein k /y receptor [Hohlfield a t a/., 1991]. However, that there is more specific reactivation of a foetal pattern of expression is supported by the

observation that in dystrophin deficient tissue, higher levels of expression of utrophin are found particularly during foetal and perinatal development [Khurana at a/., 1991, Takemitsu a t a!., 1991] and that utrophin is expressed at the extrajunctional sarcolemma in foetal tissues of normal embryonic muscle fibres [Khurana at a/., 1991, Takemitsu at a!., 1991, Nguyen thi

Man ef a/., 1991, Clerk et al., 1993]. Interestingly, analysis of foetal mouse tissue in m dx and normal mice has indicated that utrophin expression at the sarcolemma persists from foetal to adult tissue in the case of dystrophin deficient muscle but is not detected, apart from at the NMJ, in normal adult mouse muscle tissue [Khurana at a!., 1991, Nguyen thi Man at a!.,

1991, Ohiendieck at a!., 1991d, Tanaka at a!., 1991, Takemitsu at a!., 1991, Helliwell at a!., 1992b, Zhao at a!., 1992, 1993]. In addition, it has been suggested that the amount of utrophin expressed in DMD and m dx muscle may decrease from its elevated state, with age and therefore may contribute to the progression of the disease [Mitzuno at a!., 1993].

The ability of utrophin to localise to the extrajunctional sarcolemmal membrane and the observation that it is

extracted from the membrane by p H II treatment, in a similar way to dystrophin implies that it may have a similar

cytoskeletal role to dystrophin [Khurana at a!., 1990, Nguyen thi Man at a!., 1992, Matsumura at a!., 1993e]. Since current theory suggests that dystrophin is functional as a homodimer [Ervasti at a!., 1990], this raises the questions of whether utrophin can also exist as a dimer and whether it can function as a heterodimer with dystrophin. It has been suggested that in the case of female DMD-carrier muscle fibres where there is a mixture of normal and abnormal dystrophin, both

dystrophin and utrophin may mutually diffuse into neighbouring regions and the sarcolemma may be undercoated with both proteins. This may lead to the discontinuous or punctate staining seen using either anti-dystrophin or anti-utrophin antibodies [Mizuno at a!., 1993]. Furthermore, utrophin

associates with an identical or antigenically similar complex of membrane bound glycoproteins to dystrophin (dystrophin associated glycoproteins or DAPs) [Campbell at a!., 1989, Ervasti at a!., 1990, 1991a, Matsumura at a!., 1992a, Mizuno a t a!., 1993]. Utilising immunofluorescent labelled antibodies it has been demonstrated that utrophin and DAPs colocalise to the neuromuscular junction in DMD and in mdx muscle. In addition, DAPs are greatly reduced in the extrajunctional sarcolemma of dystrophin deficient muscle tissue but are not

reduced In neuromuscular junctions (NMJ) [Campbell et al., 1989, Ervasti et al., 1990, Matsumura et al., 1992a].

Significantly the NMJ Is the major site of accumulation of utrophin In normal skeletal muscle [Fardeau et al., 1990, Khurana et al., 1991, Love et al., 1991, Nguyen thi Man et al., 1991, Ohiendieck et al., 1991d, Pons et a i, 1991, Volt et al., 1991a, Takemitsu et al., 1991, Bewick et al., 1992, Helliwell et al., 1992b, Zhao et al., 1992, Fabbrizio et al., 1993b,c, Karpati et al., 1993a, Zhao et al., 1993]. Utrophin was

demonstrated to co-separate with a small fraction of DAPs from mdx muscle, using a sucrose gradient followed by Immunoprécipitation of the fractions with antl-dystrophin associated glycoproteins (DAG) and antl-utrophin antibodies [Campbell et al., 1989, Ervasti et al., 1990, Matsumura et al., 1992a]. In mdx muscle. It appears that utrophin forms a

similar sized complex with the membrane bound glycoproteins, as compared to dystrophin. However a large proportion of DAPs which normally bind to dystrophin are unassociated, this may be due to utrophin being less abundant In muscle than dystrophin Is In normal muscle, or, that utrophin has a lower affinity for DAPs than dystrophin. Interestingly, In m dx cardiac muscle, which shows no dysfunction In the mouse [Torres et al., 1987], there Is a fourfold Increase of utrophin expression and a retention of the largest DAP (glycoprotein

156DAG) to near normal levels. In contrast. In m dx quadriceps, which show some dysfunction In the mouse

[Karpati et al., 1988a], there Is a 1.3 fold increase In utrophin expression but a drastic reduction In the 156DAG [Matsumura et al., 1992a, Cox et al., 1993b].

Thus It has been proposed that. In addition to Its normal role in the cell, up regulation of DMDL (UTRN) and binding of utrophin to DAPs, may contribute to limitation of muscle damage In the dystrophin deficient mouse [Matsumura et al., 1992a, Karpati et al., 1993a, Mitzuno et al., 1993, Tachi et al., 1993]. In other words utrophin may in some way compensate for the loss of dystrophin [Khurana et al., 1991, Nguyen thi Man et al., 1991, Takematsu et al., 1991, Tanaka et al., 1991,

Matsumura ef a i, 1992a, Karpati et al., 1993a, Takemitsu a t a i, 1993, Zhao at ai., 1993].

interestingly, the highest levels of dystrophin are found in skeletal muscle and the highest levels of utrophin are found in lung [Monaco at a i, 1986, Koenig at a i, 1987, Chamberlain a t a i, 1988, Nudel at a i, 1988, Matsumura at a i, 1993e]. Both these tissues are highly stretched and need to be elastic and strong, both dystrophin and utrophin could be playing similar roles stabilising membranes under mechanical stress [Ervasti at a i, 1991a, Ibraghimov-Beskrovnaya at a i, 1992, Matsumura at a i, 1993e].

5.3 THE PRIMARY STRUCTURE OF DMDL (UTRN) AND A