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Utrophin: A potential replacement for dystrophin?
Neuromusc. Disord., Vol. 3, No. 5/6, pp. 537 539, 1993
Copyright ~" 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0960-8966/93 $6.00+ .00
Pergamon
UTROPHIN:
A POTENTIAL
REPLACEMENT
JONATHON M . TINSLEY a n d
FOR
DYSTROPHIN?
KAY E. DAVIES
M o l e c u l a r G e n e t i c s G r o u p , Institute of M o l e c u l a r Medicine, J o h n Radcliffe Hospital, H e a d i n g t o n , O x f o r d OX3 9 D U , U.K.
A~tract--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. Key words: Utrophin, dystrophin, DMD, gene therapy.
dystrophin and the DAG complex have recently been reviewed [1-3]. There are various approaches which can be adopted for the gene therapy of DMD. These include myoblast transfer, retroviral infection, adenoviral infection and direct injection of plasmid DNA. Although some progress is being made in each of these areas using the mdx mouse as a model system, there are considerable problems related to the number of muscle cells that can be made dystrophin-positive, the levels of expression of the gene and the duration of the expression. In order to circumvent some of these problems we are exploring the possibility of compensating for dystrophin loss by related proteins. This would be an alternative route to gene therapy for sufferers of DMD or BMD, which would be effective in all muscles. A similar strategy is currently being evaluated in clinical trials to upregulate fetal haemoglobin to compensate for the affected adult-globin chains in patients with sickle cell anaemia [4, 5]. Utrophin (dystrophin related protein, DRP) is a 395 kDa protein encoded by a gene located on chromosome 6q24 and has been shown to have strong sequence similarity to dystrophin [1, 6]. The actin-binding domains of dystrophin and utrophin has 85% similarity and the putative DAG binding regions have 88% similarity. Matsumara et al. [7] demonstrated that purified membranes from the mdx mouse contained complexes of utrophin and the DAGs. When quadricep muscles (which show necrosis) from these mice were analysed by immunoblotting, the level of utrophin remained approximately the same but the level of the 156DAG was drastically reduced. In cardiac muscle (which shows no pathology) the level of utrophin was elevated
INTRODUCTION
The severe muscle wasting disorders, Duchenne muscular dystrophy (DMD) and the less debilitating Becker muscular dystrophy (BMD) are due to mutations in the dystrophin gene resulting in a lack ofdystrophin or abnormal expression of truncated forms of dystrophin, respectively. Dystrophin is a large cytoskeletal protein (427 kDa with a length of 125 nm) which, in muscle, is located at the cytoplasmic surface of the sarcolemma, the neuromuscular junction (NMJ) and myotendinous junction (MTJ). The protein is composed of four domains: an actinbinding domain (shown in vitro to bind actin), a rod domain containing triple helical repeats, a cysteine-rich domain and a carboxy-terminal domain. The majority of the cysteine-rich domain and the first half of the carboxy-terminal domain bind to a complex of proteins and glycoproteins spanning the sarcolemma, called the dystrophin-associated proteins (DAPs) and dystrophin-associated glycoproteins (DAGs). This complex consists of cytoskeletal (59 kDa DAP), transmembrane (50 kDa DAG, 43 kDa DAG, 35 kDa DAG and 25 kDa DAP) and extracellular (156 kDa DAG) components. The complex links the extracellular matrix via the 156DAG binding laminin to the internal cytoskeleton via the 59DAP binding dystrophin. The breakdown of the integrity of this complex due to the loss, or impairment, of dystrophin function, leads to muscle degeneration and the DMD phenotype. The importance of the DAG complex in muscle function is also demonstrated by the fact that loss of only the 50DAG is seen in the severe childhood autosomal recessive muscular dystrophy (SCARMD). The structure of 537
538
J . M . TINSLEY and K. E. DAVIES
four fold, with no loss of the 156DAG. Immunocytochemical analysis of other m d x small calibre skeletal muscles (extraocular and toe), which also have no pathology, shows increased utrophin expression and normal levels of the 50DAG. This result suggests that the increased level of utrophin interacts with the DAG complex (or an antigenically related complex) at the sarcolemma and prevents loss of the complex so that the structure of these cells remains normal. In normal muscle, utrophin is predominantly expressed at the neuromuscular junction, localized closely to the acetylcholine receptors, and the myotendinous junctions. See reviews [1-3] for greater detail about the general tissue distribution and localization of utrophin. There is a substantial body of evidence demonstrating that utrophin is capable of localizing to the sarcolemma. During normal fetal muscle development there is increased utrophin expression, localized to the sarcolemma, during embryonic development up until 18 weeks gestation in humans and 20 days gestation in the mouse. After this time the utrophin sarcolemmal staining steadily decreases to the significantly lower adult levels shortly before birth, where utrophin is localized almost exclusively to the neuromuscular junction [8-10]. The decrease in utrophin expression coincides with the increased expression of dystrophin [10]. In the dystrophin deficient m d x mouse, utrophin levels in muscle remain elevated for longer compared to normal mice; however, once the utrophin levels have decreased to the adult levels (about 1 week after birth), the first signs of muscle fibre necrosis are detected [8, 9]. Preliminary studies have shown that utrophin is bound to the sarcolemma in D M D and BMD patients [11, 12]. Two recent, more comprehensive, studies have analysed a large number of D M D and BMD patients for utrophin expression and in all cases confirmed an increased expression of utrophin with localization at the sarcolemma. Karpati et al. [13] demonstrated by immunoblotting and quantitation, that in 13 D M D and 4 BMD patients, the utrophin levels increased 17 and 15 fold, respectively, over normal levels. Mizuno et al. [14] analysed 39 D M D and I I BMD patients by immunocytochemistry of muscle sections and in all cases demonstrated increased sarcolemmal staining by utrophin. Thus, in certain circumstances utrophin can localize to the sarcolemma probably at the same binding sites as dystrophin, namely actin and the
DAGs. If expression of utrophin is high enough, it may be able to maintain the D A G complex. It may be possible to find some agent to upregulate utrophin similar to the upregulation of fetal haemoglobin using butyrate [5]. It is unlikely that such external upregulation could be tightly controlled, giving rise to potentially high levels of utrophin within the cell. However, this may not be a problem as Cox et al. [15] have demonstrated that gross overexpression of dystrophin in transgenic m d x mice reverts the muscle pathology to normal with no obvious detrimental side effects. In summary, the recent evidence suggests that in certain circumstances utrophin may have the same functions as dystrophin in the muscle cell, but nature is unable to combat the disease by upregulating utrophin. It remains to be seen if external intervention causing either upregulation of utrophin or continued expression of utrophin (once fetal muscle development is complete) will stop the regeneration and necrosis seen in dystrophin deficient muscle. Acknowledgements The authors wish to thank Professor E. Ozawa for providing copies of manuscripts in press. We thank the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association U.S.A. and the Medical Research Council for generous support.
REFERENCES
I.
Tinsley J M, Blake D J, Pearce M, Knight A E, Kendrick-Jones J, Davies K E. Dystrophin and related proteins. Curr Opin Genet Dev 1993; 3: 484~490. 2. Love D R, Byth B C, Tinsley J M, Blake D J, Davies K E. Dystrophin and dystrophin-related proteins: a review of protein and RNA studies. Neuromusc Disord 1993; 3:5 21. 3. Ahn A H, Kunkel L M. The structural and functional diversity ofdystrophin. Nature Genet 1993; 3:283 290. 4. Rodgers G P, Dover G J, Uyesaka N, et al. Augmentation by erythropoitein of the fetal-haemoglobin response to hydroxyurea in sickle cell disease. N Engl J M e d 1993; 328:73 80. 5. Perrine S, Ginder G D, Failer D V, et al. A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders. N Engl J M e d 1993; 328:81 86. 6. Tinsley J M, Blake D J, Roche A, et al. Primary structure of dystrophin-related protein. Nature 1992; 360:591 593. 7. Matsumura K, Ervasti J M, Ohlendieck K, Kahl S D, Campbell K P. Association of the dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 1992; 360: 588-591. 8. Khurana T S, Watkins S C, Chafey P, et al. lmmunolocalization and developmental expression of dystrophin related protein in skeletal muscle. Neuromuse Disord 1991; !: 185-.194. 9. Takemitsu M, Ishiura S, Koga R, et al. Dystrophinrelated protein in the fetal and denervated skeletal muscles of normal and mdx mice. Biochem Biophys Res Commun 1991" 180: 1179-1186.
Utrophin: a Potential Replacement for Dystrophin? 10. Clerk A, Morris G E, Dubowitz V, Davies K E, Sewry C A. Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle. Histochem J 1993; 25: 554-561. 11. Nguyen T M, Ellis J M, Love D R, et al. Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines. J Cell Biol 1991; 115: 1695-1700. 12. Tanaka H, Ishiguro T, Eguchi C, Saito K, Ozawa E. Expression of a dystrophin-related protein associated with the skeletal muscle cell membrane. Histochem J
539
1991; 96: 1-5. 13. Karpati G, Carpenter S, Morris G E, et al. Localisation and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological muscle. J Neuropathol Exp Neurology 1993; 52:119-128. 14. Mizuno Y, Nonaka I, Hiral S, Ozawa E. Reciprocal expression of dystrophin and utrophin in muscles of Duchenne muscular dystrophy patients, female DMDcarriers and control subjects. J Neurol Sci 1993; 119: 4352. 15. Cox G A, Cole N M, Matsumara K, et al. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without cytotoxicity. Nature 1993; 364:725 729.
Copyright ~" 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0960-8966/93 $6.00+ .00
Pergamon
UTROPHIN:
A POTENTIAL
REPLACEMENT
JONATHON M . TINSLEY a n d
FOR
DYSTROPHIN?
KAY E. DAVIES
M o l e c u l a r G e n e t i c s G r o u p , Institute of M o l e c u l a r Medicine, J o h n Radcliffe Hospital, H e a d i n g t o n , O x f o r d OX3 9 D U , U.K.
A~tract--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. Key words: Utrophin, dystrophin, DMD, gene therapy.
dystrophin and the DAG complex have recently been reviewed [1-3]. There are various approaches which can be adopted for the gene therapy of DMD. These include myoblast transfer, retroviral infection, adenoviral infection and direct injection of plasmid DNA. Although some progress is being made in each of these areas using the mdx mouse as a model system, there are considerable problems related to the number of muscle cells that can be made dystrophin-positive, the levels of expression of the gene and the duration of the expression. In order to circumvent some of these problems we are exploring the possibility of compensating for dystrophin loss by related proteins. This would be an alternative route to gene therapy for sufferers of DMD or BMD, which would be effective in all muscles. A similar strategy is currently being evaluated in clinical trials to upregulate fetal haemoglobin to compensate for the affected adult-globin chains in patients with sickle cell anaemia [4, 5]. Utrophin (dystrophin related protein, DRP) is a 395 kDa protein encoded by a gene located on chromosome 6q24 and has been shown to have strong sequence similarity to dystrophin [1, 6]. The actin-binding domains of dystrophin and utrophin has 85% similarity and the putative DAG binding regions have 88% similarity. Matsumara et al. [7] demonstrated that purified membranes from the mdx mouse contained complexes of utrophin and the DAGs. When quadricep muscles (which show necrosis) from these mice were analysed by immunoblotting, the level of utrophin remained approximately the same but the level of the 156DAG was drastically reduced. In cardiac muscle (which shows no pathology) the level of utrophin was elevated
INTRODUCTION
The severe muscle wasting disorders, Duchenne muscular dystrophy (DMD) and the less debilitating Becker muscular dystrophy (BMD) are due to mutations in the dystrophin gene resulting in a lack ofdystrophin or abnormal expression of truncated forms of dystrophin, respectively. Dystrophin is a large cytoskeletal protein (427 kDa with a length of 125 nm) which, in muscle, is located at the cytoplasmic surface of the sarcolemma, the neuromuscular junction (NMJ) and myotendinous junction (MTJ). The protein is composed of four domains: an actinbinding domain (shown in vitro to bind actin), a rod domain containing triple helical repeats, a cysteine-rich domain and a carboxy-terminal domain. The majority of the cysteine-rich domain and the first half of the carboxy-terminal domain bind to a complex of proteins and glycoproteins spanning the sarcolemma, called the dystrophin-associated proteins (DAPs) and dystrophin-associated glycoproteins (DAGs). This complex consists of cytoskeletal (59 kDa DAP), transmembrane (50 kDa DAG, 43 kDa DAG, 35 kDa DAG and 25 kDa DAP) and extracellular (156 kDa DAG) components. The complex links the extracellular matrix via the 156DAG binding laminin to the internal cytoskeleton via the 59DAP binding dystrophin. The breakdown of the integrity of this complex due to the loss, or impairment, of dystrophin function, leads to muscle degeneration and the DMD phenotype. The importance of the DAG complex in muscle function is also demonstrated by the fact that loss of only the 50DAG is seen in the severe childhood autosomal recessive muscular dystrophy (SCARMD). The structure of 537
538
J . M . TINSLEY and K. E. DAVIES
four fold, with no loss of the 156DAG. Immunocytochemical analysis of other m d x small calibre skeletal muscles (extraocular and toe), which also have no pathology, shows increased utrophin expression and normal levels of the 50DAG. This result suggests that the increased level of utrophin interacts with the DAG complex (or an antigenically related complex) at the sarcolemma and prevents loss of the complex so that the structure of these cells remains normal. In normal muscle, utrophin is predominantly expressed at the neuromuscular junction, localized closely to the acetylcholine receptors, and the myotendinous junctions. See reviews [1-3] for greater detail about the general tissue distribution and localization of utrophin. There is a substantial body of evidence demonstrating that utrophin is capable of localizing to the sarcolemma. During normal fetal muscle development there is increased utrophin expression, localized to the sarcolemma, during embryonic development up until 18 weeks gestation in humans and 20 days gestation in the mouse. After this time the utrophin sarcolemmal staining steadily decreases to the significantly lower adult levels shortly before birth, where utrophin is localized almost exclusively to the neuromuscular junction [8-10]. The decrease in utrophin expression coincides with the increased expression of dystrophin [10]. In the dystrophin deficient m d x mouse, utrophin levels in muscle remain elevated for longer compared to normal mice; however, once the utrophin levels have decreased to the adult levels (about 1 week after birth), the first signs of muscle fibre necrosis are detected [8, 9]. Preliminary studies have shown that utrophin is bound to the sarcolemma in D M D and BMD patients [11, 12]. Two recent, more comprehensive, studies have analysed a large number of D M D and BMD patients for utrophin expression and in all cases confirmed an increased expression of utrophin with localization at the sarcolemma. Karpati et al. [13] demonstrated by immunoblotting and quantitation, that in 13 D M D and 4 BMD patients, the utrophin levels increased 17 and 15 fold, respectively, over normal levels. Mizuno et al. [14] analysed 39 D M D and I I BMD patients by immunocytochemistry of muscle sections and in all cases demonstrated increased sarcolemmal staining by utrophin. Thus, in certain circumstances utrophin can localize to the sarcolemma probably at the same binding sites as dystrophin, namely actin and the
DAGs. If expression of utrophin is high enough, it may be able to maintain the D A G complex. It may be possible to find some agent to upregulate utrophin similar to the upregulation of fetal haemoglobin using butyrate [5]. It is unlikely that such external upregulation could be tightly controlled, giving rise to potentially high levels of utrophin within the cell. However, this may not be a problem as Cox et al. [15] have demonstrated that gross overexpression of dystrophin in transgenic m d x mice reverts the muscle pathology to normal with no obvious detrimental side effects. In summary, the recent evidence suggests that in certain circumstances utrophin may have the same functions as dystrophin in the muscle cell, but nature is unable to combat the disease by upregulating utrophin. It remains to be seen if external intervention causing either upregulation of utrophin or continued expression of utrophin (once fetal muscle development is complete) will stop the regeneration and necrosis seen in dystrophin deficient muscle. Acknowledgements The authors wish to thank Professor E. Ozawa for providing copies of manuscripts in press. We thank the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association U.S.A. and the Medical Research Council for generous support.
REFERENCES
I.
Tinsley J M, Blake D J, Pearce M, Knight A E, Kendrick-Jones J, Davies K E. Dystrophin and related proteins. Curr Opin Genet Dev 1993; 3: 484~490. 2. Love D R, Byth B C, Tinsley J M, Blake D J, Davies K E. Dystrophin and dystrophin-related proteins: a review of protein and RNA studies. Neuromusc Disord 1993; 3:5 21. 3. Ahn A H, Kunkel L M. The structural and functional diversity ofdystrophin. Nature Genet 1993; 3:283 290. 4. Rodgers G P, Dover G J, Uyesaka N, et al. Augmentation by erythropoitein of the fetal-haemoglobin response to hydroxyurea in sickle cell disease. N Engl J M e d 1993; 328:73 80. 5. Perrine S, Ginder G D, Failer D V, et al. A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders. N Engl J M e d 1993; 328:81 86. 6. Tinsley J M, Blake D J, Roche A, et al. Primary structure of dystrophin-related protein. Nature 1992; 360:591 593. 7. Matsumura K, Ervasti J M, Ohlendieck K, Kahl S D, Campbell K P. Association of the dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 1992; 360: 588-591. 8. Khurana T S, Watkins S C, Chafey P, et al. lmmunolocalization and developmental expression of dystrophin related protein in skeletal muscle. Neuromuse Disord 1991; !: 185-.194. 9. Takemitsu M, Ishiura S, Koga R, et al. Dystrophinrelated protein in the fetal and denervated skeletal muscles of normal and mdx mice. Biochem Biophys Res Commun 1991" 180: 1179-1186.
Utrophin: a Potential Replacement for Dystrophin? 10. Clerk A, Morris G E, Dubowitz V, Davies K E, Sewry C A. Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle. Histochem J 1993; 25: 554-561. 11. Nguyen T M, Ellis J M, Love D R, et al. Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines. J Cell Biol 1991; 115: 1695-1700. 12. Tanaka H, Ishiguro T, Eguchi C, Saito K, Ozawa E. Expression of a dystrophin-related protein associated with the skeletal muscle cell membrane. Histochem J
539
1991; 96: 1-5. 13. Karpati G, Carpenter S, Morris G E, et al. Localisation and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological muscle. J Neuropathol Exp Neurology 1993; 52:119-128. 14. Mizuno Y, Nonaka I, Hiral S, Ozawa E. Reciprocal expression of dystrophin and utrophin in muscles of Duchenne muscular dystrophy patients, female DMDcarriers and control subjects. J Neurol Sci 1993; 119: 4352. 15. Cox G A, Cole N M, Matsumara K, et al. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without cytotoxicity. Nature 1993; 364:725 729.