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Localization of dystrophin in the Purkinje cells of normal mice
Neurosciencc L~,tters. 137 (1992) 105 108
105
Elsevier Scientific Publishers Ireland Ltd.
NSL 08472
Localization of dystrophin in the Purkinje cells of normal mice J o h n n y H u a r d and J a c q u e s P. T r e m b l a y Neurohiology Laborato~ T, Lavol University, Enlant-J&u.v Ho,~pital, Que. f ('ana&~ ) (Received 23 September 1991 : Revised version received 25 November 1991 : Accepted 25 November 1991 Key words:
Dystrophm: Normal mouse; Duchenne muscular dystrophy; Purkinje cell; m~tv mouse; hnmunocytochemistry: h n n l u n o b l o u i n g
A monoclonal antibody that reacls with a mid rod fragment of dystrophin was used to localize this protein in the central nervous syslem (CNS). Due to a low abundance of dysirophin in the CNS. an immunoperoxidase reaction amplified with a biotin avidin s_~slem ~ a s n s c d All Purkinjc cells in normal mice were dystrophin positive while the mdx mouse cerebellum was completely devoid of reaction. Dystrophin slaining was present in the soma and dendrites of Purkinje cells but not in their axons. This uniform dystrophin labelling in the normal mouse Purkinje cells indicates thai this protein is not only localized in synaptic contact regions of the CNS.
Duchenne muscular dystrophy (DMD) has been characterized by a lack of dystrophin expression in the membrane of muscle fibers [1 3, 5, 10, 11, 15, 20, 22]. This protein may have a role in maintaining muscle fiber membrane integrity [1, 19 -22]. Mdx mice provide a good animal model for D M D because these mice also have a genetic alteration which prevents dystrophin presence in their sarcolemma [1, 9, 10]. Dystrophin transcripts have also been reported in the CNS [6, 7, 18] and recently Lidov et al. [14] observed the presence of dystrophin in Purkinje cells of normal mice. The present study was aimed at investigating in more detail the distribution of dystrophin in the cerebellum of normal mice and to verify its absence in the cerebellum of mdx mice. The immunoperoxidase staining technique amplified with a biotin avidin system was used to increase the low dystrophin signal in the cerebellum. The cell body and dendritic tree of Purkinje cells in normal mice were dystrophin positive but their axons were not stained. The staining obtained was quite intense and revealed that dystrophin was uniformly distributed in the perikarya and not associated only with synaptic contacts as previously reported by Lidov et al. [14]. Normal and mdx mice were perfused with phosphatebuffered saline (PBS). In each case, a muscle sample, the extensor digitorum longus (EDL), and the brain were removed. The muscle was placed in a 30% sucrose solution overnight at 4°C and was frozen in liquid nitrogen to obtain cryostat sections (8 #in). The cerebellum was Correspomtence. P. Tremblay, Laboratoire de Ncurobiologie, H6pital de I'Enfant-Jdsus, 1401, 1Be Rue, Quc., GIJ IZ4, Canada.
dissected out and fixed by immersion in 2% paraformaldehyde in PBS for 2 h. It was then transferred to a 30% sucrose solution overnight at 4°C. The cerebellum pieces were cooled to -20°C in 30% sucrose. Cryostat sections (12#m) were blocked with 10% horse serum for 1 h. The sections were then incubated with the NCLDysl monoclonal antibody (against dystrophin's rod domain within amino acid 1180-1388) 1:30 in PBS overnight at 4°C (Novo Castra Labs, Newcastle upon Tyne). The sections were rinsed several times in PBS and reincubated with a horse antimouse antibody conjugated with biotin 1:400 (3.75 mg/ml) in PBS for 1 h (Dako, Denmark). Following several rinses, the sections were incubated with avidin conjugated with horseradish peroxidase (HRP) (ABC Elite kit, Dako, Denmark) tk~r 45 min in PBS. The peroxidase activity was revealed with 3,3'-diaminobenzidine 0.5 mg/ml and 0.015% hydrogen peroxide for approximately 7 min. For immunoblotting, small pieces of muscle and cerebellum were removed betk~re fixation and homogenized in a buffer containing 50 mM Tris HC1 pH 8, 0.3% SDS, 5 mM EDTA. 5 mM EGTA, 40 mM dithiothreilol and 5 #g/ml antipain, 5 #g/ml leupeptin, 5 #g/ml pepstatin and 5 #g/ml phenylmethylsulfonylfluoride (Sigma Co.). The homogenates containing 45 #g of total protein were mixed with a sample buffer consisting of 0.5 mM TrisHCI pH 6.8, 10% glycerol, 1(1% SDS, 5% fl-mercaptoethanol and 0.05% (w/v) Bromophenol blue and were separated in polyacrylamide SDS gels/7% separator gel and 6 % stacking gel). Transblotting was performed onto 0.45 # m nitrocellulose membrane (Biorad Lab) tk)r 12 h at a constant voltage of 70 V at 4°C. Alter transblotting,
I (t6
1
2
3
4
dyslrophin
A
Fig, 1. Dystrophin immunocytochemistry and immunoblotting using the NCLDysl antibody. A illustrates the immunoblottmg obtained with the NCLDysl antibody. A 420 kDa dystrophin band is present in the skeletal muscle (lane I) and the cerebellum (Pane 2) of normal mice. The skeletal muscle (lane 4) and cerebellum (lane 3) were devoid of this protein in mdx mice. A positive immunoperoxidase reaction was detected on the plasma membrane of muscle fibers and on soma and dendrites of Purkinje cells in normal mice (B-D). No dystrophin immunoperoxidase reaction was observed in both tissues of the rndx- mice (C, E). A hematoxylin eosin stain of a section adjacent to that of E permitted to conlirm the presence or" Purkinje cells in the region of" the mdx cerebellum corresponding within the square in E (g). Bars 25//m.
the m e m b r a n e w a s h y b r i d i z e d with the N C L D y s l a n t i b o d y . A s for i m m u m o h i s t o c h e m i s t r y , the n i t r o c e l l u l o s e m e m b r a n e was first i n c u b a t e d w i t h N C L D y s l ([:30) fol-
l o w e d by i n c u b a t i o n with a horse a n t i m o u s e a n t i b o d y c o n j u g a t e d with b i o t i n (3.75 m g / m l in PBS, 1 h). T h i s a n t i b o d y was revealed with s t r e p t a v i d i n c o n j u g a t e d with
107
alkaline phosphatase for 1 h (0.3 #g/ml). The phosphatase activity was revealed with nitroblue tetrazolium chloride NBT (495 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate p Toluidine (BCIP) (165 mg/ml). The specificity of NCLDysl antibody was verified by Western blot analysis (Fig. 1A). NCLDysl recognized a dystrophin band at 420 kDa in the skeletal muscle of a normal mouse. A less intense reaction was also obtained at the 420 kDa band in the normal mouse cerebellum. Nicholson etal. [17] also demonstrated by immunoblof ring the presence of a low dystrophin level in the normal rat and human cerebella with the NCLDysl antibody'. The skeletal muscle and cerebellum of mdv mice were negative for this 420 kDa protein band. A positive immunoperoxidase reaction was obtained with the same antibody on cryostat sections of normal mouse muscle (Fig. 1B). However, the mdx mouse muscle was completely devoid of dystrophin (Fig. IC). In the cerebellum of normal mice, a strong positive reaction in the soma and dendritic tree of Purkinje cells was observed but the axons of these cells were not stained (Fig. ID). The other cerebellar cells remained unstained. The Purkinje cells of mdx mouse was completely devoid of dystrophin (Fig. IE,F). This absence of reaction shows that the protein detected here is truly dystrophin since the mdx mice were completely devoid of this antigen, The CNS has been shown to contain dystrophin related proteins [8, 12, 13], but it seems that these proteins are not recognized by the NCLDysl antibody in the cerebellum. However such dystrophin-like proteins might be recognized in the cerebellum by other anti-dystrophin antibodies. Our immunoperoxidase technique allows the detection of a very low dystrophin signal and will probably permit us, in the near future, to identify other neurons containing this protein. Lidov etal. [14] have also observed, by immunofluorescence, the localization of dystrophin in Purkinje cells. These authors supported by electron microscopic studies suggested that dystrophin was strongly associated only with synaptic contact membranes. Our results by immunoperoxidase show a uniform distribution of dystrophin staining in the soma and dendritic tree of Purkinje cells. Given that the sections are only 12 #m thick, the soma would not appear labelled ifdystrophin was present only on the plasma membrane. Miyatake et al. [16] suggested that since dystrophin has some relationship with actin filaments it could affect cell motility, regulation of cell shape and intracellular transport in a variety of non-muscle cells. The function ofdystrophin in the cerebellum is not yet clear but this structure is strongly implicated in generating smooth coordinated movements. A dystrophin deficit in this region may be responsible for producing some pathological signs such as ataxia and tremor. In
fact, Bulfield et al. [4] have observed mild incoordinations and tremors in older mdx mice. However, in DMD patients none of these symptoms have been reported. We wish to thank Ms Lyse Laroche for secretarial assistance and Mr Franqois Tardif for technical assistance. This work was supported by a grant from the Medical Research Council of Canada and by Muscular Dystrophy Association of Canada. 1 Arahata. K.. Ishiura, S., lshiguro, T., Tsukahara, T,, Suhara, Y., Eguchi, C., lshihara, T., Nonaka, I., Ozawa, E. and Sugita, H., Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchcnne muscular dystrophy peptide, Nature, 333 (1988) 861-863. 2 Beam, K.G.. Duchenne muscular dystrophy. Localizing the gene product, Nature, 333 (1988) 798 799. 3 Bonilla, E.. Samiu, C.E., Miranda, A.F., Hays, A.P., Salviati, G., Dimauro, S., Kunkel. L.M., Hoffman, E.P. and Rowland, L.R, Duehennc muscular dystrophy: deficiency ofdystrophin at the muscle cell surface, Cell, 54 (1988) 447 452. 4 Bulfield, G., Sillcr, W.G,, Wight, P.A. and Moore, K., X-chromosome-linked muscular dystrophy (mdx)in tile mouse, Proc. Natl. Acad. Sci. U.S.A..81 (1984) 1189 1192. 5 Carpenter, S., Karpati, G., Zubrzycka-Gaarn, E.. Bulman, D.E., Ray, P.N. and Worton, R.G., Dystrophin is localized to the plasma membrane of human skeletal muscle libers by electron microscopic eytochemical stud),, Muscle Nerve, 13 (1990)(376 380. 6 Chamberlain. J.S., Pearlman, J.A.. Muzny, D.M., Gibbs, R.S., Ranier, J.E., Reeves. A.A. and Caskey, C.T.. Expression of the murine Duchenne muscular dystrophy gene in muscle and brain, Science, 239 (1988) 1416 1418. 7 Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A.. Transcription of the dystrophin gene in human muscle and nonmuscle tissues, Nature. 333 (19881 858 860. 8 Clerk, A., Muntoni, F. and Strong. P., Dystrophin and dystrophinlike proteins in muscle and brain of normal and mdx mice, Biochem. Soc. Trans., 18 (1990) 388 389. 9 Hoffman. E.P.. Brown, R.H. and Kunkcl, L.M., Dystrophin of the protein product of the Duchcnne muscular dystrophy locus, Cell, 5 [ (1987) 919 928. 10 Hoffman, E.P,. Knudson, C.M.. Campbell, K.P. and Kunkel, L.M.. Subcellular fractionation of dystrophin to the triads of skeletal muscle, Nature, 330 (1987) 754 758. I 1 Hoffman, E.P.. Hudecki, M.S.. Rosenbcrg, P.A., Pollina. C.M. and Kunkel, LM.. Ceil and fiber-type distribution of dystrophin, Neuron, 1(1988) 411 420. 12 lshiura, S., Arahata, K.. Tsukahara. T., Koga, R,, Anraku, H., Yamaguchi, M.. Kikuchi, T., Nonaka, L. and Sugita, H., Antibody against the C-terminal portion of dystrophin cross reacts with the 400 Kda protein in the pia mater of dystrophin-delicient mdx mouse brain, J. Biochem., 107 (1990) 510 513. 13 JunE, D., Pons, F., Edger, J.J., Aunis, D. and Rendon. A., Dystrophin in central nervous system: a developmental, regional distribution and subeellular localization study, Neurosc. Len., 124 (1991)87 91. 14 Lidov, H.G.W., Byers, T.J., Watkins. S.C. and Kunkel L.M., Localization of dystrophin to postsynaptic regions oi central nervous system cortical neurons. Nature 348 (1990) 725 727. 15 Miranda, A.F., Bonilla, E., Mastucci, G.. Moraes, C.T., Hays. A.P. and Dimauro, S., lmmunocytochemical study ofdystrophin in mus-
Ills
16
17
18
19
cle from patients with Duchenne muscular dystrophy and unaffected control patiens, Am. J. Pathol.. 132 (1988) 410 416. Miyatake, M., Miike, T.. Zhao, J.E., Yoshioka. K.. Uchino, M. and Usuku. G., Dystrophin localization and presumed function, Muscle Nerve. 14(1991) 113 119. Nicholson, L.~6B., Davison, K., Falkons, G., Harwood, C . O'Donnell, E., Slates, C.R. and Harris. J.B.. Dystrophin in skeletal muscle. Western blot analysis using a monoclonal antibody, ,I. Neurol. Sci., 94 {1989) 125-136. Nudel, U., Robzyk, K. and Yaffe, D., Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain. Nature, 331 (1988) 635 638. Salviati, G., Belto, R., Geoldo, S., Biasia, E., Bonilla, E., Miranda. A.F. and Dimauro, S., Cell fractionation studies indicate that dystrophin is a protein of surface of skeletal muscle, Biochem. J., 258 (1989) 837 841.
20 Sugita, H., Arahata, K., Ishiguro. E, Suhav~. Y., Tsukahara, I lshiura, S., Eguchi, L., Nonaka, [. and Oza,*a~ E.. Negative immu, nostaining of Duchenne muscular d~strophy (DMD) and mdx muscle surf'ace membrane with antibody against synthetic peptidc fragment predicted l'rom DMD eDNA.. Proc. Jpn Acad., 64 t1988) 37 39. 21 Watkins, S.C., Hoffman, E,P., Slayter, H.S. and Kunkel, I_,.M., lmmunoelectron microscopic localization of dystrophin in myofibres, Nature, 333 (1988) 863-866. 22 Zubrzycka-Gaarn, E.E., Bulman, D.E., Karpati, G., Burghes, A.H.M., Belfall, B., Klamut, H.J., Talbot, J., Hodges, R.S., Ray, P.N. and Wortom R.G., The Duchenne muscular dystrophy gene is localized in the sarcolemma of human skeletal muscle, Nature, 333 (1988) 466 469,
105
Elsevier Scientific Publishers Ireland Ltd.
NSL 08472
Localization of dystrophin in the Purkinje cells of normal mice J o h n n y H u a r d and J a c q u e s P. T r e m b l a y Neurohiology Laborato~ T, Lavol University, Enlant-J&u.v Ho,~pital, Que. f ('ana&~ ) (Received 23 September 1991 : Revised version received 25 November 1991 : Accepted 25 November 1991 Key words:
Dystrophm: Normal mouse; Duchenne muscular dystrophy; Purkinje cell; m~tv mouse; hnmunocytochemistry: h n n l u n o b l o u i n g
A monoclonal antibody that reacls with a mid rod fragment of dystrophin was used to localize this protein in the central nervous syslem (CNS). Due to a low abundance of dysirophin in the CNS. an immunoperoxidase reaction amplified with a biotin avidin s_~slem ~ a s n s c d All Purkinjc cells in normal mice were dystrophin positive while the mdx mouse cerebellum was completely devoid of reaction. Dystrophin slaining was present in the soma and dendrites of Purkinje cells but not in their axons. This uniform dystrophin labelling in the normal mouse Purkinje cells indicates thai this protein is not only localized in synaptic contact regions of the CNS.
Duchenne muscular dystrophy (DMD) has been characterized by a lack of dystrophin expression in the membrane of muscle fibers [1 3, 5, 10, 11, 15, 20, 22]. This protein may have a role in maintaining muscle fiber membrane integrity [1, 19 -22]. Mdx mice provide a good animal model for D M D because these mice also have a genetic alteration which prevents dystrophin presence in their sarcolemma [1, 9, 10]. Dystrophin transcripts have also been reported in the CNS [6, 7, 18] and recently Lidov et al. [14] observed the presence of dystrophin in Purkinje cells of normal mice. The present study was aimed at investigating in more detail the distribution of dystrophin in the cerebellum of normal mice and to verify its absence in the cerebellum of mdx mice. The immunoperoxidase staining technique amplified with a biotin avidin system was used to increase the low dystrophin signal in the cerebellum. The cell body and dendritic tree of Purkinje cells in normal mice were dystrophin positive but their axons were not stained. The staining obtained was quite intense and revealed that dystrophin was uniformly distributed in the perikarya and not associated only with synaptic contacts as previously reported by Lidov et al. [14]. Normal and mdx mice were perfused with phosphatebuffered saline (PBS). In each case, a muscle sample, the extensor digitorum longus (EDL), and the brain were removed. The muscle was placed in a 30% sucrose solution overnight at 4°C and was frozen in liquid nitrogen to obtain cryostat sections (8 #in). The cerebellum was Correspomtence. P. Tremblay, Laboratoire de Ncurobiologie, H6pital de I'Enfant-Jdsus, 1401, 1Be Rue, Quc., GIJ IZ4, Canada.
dissected out and fixed by immersion in 2% paraformaldehyde in PBS for 2 h. It was then transferred to a 30% sucrose solution overnight at 4°C. The cerebellum pieces were cooled to -20°C in 30% sucrose. Cryostat sections (12#m) were blocked with 10% horse serum for 1 h. The sections were then incubated with the NCLDysl monoclonal antibody (against dystrophin's rod domain within amino acid 1180-1388) 1:30 in PBS overnight at 4°C (Novo Castra Labs, Newcastle upon Tyne). The sections were rinsed several times in PBS and reincubated with a horse antimouse antibody conjugated with biotin 1:400 (3.75 mg/ml) in PBS for 1 h (Dako, Denmark). Following several rinses, the sections were incubated with avidin conjugated with horseradish peroxidase (HRP) (ABC Elite kit, Dako, Denmark) tk~r 45 min in PBS. The peroxidase activity was revealed with 3,3'-diaminobenzidine 0.5 mg/ml and 0.015% hydrogen peroxide for approximately 7 min. For immunoblotting, small pieces of muscle and cerebellum were removed betk~re fixation and homogenized in a buffer containing 50 mM Tris HC1 pH 8, 0.3% SDS, 5 mM EDTA. 5 mM EGTA, 40 mM dithiothreilol and 5 #g/ml antipain, 5 #g/ml leupeptin, 5 #g/ml pepstatin and 5 #g/ml phenylmethylsulfonylfluoride (Sigma Co.). The homogenates containing 45 #g of total protein were mixed with a sample buffer consisting of 0.5 mM TrisHCI pH 6.8, 10% glycerol, 1(1% SDS, 5% fl-mercaptoethanol and 0.05% (w/v) Bromophenol blue and were separated in polyacrylamide SDS gels/7% separator gel and 6 % stacking gel). Transblotting was performed onto 0.45 # m nitrocellulose membrane (Biorad Lab) tk)r 12 h at a constant voltage of 70 V at 4°C. Alter transblotting,
I (t6
1
2
3
4
dyslrophin
A
Fig, 1. Dystrophin immunocytochemistry and immunoblotting using the NCLDysl antibody. A illustrates the immunoblottmg obtained with the NCLDysl antibody. A 420 kDa dystrophin band is present in the skeletal muscle (lane I) and the cerebellum (Pane 2) of normal mice. The skeletal muscle (lane 4) and cerebellum (lane 3) were devoid of this protein in mdx mice. A positive immunoperoxidase reaction was detected on the plasma membrane of muscle fibers and on soma and dendrites of Purkinje cells in normal mice (B-D). No dystrophin immunoperoxidase reaction was observed in both tissues of the rndx- mice (C, E). A hematoxylin eosin stain of a section adjacent to that of E permitted to conlirm the presence or" Purkinje cells in the region of" the mdx cerebellum corresponding within the square in E (g). Bars 25//m.
the m e m b r a n e w a s h y b r i d i z e d with the N C L D y s l a n t i b o d y . A s for i m m u m o h i s t o c h e m i s t r y , the n i t r o c e l l u l o s e m e m b r a n e was first i n c u b a t e d w i t h N C L D y s l ([:30) fol-
l o w e d by i n c u b a t i o n with a horse a n t i m o u s e a n t i b o d y c o n j u g a t e d with b i o t i n (3.75 m g / m l in PBS, 1 h). T h i s a n t i b o d y was revealed with s t r e p t a v i d i n c o n j u g a t e d with
107
alkaline phosphatase for 1 h (0.3 #g/ml). The phosphatase activity was revealed with nitroblue tetrazolium chloride NBT (495 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate p Toluidine (BCIP) (165 mg/ml). The specificity of NCLDysl antibody was verified by Western blot analysis (Fig. 1A). NCLDysl recognized a dystrophin band at 420 kDa in the skeletal muscle of a normal mouse. A less intense reaction was also obtained at the 420 kDa band in the normal mouse cerebellum. Nicholson etal. [17] also demonstrated by immunoblof ring the presence of a low dystrophin level in the normal rat and human cerebella with the NCLDysl antibody'. The skeletal muscle and cerebellum of mdv mice were negative for this 420 kDa protein band. A positive immunoperoxidase reaction was obtained with the same antibody on cryostat sections of normal mouse muscle (Fig. 1B). However, the mdx mouse muscle was completely devoid of dystrophin (Fig. IC). In the cerebellum of normal mice, a strong positive reaction in the soma and dendritic tree of Purkinje cells was observed but the axons of these cells were not stained (Fig. ID). The other cerebellar cells remained unstained. The Purkinje cells of mdx mouse was completely devoid of dystrophin (Fig. IE,F). This absence of reaction shows that the protein detected here is truly dystrophin since the mdx mice were completely devoid of this antigen, The CNS has been shown to contain dystrophin related proteins [8, 12, 13], but it seems that these proteins are not recognized by the NCLDysl antibody in the cerebellum. However such dystrophin-like proteins might be recognized in the cerebellum by other anti-dystrophin antibodies. Our immunoperoxidase technique allows the detection of a very low dystrophin signal and will probably permit us, in the near future, to identify other neurons containing this protein. Lidov etal. [14] have also observed, by immunofluorescence, the localization of dystrophin in Purkinje cells. These authors supported by electron microscopic studies suggested that dystrophin was strongly associated only with synaptic contact membranes. Our results by immunoperoxidase show a uniform distribution of dystrophin staining in the soma and dendritic tree of Purkinje cells. Given that the sections are only 12 #m thick, the soma would not appear labelled ifdystrophin was present only on the plasma membrane. Miyatake et al. [16] suggested that since dystrophin has some relationship with actin filaments it could affect cell motility, regulation of cell shape and intracellular transport in a variety of non-muscle cells. The function ofdystrophin in the cerebellum is not yet clear but this structure is strongly implicated in generating smooth coordinated movements. A dystrophin deficit in this region may be responsible for producing some pathological signs such as ataxia and tremor. In
fact, Bulfield et al. [4] have observed mild incoordinations and tremors in older mdx mice. However, in DMD patients none of these symptoms have been reported. We wish to thank Ms Lyse Laroche for secretarial assistance and Mr Franqois Tardif for technical assistance. This work was supported by a grant from the Medical Research Council of Canada and by Muscular Dystrophy Association of Canada. 1 Arahata. K.. Ishiura, S., lshiguro, T., Tsukahara, T,, Suhara, Y., Eguchi, C., lshihara, T., Nonaka, I., Ozawa, E. and Sugita, H., Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchcnne muscular dystrophy peptide, Nature, 333 (1988) 861-863. 2 Beam, K.G.. Duchenne muscular dystrophy. Localizing the gene product, Nature, 333 (1988) 798 799. 3 Bonilla, E.. Samiu, C.E., Miranda, A.F., Hays, A.P., Salviati, G., Dimauro, S., Kunkel. L.M., Hoffman, E.P. and Rowland, L.R, Duehennc muscular dystrophy: deficiency ofdystrophin at the muscle cell surface, Cell, 54 (1988) 447 452. 4 Bulfield, G., Sillcr, W.G,, Wight, P.A. and Moore, K., X-chromosome-linked muscular dystrophy (mdx)in tile mouse, Proc. Natl. Acad. Sci. U.S.A..81 (1984) 1189 1192. 5 Carpenter, S., Karpati, G., Zubrzycka-Gaarn, E.. Bulman, D.E., Ray, P.N. and Worton, R.G., Dystrophin is localized to the plasma membrane of human skeletal muscle libers by electron microscopic eytochemical stud),, Muscle Nerve, 13 (1990)(376 380. 6 Chamberlain. J.S., Pearlman, J.A.. Muzny, D.M., Gibbs, R.S., Ranier, J.E., Reeves. A.A. and Caskey, C.T.. Expression of the murine Duchenne muscular dystrophy gene in muscle and brain, Science, 239 (1988) 1416 1418. 7 Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A.. Transcription of the dystrophin gene in human muscle and nonmuscle tissues, Nature. 333 (19881 858 860. 8 Clerk, A., Muntoni, F. and Strong. P., Dystrophin and dystrophinlike proteins in muscle and brain of normal and mdx mice, Biochem. Soc. Trans., 18 (1990) 388 389. 9 Hoffman. E.P.. Brown, R.H. and Kunkcl, L.M., Dystrophin of the protein product of the Duchcnne muscular dystrophy locus, Cell, 5 [ (1987) 919 928. 10 Hoffman, E.P,. Knudson, C.M.. Campbell, K.P. and Kunkel, L.M.. Subcellular fractionation of dystrophin to the triads of skeletal muscle, Nature, 330 (1987) 754 758. I 1 Hoffman, E.P.. Hudecki, M.S.. Rosenbcrg, P.A., Pollina. C.M. and Kunkel, LM.. Ceil and fiber-type distribution of dystrophin, Neuron, 1(1988) 411 420. 12 lshiura, S., Arahata, K.. Tsukahara. T., Koga, R,, Anraku, H., Yamaguchi, M.. Kikuchi, T., Nonaka, L. and Sugita, H., Antibody against the C-terminal portion of dystrophin cross reacts with the 400 Kda protein in the pia mater of dystrophin-delicient mdx mouse brain, J. Biochem., 107 (1990) 510 513. 13 JunE, D., Pons, F., Edger, J.J., Aunis, D. and Rendon. A., Dystrophin in central nervous system: a developmental, regional distribution and subeellular localization study, Neurosc. Len., 124 (1991)87 91. 14 Lidov, H.G.W., Byers, T.J., Watkins. S.C. and Kunkel L.M., Localization of dystrophin to postsynaptic regions oi central nervous system cortical neurons. Nature 348 (1990) 725 727. 15 Miranda, A.F., Bonilla, E., Mastucci, G.. Moraes, C.T., Hays. A.P. and Dimauro, S., lmmunocytochemical study ofdystrophin in mus-
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17
18
19
cle from patients with Duchenne muscular dystrophy and unaffected control patiens, Am. J. Pathol.. 132 (1988) 410 416. Miyatake, M., Miike, T.. Zhao, J.E., Yoshioka. K.. Uchino, M. and Usuku. G., Dystrophin localization and presumed function, Muscle Nerve. 14(1991) 113 119. Nicholson, L.~6B., Davison, K., Falkons, G., Harwood, C . O'Donnell, E., Slates, C.R. and Harris. J.B.. Dystrophin in skeletal muscle. Western blot analysis using a monoclonal antibody, ,I. Neurol. Sci., 94 {1989) 125-136. Nudel, U., Robzyk, K. and Yaffe, D., Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain. Nature, 331 (1988) 635 638. Salviati, G., Belto, R., Geoldo, S., Biasia, E., Bonilla, E., Miranda. A.F. and Dimauro, S., Cell fractionation studies indicate that dystrophin is a protein of surface of skeletal muscle, Biochem. J., 258 (1989) 837 841.
20 Sugita, H., Arahata, K., Ishiguro. E, Suhav~. Y., Tsukahara, I lshiura, S., Eguchi, L., Nonaka, [. and Oza,*a~ E.. Negative immu, nostaining of Duchenne muscular d~strophy (DMD) and mdx muscle surf'ace membrane with antibody against synthetic peptidc fragment predicted l'rom DMD eDNA.. Proc. Jpn Acad., 64 t1988) 37 39. 21 Watkins, S.C., Hoffman, E,P., Slayter, H.S. and Kunkel, I_,.M., lmmunoelectron microscopic localization of dystrophin in myofibres, Nature, 333 (1988) 863-866. 22 Zubrzycka-Gaarn, E.E., Bulman, D.E., Karpati, G., Burghes, A.H.M., Belfall, B., Klamut, H.J., Talbot, J., Hodges, R.S., Ray, P.N. and Wortom R.G., The Duchenne muscular dystrophy gene is localized in the sarcolemma of human skeletal muscle, Nature, 333 (1988) 466 469,