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Neuroscience Letters, 124 (1991) 87-91 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439401000127 NSL 07606
Dystrophin in central nervous system: a developmental, regional distribution and subcellular localization study Daniel Jung 1, Franqoise Pons 2'3, Jean J. L6ger ~, Dominique Aunis a and Alvaro R e n d o n 1 1Unitb 1NSERM U-338, Centre de Neurochimie, Strasbourg (France), 2UnitO INSERM U-300, Facult~ de Pharmacie, Montpellier (France) and 31nstitut de Pathologic Gbnbrale, Facultb de Mkdecine, Montpellier (France) (Received 23 November 1990; Revised version received 7 December 1990; Accepted 10 December 1990)
Key words: Dystrophin; Rat brain; mdx mouse; Development; Subeellular localization; Synaptic plasma membrane Dystrophin, the protein encoded by the Duchenne muscular dystrophy gene has been shown to be expressed in central nervous system. In the present study, polyclonal antibodies raised against 3 fusion proteins constructed from different structural domains of dystrophin were used to identify dystrophin in protein extracts from rat and mdx mouse brain. The developmental expression of the protein, its regional distribution in rat brain and its localization in rat brain subcellular fractions were also examined. We found that dystrophin or a 'dystrophin-related protein' is expressed in mdx mouse brain. Dystrophin is detectable at very early stages of rat brain development and is expressed in all adult brain regions examined, although quantitative regional differences were found. Subeellular distribution analysis indicates that dystrophin is absent in mitochondrial and synaptic vesicle-enriched fractions but is recovered in the synaptic plasma membrane fraction.
Dystrophin is the protein product of the Duchenne muscular dystrophy gene. This exceptionally large gene encodes a 14 kb transcript and a protein with a predicted molecular mass of 427 kDa [14, 19]. Initial studies indicated that dystrophin expression was restricted to cardiac and skeletal muscle [24]. However, more recent studies using a variety of genetic and immunological approaches have shown that dystrophin or dystrophinlike proteins are also present in smooth muscle, in nervous system and to a lesser extent in a variety of other non-muscle tissues [6, 12, 25]. The predicted amino acid sequence of dystrophin reveals four distinct domains on the molecule, three of which share various degrees of sequence homology with the cytoskeletal proteins spectrin and ~-actinin. Dystrophin has been localized by immunocytochemistry in the sarcolemma of muscle fibres [1, 29, 30], at the neuromuscular junction in rat and mouse [11, 23] and in the electric organ of Torpedo [5, 18]. In the central nervous system it has been shown that dystrophin expression is mainly neuronal in origin [7, 8, 15]. In the present study, we examined the expression of dystrophin in both rat brain and mdx mouse brain, an X chromosome-linked recessive myopathic mutant which has a nonsense mutation in the mdx locus. The developCorrespondence: A. Rendon, Centre de Neurochimie, U-338 de I'INSERM, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France.
mental expression and the regional and subcellular distribution of dystrophin in rat brain was also studied by immunoblot analysis and immunohistochemistry using polyclonal antibodies raised against fragments from 3 different regions of chicken skeletal muscle dystrophin [2, 211. Wistar rats of various ages and adult mdx mice were used. Animals were killed by decapitation; whole brain, brain regions and skeletal muscle were rapidly dissected and homogenized in isolation buffer (1 mM EDTA-K +, 10 mM Tris-HCl, pH 7.4) containing a mixture of protease inhibitors: p-tosyl, L-arginine methylester, 0.1 rag/ ml; aprotinin, 0.05 U/ml; pepstatin, 1 mM; leupeptin, 1 raM; phenylmethyl sulfonyl fluoride, 1 mM. After homogenization and protein determination [3], extracts were boiled for 5 rain in eleetrophoresis LDS loading buffer [20], centrifuged 10 rain at 12,000 g to eliminate insoluble material and stored at -70°C. Highly purified rat brain mitochondria and synaptic plasma membranes were isolated as described previously [26]. Synaptic vesicle-enriched fraction was isolated from rat brain as described in [16]. Equal amounts of protein (300/tg) were loaded on to each lane and separated on LDS 3.5-12.5% polyacrylamide gradient gels. After eleetrophoresis, separated proteins were transferred electrophoretically to a nitrocellulose membrane [28], immunoreacted with rabbit anti-dystrophin fragment antiserum and devel-
88 a
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Fig. I. Detection of 400 kDa dystrophin in rat brain and in mutant mdx mouse brain by immunoblotting with antiserum H. Lane a: crude extract of normal rat brain; lane b: crude extract of mdx mouse brain; lane c: crude extract of rat skeletal muscle. Asterisk indicates the location of dystrophin immunoreactive band. Size and location of molecular weight markers are indicated.
oped with goat anti-rabbit IgG conjugated to alkaline phosphatase. Indirect immunofluorescence was performed on cryostat sections (8-10/~m) of frozen brain tissue or skeletal muscle as previously described [10]. Anti-dystrophin fragment antisera were raised in rabbits immunized with one of three fusion proteins constructed from different fragments of chicken skeletal muscle dystrophin eDNA and spanning the N-terminal domain (serum A: residues 43-760), a part of the central spectrinlike domain (serum C: residues 1373-1728), and the entire C-terminal domain (serum H: residues 3357-3660) as described elsewhere [2, 21]. Immunoblotting experiments were first performed to reveal the presence of dystrophin in rat adult tissues. As shown in Fig. 1, a strong reactivity with the antiserum raised against the C-terminal end of chicken skeletal muscle dystrophin was found in skeletal muscle crude extract. A band corresponding to a 400 kDa protein was detected, with a molecular mass similar to that described for normal human and mouse muscle tissue extracts. Bands of lower molecular mass were also visible, an indication of endogenous active proteolysis generating
D
Fig. 2. Indirect immunofluorescent detection of dystrophin in muscle and brain of normal and mutant mdx mouse. Frozen (8-10 gm) cryostat sections of normal (A) and mdx (B) gastrocnemius mouse muscle and of normal (C) and mdx (D) mouse hippocampus were immunostained with antiserum H.
89 a
immunoreactive fragments. As shown in Fig. 1 (lane a), a 400 kDa band was also detected in rat brain extract; this band displayed a fainter immunoreactivity representing approximately one tenth of the reactivity found in skeletal muscle. These data are consistent with recent results showing that the level of expression of human dystrophin gene and its transcript are lower in brain than in skeletal muscle [6, 25]. With regard to the recent findings showing the existence of an autosomal gene for dystrophin [22] and the presence in brain of a dystrophin gene promoter which seems to be different from the promoter present in muscle [7], it was interesting to determine whether anti-dystrophin immunoreactivity could be found in the brain of mdx mouse. Surprisingly, we found that the antiserum H raised against the C-terminal end of dystrophin recognizes a band of 400 kDa and that the immunoreactivity pattern associated with smaller protein species was almost identical to that found in the normal rat brain (in Fig. 1, compare lanes a and b). In addition, immunohistochemical study performed on normal and mutant mdx mouse tissues showed an intense fluorescent labelling in skeletal muscle from normal mouse while no immunofluorescence was detectable in skeletal muscle from the mdx mouse (Fig. 2A,B). In contrast, the hippocampus was found to be moderately labelled in both normal and mdx mice (Fig. 2C,D). These results seem to indicate that dystrophin, or a 'dystrophin-related protein', is present in the normal rat and mouse brain and also in the brain of the mdx mouse; this is in apparent contradiction with the absence of dystrophin in mdx mouse brain tissue reported by Hoffman et al. [15]. It is possible that our antiserum H raised against the C-terminal domain of dystrophin is able to cross-react with a family of dystrophin-related proteins, as has been suggested for other antidystrophin antibodies [9, 17]. However as similar immunoreactive patterns were obtained with the two other sera specific to the central part and to the N-terminal domain of dystrophin (data not shown), the immunoreactivity detected in the rndx mouse brain is likely to be specific for the dystrophin molecule. The developmental expression of dystrophin in crude rat brain extracts was further examined. As shown in Fig. 3A, the serum H raised against the C-terminal domain of dystrophin reacts weakly but unambiguously with a protein displaying an electrophoretic mobility similar to that of rat muscle dystrophin. Dystrophin immunoreactivity was detectable at the earliest stage of brain development assayed here (day 18 of gestation) and its level relative to total protein was the same as in the adult. To examine the possibility that dystrophin might be
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Fig. 3. Dystrophin in rat brain. A: dystrophin detection in developing normal rat brain by immunoblotting with antiserum H. Crude rat brain extracts at different ages were tested: 18 days of gestation (a), newborn (b), 10 day-old (c), 15 day-old (d), 21 day-old (e), adult (t'); crude rat skeletal muscle extract (g). Asterisk indicates the location of the 400 kDa immunoreactive dystrophin band. B: regional distribution of dystrophin in normal adult rat brain. After electrophoretic separation and transfer of proteins onto nitrocellulose membrane, immunodetection was carried out with antiserum C and developed with t25I-conjugated goat anti-rabbit IgG. The nitrocellulose membrane was autoradiographed and each 400 kDa dystrophin band was cut out and radioactivity determined. The semi-quantitative assay was normalized by loading each lane with an equal protein amount (300 pg). Values represent dystrophin level expressed as the percentage of that found in whole brain. Data from one of three independent experiments are shown.
preferentially expressed in brain regions, we have determined the relative levels of dystrophin in ten regions dissected from adult rat brain. As shown in Fig. 3B, the levels of dystrophin expressed relative to the protein in whole brain were substantially different between rat brain regions; an enrichment of dystrophin seems to occur in regions known to contain nerve processes at a high density. Cerebellum, olfactory bulb, olfactory tubercles and striatum contained less dystrophin than hypothalamus, thalamus or hippocampus (a ratio ranging from 1.3 to 1.7). This non uniform distribution may be related to its functional role in nervous system yet to be elucidated. In muscle cells dystrophin is localized in the sarcolemma [1, 29, 30]. Here we addressed the question of the subcellular localization of dystrophin in neurons, the only non-myogenic cell expressing dystrophin. Rat brain tissue was fractionated into its subcellular components and the presence of dystrophin was examined in each fraction by immunoblotting using antiserum C. As shown in Fig. 4, no reactivity could be detected in the synaptic vesicle-enriched fraction or in the mitochondrial fraction. In contrast dystrophin was found to be
90
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Fig. 4. Dystrophin detection in adult rat brain subeellular fractions by immunoblotting with antiserum C. Lane a: mitochondria-purified fraction; lane b: synaptic vesicle-enriched fraction; lane c: synaptic plasma membrane fraction; lane d: whole rat brain crude extract; lane e: rat skeletal muscle extract. Asterisk indicates the position of the 400 kDa dystrophin band. Size and location of molecular weight markers are indicated.
enriched in the plasma m e m b r a n e fraction. In skeletal muscle extract, antisera against the spectrin-like d o m a i n (antiserum C: Fig. 4) or against the C-terminal d o m a i n (antiserum H: data not shown) detected a main c o m p o nent with a molecular mass o f 400 k D a and a fainter c o m p o n e n t with a slightly lower molecular mass; a similar finding was obtained in the brain synaptic plasma m e m b r a n e fraction suggesting a high h o m o l o g y between skeletal muscle dystrophin and brain dystrophin. The structure typical o f spectrin with its repeated domain [27] is not exclusive to this protein but is n o w recognized to characterize a protein superfamily that includes ~t-actinin and dystrophin. On the basis o f structural homologies and similarities regarding subcellular localization it has been suggested that dystrophin could play a functional role in muscle cell sarcolemma similar to that o f spectrin and ~-actinin. However the situation seems to be different in central nervous system; in rat brain, spectrin has been f o u n d to be associated with the cytoplasmic surface o f plasma membranes, mitochondrial membranes and synaptic vesicles (for review see ref. 13). Therefore, the apparent exclusive localization o f dystrophin on the neuronal plasma membrane, possibly resulting from specific protein-protein interactions [4], m a y confer a specific function to this protein. One o f the manifestations o f D u c h e n n e muscular dystrophy which is not yet understood concerns the varying
degree o f associated mental disorders. Further detailed studies on the localization and on the function o f brain dystrophin is required to elucidate the relationship between the lack o f expression o f this protein and the appearance o f neurological mental disorders. This work was supported by the Centre National de la Recherche Scientifique, the Institut National p o u r la Sant6 et la Recherche Mrdicale and by grants from the Association Franqaise contre les Myopathies. D.J. is the recipient o f a 'contrat D R E T 89/1561', France. We thank Dr. R o l a n d Heilig and Dr. Dominique M o r n e t for their help in the p r o d u c t i o n o f antisera, S. Gobaille for dissecting brains, D. Filliol and D. Ledesma for their excellent technical assistance and Dr. O.K. Langley for correcting the manuscript.
1 Arahata, K., Ishiura, S., Ishiguro, T., Tsukahara, T., Suhara, Y., Eguchi, C., Ishihara, T., Nonaka, I., Ozawa, E. and Sugita, H., Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide, Nature, 333 (1988) 861-863. 2 Augier, N., Heilig, R., Boucraut, J., Lrger, J., Lemaire, C., Georgesco, M., Eschene, B., Mandel, J.L., Pons, F., Prlissier, J.F. and L~ger, J.J., Dystrophin detection in diverse neuromuscular dystrophies by immunofluorescence and immunoblot analyses with polyclonal antibodies directed against three different regions of the dystrophin molecule, J. Clin. Invest., submitted. 3 Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1970) 248-254. 4 Campbell, K.P. and Kahl, S.D., Association of dystrophin and an integral membrane glycoprotein, Nature, 338 (1989) 259-262. 5 Chang, H.W., Bock, E. and Bonilla, E., Dystrophin in electric organ of Torpedo californica homologous to that in human muscle, J. Biol. Chem., 264 (1989) 20831-20834. 6 Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A., Transcription of dystrophin gene in human muscle and non-muscle tissues, Nature, 333 (1988) 858-860. 7 Chelly, J., Hamard, G., Koulakoff, A., Kaplan, J.C., Kahn, A. and Berwald-Netter, Y., Dystrophin gene transcribed from different promoters in neuronal and glial cells, Nature, 344 (1990) 64-65. 8 Chelly, J., Montarras, D., Pinset, C., Berwald-Netter, Y., Kaplan, J.C. and Kahn, A., Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells, Eur. J. Biochem., 187 (1990) 611145118. 9 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. 10 Dechesne, C.A., Lrger, J.O.C., Bouvagnet, P., Mairhofer, H. and Lrger, J.J., Local diversity of myosin expression in mammalian atrial muscles, Circ. Res., 57 (1985) 767-775. 11 Fardeau, M., Tomr, F.M.S., Collin, H., Augier, N., Pons, F., Lrger, J. and Lrger, J.J., Prrsence d'une protrine du type dystrophine au niveau de la jonction neuromusculaire dans la dystrophic musculaire de Duchenne et la souris mutante mdx, C.R. Acad. Sci. Paris, in press.
91 12 Feener, C.A., Koenig, M. and Kunkel, L.M., Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus, Nature, 338 (1989) 509-511. 13 Goodman, S.R., Krebs, K.E., Whitfield, C.F., Riederer, B.M. and Zagon, I.S., Spectrin and related proteins, CRC Crit. Rev. Biochem., 23 (1988) 171-234. 14 Hoffman, E.P., Brown, Jr. R.H. and Kunkel, L.M., Dystrophin: The protein product of the Duchenne muscular dystrophy locus, Cell, 51 (1987) 919-1128. 15 Hoffman, E.P., Hudecki, M.S., Rosenberg, P.A., Pollina, C.M. and Kunkel, L.M., Cell and fiber-type distribution of Dystrophin, Neuron, 1 (1988) 411-420. 16 Huttner, W.B., Schiebler, W., Greengard, P. and De Camilli, P., Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation, J. Cell Biol., 116 (1983) 1274-1283. 17 Ishiura, S., Arahata, K., Tsukahara, T., Koga, R., Anraku, H., Yamaguchi, M., Kikuchi, T., Nonaka, I. and Sugita, H., Antibody against the C-terminal portion of dystrophin crossreacts with the 400 kDa protein in the pia mater of dystrophin-deficient mdx mouse brain, J. Biochem., 107 (1990) 510-513. 18 Jasmin, B.J., Cartaud, A., Ludosky, M.A., Changeux, J.P. and Cartaud, J., Asymmetric distribution of dystrophin in developing and adult Torpedo marmorata electrocyte: evidence for its association with the acetylcholine receptor-rich membrane, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 3938-3941. 19 Koenig, M., Monaco, A.P. and Kunkel, L.M., The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein, Cell, 53 (1988) 219-228. 20 Laemmli, U.K., Cleavage of structural proteins during the assembly of the bacteriophage T4, Nature, 227 (1970) 680-688. 21 Lemaire, C., Heilig, R. and Mandel, J.L., The chicken dystrophin cDNA: striking conservation of the C-terminal coding and 3' untranslated regions between man and chicken, EMBO J., 7 (1988) 4157-4162.
22 Love, D.R., Hill, D.F., Dickson, G., Spurr, N.K., Byth, C., Marsden, R.F., Walsh, F.S., Edwards, Y.H. and Davies, K.E., An autosomal transcript in skeletal muscle with homology to dystrophin, Nature, 339 (1989) 55-58. 23 Miike, T., Miyatake, M., Zhao, J., Yoshioka, K. and Uchino, M., Immunohistochemical dystrophin reaction in synaptic regions, Brain Dev., 11 (1989) 344-346. 24 Monaco, A.P., Neve, R.L., Colletti-Feener, C., Bertelson, C.J., Kurnit, D.M. and Kunkel, L.M., Isolation of candidate c DNAs for portions of the Duchenne muscular dystrophy gene, Nature, 323 (1986) 646-650. 25 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) 635qi38. 26 Rendon, A. and Masmoudi, A., Purification of non-synaptic and synaptic mitochondria and plasma membranes from rat brain by a rapid Percoll gradient procedure, J. Neurosci. Methods, 14 (1985) 41-51. 27 Speicher, D.W. and Marchesi, V.T., Erythrocyte spectrin is comprised of many homologous triple helical segments, Nature, 311 (1984) 177-180. 28 Towbin, H., Staehlin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A., 70 (1979) 765-768. 29 Watkins, S.C., Hoffman, E.P., Slayter, H.S. and Kunkel, L.M., Immunoelectron microscopic localization of dystrophin in myofibres, Nature, 333 (1988) 863-866. 30 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 Worton, R.G., The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle, Nature, 333 (1988) 466~69.