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Microheterogeneity of myelin basic proteins in the partially myelinated spinal roots of the Bar Harbor 129 ReJ muscular dystrophic mouse
NeuroscienceLetters, 109 (1990) 321-324
321
Elsevier Scientific Publishers Ireland Ltd. NSL 06647
Microheterogeneity of myelin basic proteins in the partially myelinated spinal roots of the Bar Harbor 129 ReJ muscular dystrophic mouse H u w A. J o h n
Deparirnentof Genetics, Universityof Edinburgh, Edinburgh (U.K.) (Received 11 September 1989; Revisedversion received 10 October 1989; Accepted 11 October 1989)
Key words: Bar Harbor 129 ReJ dystrophic mouse; Spinal root; Myelin protein; Two-dimensional gel electrophoresis The protein composition of normal spinal roots and the partially myelinatedspinal roots of Bar Harbor 129 ReJ dystrophic mice was analyzed by 2D-gel electrophoresiswhich resolved basic proteins. The normal roots contained proteins with mobilities identical to two of the three 18.5 kDa and two of the three 14 kDa myelin basic protein spots resolved in purified spinal cord myelin suggesting that normal root myelin may have some of the characteristics of CNS myelin. In contrast dystrophic roots contained spots with mobilities identical to only one of the spots resolved for each myelin basic protein. The possibility that the differencein microheterogeneitymay be responsible for the decreased myelination is discussed. The spinal roots of adult dystrophic mice (Bar H a r b o r 129 ReJ dydy) contain large groups of amyelinated axons and examples of bizarre myelination [2]. N a k e d and thinly myelinated axons along with heavily myelinated axons were also demonstrated in the mutant with the more benign allele (C57 B1 6J dy es dy 2s) [3, 17, 18]. Myelination of CNS type involving oligodendroglial-like cells was observed in both mutants [2, 18]. The myelin of the thinly myelinated axons was more compact than that of the heavily myelinated axons and had a periodicity similar to CNS myelin [18]. Recent evidence suggests that different charged isomers of myelin basic proteins correlate with the degree of compacting of myelin [14]. In the present investigation the high resolution non equilibrium p H gradient electrophoresis ( N E P H G E ) technique [16] which is particularly appropriate for the analysis of the basic proteins present in myelin, was used to reinvestigate the protein composition of dystrophic roots. N o r m a l ( + + or +dy) and dystrophic (dydy) mice (3-10 weeks old) were littermates from heterozygous matings of strain Bar H a r b o r 129 ReJ + dy. Mice were killed by cervical dislocation and skinned. The lumbosacral spinal cord with attached roots was removed by the dorsal approach, placed in cold Dulbecco A physiological
Correspondence: H.A. John, Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh EH9 3JN, U.K. 0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
322
saline and dorsal and ventral spinal roots L2-L 6 were dissected free from the spinal cord. Roots from equivalent anatomical areas of individual mice were homogenized in 20/tl 0.5% sodium dodecyl sulfate (SDS). 5% fl-mercaptoethanol, 10% glycerol, 0.0625 M Tris-HCl pH 6.8 [16] in a microhomogenizer (Uniform 0.1 ml), incubated at 100°C for 2 min and microfuged to remove SDS insoluble proteins. Protein concentration was estimated by measuring the absorbance at 280 and 260 nm in a 1 mm cuvette [7]. Prior to electrophoresis an equal volume of 9.5 M urea, 0.5 mM dithiothreitoi, 2% Nonidet P 40, 2% ampholine (pH 3-10) was added. First-dimension focusing of 20/~1 of this mixture was carried out as described [16] for 70 min. Proteins were run in the second dimension on a discontinuous SDS-polyacrylamide slab gel [12] with a 10% w/v acrylamide slab separating gel (300 × 150 x 1 mm) for 5 h at 30 mA and stained with Coomassie brilliant blue R250. Molecular weights were determined as described previously [11]. Myelin and neurofilaments were purified from normal mouse sciatic nerves (4-9 pairs, vertebral column to knee) or spinal cord with spinal roots removed [8, 15] and lyophylized. Histones (calf thymus) were supplied by Sigma. Prior to electrophoresis weighed samples were treated as described above. Total protein recovered from roots were analysed using NEPHGE (Fig. la,b) and myelin, neurofilament and histone proteins were identified by comparison and coelectrophoresis with the purified proteins. Neurofilament and histone proteins were at comparable levels in both normal and dystrophic roots. In purified normal spinal cord myelin the 18.5 kDa (18.5K) myelin basic protein resolved into 3 spots, 18.5Ka,b,c and the 14K myelin basic protein resolved into 3 spots 14Ka,b,c (Fig. lc). In contrast the basic proteins which accounted for a smaller proportion of total protein in purified sciatic nerve myelin, resolved into one major spot each with mobilities identical to the more basic spots 18.5Kc and 14Kc of spinal cord myelin (Figure ld). In normal roots spots with mobilities identical to 18.5Kb and c and 14Kb and c were detectable (Fig. la) suggesting that the myelin may have some of the characteristics of CNS myelin. However, CNS specific proteolipid protein which was prominent in spinal cord myelin (Figure lc), was not detectable (Fig. la) suggesting that a myelin of intermediate type may normally occur in the transitional zone between CNS and PNS. In contrast to normal roots, dystrophic roots contained only spots with mobilities identical to the 18.5Kb and 14Kc spots of the spinal cord myelin preparation and these were present in decreased amounts compared to normal (Fig. lb). When protein loadings of dystrophic roots were increased no additional myelin basic protein spots were detected. P0 glycoprotein and a high molecular weight myelin protein which are characteristic of PNS myelin (Fig. ld) were decreased in dystrophic roots (Fig. 1b). Myelin basic proteins are localized in the major electron dense line in compact CNS myelin and are considered to be responsible for the compacting and stability of the myelin [5]. Charge microheterogeneity of myelin basic protein has been reported previously and may result from post-translational modifications such as phosphorylation, deamidation and C-terminal arginine loss [6]. Decreased levels of
323 b
G
Po
b 18-5K
Fig. 1.2D-NEPHGE of spinal root and myelin proteins, a: normal roots, b: dystrophic roots, c: spinal cord myelin, d: sciatic nerve myelin. 18.5Ka,b,c, 18.5 kDa myelin basic protein isomers; 14Ka,b,c, 14 kDa myelin basic protein isomers; PLP, proteolipid protein; P0, P0 glycoprotein; HMW, PNS myelin high molecular weight protein; P200, P150, P68, neurofilament subunits. H, histories. In (a) and (b) proteins from equivalent anatomical areas (lumbrosacral dorsal and ventral roots L2-Lr) were analyzed and in (c) and (d) 100/~g of lyophylized protein. Basic proteins to the right.
the more basic isomers have been shown to correlate with the presence of less compact myelin [14]. These observations suggest that the decreased amount of myelin basic protein, in particular of the most basic form 18.5Kc, in dystrophic rootsmay account for the substantially decreased myelination and abnormally thin myelin sheaths. However, Weinberg et al. [18] reported that the myelin of the more thinly myelinated axons of the dystrophic roots was compact with a periodicity of 10.3 nm characteristic of CNS myelin compared with 11.7 nm for myelin of PNS type in heavily myelinated axons. Such compact myelin might be expected to contain an increased amount of the more basic myelin basic protein but this was not the case. Perhaps the difference in periodicity observed by Weinberg et al. [18] was due to the decreased amount of P0 glycoprotein in dystrophic roots. The decreased amount of myelin basic proteins, particularly the basic isomers, in dystrophic roots is unlikely to be a primary defect because the failure of Schwann cells to ensheath axons is corrected when the dystrophic spinal roots are transplanted into more distal regions of the peripheral nerves of normal or dystrophic mice [1]. The ability of Schwann cells to ensheath axons is linked to the production of a base-
324
ment membrane [4]. Dystrophic spinal roots have previously been shown to be deficient in basement membranes [9, 10, 13] and collagen [11]. Perhaps a normal level of basement membrane is necessary to trigger the appearance of the more basic forms of the myelin basic proteins during myelination. This work was supported by the Muscular Dystrophy Group, U.K. I thank Frank Johnson for photography. 1 Aguayo, A.J., Perkins, C.S. and Bray, G.M., Cell interactions in nerves of dystrophic mice. In D.L. Schotland (Ed.), Disorders of the Motor Unit, Wiley, New York, 1982, pp. 37 50. 2 Bradley, W.G. and Jenkinson, M., Abnormalities in peripheral nerves in murine muscular dystrophy, J. Neurol. Sci., 18 (1973) 227-247. 3 Bradley, W.G. and Jenkinson, M., Neural abnormalities in the dystrophic mouse, J. Neurol. Sci., 25 (1975) 249-255. 4 Bunge, R.P., Bunge, M.B. and Eldridge, C.F., Linkage between axonal ensheathment and basal lamina production by Schwann cells, Annu. Rev. Neurosci., 9 (1986) 305-328. 5 Campagnoni, A.T., Molecular biology of myelin proteins from the central nervous system, J. Neurochem., 51 (1988) 1 14. 6 Cheifetz, S. and Moscarello, M.A., Effect of bovine basic protein charge microheterogeneity on protein-induced aggregation of unitamellar vesicles containing a mixture of acidic and neutral phospholipids, Biochemistry, 24 (1985) 1909-1914. 7 Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M., Data for Biochemical Research, Clarendon, Oxford, 1969, p. 625. 8 de Vries, E.H., Norton, W.T. and Raine, C.S., Axons - isolation from mammalian central nervous system, Science, 175 (1972) 1376-1372. 9 Jaros, E. and Bradley, W.G. Development of the amyelinated lesion in the ventral root of the dystrophic mouse. Ultrastructural, quantitative and autoradiographic study, J. Neurol. Sci., 36 (1978) 317-339. 10 Jaros, E. and Bradley, W.G. Atypical axon - Schwann cell relationships in the common peroneal nerve of the dystrophic mouse an ultrastructural study, Neuropathol. Appl. Neurobiol., 5 (1979) 133 -147. 11 John, H.A. and Purdom, I.F., Myelin proteins and collagen in the spinal roots and sciatic nerves of muscular dystrophic mice, J. Neurol. Sci., 65 (1984) 69-80. 12 Laemmli, U.K., Cleavage of structural proteins during the assembly of bacteriophage T4, Nature (Lond.), 227 (1970) 680~85. 13 Madrid, R.E., Jaros, E., Cullen, M.J. and Bradley, W.E., Genetically determined defect of Schwann cell basement membrane in dystrophic mouse, Nature (Lond.), 257 (1975) 319-321. 14 Moscarello, M.A., Brady, G.W., Fein, D.B., Wood, D.D. and Cruz, T.F., The role of charge microbeterogeneity of basic protein in the formation and maintenance of the multilayered structure of myelin: a possible role in multiple sclerosis, J. Neurosci. Res., 15 (1986) 87-99. 15 Norton, W.T. and Poduslo, S.E., Myelination in rat brain - method of myelin isolation, J. Neurochem., 21 (1973) 759-773. 16 O'Farrell, P.Z., Goodman, H.M. and O'Farrell, P.H., High resolution two-dimensional electrophoresis o f basic as well as acidic proteins, Cell, 12 (1977) I 133-1142. 17 Stirling, C.A., Abnormalities in Schwann cell sheaths in spinal nerve roots of dystrophic mice, J. Anat., 119 (1975) 169-180. 18 Weinberg, H.J., Spencer, P.S. and Raine, C.S., Aberrant PNS development in dystrophic mice, Brain Res., 88 (1975) 532-537.
321
Elsevier Scientific Publishers Ireland Ltd. NSL 06647
Microheterogeneity of myelin basic proteins in the partially myelinated spinal roots of the Bar Harbor 129 ReJ muscular dystrophic mouse H u w A. J o h n
Deparirnentof Genetics, Universityof Edinburgh, Edinburgh (U.K.) (Received 11 September 1989; Revisedversion received 10 October 1989; Accepted 11 October 1989)
Key words: Bar Harbor 129 ReJ dystrophic mouse; Spinal root; Myelin protein; Two-dimensional gel electrophoresis The protein composition of normal spinal roots and the partially myelinatedspinal roots of Bar Harbor 129 ReJ dystrophic mice was analyzed by 2D-gel electrophoresiswhich resolved basic proteins. The normal roots contained proteins with mobilities identical to two of the three 18.5 kDa and two of the three 14 kDa myelin basic protein spots resolved in purified spinal cord myelin suggesting that normal root myelin may have some of the characteristics of CNS myelin. In contrast dystrophic roots contained spots with mobilities identical to only one of the spots resolved for each myelin basic protein. The possibility that the differencein microheterogeneitymay be responsible for the decreased myelination is discussed. The spinal roots of adult dystrophic mice (Bar H a r b o r 129 ReJ dydy) contain large groups of amyelinated axons and examples of bizarre myelination [2]. N a k e d and thinly myelinated axons along with heavily myelinated axons were also demonstrated in the mutant with the more benign allele (C57 B1 6J dy es dy 2s) [3, 17, 18]. Myelination of CNS type involving oligodendroglial-like cells was observed in both mutants [2, 18]. The myelin of the thinly myelinated axons was more compact than that of the heavily myelinated axons and had a periodicity similar to CNS myelin [18]. Recent evidence suggests that different charged isomers of myelin basic proteins correlate with the degree of compacting of myelin [14]. In the present investigation the high resolution non equilibrium p H gradient electrophoresis ( N E P H G E ) technique [16] which is particularly appropriate for the analysis of the basic proteins present in myelin, was used to reinvestigate the protein composition of dystrophic roots. N o r m a l ( + + or +dy) and dystrophic (dydy) mice (3-10 weeks old) were littermates from heterozygous matings of strain Bar H a r b o r 129 ReJ + dy. Mice were killed by cervical dislocation and skinned. The lumbosacral spinal cord with attached roots was removed by the dorsal approach, placed in cold Dulbecco A physiological
Correspondence: H.A. John, Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh EH9 3JN, U.K. 0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
322
saline and dorsal and ventral spinal roots L2-L 6 were dissected free from the spinal cord. Roots from equivalent anatomical areas of individual mice were homogenized in 20/tl 0.5% sodium dodecyl sulfate (SDS). 5% fl-mercaptoethanol, 10% glycerol, 0.0625 M Tris-HCl pH 6.8 [16] in a microhomogenizer (Uniform 0.1 ml), incubated at 100°C for 2 min and microfuged to remove SDS insoluble proteins. Protein concentration was estimated by measuring the absorbance at 280 and 260 nm in a 1 mm cuvette [7]. Prior to electrophoresis an equal volume of 9.5 M urea, 0.5 mM dithiothreitoi, 2% Nonidet P 40, 2% ampholine (pH 3-10) was added. First-dimension focusing of 20/~1 of this mixture was carried out as described [16] for 70 min. Proteins were run in the second dimension on a discontinuous SDS-polyacrylamide slab gel [12] with a 10% w/v acrylamide slab separating gel (300 × 150 x 1 mm) for 5 h at 30 mA and stained with Coomassie brilliant blue R250. Molecular weights were determined as described previously [11]. Myelin and neurofilaments were purified from normal mouse sciatic nerves (4-9 pairs, vertebral column to knee) or spinal cord with spinal roots removed [8, 15] and lyophylized. Histones (calf thymus) were supplied by Sigma. Prior to electrophoresis weighed samples were treated as described above. Total protein recovered from roots were analysed using NEPHGE (Fig. la,b) and myelin, neurofilament and histone proteins were identified by comparison and coelectrophoresis with the purified proteins. Neurofilament and histone proteins were at comparable levels in both normal and dystrophic roots. In purified normal spinal cord myelin the 18.5 kDa (18.5K) myelin basic protein resolved into 3 spots, 18.5Ka,b,c and the 14K myelin basic protein resolved into 3 spots 14Ka,b,c (Fig. lc). In contrast the basic proteins which accounted for a smaller proportion of total protein in purified sciatic nerve myelin, resolved into one major spot each with mobilities identical to the more basic spots 18.5Kc and 14Kc of spinal cord myelin (Figure ld). In normal roots spots with mobilities identical to 18.5Kb and c and 14Kb and c were detectable (Fig. la) suggesting that the myelin may have some of the characteristics of CNS myelin. However, CNS specific proteolipid protein which was prominent in spinal cord myelin (Figure lc), was not detectable (Fig. la) suggesting that a myelin of intermediate type may normally occur in the transitional zone between CNS and PNS. In contrast to normal roots, dystrophic roots contained only spots with mobilities identical to the 18.5Kb and 14Kc spots of the spinal cord myelin preparation and these were present in decreased amounts compared to normal (Fig. lb). When protein loadings of dystrophic roots were increased no additional myelin basic protein spots were detected. P0 glycoprotein and a high molecular weight myelin protein which are characteristic of PNS myelin (Fig. ld) were decreased in dystrophic roots (Fig. 1b). Myelin basic proteins are localized in the major electron dense line in compact CNS myelin and are considered to be responsible for the compacting and stability of the myelin [5]. Charge microheterogeneity of myelin basic protein has been reported previously and may result from post-translational modifications such as phosphorylation, deamidation and C-terminal arginine loss [6]. Decreased levels of
323 b
G
Po
b 18-5K
Fig. 1.2D-NEPHGE of spinal root and myelin proteins, a: normal roots, b: dystrophic roots, c: spinal cord myelin, d: sciatic nerve myelin. 18.5Ka,b,c, 18.5 kDa myelin basic protein isomers; 14Ka,b,c, 14 kDa myelin basic protein isomers; PLP, proteolipid protein; P0, P0 glycoprotein; HMW, PNS myelin high molecular weight protein; P200, P150, P68, neurofilament subunits. H, histories. In (a) and (b) proteins from equivalent anatomical areas (lumbrosacral dorsal and ventral roots L2-Lr) were analyzed and in (c) and (d) 100/~g of lyophylized protein. Basic proteins to the right.
the more basic isomers have been shown to correlate with the presence of less compact myelin [14]. These observations suggest that the decreased amount of myelin basic protein, in particular of the most basic form 18.5Kc, in dystrophic rootsmay account for the substantially decreased myelination and abnormally thin myelin sheaths. However, Weinberg et al. [18] reported that the myelin of the more thinly myelinated axons of the dystrophic roots was compact with a periodicity of 10.3 nm characteristic of CNS myelin compared with 11.7 nm for myelin of PNS type in heavily myelinated axons. Such compact myelin might be expected to contain an increased amount of the more basic myelin basic protein but this was not the case. Perhaps the difference in periodicity observed by Weinberg et al. [18] was due to the decreased amount of P0 glycoprotein in dystrophic roots. The decreased amount of myelin basic proteins, particularly the basic isomers, in dystrophic roots is unlikely to be a primary defect because the failure of Schwann cells to ensheath axons is corrected when the dystrophic spinal roots are transplanted into more distal regions of the peripheral nerves of normal or dystrophic mice [1]. The ability of Schwann cells to ensheath axons is linked to the production of a base-
324
ment membrane [4]. Dystrophic spinal roots have previously been shown to be deficient in basement membranes [9, 10, 13] and collagen [11]. Perhaps a normal level of basement membrane is necessary to trigger the appearance of the more basic forms of the myelin basic proteins during myelination. This work was supported by the Muscular Dystrophy Group, U.K. I thank Frank Johnson for photography. 1 Aguayo, A.J., Perkins, C.S. and Bray, G.M., Cell interactions in nerves of dystrophic mice. In D.L. Schotland (Ed.), Disorders of the Motor Unit, Wiley, New York, 1982, pp. 37 50. 2 Bradley, W.G. and Jenkinson, M., Abnormalities in peripheral nerves in murine muscular dystrophy, J. Neurol. Sci., 18 (1973) 227-247. 3 Bradley, W.G. and Jenkinson, M., Neural abnormalities in the dystrophic mouse, J. Neurol. Sci., 25 (1975) 249-255. 4 Bunge, R.P., Bunge, M.B. and Eldridge, C.F., Linkage between axonal ensheathment and basal lamina production by Schwann cells, Annu. Rev. Neurosci., 9 (1986) 305-328. 5 Campagnoni, A.T., Molecular biology of myelin proteins from the central nervous system, J. Neurochem., 51 (1988) 1 14. 6 Cheifetz, S. and Moscarello, M.A., Effect of bovine basic protein charge microheterogeneity on protein-induced aggregation of unitamellar vesicles containing a mixture of acidic and neutral phospholipids, Biochemistry, 24 (1985) 1909-1914. 7 Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M., Data for Biochemical Research, Clarendon, Oxford, 1969, p. 625. 8 de Vries, E.H., Norton, W.T. and Raine, C.S., Axons - isolation from mammalian central nervous system, Science, 175 (1972) 1376-1372. 9 Jaros, E. and Bradley, W.G. Development of the amyelinated lesion in the ventral root of the dystrophic mouse. Ultrastructural, quantitative and autoradiographic study, J. Neurol. Sci., 36 (1978) 317-339. 10 Jaros, E. and Bradley, W.G. Atypical axon - Schwann cell relationships in the common peroneal nerve of the dystrophic mouse an ultrastructural study, Neuropathol. Appl. Neurobiol., 5 (1979) 133 -147. 11 John, H.A. and Purdom, I.F., Myelin proteins and collagen in the spinal roots and sciatic nerves of muscular dystrophic mice, J. Neurol. Sci., 65 (1984) 69-80. 12 Laemmli, U.K., Cleavage of structural proteins during the assembly of bacteriophage T4, Nature (Lond.), 227 (1970) 680~85. 13 Madrid, R.E., Jaros, E., Cullen, M.J. and Bradley, W.E., Genetically determined defect of Schwann cell basement membrane in dystrophic mouse, Nature (Lond.), 257 (1975) 319-321. 14 Moscarello, M.A., Brady, G.W., Fein, D.B., Wood, D.D. and Cruz, T.F., The role of charge microbeterogeneity of basic protein in the formation and maintenance of the multilayered structure of myelin: a possible role in multiple sclerosis, J. Neurosci. Res., 15 (1986) 87-99. 15 Norton, W.T. and Poduslo, S.E., Myelination in rat brain - method of myelin isolation, J. Neurochem., 21 (1973) 759-773. 16 O'Farrell, P.Z., Goodman, H.M. and O'Farrell, P.H., High resolution two-dimensional electrophoresis o f basic as well as acidic proteins, Cell, 12 (1977) I 133-1142. 17 Stirling, C.A., Abnormalities in Schwann cell sheaths in spinal nerve roots of dystrophic mice, J. Anat., 119 (1975) 169-180. 18 Weinberg, H.J., Spencer, P.S. and Raine, C.S., Aberrant PNS development in dystrophic mice, Brain Res., 88 (1975) 532-537.