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Pattern of myelin proteins in moderate hypoxia
Exp. Pathol. 1990; 39: 45-48 VEB Gustav Fischer Verlag Jena
Department of Neurology (Head: Prof. Dr. med. M. WENDER), School of Medicine, Poznan, Poland
Pattern of myelin proteins in moderate hypoxia By M. WENDER, Z. ADAMCZEWSKA-GONCERZEWICZ, D. TALKOWSKA, J. PANKRAC and A. GROCHOWALSKA With 2 figures
Address for correspondence: Prof. Dr. M. WENDER, Department of Neurology, School of Medicine, 49 Przybyszewskiego Str., 60-355 Poznan, Poland Key words: myelin proteins; proteins, myelin; hypoxia, brain; brain, myelin protein; demyelination; oxygen deficiency, brain
Summary Myelin proteins isolated from the brain of Wi star rats subjected to moderate hypoxia (7% of oxygen in a respiration gaseous mixture) for 30 min were investigated. Among the various protein fractions Wolfgram protein happened to be most markedly affected, demonstrating considerably lowered percentages in all experimental groups, starting from 24 h after hypoxia. Our findings compared with those obtained in other experimental conditions lead to the conclusion that changes in the myelin protein pattern are characteristic for particular morbid processes evoked by various pathogenic factors. The demyelination is concomitant with severe loss of basic proteins, whereas myelin lesions of other types are connected with changes in protein of higher molecular mass.
Introduction Lesions of the oligodendroglia-myelin complex and axons in hypoxia are known from various clinico-morphological observations and experimental studies (RAINE 1984; SMITH and BENJAMINS 1984). Results of our karyometric and cytophotometric studies show great sensitivity of oligodendroglia to oxygen deficiency (WENDER et al. 1988a). The structure of myelin in mild hypoxia is apparently normal. However morphometric evaluation has revealed selective destruction of bigger axons featuring relatively smaller numbers of myelin lamellae in this experimental condition (WENDER et al. 1988). The question arises whether the early physicochemical changes occurring in the myelin sheaths depend on factors affecting any particular component of the protein or depend on the lipid moiety of the complex multilayer structure of the myelin. Moderate hypoxia induces only minor changes in lipid chemistry of myelin (WENDER et al. 1988b). Therefore, we have examined the pattern of myelin protein changes.
Material and Methods Experiments were performed on white rats of Wi star strain, each weighing 200-250 g. The
animals were placed for 30 min in a gaseous mixture of 7% oxygen, 92.9% nitrogen and 0.1 % carbon dioxide. Appropriate composition of gases in a specially constructed glass chamber was Exp. Pathol. 39 (1990) 1
45
secured by feeding the gases through flowmeter and rotometer produced by Junkaler, Dessau. The animals in groups of 10 each were sacrificed after 4 h, 24 h, 14 days or 2 months after hypoxia, by decapitation in a light halothan anaesthesia. The heads were immediately placed in liquid nitrogen. Biochemical methods The myelin fraction was obtained by fractionation in the discontinuous sucrose density gradient by the method of NORTON and PODUSLO (1973). Polyacrylamide gel electrophoresis was performed according to AGRAWAL (1974). The separated proteins were then stained by means of Coomasie Brilliant Blue B-250 (fig. 1). The obtained gels were scanned by means of densitometry at 610 nm, using a microdensitometer of Kipp-Zonen, Holland, with integrating device, and the results were expressed in percentage of total proteins. Myelin proteins separated on polyacrylamide gels were identified using standard proteins of known molecular mass (fig. 2).
Results The clinical and morphological observations of experimental animals after moderate hypoxia were identical to those described in our previous publication (WENDER et al. 1988 c). Histological studies failed to demonstrate any changes of myelin stainability. Results obtained in electrophoretic studies of myelin proteins are presented in table 1. Most pronounced alterations concern the high molecular weight Wolfgram protein which demonstrates the beginning of the decrease as soon as 4 h after the experimental hypoxia and the decrease lasts until the latest experimental period (2 months after hypoxia). The main myelin protein - Folch Lees proteolipid and Agrawal protein decreased in the later period of the experiment (2 months after hypoxia). Both fractions of basic proteins (LBP and SEP) showed a relative increase in the majority of the experimental groups starting 24 h after hypoxia.
Discussion Myelin as an assembly of biological membranes consists of specific proteins and lipids in its bilayers. In pathological conditions leading to myelin lesion or decay, the fundamental question is which of its components is the weak point of myelin molecular structure, serving as a target for the action of the noxious agents. The answer to this question would be of crucial importance for the understanding of the pathomechanism of myelin lesions and decomposition. There exists a hypothesis, not yet entirely documented, that the basic myelin proteins are primarily responsible for the development of inflammatory demyelination. NORTON et al. (1978) advocated the view that neutral proteinases excreted by macrophages should be primarily involved in this process. Anyway the question whether the basic proteins of the central myelin function as a general target component in all processes leading to myelin lesion or decay or whether the role of basic proteins is limited only to those evoked by neuroallergic processes remains still open. Hexachlorophine intoxication connected with myelin breakdown is characterized by the great loss of myelin assiciated glycoprotein (MATTHIEU et al. 1974). At the margin of the developing plaque in multiple sclerosis the great decrease of MAG immunostaining was reported by Iyoy AMA et al. (1980). In triethyltin (TET) intoxication the Agrawal protein of the myelin sheath happened to be most evidently affected (WENDER et al. 1983 a). The protein spectrum of myelin in central Wallerian degeneration demonstrates considerably reduced percentage of basic proteins in the late stage of the experimental process (WENDER et al. 1983 b). A totally different pattern of deviations of myelin proteins was found as a result of moderate hypoxia with a sligthly decreased amount of Wolfgram protein and in the late period after hypoxia also of Folch-Lees proteolipid and Agrawal protein. The changes in both fractions of basic proteins are of minor importance, they are due to shifts of the proteins. It should be stressed that in acute, shorter lasting hypoxia similar but milder changes occur. The findings presented above in the study of hypoxia, and those obtained under other experimental conditions lead to the conclusion that various noxious agents affect different proteins 46
Exp. Pathol. 39 (1990) 1
PLP Wolfgram protein (WP) Folch- Lees proteolipid (PLP) Agrawal pratein ( A P ) Large basic protein (LBP) Small basic protein (S8P)
SBP WP
Fig. 1. Densitogram of myelin proteins of normal rat. I/)
c:
o ~ 10
8 '5:? 6
,,\J
o standard proteins • myelin proteins of control rats
....
~ 4 o
EO 3
1 09 08 07 06 05 04
03 02 01
a
Rf
Fig. 2. Estimation of molecular mass of myelin proteins of normal rat. Log 10 of molecular mass of protein standard presented as R f function. Every point represents the mean of 10 estimations from myelin of control rats: 1. phosphorylase, 2. bovine serum albumin, 3. ovalbumin, 4. carbonic anhydrase, 5. inhibitor of topsoil tripsine, 6.
of the myelin sheath and the resulting changes in the protein pattern, even when not fully specific, are characteristic of the particular morbid processes evoked by various pathogenic factors. Therefore, it seems improbable to suppose that a special molecular part of the myelin bilayers is the general weak point in its architecture. It should be also stressed that demyelination is, at least, concomitant with the severe loss of basic proteins, whereas the myelin lesions of other types, not leading to decomposition of myelin bilayers, are connected with changes of proteins of higher molecular mass.
Exp. Pathol. 39 (1990) 1
47
Table 1. Myelin proteins in rats subjected to chronic hypoxia (in % of total myelin proteins). Control
24 h
4h
2 months
Wolfgram Protein (WP)
30.1
23.2
± 0.9**
23.8
± 1.2**
21.2
± 1.5**
Folch-LeesProteolipid (PLP) Agrawal Protein (AP) Large Basic Protein (LBP) Small Basic Protein (SBP) SBP/LBP rate
40.7 ± 2.1 45.6 ± 1.0** 48.0
± 1.1**
39.1
± 0.8
35.5
± 1.7**
± 1.6
12.0 ± 2.1 5.8 ± 1.0
24.4
± 1.3**
2 weeks
8.8 ± 1.2**
8.3 ± 0.9** 14.5 ± 0.5
6.8 ± 0.9
9.7 ± 0.8**
11.4 ± 0.7 14.4 ± 1.5 2.0 ± 0.5
2.1 ± 0.4
9.6 ± 1.5*
10.8 ± 0.5** 13.0 ± 0.7 1.1 ± 0.2
1.4 ± 0.2*
9.7 ± 0.8** 12.2 ± 1.1** 21.4 ± 2.0** 1.8 ± 0.3
Results are presented in % as mean from 10 estimations ± SEM. * Statistically significant differences in comparison with normal values at the level p
References AGRAWAL, H.: Analysis of membrane proteins by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. In: Fundamentals of lipid chemistry (eds. BURTON, R., GUERRA, F.), pp. 511-543. Science Publ. Div. 1974. IYOYAMA, Y., STERNBERGER, N., DE WEBSTER, M., QUARLES, R., COHEN, S., RICHARDSON, J.: Immunocytochemical observation on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions. Ann. Neurol. 1980; 7: 167-177. MATTHIEU, 1., ZIMMERMAN, A., DE WEBSTER, H., ULSOMER, B., BRADY, R., QUARLES, R.: Hexachlorophenointoxication, characterisation of myelin and myelin related fractions in rats during early postnatal development. Exp. Neurol. 1974; 45: 558-574. NORTON, N., PODUSLO, S.: Myelination in rat brain: method of myelin isolation. 1. Neurochem. 1973; 21: 749-758. NORTON, W., CAMMER, W., BLOOM, B., GORDON, S.: Neutral proteinase secreted by macrophages degrade basic protein: a possible mechanism of inflammatory demyelination. In: Myelination and Demyelination. (ed. PALO, 1.), pp. 365-387. Plenum Publ. Co., New York 1978. RAINE, C.: The neuropathology of myelin diseases. In: Myelin (ed. MORELL, P.), pp. 259-310. Plenum Press, New York-London 1984. SMITH, M., BENJAMINS, 1.: Model systems for study of perturbations of myelin metabolism. In: Myelin (ed. MORELL, P.), pp. 441-487. Plenum Press, New York-London 1984. WENDER, M., ZGORZALEWICZ, B., PIECHOWSKI, A., SPIESZALSKI, W., BUCHOLC, M.: The pattern of myelin proteins in triethyltin (TET) intoxication. Exp. Pathol. 1983a; 23: 193-195. - SNIATALA-KAMASA, M., PIECHOWSKI, A.: Myelin proteins in Wallerian degeneration of the optic nerve. Exp. Pathol. 1983b; 23: 215-217. SZCZECH, 1., GODLEWSKI, A., GROCHOWALSKA, A.: Karyometric and cytophotometric studies of the oligodendroglia in the corpus callosum of the rat after hypoxia. Exp. Pathol. 1988a; 33: 249-255. ADAMCZEWSKA-GONCERZEWICZ, Z., STANISLAWSKA, 1., PANKRAC, 1., TALKOWSKA, D., GROCHOWALSKA, A.: Myelin lipids of the rat brain in experimental hypoxia. Exp. Pathol. 1988b; 33: 59-63. HEJDUK-HANTKE, H., GONCERZEWICZ, A., WYGL~DALSKA-1ERNAS, H.: Morphometric studies of the nervous fibers in corpus callosum of rats exposed to mild hypoxia. Neuropatol. Pol. 1988c; in press. ADAMCZEWSKA-GONCERZEWICZ, Z., TALKOWSKA, D., PANKRAC, 1., SROCZYNSKI, E., GROCHOWALSKA, A.: Influence of experimental acute hypoxia on myelin proteins. Neuropatol. Pol. 1988d; in press. (Received November, 6, 1988; Accepted November II, 1988)
48
Exp. Pathol. 39 (1990) I
Department of Neurology (Head: Prof. Dr. med. M. WENDER), School of Medicine, Poznan, Poland
Pattern of myelin proteins in moderate hypoxia By M. WENDER, Z. ADAMCZEWSKA-GONCERZEWICZ, D. TALKOWSKA, J. PANKRAC and A. GROCHOWALSKA With 2 figures
Address for correspondence: Prof. Dr. M. WENDER, Department of Neurology, School of Medicine, 49 Przybyszewskiego Str., 60-355 Poznan, Poland Key words: myelin proteins; proteins, myelin; hypoxia, brain; brain, myelin protein; demyelination; oxygen deficiency, brain
Summary Myelin proteins isolated from the brain of Wi star rats subjected to moderate hypoxia (7% of oxygen in a respiration gaseous mixture) for 30 min were investigated. Among the various protein fractions Wolfgram protein happened to be most markedly affected, demonstrating considerably lowered percentages in all experimental groups, starting from 24 h after hypoxia. Our findings compared with those obtained in other experimental conditions lead to the conclusion that changes in the myelin protein pattern are characteristic for particular morbid processes evoked by various pathogenic factors. The demyelination is concomitant with severe loss of basic proteins, whereas myelin lesions of other types are connected with changes in protein of higher molecular mass.
Introduction Lesions of the oligodendroglia-myelin complex and axons in hypoxia are known from various clinico-morphological observations and experimental studies (RAINE 1984; SMITH and BENJAMINS 1984). Results of our karyometric and cytophotometric studies show great sensitivity of oligodendroglia to oxygen deficiency (WENDER et al. 1988a). The structure of myelin in mild hypoxia is apparently normal. However morphometric evaluation has revealed selective destruction of bigger axons featuring relatively smaller numbers of myelin lamellae in this experimental condition (WENDER et al. 1988). The question arises whether the early physicochemical changes occurring in the myelin sheaths depend on factors affecting any particular component of the protein or depend on the lipid moiety of the complex multilayer structure of the myelin. Moderate hypoxia induces only minor changes in lipid chemistry of myelin (WENDER et al. 1988b). Therefore, we have examined the pattern of myelin protein changes.
Material and Methods Experiments were performed on white rats of Wi star strain, each weighing 200-250 g. The
animals were placed for 30 min in a gaseous mixture of 7% oxygen, 92.9% nitrogen and 0.1 % carbon dioxide. Appropriate composition of gases in a specially constructed glass chamber was Exp. Pathol. 39 (1990) 1
45
secured by feeding the gases through flowmeter and rotometer produced by Junkaler, Dessau. The animals in groups of 10 each were sacrificed after 4 h, 24 h, 14 days or 2 months after hypoxia, by decapitation in a light halothan anaesthesia. The heads were immediately placed in liquid nitrogen. Biochemical methods The myelin fraction was obtained by fractionation in the discontinuous sucrose density gradient by the method of NORTON and PODUSLO (1973). Polyacrylamide gel electrophoresis was performed according to AGRAWAL (1974). The separated proteins were then stained by means of Coomasie Brilliant Blue B-250 (fig. 1). The obtained gels were scanned by means of densitometry at 610 nm, using a microdensitometer of Kipp-Zonen, Holland, with integrating device, and the results were expressed in percentage of total proteins. Myelin proteins separated on polyacrylamide gels were identified using standard proteins of known molecular mass (fig. 2).
Results The clinical and morphological observations of experimental animals after moderate hypoxia were identical to those described in our previous publication (WENDER et al. 1988 c). Histological studies failed to demonstrate any changes of myelin stainability. Results obtained in electrophoretic studies of myelin proteins are presented in table 1. Most pronounced alterations concern the high molecular weight Wolfgram protein which demonstrates the beginning of the decrease as soon as 4 h after the experimental hypoxia and the decrease lasts until the latest experimental period (2 months after hypoxia). The main myelin protein - Folch Lees proteolipid and Agrawal protein decreased in the later period of the experiment (2 months after hypoxia). Both fractions of basic proteins (LBP and SEP) showed a relative increase in the majority of the experimental groups starting 24 h after hypoxia.
Discussion Myelin as an assembly of biological membranes consists of specific proteins and lipids in its bilayers. In pathological conditions leading to myelin lesion or decay, the fundamental question is which of its components is the weak point of myelin molecular structure, serving as a target for the action of the noxious agents. The answer to this question would be of crucial importance for the understanding of the pathomechanism of myelin lesions and decomposition. There exists a hypothesis, not yet entirely documented, that the basic myelin proteins are primarily responsible for the development of inflammatory demyelination. NORTON et al. (1978) advocated the view that neutral proteinases excreted by macrophages should be primarily involved in this process. Anyway the question whether the basic proteins of the central myelin function as a general target component in all processes leading to myelin lesion or decay or whether the role of basic proteins is limited only to those evoked by neuroallergic processes remains still open. Hexachlorophine intoxication connected with myelin breakdown is characterized by the great loss of myelin assiciated glycoprotein (MATTHIEU et al. 1974). At the margin of the developing plaque in multiple sclerosis the great decrease of MAG immunostaining was reported by Iyoy AMA et al. (1980). In triethyltin (TET) intoxication the Agrawal protein of the myelin sheath happened to be most evidently affected (WENDER et al. 1983 a). The protein spectrum of myelin in central Wallerian degeneration demonstrates considerably reduced percentage of basic proteins in the late stage of the experimental process (WENDER et al. 1983 b). A totally different pattern of deviations of myelin proteins was found as a result of moderate hypoxia with a sligthly decreased amount of Wolfgram protein and in the late period after hypoxia also of Folch-Lees proteolipid and Agrawal protein. The changes in both fractions of basic proteins are of minor importance, they are due to shifts of the proteins. It should be stressed that in acute, shorter lasting hypoxia similar but milder changes occur. The findings presented above in the study of hypoxia, and those obtained under other experimental conditions lead to the conclusion that various noxious agents affect different proteins 46
Exp. Pathol. 39 (1990) 1
PLP Wolfgram protein (WP) Folch- Lees proteolipid (PLP) Agrawal pratein ( A P ) Large basic protein (LBP) Small basic protein (S8P)
SBP WP
Fig. 1. Densitogram of myelin proteins of normal rat. I/)
c:
o ~ 10
8 '5:? 6
,,\J
o standard proteins • myelin proteins of control rats
....
~ 4 o
EO 3
1 09 08 07 06 05 04
03 02 01
a
Rf
Fig. 2. Estimation of molecular mass of myelin proteins of normal rat. Log 10 of molecular mass of protein standard presented as R f function. Every point represents the mean of 10 estimations from myelin of control rats: 1. phosphorylase, 2. bovine serum albumin, 3. ovalbumin, 4. carbonic anhydrase, 5. inhibitor of topsoil tripsine, 6.
of the myelin sheath and the resulting changes in the protein pattern, even when not fully specific, are characteristic of the particular morbid processes evoked by various pathogenic factors. Therefore, it seems improbable to suppose that a special molecular part of the myelin bilayers is the general weak point in its architecture. It should be also stressed that demyelination is, at least, concomitant with the severe loss of basic proteins, whereas the myelin lesions of other types, not leading to decomposition of myelin bilayers, are connected with changes of proteins of higher molecular mass.
Exp. Pathol. 39 (1990) 1
47
Table 1. Myelin proteins in rats subjected to chronic hypoxia (in % of total myelin proteins). Control
24 h
4h
2 months
Wolfgram Protein (WP)
30.1
23.2
± 0.9**
23.8
± 1.2**
21.2
± 1.5**
Folch-LeesProteolipid (PLP) Agrawal Protein (AP) Large Basic Protein (LBP) Small Basic Protein (SBP) SBP/LBP rate
40.7 ± 2.1 45.6 ± 1.0** 48.0
± 1.1**
39.1
± 0.8
35.5
± 1.7**
± 1.6
12.0 ± 2.1 5.8 ± 1.0
24.4
± 1.3**
2 weeks
8.8 ± 1.2**
8.3 ± 0.9** 14.5 ± 0.5
6.8 ± 0.9
9.7 ± 0.8**
11.4 ± 0.7 14.4 ± 1.5 2.0 ± 0.5
2.1 ± 0.4
9.6 ± 1.5*
10.8 ± 0.5** 13.0 ± 0.7 1.1 ± 0.2
1.4 ± 0.2*
9.7 ± 0.8** 12.2 ± 1.1** 21.4 ± 2.0** 1.8 ± 0.3
Results are presented in % as mean from 10 estimations ± SEM. * Statistically significant differences in comparison with normal values at the level p
References AGRAWAL, H.: Analysis of membrane proteins by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. In: Fundamentals of lipid chemistry (eds. BURTON, R., GUERRA, F.), pp. 511-543. Science Publ. Div. 1974. IYOYAMA, Y., STERNBERGER, N., DE WEBSTER, M., QUARLES, R., COHEN, S., RICHARDSON, J.: Immunocytochemical observation on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions. Ann. Neurol. 1980; 7: 167-177. MATTHIEU, 1., ZIMMERMAN, A., DE WEBSTER, H., ULSOMER, B., BRADY, R., QUARLES, R.: Hexachlorophenointoxication, characterisation of myelin and myelin related fractions in rats during early postnatal development. Exp. Neurol. 1974; 45: 558-574. NORTON, N., PODUSLO, S.: Myelination in rat brain: method of myelin isolation. 1. Neurochem. 1973; 21: 749-758. NORTON, W., CAMMER, W., BLOOM, B., GORDON, S.: Neutral proteinase secreted by macrophages degrade basic protein: a possible mechanism of inflammatory demyelination. In: Myelination and Demyelination. (ed. PALO, 1.), pp. 365-387. Plenum Publ. Co., New York 1978. RAINE, C.: The neuropathology of myelin diseases. In: Myelin (ed. MORELL, P.), pp. 259-310. Plenum Press, New York-London 1984. SMITH, M., BENJAMINS, 1.: Model systems for study of perturbations of myelin metabolism. In: Myelin (ed. MORELL, P.), pp. 441-487. Plenum Press, New York-London 1984. WENDER, M., ZGORZALEWICZ, B., PIECHOWSKI, A., SPIESZALSKI, W., BUCHOLC, M.: The pattern of myelin proteins in triethyltin (TET) intoxication. Exp. Pathol. 1983a; 23: 193-195. - SNIATALA-KAMASA, M., PIECHOWSKI, A.: Myelin proteins in Wallerian degeneration of the optic nerve. Exp. Pathol. 1983b; 23: 215-217. SZCZECH, 1., GODLEWSKI, A., GROCHOWALSKA, A.: Karyometric and cytophotometric studies of the oligodendroglia in the corpus callosum of the rat after hypoxia. Exp. Pathol. 1988a; 33: 249-255. ADAMCZEWSKA-GONCERZEWICZ, Z., STANISLAWSKA, 1., PANKRAC, 1., TALKOWSKA, D., GROCHOWALSKA, A.: Myelin lipids of the rat brain in experimental hypoxia. Exp. Pathol. 1988b; 33: 59-63. HEJDUK-HANTKE, H., GONCERZEWICZ, A., WYGL~DALSKA-1ERNAS, H.: Morphometric studies of the nervous fibers in corpus callosum of rats exposed to mild hypoxia. Neuropatol. Pol. 1988c; in press. ADAMCZEWSKA-GONCERZEWICZ, Z., TALKOWSKA, D., PANKRAC, 1., SROCZYNSKI, E., GROCHOWALSKA, A.: Influence of experimental acute hypoxia on myelin proteins. Neuropatol. Pol. 1988d; in press. (Received November, 6, 1988; Accepted November II, 1988)
48
Exp. Pathol. 39 (1990) I