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Myelin protein metabolism in demyelination and remyelination in the sciatic nerve
Brain Research, 270 (1983) 37-44
37
Elsevier
M y e l i n P r o t e i n M e t a b o l i s m in D e m y e l i n a t i o n a n d R e m y e l i n a t i o n in t h e S c i a t i c N e r v e
MARION EDMONDS SMITH*, J E F F E R Y D. KOCSIS and STEPHEN G. WAXMAN
Department of Neurology, Veterans Administration Medical Center, Palo A ho, CA 94304 and Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Accepted November 23rd, 1982)
Key words: peripheral nerve - lysolecithin - demyelination - remyelination - PNS myelin protein - P0
Lysophosphatidylcholine (LPC) was injected intraneurally into the right sciatic nerve of a series of rats, leaving the left nerve as a control. At various time points up to 30 days after LPC treatment, the injected and control nerves were removed and incubated with [3H]amino acids, then purified myelin was prepared from the nerves. At all time points investigated the uptake of labeled amino acids was much higher in myelin proteins from the LPC-treated nerve than in those from the intact control nerve. [3H]Fucose uptake was also slightly increased. In the first week after LPC injection the increased amino acid incorporation was much greater in the myelin proteins of molecular weight higher than P0. From 10 days on, the smaller myelin proteins including P0, 19K, P1 and P2 showed the largest increase. From comparison of the morphology and biochemistry at 3 and 7 days after LPC injection, we propose that in the first 7 days, while myelin is degenerating, those proteins associated with Schwann cell or myelin reaction, interaction, and recognition functions are most stimulated metabolically, while after ten days the structural myelin proteins are actively resynthesized and the axon is remyelinated. INTRODUCTION
Morphological studies have shown that intraneural injection of lysophosphatidylcholine (LPC) into the mouse sciatic nerve leads to demyelination3,4. Hall and Gregson 4 have shown a drastic disruption of the myelin sheath at the site of LPC injection, with phagocytosis of the myelin debris by cells lying parallel to the axons. Demyelination begins within lh after intraneural LPC injection3. After the myelin is removed, remyelination begins about one week after the initial insult and is well established by 14 days ~2. The site-specific characteristics of this primary demyelinative lesion and the predictable course of demyelination and remyelination make this an excellent model with which to examine various aspects of the demyelinative lesion and subsequent remyelination. Electrophysiological studies indicate that conduction block occurs after LPC-induced demyelination, and that im-
pulse conduction reappears approximately one week later ja.t3. In the experiments reported here, we have investigated the metabolism of PNS myelin protein during different phases of myelin breakdown and resynthesis, and attempted to correlate the metabolic changes with those observed morphologically. METHODS
Injection of LPC The right sciatic nerve was exposed in adult male rats anesthetized with sodium barbital (50 mg/kg) and a total of 10/zl of 1% LPC (Sigma, St. Louis, MO) was delivered into two sites just above the tibial branch of the sciatic nerve by means of a glass micropipette attached to a Hamilton 100 ~1 syringe. The tip of the micropipette was inserted through the perineurium into the nerve, and the LPC was delivered over a minute. Following injection of LPC, the incision
* To whom all correspondence should be addressed at: Department of Neurology (127A) Veterans Administration Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304, U.S.A. 0006-8993 / 83 / $03.00 ~ 1983 Elsevier Science Publishers B.V.
38 was closed by suture. The left intact sciatic nerve served as a control. At various times after injection, from 1 day up to 30 days, the animals were killed by decapitation, and the portions of the nerve from the tibial branch to the sciatic notch were removed from the experimental and control sciatic nerve.
Preparation and incubation of tissues Four experimental or control nerves from each post-injection interval were pooled to make a total of about 75 mg wet weight of tissue. These were chopped into small pieces and incubated for 3 h at 37 °C in 1.5 ml Krebs-Ringer bicarbonate containing 10 mM glucose and 50 /zCi [3H]amino acid mixture (New England Nuclear, NY) as previously described H. One series was double-labeled with U4C]amino acid mixture and [3H]fucose. At the end of the incubation period the mixture was centrifuged, the medium decanted, the nerves washed in distilled water, and again centrifuged. After homogenization in 30% sucrose with the aid of a Polytron homogenizer (Brinkman, Westbury, NY) purified myelin was prepared from the tissue as previously described t4. The final washed myelin pellet was lyophilized, partially delipidated with several washes of ethyl ether, and dissolved in 1% sodium dodecyl sulfate (SDS)-8% sucrose solution containing 1.5% dithiothreitol. The proteins were separated by SDS polyacrylamide gel electrophoresis on 10~20% gradient slabs using the buffer system of LaemmlP and a Protean electrophoresis cell (Biorad, Albany, CA). Thirty-five ~g protein were placed in each well, the slabs were run at 25 mA for 18 h, then stained with Coomassie Blue, destained, and scanned at 550 nm with a Gilford spectrophotometer equipped with a linear transport system. The separated proteins were measured by cutting out and weighing the scanned peaks. Radioactivity determination The gels were sliced into 2 mm slices, and each slice was transferred to a counting vial, finely chopped with scissors, and the proteins eluted with Protosol (New England Nuclear, Albany, NY), overnight at 50"C. After addition of scin-
Fig. 1. A: demyelinated fiber, 3-days post-LPC injection. Myelin sheath is vesiculated (V) throughout its entire thickness. The axon (a) appears intact. Longitudinal section, x 4400. B: transverse sectionsof fiber at 3 days post-LPC injection. Myelin debris (D) is present in the fiber the right. Note dark shrunken axon (a)~with lossof continuity ofaxolemma. x 4000. tillation cocktail, the vials were counted in a Delta 300 liquid scintillation counter using the channels ratio method for quench correction, We examined 3 full series of treated nerves with at least 7 time points in each series, as well as two other series with fewer time points. RESULTS
Ultrastructure The areas of sciatic nerve to which LPC was applied were examined by electron microscopy at various times after EPC injection. Although the morphology of the degradative events has been previously described in the mouse 4, it was important to establish the time course of demye-
39 lination and remyelination in the rat. At the earliest time point examined, 3 days post-injection, myelin vesiculation was well advanced (Fig. 1A, B). The myelin lamellae were disrupted to form a network of ovoid membrane profiles. In some fascicles the myelin was completely vesiculated, with loss of compaction from the axonal surface
to the surrounding basement membrane. Myelin debris were in some cases observed within the Schwann cell cytoplasm. Occasionally some evidence of axonal damage was observed (Fig. 1B). At 7 days post-injection, the vesiculated myelin was completely removed, although in most axons phagocytosed membranous material
Fig. 2. A: demyelinated axon, 7 days post-LPC injection. A Schwann cell (SC), covered by a basement membrane, surrounds the axon (a), although, in this case, no compact myelin is present. × 9100. B: remyelinated axon (A), 7 days post-LPC injection. Note the relatively thin myelin sheath (m) surrounded by a Schwann cell (SC). e, extracellular space. × 9620.
40
~ii~
HMW
was observed within a few Schwann cells. Elongated cells surrounded by a basement membrane, probably Schwann cells, were lined up along the bare axons and, in many cases, these cells surrounded the axons (Fig. 2A). In a few areas compact myelin lamellae are discernible. These myelin sheaths were inappropriately thin, compared to fiber diameter, and probably represent remyelinated segments (Fig. 2B).
~. . . . . . . . . . .
'
P023
and
19K PI~ S m a l l BP__, + P2
Control LPC day 1
Control LPC day 19
Fig. 3. PNS myelinproteins separated by polyacrylamidegel electrophoresis.Each lane contains 35 ~g protein. Separated proteins are stained with Coomassie Blue. Each pair represents myelin from control (left) and LPC-treated (right) nervesat 1and 19days post-LPC injection.
Day 1 450,
PO !~
400
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? !
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Fig. 4. Radioactivity in slices of gels containing myelin proteins separated by polyacrylamide gel electrophoresis as in Fig. 3 at one day after LPC injection.
Biochemical studies The separated myelin proteins visualized on the SDS polyacrylamide gel slabs were identified as the glycoprotein P0, which is the main structural protein, 23K and 19K, which are also glycoproteins (nomenclature of Eylar et al?), and the 3 basic proteins (P~, the small basic protein (SBP) seen in rodents, and P2 which comigrates with the small basic protein) (Fig. 3). Very little stained material was visible above the P0 protein. There was no difference in appearance of the bands containing the myelin proteins of the control and the LPC-treated nerves. The proteins appeared to be identical qualitatively, and approximately the same amounts of each protein were present. The area to which LPC was applied was a relatively small proportion of the total nerve segment taken; therefore, there was no appreciable diminution in myelin amounts in spite of the LPC-elicited disruption. When the radioactivity of the sliced gels was measured, it was apparent that the uptake of radioactive amino acids into the proteins was considerably higher in myelin from the LPC treated animals than in the control, even one day after administration of LPC (Fig. 4). The increased incorporation was undoubtedly partially due to increased permeability and access of the amino acids to the sites of synthesis and exchange after damage to the nerve. A considerable amount of amino acid uptake was seen in the high molecular weight region above the P0, even though very little stained protein material was visible. The pattern of increased uptake of amino acids was quite different, however, in the early time period up to one week after LPC injection compared to that in the later period after 10 days. The early high incorporation into the myelin from the ex-
41
perimental nerve was most marked in the high molecular weight proteins compared to the recognizable myelin proteins (Fig. 4), and this pattern was retained for about a week. F r o m 10 days on, P0, the other glycoproteins and the basic proteins of LPC-treated nerve, incorporated proportionately more radioactivity, while the uptake into the high molecular weight proteins decreased (Fig. 5). This two-phase process is well illustrated when the total amino acid uptake into the high molecular weight proteins and that of the P0 compared to the analogous fractions of the control nerves are plotted over the 30 day time period. Two time courses representing different experiments, with the uptake expressed as percent of control, are shown on Fig. 6. Although each set of animals yielded data showing a somewhat different course of activation, a two stage stimulatory pattern was evident in all experiments. The degree of activation depended on the activity of the control which we have shown earlier to
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Fig. 6. A and B: time course of two series of rats injected with LPC and using [3H]amino acids as the protein precursor. The total counts in the high molecular weight proteins and in the P0 proteins are expressed as percent of the counts in the analogous control proteins. Note the increased uptake into high molecular weight proteins, relative to that for P0, prior to I0 days post-LPC injection.
400-
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Fig. 5. Radioactivity in slices of gels containing separated myelin proteins 19 days after LPC injection.
be age-dependent ~4. Thus in Fig. 6A somewhat younger animals were used (about 8 weeks), while in Fig. 6B the rats were older. The percent activation appeared to be larger with older animals, especially with P0 during remyelination. In both cases, however, the curves for high molecular weight and P0 cross at 10 days post-injection. Generally, the other myelin proteins (19K, P~, and SBP + P2) were metabolically similar to P0, with the rates of amino acid uptake increasing to very high levels in these proteins on day 10, and gradually decreasing thereafter (Table I). In one experiment where the incorporation of [3H]fucose into glycoproteins was measured, the pattern was not as striking, although the largest increase in uptake of fucose into P0 occurred on day 10, the same time as that for radioactive amino acids (Fig. 7). In general, an increased incorporation of fucose was evident, but it was not as striking as that for amino acids.
42 TABLE I
Percent of control (dpm LPC/dpm Control) uptake of [~H]amino acids into sciatic nerve myelin proteins in a typical experiment Slab gels with the myelin proteins separated, as shown in Fig. 3, were sliced and the slices counted for radioactivity.
Dav
HM W
Po
19K
PI
P,
l 6 8 10 14 30
186 207 340 295 249 156
148 152 273 349 328 179
138 130 206 418 447 199
115 138 191 428 236 179
143 171 196 318 215 180
DISCUSSION
Lysolecithin-induced demyelination affords an excellent model with which to study metabolic reaction of nerve to a monophasic insult. Although a small amount of axonal damage was involved, the demyelination was, for the most part, of the primary type. Most metabolic studies of peripheral nerve demyelination remyelination so far published utilize the Wallerian degeneration model 7.
225-
-~
200-
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175-
2
go
_ _ _,..o. . . .
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.-o P0
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Fig. 7. Time course of uptake of [3 H]fucose into myelin proteins in high molecular weight fractions and P0 expressed as percent of control.
rineural injection of LPC, with all traces of the myelin sheath gone by 96 h. In experiments reported here in the rat, where somewhat larger amounts of LPC were injected, the sequence of events appears to be similar, although a large amount of disrupted myelin was still present at 3 days after LPC administration. Gregson and HalP reported that remyelination in the mouse had begun by day 14. Smith and HalP 2 noted some remyelination one week following intraneural LPC injection in mouse sciatic nerve. The present results show morphological evidence of remyelination as early as 7 days after intraneural LPC injection in rat peripheral nerve. The choice of the appropriate control nerve was a major problem in this study. After some experimentation with sham-operated nerves and perineural injection of saline into the opposite nerve, it became evident that any manipulation of the control nerve resulted in some degree ofdemyelination. The decision was made, therefore, to use intact nerves which had undergone no invasive procedures as controls. Another difficulty was the possibility of variability of the injection procedure and nerve sampling. For this reason, 4 nerves were pooled in each experiment, and attempts were made to observe the anatomical landmarks precisely in selecting the nerve segment to be studied. The lesion in the nerve, however, represented a small part of the entire sample taken, probably less than 10%, with the bulk of the myelin serving as carrier of the metabolically disturbed area which may extend beyond the lesion. In studying the metabolism of this model of demyelination remyelination, the role of proteins, especially of the glycoproteins, was of greatest interest. Glycoproteins have been associated with the role of 'recognition' in cell cell interaction, and we considered that such interactive processes should be stimulated during disruption of the myelin--axonal interactions and the subsequent re-establishment of relatively normal relationships. Glycoproteins are known to comprise at least 65% of the PNS myelin protein. The most abundant of these is the P0 protein, the main structural myelin protein which constitutes over half the total. It contains a 9-
43 sugar chain linked by N-asparaginyl linkage to the protein. In addition, fucose is well incorporated into high molecular weight myelin proteins of young rats, although less well into those of older rats ~4. High molecular weight proteins of PNS myelin, therefore, appear to contain a number of glycoproteins, only one of which has been identified 2. We have previously shown that the high molecular weight proteins contain over 40% of the total in vitro incorporated radioactivity in myelin proteins of young animals and 60-65% of that in older animals after incubating chopped nerves with [3H]amino acids 14. In view of their smaller amounts (based on staining characteristics), the high molecular weight proteins probably contain components which are more metabolically active than the prominent myelin proteins. It is likely that they represent Schwann cell membrane fragments, and interactive and recognition entities which retain more metabolic activity than the structural elements of the myelin sheath. We have tentatively interpreted our results as indicating that the demyelinative phase (up to 7 days after LPC) is accompanied by a large increase in metabolic activity of the Schwann cell membranes and recognition factors as the myelin sheath becomes vesiculated and removed from the axon. During the next phase, in which remyelination takes place (1030 days), the high molecular weight proteins diminish somewhat in their metabolic activity, while the myelin proteins are resynthesized and deposited resulting in a large increase in amino acide uptake into P0 and the other myelin proteins. We have seen morphological evidence of the beginning of remyelination as early as seven days, and others have found that conduction is beginning to be restored around six days when REFERENCES 1 Eylar, E. H., Uyemura, K., Brostoff, S. W., Kitamura, K., lshaque, A. and Greenfield, L., Proposed nomenclature for PNS myelin proteins, Neurochem. Res., 4 (1979) 289~ 293. 2 Figlewicz, D. A., Quarles, R. H., Johnson, D., Barbarash, G. R. and Sternberger, N. H., Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system, J. Neurochem., 37 (1981) 749~ 758.
the Schwann cells reassociate with the axon ~2, but have not yet begun to form myelin. The fucose uptake in the two fractions was much less dramatic than that of amino acid incorporation. Actually, the in vitro uptake of fucose in PNS myelin of adult animals is very low H, and this may be due to a decreased permeability or transport rate. It is also possible that fucose may be retained within the Schwann cells after myelin digestion and reutilized, making transport from an exogenous source less necessary than in the case of amino acids. Peterson et al? found, in degenerating nerve segments distal to a crush that the ratio pH]fucose/[14C]leucine incorporation in vitro into sciatic nerve myelin was decreased due to a significant increase in leucine uptake relative to fucose. Reutilization of myelin cholesterol during remyelination after Wallerian degeneration has been shown by others ~1. A more detailed study of the high molecular weight myelin proteins is clearly needed to identify the individual proteins, their function, and location. From results obtained with this model of demyelination, it appears that they may be important indicators of the dynamics of cell interaction and recognition. ACKNOWLEDGEMENTS
The authors are grateful to Paul Somera for running the slab gels and providing other excellent technical assistance, and to Mary Smith for providing the electron micrographs. This research is supported by the Medical Research Service of the Veterans Administration, and by Grants NS-02785 (to M.E.S.) and NS 15320 (to S.G.W.) from the NIH, and by grants from the National Multiple Sclerosis Society. 3 Gregson, N. A. and Hall, S. M., A quantitative analysis of the effects of the intraneural injection oflysophosphatidyl choline, J. Cell Sci., 13 (1973) 257 277. 4 Hall, S. M. and Gregson, N. A., The in vivo and ultrastructural effects of injection of lysophosphatidyl choline into myelinated peripheral nerve fibers of the adult mouse, J. CellSci.. 9 (1971) 76~789. 5 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T, Nature (Lond.), 227 (1970) 68(P 685.
44 6 Margolis, R. U. and Margolis, R. K.. Perspectives and functional implications. In R. U. Margolis and R. K. Margolis (Eds.). Complex Carbohydrates of Nervous Tissue. Plenum Press. New York. 1979. pp. 37~386. 7 Majno. G. and Karnovsky. M. k., A biochemical and morphological study of myelination and demyelination. II. kipogenesis in vitro by rat nerves following transection.J, exp. Med., 108 (1958) 197 208. 8 Peterson. R. G.. Baughman. S. and Scheidler, D. M., Incorporation of fucose and teucine into PNS myelin in nerves undergoing early Wallerian degeneration, Neurochem. Res.. 6(1981)213-223. 9 Pritchard. E. T. and Rossiter, R. J., Chemical studies of peripheral nerve during Wallerian degeneration. XI. In vitr¢~ incorporation of 14C-labelled precursors into phosphatides. J. Neurochem., 3 (1959) 341 o346. 10 Rawlins. F. A. and Smith, M. E., Metabolism of sciatic
11
12 13
14
nerve myelin in Wallerian degeneration, Neurobiologv, 1 (1971 ) 225-230. Rawlins, F. A., Villegas, G. M., Hedley-Whyte, E. T. and Uzman, B. G., Fine structural localization of cholesterol[1,2-3H] in degenerating and regenerating mouse sciatic nerve, J. Cell. Biol., 52(1972)615 625, Smith, K. J. and Hall, S. M., Nerve conduction during peripheral demyelination and remyelination, J. neurol. Sci., 48 (1980) 201-219. Smith, K. J., Bostock, H. and Hall, S. M., Saltatory conduction precedes remyelination in axons demyelinated with lysophosphatidyl choline, J. neurol. Sci., 54 (1982) 13 31. Smith. M. E., Biosynthesis of peripheral nervous system myelin proteins in vitro, J. Neurochem., 35 (1980) 11831189.
37
Elsevier
M y e l i n P r o t e i n M e t a b o l i s m in D e m y e l i n a t i o n a n d R e m y e l i n a t i o n in t h e S c i a t i c N e r v e
MARION EDMONDS SMITH*, J E F F E R Y D. KOCSIS and STEPHEN G. WAXMAN
Department of Neurology, Veterans Administration Medical Center, Palo A ho, CA 94304 and Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Accepted November 23rd, 1982)
Key words: peripheral nerve - lysolecithin - demyelination - remyelination - PNS myelin protein - P0
Lysophosphatidylcholine (LPC) was injected intraneurally into the right sciatic nerve of a series of rats, leaving the left nerve as a control. At various time points up to 30 days after LPC treatment, the injected and control nerves were removed and incubated with [3H]amino acids, then purified myelin was prepared from the nerves. At all time points investigated the uptake of labeled amino acids was much higher in myelin proteins from the LPC-treated nerve than in those from the intact control nerve. [3H]Fucose uptake was also slightly increased. In the first week after LPC injection the increased amino acid incorporation was much greater in the myelin proteins of molecular weight higher than P0. From 10 days on, the smaller myelin proteins including P0, 19K, P1 and P2 showed the largest increase. From comparison of the morphology and biochemistry at 3 and 7 days after LPC injection, we propose that in the first 7 days, while myelin is degenerating, those proteins associated with Schwann cell or myelin reaction, interaction, and recognition functions are most stimulated metabolically, while after ten days the structural myelin proteins are actively resynthesized and the axon is remyelinated. INTRODUCTION
Morphological studies have shown that intraneural injection of lysophosphatidylcholine (LPC) into the mouse sciatic nerve leads to demyelination3,4. Hall and Gregson 4 have shown a drastic disruption of the myelin sheath at the site of LPC injection, with phagocytosis of the myelin debris by cells lying parallel to the axons. Demyelination begins within lh after intraneural LPC injection3. After the myelin is removed, remyelination begins about one week after the initial insult and is well established by 14 days ~2. The site-specific characteristics of this primary demyelinative lesion and the predictable course of demyelination and remyelination make this an excellent model with which to examine various aspects of the demyelinative lesion and subsequent remyelination. Electrophysiological studies indicate that conduction block occurs after LPC-induced demyelination, and that im-
pulse conduction reappears approximately one week later ja.t3. In the experiments reported here, we have investigated the metabolism of PNS myelin protein during different phases of myelin breakdown and resynthesis, and attempted to correlate the metabolic changes with those observed morphologically. METHODS
Injection of LPC The right sciatic nerve was exposed in adult male rats anesthetized with sodium barbital (50 mg/kg) and a total of 10/zl of 1% LPC (Sigma, St. Louis, MO) was delivered into two sites just above the tibial branch of the sciatic nerve by means of a glass micropipette attached to a Hamilton 100 ~1 syringe. The tip of the micropipette was inserted through the perineurium into the nerve, and the LPC was delivered over a minute. Following injection of LPC, the incision
* To whom all correspondence should be addressed at: Department of Neurology (127A) Veterans Administration Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304, U.S.A. 0006-8993 / 83 / $03.00 ~ 1983 Elsevier Science Publishers B.V.
38 was closed by suture. The left intact sciatic nerve served as a control. At various times after injection, from 1 day up to 30 days, the animals were killed by decapitation, and the portions of the nerve from the tibial branch to the sciatic notch were removed from the experimental and control sciatic nerve.
Preparation and incubation of tissues Four experimental or control nerves from each post-injection interval were pooled to make a total of about 75 mg wet weight of tissue. These were chopped into small pieces and incubated for 3 h at 37 °C in 1.5 ml Krebs-Ringer bicarbonate containing 10 mM glucose and 50 /zCi [3H]amino acid mixture (New England Nuclear, NY) as previously described H. One series was double-labeled with U4C]amino acid mixture and [3H]fucose. At the end of the incubation period the mixture was centrifuged, the medium decanted, the nerves washed in distilled water, and again centrifuged. After homogenization in 30% sucrose with the aid of a Polytron homogenizer (Brinkman, Westbury, NY) purified myelin was prepared from the tissue as previously described t4. The final washed myelin pellet was lyophilized, partially delipidated with several washes of ethyl ether, and dissolved in 1% sodium dodecyl sulfate (SDS)-8% sucrose solution containing 1.5% dithiothreitol. The proteins were separated by SDS polyacrylamide gel electrophoresis on 10~20% gradient slabs using the buffer system of LaemmlP and a Protean electrophoresis cell (Biorad, Albany, CA). Thirty-five ~g protein were placed in each well, the slabs were run at 25 mA for 18 h, then stained with Coomassie Blue, destained, and scanned at 550 nm with a Gilford spectrophotometer equipped with a linear transport system. The separated proteins were measured by cutting out and weighing the scanned peaks. Radioactivity determination The gels were sliced into 2 mm slices, and each slice was transferred to a counting vial, finely chopped with scissors, and the proteins eluted with Protosol (New England Nuclear, Albany, NY), overnight at 50"C. After addition of scin-
Fig. 1. A: demyelinated fiber, 3-days post-LPC injection. Myelin sheath is vesiculated (V) throughout its entire thickness. The axon (a) appears intact. Longitudinal section, x 4400. B: transverse sectionsof fiber at 3 days post-LPC injection. Myelin debris (D) is present in the fiber the right. Note dark shrunken axon (a)~with lossof continuity ofaxolemma. x 4000. tillation cocktail, the vials were counted in a Delta 300 liquid scintillation counter using the channels ratio method for quench correction, We examined 3 full series of treated nerves with at least 7 time points in each series, as well as two other series with fewer time points. RESULTS
Ultrastructure The areas of sciatic nerve to which LPC was applied were examined by electron microscopy at various times after EPC injection. Although the morphology of the degradative events has been previously described in the mouse 4, it was important to establish the time course of demye-
39 lination and remyelination in the rat. At the earliest time point examined, 3 days post-injection, myelin vesiculation was well advanced (Fig. 1A, B). The myelin lamellae were disrupted to form a network of ovoid membrane profiles. In some fascicles the myelin was completely vesiculated, with loss of compaction from the axonal surface
to the surrounding basement membrane. Myelin debris were in some cases observed within the Schwann cell cytoplasm. Occasionally some evidence of axonal damage was observed (Fig. 1B). At 7 days post-injection, the vesiculated myelin was completely removed, although in most axons phagocytosed membranous material
Fig. 2. A: demyelinated axon, 7 days post-LPC injection. A Schwann cell (SC), covered by a basement membrane, surrounds the axon (a), although, in this case, no compact myelin is present. × 9100. B: remyelinated axon (A), 7 days post-LPC injection. Note the relatively thin myelin sheath (m) surrounded by a Schwann cell (SC). e, extracellular space. × 9620.
40
~ii~
HMW
was observed within a few Schwann cells. Elongated cells surrounded by a basement membrane, probably Schwann cells, were lined up along the bare axons and, in many cases, these cells surrounded the axons (Fig. 2A). In a few areas compact myelin lamellae are discernible. These myelin sheaths were inappropriately thin, compared to fiber diameter, and probably represent remyelinated segments (Fig. 2B).
~. . . . . . . . . . .
'
P023
and
19K PI~ S m a l l BP__, + P2
Control LPC day 1
Control LPC day 19
Fig. 3. PNS myelinproteins separated by polyacrylamidegel electrophoresis.Each lane contains 35 ~g protein. Separated proteins are stained with Coomassie Blue. Each pair represents myelin from control (left) and LPC-treated (right) nervesat 1and 19days post-LPC injection.
Day 1 450,
PO !~
400
HMW
I0
3OO
SBP* P2
,v I b *, I
o"
- - Control - - - LPC
,
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i
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,
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150.
100.
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? !
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50 ¸
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2'0
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Slice number
Fig. 4. Radioactivity in slices of gels containing myelin proteins separated by polyacrylamide gel electrophoresis as in Fig. 3 at one day after LPC injection.
Biochemical studies The separated myelin proteins visualized on the SDS polyacrylamide gel slabs were identified as the glycoprotein P0, which is the main structural protein, 23K and 19K, which are also glycoproteins (nomenclature of Eylar et al?), and the 3 basic proteins (P~, the small basic protein (SBP) seen in rodents, and P2 which comigrates with the small basic protein) (Fig. 3). Very little stained material was visible above the P0 protein. There was no difference in appearance of the bands containing the myelin proteins of the control and the LPC-treated nerves. The proteins appeared to be identical qualitatively, and approximately the same amounts of each protein were present. The area to which LPC was applied was a relatively small proportion of the total nerve segment taken; therefore, there was no appreciable diminution in myelin amounts in spite of the LPC-elicited disruption. When the radioactivity of the sliced gels was measured, it was apparent that the uptake of radioactive amino acids into the proteins was considerably higher in myelin from the LPC treated animals than in the control, even one day after administration of LPC (Fig. 4). The increased incorporation was undoubtedly partially due to increased permeability and access of the amino acids to the sites of synthesis and exchange after damage to the nerve. A considerable amount of amino acid uptake was seen in the high molecular weight region above the P0, even though very little stained protein material was visible. The pattern of increased uptake of amino acids was quite different, however, in the early time period up to one week after LPC injection compared to that in the later period after 10 days. The early high incorporation into the myelin from the ex-
41
perimental nerve was most marked in the high molecular weight proteins compared to the recognizable myelin proteins (Fig. 4), and this pattern was retained for about a week. F r o m 10 days on, P0, the other glycoproteins and the basic proteins of LPC-treated nerve, incorporated proportionately more radioactivity, while the uptake into the high molecular weight proteins decreased (Fig. 5). This two-phase process is well illustrated when the total amino acid uptake into the high molecular weight proteins and that of the P0 compared to the analogous fractions of the control nerves are plotted over the 30 day time period. Two time courses representing different experiments, with the uptake expressed as percent of control, are shown on Fig. 6. Although each set of animals yielded data showing a somewhat different course of activation, a two stage stimulatory pattern was evident in all experiments. The degree of activation depended on the activity of the control which we have shown earlier to
Day 19 450-
--
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---
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,
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200
150
I
I
l
l
I
2 4 6 8 101
I
I
141618 2o
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Days after LPC injection
Fig. 6. A and B: time course of two series of rats injected with LPC and using [3H]amino acids as the protein precursor. The total counts in the high molecular weight proteins and in the P0 proteins are expressed as percent of the counts in the analogous control proteins. Note the increased uptake into high molecular weight proteins, relative to that for P0, prior to I0 days post-LPC injection.
400-
350-
o=
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E 250" SBP ÷P2 HMW
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o
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8,
,~[~ 6 q ' ~ ' ; . / olk-~&mA I
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,
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Slice number
Fig. 5. Radioactivity in slices of gels containing separated myelin proteins 19 days after LPC injection.
be age-dependent ~4. Thus in Fig. 6A somewhat younger animals were used (about 8 weeks), while in Fig. 6B the rats were older. The percent activation appeared to be larger with older animals, especially with P0 during remyelination. In both cases, however, the curves for high molecular weight and P0 cross at 10 days post-injection. Generally, the other myelin proteins (19K, P~, and SBP + P2) were metabolically similar to P0, with the rates of amino acid uptake increasing to very high levels in these proteins on day 10, and gradually decreasing thereafter (Table I). In one experiment where the incorporation of [3H]fucose into glycoproteins was measured, the pattern was not as striking, although the largest increase in uptake of fucose into P0 occurred on day 10, the same time as that for radioactive amino acids (Fig. 7). In general, an increased incorporation of fucose was evident, but it was not as striking as that for amino acids.
42 TABLE I
Percent of control (dpm LPC/dpm Control) uptake of [~H]amino acids into sciatic nerve myelin proteins in a typical experiment Slab gels with the myelin proteins separated, as shown in Fig. 3, were sliced and the slices counted for radioactivity.
Dav
HM W
Po
19K
PI
P,
l 6 8 10 14 30
186 207 340 295 249 156
148 152 273 349 328 179
138 130 206 418 447 199
115 138 191 428 236 179
143 171 196 318 215 180
DISCUSSION
Lysolecithin-induced demyelination affords an excellent model with which to study metabolic reaction of nerve to a monophasic insult. Although a small amount of axonal damage was involved, the demyelination was, for the most part, of the primary type. Most metabolic studies of peripheral nerve demyelination remyelination so far published utilize the Wallerian degeneration model 7.
225-
-~
200-
g o
175-
2
go
_ _ _,..o. . . .
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.-o P0
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~
iI
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~ o
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~
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1'5
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Days after LPC
Fig. 7. Time course of uptake of [3 H]fucose into myelin proteins in high molecular weight fractions and P0 expressed as percent of control.
rineural injection of LPC, with all traces of the myelin sheath gone by 96 h. In experiments reported here in the rat, where somewhat larger amounts of LPC were injected, the sequence of events appears to be similar, although a large amount of disrupted myelin was still present at 3 days after LPC administration. Gregson and HalP reported that remyelination in the mouse had begun by day 14. Smith and HalP 2 noted some remyelination one week following intraneural LPC injection in mouse sciatic nerve. The present results show morphological evidence of remyelination as early as 7 days after intraneural LPC injection in rat peripheral nerve. The choice of the appropriate control nerve was a major problem in this study. After some experimentation with sham-operated nerves and perineural injection of saline into the opposite nerve, it became evident that any manipulation of the control nerve resulted in some degree ofdemyelination. The decision was made, therefore, to use intact nerves which had undergone no invasive procedures as controls. Another difficulty was the possibility of variability of the injection procedure and nerve sampling. For this reason, 4 nerves were pooled in each experiment, and attempts were made to observe the anatomical landmarks precisely in selecting the nerve segment to be studied. The lesion in the nerve, however, represented a small part of the entire sample taken, probably less than 10%, with the bulk of the myelin serving as carrier of the metabolically disturbed area which may extend beyond the lesion. In studying the metabolism of this model of demyelination remyelination, the role of proteins, especially of the glycoproteins, was of greatest interest. Glycoproteins have been associated with the role of 'recognition' in cell cell interaction, and we considered that such interactive processes should be stimulated during disruption of the myelin--axonal interactions and the subsequent re-establishment of relatively normal relationships. Glycoproteins are known to comprise at least 65% of the PNS myelin protein. The most abundant of these is the P0 protein, the main structural myelin protein which constitutes over half the total. It contains a 9-
43 sugar chain linked by N-asparaginyl linkage to the protein. In addition, fucose is well incorporated into high molecular weight myelin proteins of young rats, although less well into those of older rats ~4. High molecular weight proteins of PNS myelin, therefore, appear to contain a number of glycoproteins, only one of which has been identified 2. We have previously shown that the high molecular weight proteins contain over 40% of the total in vitro incorporated radioactivity in myelin proteins of young animals and 60-65% of that in older animals after incubating chopped nerves with [3H]amino acids 14. In view of their smaller amounts (based on staining characteristics), the high molecular weight proteins probably contain components which are more metabolically active than the prominent myelin proteins. It is likely that they represent Schwann cell membrane fragments, and interactive and recognition entities which retain more metabolic activity than the structural elements of the myelin sheath. We have tentatively interpreted our results as indicating that the demyelinative phase (up to 7 days after LPC) is accompanied by a large increase in metabolic activity of the Schwann cell membranes and recognition factors as the myelin sheath becomes vesiculated and removed from the axon. During the next phase, in which remyelination takes place (1030 days), the high molecular weight proteins diminish somewhat in their metabolic activity, while the myelin proteins are resynthesized and deposited resulting in a large increase in amino acide uptake into P0 and the other myelin proteins. We have seen morphological evidence of the beginning of remyelination as early as seven days, and others have found that conduction is beginning to be restored around six days when REFERENCES 1 Eylar, E. H., Uyemura, K., Brostoff, S. W., Kitamura, K., lshaque, A. and Greenfield, L., Proposed nomenclature for PNS myelin proteins, Neurochem. Res., 4 (1979) 289~ 293. 2 Figlewicz, D. A., Quarles, R. H., Johnson, D., Barbarash, G. R. and Sternberger, N. H., Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system, J. Neurochem., 37 (1981) 749~ 758.
the Schwann cells reassociate with the axon ~2, but have not yet begun to form myelin. The fucose uptake in the two fractions was much less dramatic than that of amino acid incorporation. Actually, the in vitro uptake of fucose in PNS myelin of adult animals is very low H, and this may be due to a decreased permeability or transport rate. It is also possible that fucose may be retained within the Schwann cells after myelin digestion and reutilized, making transport from an exogenous source less necessary than in the case of amino acids. Peterson et al? found, in degenerating nerve segments distal to a crush that the ratio pH]fucose/[14C]leucine incorporation in vitro into sciatic nerve myelin was decreased due to a significant increase in leucine uptake relative to fucose. Reutilization of myelin cholesterol during remyelination after Wallerian degeneration has been shown by others ~1. A more detailed study of the high molecular weight myelin proteins is clearly needed to identify the individual proteins, their function, and location. From results obtained with this model of demyelination, it appears that they may be important indicators of the dynamics of cell interaction and recognition. ACKNOWLEDGEMENTS
The authors are grateful to Paul Somera for running the slab gels and providing other excellent technical assistance, and to Mary Smith for providing the electron micrographs. This research is supported by the Medical Research Service of the Veterans Administration, and by Grants NS-02785 (to M.E.S.) and NS 15320 (to S.G.W.) from the NIH, and by grants from the National Multiple Sclerosis Society. 3 Gregson, N. A. and Hall, S. M., A quantitative analysis of the effects of the intraneural injection oflysophosphatidyl choline, J. Cell Sci., 13 (1973) 257 277. 4 Hall, S. M. and Gregson, N. A., The in vivo and ultrastructural effects of injection of lysophosphatidyl choline into myelinated peripheral nerve fibers of the adult mouse, J. CellSci.. 9 (1971) 76~789. 5 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T, Nature (Lond.), 227 (1970) 68(P 685.
44 6 Margolis, R. U. and Margolis, R. K.. Perspectives and functional implications. In R. U. Margolis and R. K. Margolis (Eds.). Complex Carbohydrates of Nervous Tissue. Plenum Press. New York. 1979. pp. 37~386. 7 Majno. G. and Karnovsky. M. k., A biochemical and morphological study of myelination and demyelination. II. kipogenesis in vitro by rat nerves following transection.J, exp. Med., 108 (1958) 197 208. 8 Peterson. R. G.. Baughman. S. and Scheidler, D. M., Incorporation of fucose and teucine into PNS myelin in nerves undergoing early Wallerian degeneration, Neurochem. Res.. 6(1981)213-223. 9 Pritchard. E. T. and Rossiter, R. J., Chemical studies of peripheral nerve during Wallerian degeneration. XI. In vitr¢~ incorporation of 14C-labelled precursors into phosphatides. J. Neurochem., 3 (1959) 341 o346. 10 Rawlins. F. A. and Smith, M. E., Metabolism of sciatic
11
12 13
14
nerve myelin in Wallerian degeneration, Neurobiologv, 1 (1971 ) 225-230. Rawlins, F. A., Villegas, G. M., Hedley-Whyte, E. T. and Uzman, B. G., Fine structural localization of cholesterol[1,2-3H] in degenerating and regenerating mouse sciatic nerve, J. Cell. Biol., 52(1972)615 625, Smith, K. J. and Hall, S. M., Nerve conduction during peripheral demyelination and remyelination, J. neurol. Sci., 48 (1980) 201-219. Smith, K. J., Bostock, H. and Hall, S. M., Saltatory conduction precedes remyelination in axons demyelinated with lysophosphatidyl choline, J. neurol. Sci., 54 (1982) 13 31. Smith. M. E., Biosynthesis of peripheral nervous system myelin proteins in vitro, J. Neurochem., 35 (1980) 11831189.