Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture

Neurochem. Int. Vol. 13, No. 1, pp. 111-118, 1988 Printed in Great Britain. All rights reserved

0197-0186/88 $3.00+0.00 Copyright © 1988 Pergamon Press plc

LIPID SYNTHESIS BY OLIGODENDROCYTES FROM A D U L T PIG BRAIN M A I N T A I N E D IN LONG-TERM CULTURE P. BURGISSER,* H.-H. ALTHAUS, A. ROHMANN and V. NEUHOFF Max Planck Institute for Experimental Medicine, Department of Neurochemistry, G6ttingen, FR.G. (Received 1 December 1987; accepted 18 February 1988)

Abstract---Oligodendrocytes were isolated from adult pig brain and cultivated for 18-24 days. [~4C]acetate, [3H]galactose or [35S]sulfatewere added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [~4C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [~4C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [~4C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [3H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [35S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.

A method has been developed in our laboratory which allows the bulk isolation of oligodendrocytes from adult pig brain using a Percoll gradient. About 90-95% of the freshly isolated cells are stained with an anti-galactocerebroside antiserum. After 6 weeks in culture, more than 80% of the cells are still galactocerebroside-positive. In two previous papers (Gebicke-H/irter et al., 1984a, b), we described the morphological and immunocytochemical properties of these cells, as well as their protein pattern and synthetic activity. In the present study, we investigated the ability of those oligodendrocytes to synthesize lipids at 18-24 days in vitro. Whereas lipid synthesis by oligodendrocytes in long-term culture has already been studied with cells originating from lamb (Mack and Szuchet, 1981; Mack et al., 1981; Szuchet et al., 1983), this has not yet been done with cells isolated from pig brain. Furthermore, earlier reports focused on oligodendroglia maintained in culture for a few days only (Poduslo et al., 1978). A n important question is whether or not mature oligodendrocytes can be induced to resuming their

program of myelination. The answer is not yet definitely known although the cells synthesize myelinspecific proteins (Gebicke-H/irter et aL, 1984b) and form myelin-like structures when carbon fibers are added to the culture system (Althaus et al., 1984, 1987). Since myelin is characterized by a high proportion of lipids, we wanted to know whether the oligodendrocytes could synthesize lipids constitutive of myelin or indicative of myelination as well. In addition, we investigated the synthesis of some neutral lipids. Parts of the results were already presented in an abstract form (Bfirgisser et al., 1984). EXPERIMENTAL PROCEDURES

Materials

All chemicals were of analytical grade or chromatographically pure. Most lipids standards originated from bovine brain and were obtained from Sigma (sphingomyelin, L-~-phosphatidylcholine, L-~-phosphatidyl-L-serine, L-~-phosphatidylethanolamine, galactocerebrosides) or Serva (sulfatides). Other standards were L-~-phosphatidylinositol from soybean (Sigma), L-~-phosphatidic acid from egg lecithin (Sigma), glucocerebrosides from "Gaucher's" spleen (Sigma), monogalactosyl diglyceride (Serva), cholesterol, cholesterol oleate, palmitic acid, *Address all correspondence and reprint requests to: P. 8,11,14-eicosatrienoic acid, and trierucin (Sigma). RetroBiirgisser, Laboratoire de Neurochimie, Service de P6di- peritoneal fat of guinea pig was homogenized in chloroform, atrie, CHUV, CH-1011 Lausanne, Switzerland. sonicated and filtered to give a crude triglyceride fraction. 111

112

P. BORGISSERet al.

Oligodendroglial cultures and immunocTtochemislry Oligodendrocytes were isolated from adult pig brains and cultured on poty-L-lysine-coated Petri dishes as described previously (Gebicke-Hfirter et al., 1984a), except that the medium was supplemented with 10% fetal calf serum instead of 20% and the concentration of cytosine arabinofuranoside was lowered to 16ttM. lmmunofluorescent staining was performed using anti-galactocerebroside and anti-Wolfgram protein (a gift from Dr Norman Karin) antisera (Gebicke-Hfirter et al., 1984a). Incorporation ¢~f precursors into lipid~ attd [ipid extraction Cultures at 18 24 days in vitro were incubated for 24 h with one of the following precursors dissolved in culture medium (2 ml per dish): 2tlCi/ml D-[6-3H]gatactose (32 Ci/mmol, Amersham); 1.5 ~Ci/ml [2-14C]acetate (57 mCi/mmol, Amersham): 15/~ Ci/ml [3SS]sulfate (801-1282 Ci/mmol, Amersham); 50 nCi/ml myo[14C(U)]inositol (240mCi/mmol, New England Nuclear). Subsequently, the cells were washed 3 times with phosphatebuffered saline, scraped off with a rubber policeman and collected by centrifugation (700g for 15 min). The pellets were stored at -20'~C. Lipids were extracted twice (Folch et al., 1957) in 0.8 ml chloroform/methanol 2:1 (vol/vol) containing 4% water using a sonicating water bath. Insoluble material was removed by centrifugation. The pooled supernatants were partitioned with aqueous KC1 and the lower phase was washed once in the presence of KCI to avoid losses of sulfatides (Folch et al., 1957). The final lower phase was stored overnight at - 2 0 C . High-perlormance thin-layer chromatograph) The lower phase was dried under a stream of N 2 and the lipids were redissolved in 0.1 ml of chloroform/methanol 2:1 (vol/vol) to be spotted on high-performance thin-layer chromatography plates (silica gel 60, 10× 10cm, from Merck). In later experiments, LHP-K plates with preadsorbant area (Whatman) were used (Yao and Rastetter, 1985; BiJrgisser et al., 1986). Plates were developed according to Vitiello and Zanetta (1978) with methyl acetate/ 1-propanol/chloroform/methanol/aqueous KCI 2.5 g/1 (25 : 25 : 25 : 10: 9, by vol). When neutral lipids were studied, the chromatography was stopped when the solvent front had reached two-thirds of the plate height. The plates were then dried and submitted to a second run in the same direction either in solvent A (petroleum ether, b.p. 60-80' C/diethyl ether/acetic acid, 80:20:1 by vol) or solvent B (benzene/diethyl ether/ethyl acetate/acetic acid, 80: 10:10 : 0.2 by vol) (Jagannatha and Sastry, 1981) until the solvent front was at 0.5 cm from the top. Spots were visualized by exposure to 12 vapors. For fluorography, the plates were sprayed 3 5 times with ENSHANCE spray (New England Nuclear) and exposed to an X-Omat S film (Kodak) at -80~C. A thin transparent plastic sheet was sandwiched between the plate and the film. For quantification of the radioactivity, the bands were scraped into scintillation vials and counted after the addition of 15 ml of Aquasol (New England Nuclear). All percentages given in the text represent the mean values of 3-7 measurements, involving one to two different preparations. The coefficients of variation (standard error expressed as percent of the mean) never exceeded 13%.

Incorporation ~]' [t4C]acetate into plasmalogens Chromatography was first performed according to Vitiello and Zanetta (1978). "[he dried plate was then exposed to HCI vapors (Horrocks, 1968) and developed in a second solvent system (Horrocks and Sun, 1972) at a right angle. Methanolysis oj [~H]galactose-laheled lipids For alkaline methanolysis, lipids (puritied and washed as indicated above) were dissolved in chloroform/methanol 1:4 containing 0.1 M KOH and incubated for 15 rain at 26 C [Kates. 1972). Then chloroform, methanol and water fincluding a neutralizing amount of HC1) were introduced to form a biphasic system. An aliquot of the resulting upper phase was withdrawn for scintillation counting in Aquasol. For quantitative data, control experiments were also performed in which KOH was omitted. Thus, a suitable amount of KC1 was added at the end of the incubation to give the same concentration as in the test tubes. Following two more washes (Kates, 1972), the lipids remaining in the lower phase were examined by thin-layer chromatograph>,. Alternatively, they were submitted to acidic methanolysis for 18 h at 72'C in I M HC1 and I0 M H,O (Gaver and Sweely, 1965). Afterwards. a biphasic system was reconstituted as above (except that the neutralizing agent was NaOH) and the lower phase similarly washed and used for chromatography. RESULTS

]mrnunoc.t'tochemistry After 3 weeks in culture, 85 0 0 % of the cells were positive for anti-galactocerebroside surface staining (Fig. 1). Almost the same percentage of cells was positively stained by anti-Wolfgram protein antiserum after fixation-permeabilization.

F4C]acetate incorporation A l t h o u g h [~4C]acetate was incorporated predominantly into neutral lipids (58.9%), this precursor labeled all of the main polar lipids k n o w n to be present in brain (Fig. 2). A m o n g them, phosphatidylcholine was the most strongly labeled (17.5%), followed by the e t h a n o l a m i n e p h o s p h a t i d e s (6.0%), galactocerebrosides (5.8%), phosphatidylinositol (3.2%), phosphatidylserine (3.1%), sulfatides (1.8%), and sphingomyelin (1.7%). Two additional b a n d s were labeled: (1) one referred to as X in Fig. 2 (0.8%), which could consist of phosphatidic acid a n d / o r diphosphatidylglycerol (Vitiello and Z a n e t t a , 1978); (2) an unidentified c o m p o u n d referred to as Y (1.4%). The identity of all these b a n d s was established by comigration with k n o w n standards. The presumed phosphatidylinositol and phosphatidylserine had a slightly different mobility t h a n the corresponding standard. Phosphatidylserine was definitely identified by its positive staining with ninhydrin. F l u o r o g r a p h y

i~ii~~ ~

J

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P. BI]RGISSERet

of lipids from oligodendrocytes incubated with [~4C]inositol showed only one band comigrating exactly with the presumed phosphatidylinositol labeled with [14C]acetate (not shown). When the plates were subjected to a second development for the separation of neutral lipids, most of the [~4C]acetate was incorporated into bands comigrating with cholesterol and triglycerides in two solvent systems (Fig. 3). No cholesterol esters were synthesized. A very broad band around (solvent A) or below (solvent B) cholesterol was also present and could represent fatty acids of various chain lengths. The incorporation of [~4C]acetate into ethanolamine phosphatides was further investigated, taking advantage of the selective hydrolysis of the alkenyl-ether bond by HCI vapors (Horrocks, 1968). Plasmalogens contained 71.6% of the radioactivity present in all of the ethanolamine phosphatides (after acid hydrolysis, 46.1% were in the lysophosphatidylethanolamine and 25.5% in the fatty aldehydes). The

I

NL

al.

sod

YE

TG

PE CHE X

TG

PI E Ps ["

,¢

PoE SM

O

20:3

CH

CH

--

-

GC Fig. 2. Fluorograms of the lipids synthesized by oligodendrocytes at 24 days in culture. Two different precursors were given for 24 h: [t4C]acetate (left-hand lane, containing 9800 dpm, corresponding to 27/~g protein; exposure time: 140 h) and [35S]sulfate (right-hand lane, containing 2800 dpm, corresponding to 17 #g protein; exposure time: 5 h). Lipids were extracted and separated by thin-layer chromatography according to Vitiello and Zanetta (1978). NL--neutral lipids; GC--galactocerebrosides, containing ct-hydroxylated (lower band) or unsubstituted (upper band) fatty acids; SU--sulfatides (two bands as for GC); PE-ethanolamine phosphoglycerides; Pl--phosphatidylinositol; PS--phosphatidylserine; PC--phosphatidylcholine; SM-sphingomyelin; X and Y--two components discussed in the text under Results; O~origin.

GC

Fig. 3. Fluorograms of the neutral lipids synthesized by oligodendrocytes at 20 days in culture using [~4C]acetate as precursor. The plates were first developed according to Vitiello and Zanetta (1978) for the separation of polar lipids; they were then dried and developed further with solvent A (left-hand lane) or B (right-hand lane) for the separation of neutral lipids. Only the upper part of the fluorogram is shown. Dashes indicate the position of standards. CHE-cholesterol ester (oleate); TG--triglycerides from adipose tissue and trierucin; 20:3--8,11,14-eicosatrienoic acid; CH ----cholesterol; GC--galactocerebrosides.

In vitro synthesis of oligodendroglial lipids

2

115

3

Fig. 4. Fluorogram of the lipids synthesized by oligodendrocytes at 18 days in culture using [3H]galactose as precursor. Lanes 1 and 2: control experiment in which the extracted lipids were chromatographed without prior treatment (136,000 dpm, corresponding to 120 #g protein; exposure time: 20 h for lane 1, 21 days for lane 2). Lane 3: lipids were subjected to alkaline methanolysis prior to chromatography (21 days exposure). Lane 4: lipids were subjected to alkaline followed by acidic methanolysis (21 days exposure). Same abbreviations as in Fig. 2. Quantitative data (Table 1) showed that more than 92% of the radioactivity in Z was present as monogalactosyl diglyceride. The identity of the minor alkali-resistant band (arrow in lane 3) is discussed in the text.

remaining 28.4% were found in the acid-resistant diacyl and alkylacyl species. On the other hand, radioactivity was present in choline plasmalogens, amounting to 14,8% of the total radioactivity incorporated into choline phosphatides, whereas no synthesis of serine plasmalogens was detected. [3H]galactose incorporation

As illustrated in Fig. 4, this precursor labeled primarily galactocerebrosides (65.9%), sulfatides (15.0%), and the unidentified band(s) Z (14.4%). The remaining radioactivity distributed into phospholipids (2.8%) and neutral lipids (I.9%). The incorporation was twice as high in galactocerebrosides containing unsubstituted fatty acids than in those containing ~-hydroxylated fatty acids. Sulfatides with normal fatty acids were also preferentially labeled. Two standards, glucocerebrosides and monogalactosyl diglyceride, had Rs values close to that of band Z. To further identify this compound, lipids

were subjected to mild alkaline methanolysis. Diacyl phosphoglycerides were completely cleaved, as evidenced by the complete disappearance of phosphatidylcholine, phosphatidylserine and phosphatidylinositol upon 12 staining of the chromatograms (not shown) and on fluorograms (Fig. 4). Due to the relative higher resistance of their 2-ester bond (Wells and Dittmer, 1966), 1-alkenyl/alkyl-2-acyl ethanolamine phosphatides were only partially converted into their 2-1yso derivative (Fig. 4). Under these conditions, 92.3% of the radioactivity present in Z was no longer seen after alkaline methanolysis, while the remaining 7.7% appeared as a band of lower R r (arrow, Fig. 4; Table 1) previously masked by the main component. Galactocerebrosides and sulfatides were not degraded during alkaline methanolysis, as expected for sphingolipids. Upon subsequent acidic methanolysis, they gave rise to two bands (Fig. 4). The faintly labeled but more abundant one had the expected R r for sphingosine, while the highly labeled

116

P. BURGISSER et al. Table I. Characterization of components(s) Z (Fig. 4) using mild alkaline methanolysis of [~H]galactose-labeled lipids Alkaline methanolysis (A) Methanol-water phase Band Z (Fig. 4) Galactocerebrosides (GC) Ethanolamine phosphoglycerides (PE) Phospholipids except PE (SM + PC + PS + PI) Neutral lipids (NL)

8951 ± 111 519 ± 156 29,255 ± 1596 NM NM 905 ± 45

Control (B) 168 ± 6729 ± 27,654 ± 333 ± 848 ± 813 ±

II 1142 4492 67 134 117

Difference (A - B) 8783*** 6210"** 1601 NS

92 NS

Results are expressed as cpm (mean of three measurements + SD). Oligodendrocytes at 18 days in culture were incubated for 24 h in the presence of [3H]galactose. Lipids were extracted and washed (Folch et al., 1957). The whole extract was then incubated in chloroform/methanol 1:4 in the presence (column A) or the absence (column B) of 0.1 M KOH, as explained in Experimental Procedures. Afterwards, a biphasic system was established and the radioactivity present in the methanol-water phase was measured. Following two additional washes, lipids present in the lower phase were separated by thin-layer chromatography (Vitiello and Zanetta, 1978) and the radioactivity in some of them was determined by scintillation counting. The statistical significance of the difference between methanolized and control samples was computed using the Student's t-test (last column): ***P <0.001; N S - - n o t significant (P >0.3). N M - - n o t measured. Other abbreviations as in Fig. 2.

but minor one had the expected Rt for psychosine (Yao and Rastetter, 1985). The band Y, labeled with [J4C]acetate, was not detectable when [3H]galactose was used as the precursor. [~SS]sulfate incorporation

[35S]sulfate labeled only the sulfatide doublet (Fig. 2). Even the exposure of the film for a much longer time (46 h instead of 5 h) failed to reveal any other labeled lipid.

DISCUSSION

[J4C]acetate can be incorporated into fatty acids and steroids via acetyl-CoA, and into sphingosine via palmitoyl-CoA. Therefore, it was chosen as a general lipid precursor. Using this substrate, the cultured oligodendrocytes were able to synthesize the major glycerolipids and sphingolipids found in the central nervous system, as well as cholesterol. Our quantitative data do not fit with those published by Szuchet et al. (1983) using [14C]acetate for 72 h at the same time in culture and showing sphingomyelin and phosphatidylinositol to be the most strongly labeled, followed by phosphatidylcholine. Possible explanations include species differences (sheep vs pig) and selection of a special subtype of oligodendrocyte by these authors (Szuchet and Yim, 1984), which could result in a different pool size and/or molar ratio of the lipids present. In our system, an increase of the incorporation time up to 72h did not alter significantly the distribution of the radioactivity. On the other hand, our results differ strikingly from those

obtained by Bourre et al. (1981) with cultured Scbwann cells. In the latter, most of the [L4C]acetate was incorporated into phosphatidylcholine, while cerebrosides and sulfatides contained about 100 times less radioactivity. The high proportion of [J4C]acetate that we found incorporated into ethanolamine ptasmalogens is noteworthy because, in bovine brain myelin, plasmalogens represent as much as 72-77% of the ethanolamine phosphatides (Norton and Autilio, 1966; Pleasure et al., 1981). The presence of the label nol only in the 1-1yso phosphatidylethanolamine produced by the acid hydrolysis but also in the fatty aldehydes demonstrates the expression by our cells of the reductase responsible tbr the synthesis of fatty alcohols, which are the substrates of the alkyl dihydroxyacetone phosphate synthase (Bishop and Hajra, 1978, 1981). The high proportion of the label incorporated into choline plasmalogens is surprising since only 3.6% of the total choline phosphatides in the bovine brain myelin is in the 1-alkenyl form (Norton and Autilio. 1966). We decided to investigate further the synthesis of neutral lipids because they appeared (Fig. 2) to be made up of several components. A large proportion of these neutral lipids comigrated with triglycerides using two different solvent mixtures. To our knowledge, only one publication (Carey et al., 1980) describes triglyceride synthesis by oligodendrocytes isolated from myelinating rat brain. Even though the enzymatic systems responsible for the occurrence of small amounts of triglycerides in brain have been characterized (Bishop and Hajra, 1984), the role of these products remain obscure. One possibility is that

117

In vitro synthesis of oligodendroglial lipids

triglyceride synthesis represent a metabolic adaptation of cells exposed to an abnormally high concentration of fatty acids originating from the fetal calf serum. Indeed, Shah and Johnson (1986) observed that dissociated cells from newborn rat brain accumulated cholesterol esters when cultured in the presence of fetal calf serum, a finding absent when the latter was replaced by lipoprotein-deficient serum. Nonetheless, it is interesting to note that our cells synthesized triglycerides rather than cholesterol esters, in view of the potential expression of cholesterol ester hydrolase by oligodendrocytes (Norton, 1981). [3H]galactose labeled primarily the galactolipids, galactocerebrosides and sulfatides. A third component (Z) incorporated this precursor very actively. It could consist of: (I) monogalactosyl diglyceride, which is enriched in myelin (Pieringer et al., 1973); (2) glucocerebrosides, detected in bovine and synthesized by ovine oligodendrocytes (Abe and Norton, 1979; Szuchet and Yim, 1984); (3) galactocerebroside esters, present in bovine oligodendrocytes (Abe and Norton, 1979). Since fatty acid ester, but not amide bonds are alkali-labile, alkaline methanolysis should help for identification. Table 1 indicates that the radioactivity which disappeared from the Z spot under this treatment was recovered in the methanol-water phase. This identifies the main component as monogalactosyl diglyceride (diacyl form). Glucocerebrosides, which are insensitive to alkaline methanolysis and have a slightly lower R/than monogalactosyl diglyceride, can account for the small, persisting activity (Fig. 4). This is consistent with the finding of Brammer (1984) that galactose is readily converted into glucose by oligodendrocytes. Galactocerebroside esters do not represent a major component of the Z spot because radioactivity did not increase significantly in the galactocerebrosides after alkaline methanolysis. [3H]galactose also labeled glycerophospholipids. Incorporation could occur either via fatty acids or glycerol. In the former hypothesis, alkaline methanolysis should liberate approx. 1000 cpm as fatty acid methyl esters (from Table 1, assuming that 35% of the ethanolamine phosphoglycerides are under diacyl form, Pleasure et al., 1981). Such an increase of activity in the neutral lipids after alkaline methanolysis was not observed. On the other hand, if glycerol carried the label, alkaline methanolysis would release 964 cpm in the methanol-water phase which would add to the 6210cpm originating from monogalactosyl diglyceride. The measured value (8783 cpm) is consistent with the second hypothesis

and confirms previous results (Gibson and Brammer, 1984). Figure 4 shows that sphingosine was weakly labeled. Given the above considerations, it is likely that the label originated from serine, which can be synthesized from 3-phosphoglycerate. In adult rat brain, more than 60% of monogalactosyl diglyceride is found in the myelin fraction (Pieringer et aL, 1973), where it represents a minor lipid. In rat brainstem, there is 6 times less monogalactosyl diglyceride than sulfatides (Nonaka and Kishimoto, 1979). Therefore, our finding that adult oligodendrocytes incorporated almost as much [3H]galactose into monogalactosyl diglyceride as into sulfatides is surprising. This could reflect an attempt of these cells to resume myelination, since monogalactosyl diglyceride is synthesized at a maximum rate during myelination in vivo (Pieringer et al., 1973). Using cultured fetal brain cells, Singh and Pfeiffer (1985) also noticed a very active synthesis of this lipid at a time when myelination occurs in vivo. In conclusion, oligodendrocytes in long-term culture were able to synthesize not only lipids present in membranes in general, but also lipids which are enriched in myelin, such as galactocerebrosides, sulfatides, and ethanolamine plasmalogens (Norton, 1981). Thus, these and the previous results (GebickeH/irter et al., 1984b) demonstrate that adult oligodendrocytes can produce the basic components of myelin. Secondly, we demonstrated that oligodendrocytes in long-term culture synthesize triglycerides and monogalactosyl diglyceride at a high rate. The significance of these two findings remains to be elucidated. Whether the biosynthesis of lipids in adult cells can be modulated by certain factors requires further investigation. First results indicate that hydrocortisone can induce glycerol phosphate dehydrogenase, an important enzyme for the synthesis of glycerolipids (Montz et al., 1985). are grateful to Professor J.-M. Matthieu for having given us the possibility to perform some experiments in his laboratory. Acknowledgement--We

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