Lipid synthesis by an oligodendroglial fraction in suspension culture

Brain Research, 134 (1977) 0 ElsevierjNorth-Holland

Lipid synthesis

DAVID JUDY

PLEASURE, PARRIS and

377-382 Biomedical

by an oligodendroglial

ODED TAKAHIKO

ABRAMSKY, SAIDA

Experimental Child Neurology, Children’s D.S. and TS.) University of Pennsylvania (Accepted

June

9th,

377 Press

fraction

DONALD

in suspension

SILBERBERG,

culture

BARBARA

QUINN,

Hospital of Philadelphia ( D. P., B. Q. and J. P.) and (0. A., School of Medicine, Philadelphia, Pa. 19104 I U.S.A.)

1977)

Methods now available for isolation of an oligodendrogial fraction (OF) from mammalian brain7.11~22*2sJ6 and for culture of these cells in vitro7~s~is~*eJr, should facilitate analysis of oligodendroglial membrane-synthetic pathways. In the present paper, we describe morphological and enzymatic features of OF prepared from calf cerebral white matter, and studies of cholesterol and sulfatide synthesis by this fraction during suspension culture. Fresh calf brains were obtained from a slaughter house, and OF was isolated from cerebral white matter by the method of Poduslo and Norton23, with the following minor modifications: all media contained 100 units of penicillin, 100,~g of streptomycin, and 5 ,ug of Mycostatin per ml, and were sterilized by millipore filtration. All procedures were carried out in a laminar air flow hood. Highest yields of oligodendrocytes (3-5 x lo6 cellsig white matter) were obtained when trypsin digestion of the minced cerebral white matter was for 75 min at 37 “C, with 0.25 ‘A trypsin (w/v, Sigma Chemicals). These yields were 30-50% of that reported by Poduslo and Norton’s. After sucrose gradient centrifugation 23, the fraction was diluted to 1000 ml with medium A (10 mM potassium phosphate, pH 6.0, containing 5 “/, glucose, 5 ‘? fructose and I % bovine serum albumin, all w/v), and concentrated by centrifugation at 700 x g for 10 min. A portion of the concentrated OF was taken for phase and Normarski interference contrast light microscopy, and for transmission and scanning electron microscopy. Other portions of concentrated OF were suspended in medium B (Eagle’s spinner minimum essential medium with Earle’s salts, 2 mM L-glutamine and 10% (v/v) fetal calf serum from Gibco). Each ml of medium B, containing 10’ cells (by bright-field light microscopic hemocytometer count), was placed in an acidwashed centrifuge tube (Corex glass) and maintained for up to 48 h in 5 y/, C&/95 ‘j,: air at 37 “C in an humidified CO2 incubator. Radioactive precursors (ssSO1, [ I-i4C]acetate, from New England Nuclear) or puromycin were added to the medium as required. For biochemical assays, portions of OF were washed three times with 0.90,/, NaCl (w/v), with centrifugation at 700 x g for 10 min after each wash. Protein was assayed by the Lowry method’s, glutamate decarboxylase by the GABA isolation

378

Fig. 1. Morphology of an oligodendroglial fraction isolated from calf cerebral white matter. A : phase contrast microscopy or oligodendrocyte cell suspension in medium A. Original magnification x 600. B: Normarski interference contrast microscopy of oligodendrocyte cell suspension in medium A. Original magnification x 600. C: transmission electron microscopy; glutaraldehyde and osmium tetroxide fixed, as described by Raine et a1.25, and embedded in Araldite. Original magnification x 10,300. Note microtubules in cytoplasm. D : scanning electron microscopy ; glutaraldehyde-fixed, critical point dried, gold-palladium coated. Original magnification x 10,000. White line equals 1 itm.

method of Kanazawa et al.‘“, choline acetyltransferase by the method of Wilson et a1.29, acetylcholinesterdse by the method of Johnson and Russelll2, 2’,3’-cyclic nucleotide-3’-phosphohydrolase (CNP) by a modificationls of the method of Prohaska et a1.24, cerebroside sulfotransferase by the method of Siegrist et al.*’ and 3-hydroxy-

379 3-methylglutaryl coenzyme A reductase (HMG CoA reductase) by a modification’s of the method of Brown et aL3. Lipids were extracted with two portions of 3 ml each of chloroform/methanol (2/l, v/v); the pooled organic extract, with added carrier sulfatide and unesterified cholesterol, was washed once with upper phase and 4 times with theoretical upper phase lo. Lipids were resolved by silicic acid thin layer chromatography, using sequential development in one dimension with chloroform/methanol/ water (65/25/4, v/v/v) followed by hexane/diethyl ether/acetic acid (90/10/l, v/v/v). Radioactivity was measured in a liquid scintillation spectrometer. As previously reported by Raine et al. asJ6, light and electron microscopy of OF showed an homogeneous population of oligodendrocytes, with occasional myelin fragments (Figure I). Phase contrast light microscopy and scanning electron microscopy of OF after 24 and 48 h in suspension culture showed essentially the same morphological features as at the time of original isolation of the fraction. Assays of OF for neurotransmitter synthetic enzymes (glutamate decarboxylase and choline acetyltransferase) and acetylcholinesterase indicated relatively little contamination of the fraction by neuronal elements (Table I). Specific activity of CNP in OF was more than twice that reported by Poduslo in 1975 19, but only one-third that noted in Poduslo and Norton’s original report22. In our study, OF CNP specific activity was 22% of that in calf cerebral white matter (Table I). Poduslo has demonstrated that this enzyme is a constituent of the oligodendroglial plasma membrane per se, as well as of myelinrg. As previously reported by Benjamins et al. 2, OF was considerably enriched over white matter in the sulfatide-synthetic microsomal enzyme, cerebroside sulfotransferase (Table I). Enrichment of OF in HMG CoA reductase, the microsomal enzyme rate-limiting in cholesterol synthesis’,4~ls,ls,2s was considerably less than for cerebroside sulfotransferase (Table I). This discrepancy may reflect the lability of HMG CoA reductase (see below), with loss of activity during the 7-h isolation procedure. Cohen and Bernsohns reported that, during short-term in vitro incubation, OF incorporated fatty acids into phospholipids, and Deshmukh et al.6 noted incorporation of TABLE

1

Specific activities of enzymes in an oligodendroglialfraction the method of Pod&o and Norton

isolated

from

calf cerebral

white

matter

by

Values given are means + standard errors, and figures in parentheses denote numbers of assays. Methods are given in the text. Enzyme

White

matter

Oligo fraction

.~~-

Glutamate decarboxylase* Choline acetyltransferase* Acetylcholinesterase* 2’,3’-cyclic nucleotide-3’-phosphohydrola.se** Cerebroside sulfotransferase* 3-hydroxy-3-methylglutaryl coenzyme A reducta.se* .-pmoles/mg proteimmin. * nmoles/mg proteimmin.

l l

418 -I. 165 (n = 4) I7 f IS (n = 4) 9.2 rtr 4.5 (n - 3) 0.6 f 0.5 (n = 6) 1050 f 235 (n - 5) 110 :I: 55 (n -- 3) 6800+26OO(n-5) 1500+7OO(n-5) 0.023 .Z 0.010 (n r 3) 0.21 f 0.08 (n - 3) 1.7 + 0.8 (n = 4) 5.6 + 2.7 (n 7 4) -.

380

TABLE

II

Incorporation of rudioactive precursors from calf cerebral white matter

into lipids

during

suspension

culture

of oligodendroglial

fraction

Values given are means of 4 experiments (* standard errors). Each tubecontained SO@ of [I-‘“Clacetate (specific activity 59 ~Ci/~mole) or 100 PCi of 3s!SO~(specific activity 350 &i/pmole) in I ml of medium. Lipid isolation and quantitation of radioactivity are described in the text. Interval during with radioactive

Incorporation Incorporation

of (IJ4C]acetate into unesterilied sterol* of 35SO4 into sulfatide+

* Disint./min/mg

which cells precursor

were

O-24 h

24-413 h

381 :f 18 8510 .+ 550

1079 -f- I21 2548 t 332

incubated

protein.

[Wlgalactose by OF into galactosyldiglyceride. Subsequently, Poduslo et al.20.21 maintained OF in suspension culture for up to 48 h, and noted incorporation of [l%]acetate and other radioactive precursors into sterol and other lipids. In the present study, OF incorporated ssSO4 into sulfatide, and [114C]acetat.e into unesterified sterol during suspension culture; the extent of incorporation of radioactive precursors during 6-h labeling periods (not shown) was about one-quarter that observed with the 24 h labeling periods (Table II). Incorporation of 35SO4 into sulfatide fell 3-fold during the second day of culture (Table II), and there was also a decrease in the specific activity of cerebroside sulfotransferase during suspension culture (Table 111). In contrast, the rate of incorporation of [IJ4C]acetate into unesterified sterol, and the specific activity of HMG CoA reductase, rose more than 2-fold during culture (Tables II and III). The reasons for the divergent behavior of the sterol and sulfatide- synthetic pathways during suspension culture are not clear. The half-life of HMG CoA reductase in fibroblasts, hepatoma cells, C6 glioma TABLE

III

SpeciJic activities during suspension

of 3-hydroxy-3-methylglutaryl culture of oligodendroglial

coenzyme fraction from

A reductase calf white

and cerebrosi& matter

sulfotrunsjkruse

Values given are means 2 standard errors, and figures in parentheses denote number of assays. Assay methods are described in the text. Hours

in culture

0

24 48 48 (last 270 min with 100 pg/rnl puromycin) * pmoles/mg protein/min

3-hy&oxy-3-methylglutaryl coenzyme A reductase*

Cerebroside sulfotransferase* -

5.6 12.7 12.9 5.7

0.21 + 0.08 (n ::- 3) 0.15 * 0.07 (n T 3) 0.14 f 0.08 (n : 3)

I ir + +

2.7 (n 7.4 (n 4.9 (n 3.1 (n

-7 4) =- 4) : 6) = 3)

381 and neuroblastoma is only 2-4 hr~4~rQs. Wh en OF was incubated with 100,~g of puromycin/ml medium for 270 min prior to cell harvest, activity of HMG CoA reductase was less than half that in controls (Table III), suggesting that the half-life of this enzyme in oligodendrocytes in suspension is also relatively short. The present morphological and biochemical data on OF isolated from calf cerebral white matter by the method of Poduslo and Norton23 confirm previous reportsZ3JjJ” that the fraction is relatively homogeneous. The suspension culture studies indicate that a proportion of the cells in the fraction are viable, and capable of continued synthesis of membrane lipids. Oligodendroglial suspension culture should prove useful for further investigations of oligodendroglial lipid and protein-synthetic pathways. A preliminary report of some of this data was presented at the 8th annual meeting of the American Society for Neurochemistry 17. We are grateful for the skilled technical assistance of Ms. Marge Lieb. This research was supported by grants from the National Foundation-March of Dimes (l-367), the National Multiple Sclerosis Society (NMS 894-B-2), and by NIH Grants I-POl-HO-08536, NS 11037 and NS 08075. Dr. Abramsky received support from an NIH Fogarty International Research Fellowship and a FulbrightHays International Exchange Scholarship.

1 Beirne, 0. R., Heller, R. and Watson, J. A., Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in minimal deviation hepatoma 7288C. Immunological measurements in hepatoma tissue culture cells, J. biol. Chem., 252 (1977) 95&954. 2 Benjamins, J. A., Guarnieri, M., Miller, K., Sonneborn, M. and McKhann, G. M., Sulphatide synthesis in isolated oligodendroglial and neuronal cells, J. Neurochem., 23 (1974) 751-757. 3 Brown, M., Dana, S. and Goldstein, J., Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by lipoproteins, Proc. nut. Acod. Sci. (U’ush.), 70 (1973) 2162-2166. 4 Brown, M. S., Goldstein, J. L. and Siperstein, M. D., Regulation of cholesterol synthesis in normal and malignant tissue, Fed. Proc., 32 (1973) 2168-2173. 5 Cohen, S. R. and Bernsohn, J., Incorporation of I-*% labelled fatty acids into isolated neuronal soma, astroglia and oligodendroglia from calf brain, Bruin Reseurch, 60 (1973) 521-525. 6 Deshmukh, D. S., Flynn, T. J. and Pieringer, R. A., The biosynthesis of galactosyldiglyceride in glial and neuronal enriched fractions of actively myelinating rat brain, J. Neurochem., 22 (1974) 479485. 7 Fewster, M. E. and Blackstone, S., In vitro study of bovine oligodendroglia, Neurobiology, 5 (1975) 316328. 8 Fewster, M. E., Blackstone, S. C. and Ihrig, T. J., The preparation and characterization of isolated oligodendroglia from bovine white matter, Bruin Reseurch, 63 (1973) 263-271. 9 Fewster, M. E., Ihrig, T. J. and Mead, J. F., Biosynthesis of long chain fatty acids by oligodendroglia isolated from bovine white matter, J. Neurochem., 25 (1975) 207-213. IO Folch, J., Lees, M. and Sloane-Stanley, G., A simple method for the isolation and purification of total lipids from animal tissues, J. biol. Chem., 226 (1957) 497-509. I I Iqbal, K., Gundke-lqbal, I. and Wisniewski, H. M., Oligodendroglia from human autopsied brain: bulk isolation and some chemical properties, J. Neurochem., 28 (1977) 707-716. I2 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations, Anulyr. Biochem., 64 (1975) 229-238. I3 Jones, J. P., Nicholas, H. J. and Ramsey, R. B., Rate of sterol formation by rat brain glia and neurons in vitro and in vivo, J. Neurochem., 24 (1975) 123-126.

382 14 Kanazawa, I., Iversen, L. L. and Kelly, J. S., Glutamate decarboxylase activity in the rat posterior pituitary, pineal gland, dorsal root ganglion and superior cervical ganglion, J. Neurochem.. 27 (1976) 1267-1269. IS Kirsten, E. S. and Watson, J. A., Regulation of 3-hydroxy-3-methylglutaryl ccenzyme A reductase in hepatoma tissue culture cells by serum lipoproteins, J. biol. Chem., 249 (1974) 6104-6109. 16 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 17 Pleasure, D., Abramsky, O., Silberberg, D., Quinn, B. and Parris, J., Biochemical studies of oligodendrocytes from calf brain, Trans. Amer. Sot. Neurochem., 8 (1977) 143. 18 Pleasure, D. and Kim, S. U., Sterol synthesis by myelinating cultures of mouse spinal cord, Bruin Research, 103 (1976) 117-126. 19 Poduslo, S. E., The isolation and characterization of a plasma membrane and a myelin fraction derived from oligodendrogha of calf brain, J. Newochem., 24 (1975) 647-654. 20 Poduslo, S. E., McFarland, H. F., Miller, K., Krcen, C. and McKhann, G. M., Maintenance of bulk isolated oligodendroglia, Trans. Amer. Sot. Neurochem., 7 (1976) 90. 21 Poduslo, S. E., Miller, K., Piasecki, B. and McKhann, G. M., Metabolic properties of maintained oligodendroglia, Trans. Amer. Sot. Neurochem., 8 (1977) 141. 22 Poduslo, S. E. and Norton, W. T., Isolation and some chemical properties of oligodendroglia from calf brain, J. Neurochem., 19 (1972) 727-736. 23 Poduslo, S. E. and Norton, W. T., Isolation of specific brain cells, in J. M. Lowenstein (Ed.), Methods in Enzymology, Vol. 35, Academic Press, New York, 1975, pp. 561-579. 24 Prohaska, J., Clark, D. and Wells, W., Improved rapidity and precision in the determination of brain 2’,3’-cyclic nucleotide-3’-phosphohydrolase, Anulyf. &o&em., 56 (1973) 275-282. 25 Raine, C. S., Poduslo, S. E. and Norton, W. T., The ultrastructure of purified preparations of neurons and glial cells, Bruin Research, 27 (1971) 1l-24. 26 Raine, C. S., Wisniewski, H. M., Iqbal, K., Grundke-Iqbal, I. and Norton, W. T., Studies on the encephalitogenic effects of purified preparations of human and bovine oligodendrocytes, Bruin Research, I20 ( 1977) 269-286. 27 Siegrist, H. P., Burkart, T., Steck, A. J., Wiesmann, U. and Herschkowitz, N. N., Influence of lipids on the activity of cerebroside-sulfotransferase in mouse brain: a comparative study of Jimpy and normal mouse brains, J. Neurochem., 27 (1976) 599-604. 28 Volpe, J. J. and Hennessy, S. W., Regulation of cholesterol biosynthesis in cultured glial and neuronal cells, Trans. Amer. Sot. Neurochem., 8 (1977) 144. 29 Wilson, S. H., Schrier, B. K., Farber, J. L., Thompson, E. J., Rosenberg, R. N., Blume, A. J. and Nirenberg, M. W., Markers for gene expression in cultured cells from the nervous system, J. bio/. Chem., 247 (1972) 3159-3169.