Neurochem. Int. Vol. 21, No. 2, pp. 287-291, 1992 0197-0186/92 $5.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1992 Pergamon Press Ltd

PHOSPHOLIPID COMPOSITION OF OLIGODENDROGLIAL CELLS DURING NORMAL

DEVELOPMENT AND IN 18 DAY OLD HYPERTHYROID A N D MALNOURISHED RATS

SILVIA M. KREDA,* JUANA M. PASQUINI and EDUARDO F. SOTO Departamento de Quimica Biolbgica-IQUIFIB (UBA-CONICET), Facultad de Farmacia y

Bioquimica, Universidad de Buenos Aires, Junin 956-Ill 3 Buenos Aires, Argentina

(Received 29 July 1991 ; accepted 16 December 1991)

Abstract--The phospholipid composition of isolated oligodendroglial cell perikarya was studied in normal rats during development and in 18 day old malnourished and hiperthyroid rats. Phosphatidyl choline and phosphatidyl ethanolamine were found to be the major phospholipid constituents of oligodendroglial cells. Phospholipid content increased during development, mainly due to an increase of the above mentioned phospholipids. The major changes were observed in sphingomyelin, phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine between 18 and 30 days of age. The phospholipid and protein content per cell was significantly decreased in the oligodendroglial cells isolated from malnourished rats as compared to controls. When data were expressed as a function of total proteins, the composition was similar to that of normal animals. In the hyperthyroid rats on the other hand, there were no changes in the amount of phospholipids per cell, while phospholipids per milligram of total oligodendroglial cell protein were markedly decreased. The changes in myelin composition produced by hyperthyroidism that we have previously described, do not follow closely those produced by this experimental condition in oligodendroglial cells, suggesting that the metabolism of myelin might be, to a certain extent, independent of that in the parent cell.

During the last decade, techniques for the bulk iso- lation of oligodendroglial cell (OGC) perikarya have been developed in a number of laboratories (Poduslo and Norton, 1972 ; Fewster et al., 1973 ; Snyder et al., 1980; Szuchet et al. 1980; Farooq et al., 1981 ; Berti Mattera et al., 1984) and have facilitated the study of the properties of this characteristic type of cell. One of the main functions of the OGC is to myelinate axons in the CNS (Wood and Bunge, 1984). Because differentiation of these cells in the developing brain is accompanied by major elaborations of their plasma membrane, it is reasonable to suspect that membrane phospholipids (PLs) must play an important role in

* Author to whom all correspondence should be sent. Abbreviations: CNS, Central Nervous System; Ht, hyper-

thyroid/ism ; Mn, Malnutrition/shed ; OGC, Oli- godendroglial cells; UP, Unidentified PLs (probably phosphatidic acid and lyso PLs, as well as those remain- ing in the TLC at the origin) ; PC, Phosphatidyl choline; PE, Phosphatidyl ethanolamine ; PI, Phosphatidyl inosi- tol ; PL/s, Phospholipid/s ; PS, Phosphatidyl serine ; SPH, Sphingomyelin ; TPL, Total phospholipids.

this event (Volpe et al., 1986). Phospholipids represent the major lipid components of cellular membranes in the brain as well as in other tissues, but the functional role of these lipids in the developing CNS remains to be defined.

Although the PL composition of isolated OGC has been studied by several investigators (Farooq et al., 1981 ; Poduslo and Norton, 1972 ; Macala et al. 1983), it has not been worked over as a function of age, nor in pathological states such as neonatal malnutrition (Mn) and hyperthyroidism (Ht), two conditions which are known to produce important alterations of myelin during development of the central nervous system (Pasquini et al., 1981; Walters and Morell, 1981). Very little is known about the metabolic mechanisms through which these pathologies exert their effects on myelin and there is no doubt that knowledge about changes in the composition of OGC in normal and pathological states as well as during normal devel- opment will lead to a better understanding of the complex relationship between the myelin and its mother cell, the OGC.

In order to obtain further information regarding 287

288 SILVIA M. KREDA et al.

the biochemical and metabol ic events occurr ing in rat brain dur ing myelin format ion, we carried out a study of O G C isolated from normal rat bra in at different ages and in the two experimental condi t ions men- t ioned above, at the period of active myel inat ion (18 days of age).

EXPERIMENTAL PROCEDURES

Reagents Trypsin (Type III); HEPES (N-Hydroxy ethyl piperazme-

N' sulfonic acid); Deoxyribonuclease (bovine pancreas), Tween 20 and L-Hystidine monohydrochloride were obtained from Sigma Chemical Co. (St. Louis, Missouri, U.S.A.). Percoll was from Pharmacia (Uppsala, Sweden). Malachite Green O.R. was from Mallinckrodt Chemical Works (New York, U.S.A.). Precoated silicagel 60 Thin Layer Chromatography (TLC) plates were from Merck (Argentina). Silicagel High Performance TLC plates (HPTLC) were from Sigma Chemical Co (St. Louis, Missouri, U.S.A.). Levo-Thyroxine (T3) was a generous gift of Laboratorios GIaxo (Argentina). All other chemical reagents used were of the best analytical grade obtainable from commercial sources.

Animals Wistar rats of either sex were used throughout. To inves-

tigate the age dependent changes in PL composition of bulk- isolated rat OGC, studies were carried out in animals in the period of active myelination (18 day old animals), in young rats (30 day old) and in adult animals (60 and 100 days of age). The effect of Mn and Ht upon OGC composition was investigated in 18 day old animals. Newborn animals were made Ht using the method of Cocks et al. (1970). Neonatal malnutrition (Mn) was produced by increasing the litter size as described by Widdowson and McCance (t960).

Isolation of ol(qodendroglial cell Animals were killed by decapitation and the brains were

quickly removed and placed in ice cold isolation medium. The method of isolation of OGC used in this study, was developed in our laboratory by Berti Mattera et al. in 1984. This procedure, in which the final step involves the use of an "'in situ generated Percoll density gradient, permits the separation of OGC from the brains of very young (> 8 days) and old ( < 150 days) rats without prior separation of white matter". Counting of the cells was carried out on an hemo- cytometer.

Lipid extraction and analysis The pellet containing OGC was extracted with 2 : 1 (v/v)

chloroform-methanol according to Folch Pi et al. (1957) and the washed total lipid extract was used for the analysis of PLs. Lipids were separated by TLC or HPTLC according to Weiss et al. (1982) using two different solvent mixtures. The spots corresponding to the various PL species were visualized with Is vapors and assayed for lipid phosphorus as described below.

Chemical determinations The PL species were scraped off the plate and the silicagel

transferred into glass tubes. Fifty microliters of 70% per-

chloric acid were added and the tubes were put on a heating block and digested at 300'~C during 25 min as described by Chalvardjian and Rudnicki (1970). Inorganic phosphorus was eluted as described by Van Veldhoven and Mannaerts (1987) with slight modifications. For this purpose, 1 ml of bidistilled water was added to each tube. The tubes were vigorously vortexed and placed in an incubation bath at 60C. After 40 min of incubation they were centrifuged at 1000 rpm to sediment the silicagel. The supernatants were removed and evaporated to a volume of approximately 50 100 Id.

We chose two inorganic phosphorus quantification methods based on the formation of a complex between mala- chite green and phosphomolybdate at low pH. For the sam- ples ranging between 3 and 25 nmol of phosphorus, the determination was performed according to Chalvardjian and Rudnicki (1970). For samples containing less than 3 nmol of inorganic phosphorus we used the method described by Shatton el al. (1983) with slight modifications as follows: to each tube was added 400 ,ul of buffer solution (25% 0.2 M L-Hystidine HCI, adjusted pH 7.20) and 100 gl of water. The tubes were mixed and 450/d of the color reagent, prepared as described in the original paper, were added. The solution was transferred to glass cuvettes and read at 660 nm. The average recovery of PLs ranged between 88 and 99%. Pro- teins were determined by the method of Lowry eta/. ( 1951 ). Statistical evaluation of the results was carried out by the Student's t test and by the ANOVA and Scheffe tests.

RESULTS

Isolation of oligodendro#lial cell

Purity of the isolated cells, based on light and elec- t ron microscopy examinat ions and on immuno- cytochemical controls was found to range between 9 2 - 9 7 % (for a detailed descript ion of the mor- phological studies see Berti Mate ra et aL, 1984). The microscopic appearance of the cells isolated f rom the brains of the experimental animals, was comparab le to tha t of normal cells (see also Larocca et al., 1985). In normal controls , the cell yield per bra in was quite similar at all ages studied and the prote in content did not change with age in the t ime period under investigation (data not shown). In the experimental groups on the other hand , a l though the yield of cells per bra in was similar to tha t of controls , the protein content (expressed as pg/cell) was significantly higher in Ht rats and lower in Mn. Wi th reference to the phospholipids, the content per cell was quite com- parable in the three groups of studied animals, a l though in M n it was significantly lower. Some of the characterist ics of the cells isolated f rom 18 day old normal and experimental animals are shown in Table 1.

Phospholipid composition o f oligodendroqlial cell dur- in 9 development

Table 2 show the PL composi t ion of O G C from animals of different ages. The conten t per cell showed

Phospholipids in oligodendroglial cells

Table 1. Properties of isolated OGC from the brain of 18 day old hyperthyroid and malnourished rats

289

Control (8) Hyperthyroid (6) Malnourished (6)

YieM of cells : per g fresh tissue 1.40__.0.07 × 106 1.6_+0.07 x 106 1.6-+0.06 x 106 per brain 1.50-+0.06 × 106 1.5 -+0.06 x 106 1.3 __. 0.05 x 106 Protein (pg/cell) 74.7 + 2.0 116.2 -+ 8.5 51.0 + 2.7 Phospholipids (pg/cell) 5.87 -+ 0.34 5.58 -+ 0.42 4.77 _+ 0.17 Phospholipids (pg/pg protein) 0.078 + 0.004 0.048 + 0.003 0.093 -+ 0.003

Values between parenthesis indicate number of experiments. Data are expressed as mean + SEM.

an increase of 20% between 18 and 30 days of age. No significant changes in the a m o u n t of pho- spholipids were observed after 30 days of age. A m o n g the individuals PLs, PC and PE were found to be ma jo r const i tuents , while SPH, PI, PS were present in mino r amounts . Mos t of the increase in total PLs was the consequence of an increase in PC and PE (20 and 45% over the values ob ta ined at 18 days respectively), a l though the ma jo r increases were observed in PE, PI, PS and SPH (45, 40, 55 and 130% respectively). The conten t of individual PLs did not change significantly after 30 days of age. The percent PL composi t ion was a lmost the same in the four studied ages.

Phospholipid composition o f oli9odendroglial cell iso- lated from 18 day oM malnourished rat brain

The TPL conten t per cell [Fig. l(a)] was statistically significantly decreased (20%) in the O G C isolated from M n rats as compared to controls . The major PL const i tuents , PC and PE, were diminished (20 and 27% respectively) as compared to normals , and minor ones (PI, PS and SPH) also showed a tendency to decrease. The percent PL composi t ion in M n ra t O G C was a lmost similar to tha t of 18 day old normal rat OGC. W h e n the PL da ta were expressed as nmol of

inorganic phosphorus per mg of O G C total proteins, the TPLs and the individual PLs in the exper imental model were similar to those of controls [Fig. l(b)].

Phospholipid composition o f 18 day old hyperthyroid rat oligodendroglia

The T P L con ten t per cell was similar in H t and control rats. A compar i son of the PL composi t ion o f O G C from normal and exper imental animals showed tha t there were no statistically significant changes in the a m o u n t of PLs [Fig. l(a)]. The percent PL com- posi t ion did not change significantly in the O G C iso- lated f rom Ht rat brain. It should be pointed out tha t when the PL da ta were expressed as nmol of inorganic Phosphorus per mg of O G C total protein, the total PLs as well as the ma jo r PL const i tuents such as PC and PE were significantly diminished. PI, PS and SPH were not affected by this exper imental condi t ion [Fig. l(b)].

DISCUSSION

The study of the age dependen t changes in the PL composi t ion o f O G C is one o f the biochemical tools tha t can be used to reveal the developmenta l events

Table 2. Phospholipid composition of OGC during development

Age (days)

18 (8)* 30 (7) 60 (8) 100(6)

Data from bovine OGC

(Farooq et al.)

TPL 7.58+0.03 9.12 +0.45~ 8.94_+ 0.24~ 9,81 + 0.28~ 4.10 PC 2.81 +0.13 (42)t 3.14+0.21 (35)~ 2.92+0.07 (34) 3.40+0.13 (36)~ 2.05 (47) PE 1.37+0.08 (20) 1.98+0.15 (22)~ 1.82+0.13 (21)~ 2,07+0.11 (22)~ 1.10 (24) PI 0.69+0.13(10) 0.95 +0.07 (10)~ 0.96+0.03 (11)~ 0,90+0.07(10)2 0.27 (7) PS 0.52+0.08 (8) 0.82+0.06 (9)~ 0.74+0.04 (9)~ 0.81+0.03 (8)~ 0.29 (7) SPH 0.42+0.03 (6) 0.98_+0.06 (ll)ll 0.96+0.03 (ll)ll 0.94+0.21 (10)ll 0.21 (5) UP 0 .97-+0 .04(14) 1.13-+0.11 (13) 1.24_+0.08(14) 1.34_+0.10(14) 0.30 (8)

Recovery % 90 99 97 96

Quantitation of PLs in OGC was carried out as described in the text. Results are expressed as fmol of inorganic phosphorus/cell and are the mean _+ SEM. Values in parentheses show: * the number of experiments; t moles %. Statistical analysis was carried out using the Student's t Test, ANOVA and Scheff6's Test. There are significant differences between 18-30, 18-60 and 18-100 days. Significant differences are shown for the Student's t test used to compare data at 18 and other ages. :~ P < 0.05 ; §P < 0.01 ; IIP < 0.001. Results of bovine OGC shown were recalculated using data of cell pools from the original paper of Farooq et aL (1981).

290 SILV=A M. KREDA et al.

81t con,ro, (o1 J I~1 HT I I ~ * 1

=:I "N III • i lb I § 1c~

~ 8 0

E x 6 O

if. 4o o E 2 0 c

0 SPH PI PS PC PE TPL

Fig. 1. Phospholipid composition of OGC isolated from the brain of 18 day old hyperthyroid and malnourished rats. Oligodendroglial cells were isolated from 18 day old rat brain and analyzed as described in Fig. 1. Data were analyzed by the Student's t test. *P < 0.01. (a) Results are expressed as fmol of inorganic phosphorus/cell. (c) Results are expressed in nmol of inorganic phosphorus/mg of OGC total protein.

occurring in these cells before, during and after myelin formation. The method for the isolation of OGC developed in our laboratories (Berti Mattera et al.,

1984) has been shown to be applicable to the isolation of OGC from whole forebrain of young pups or adult rats. This has enabled us to carry out, for the first time, a developmental study of the phospholipid com- position of normal animals and a comparative study in two different experimental situations, viz. neonatal hyperthyroidism and malnutrition.

The data obtained by us regarding the PL content and composition of OGC isolated from adult rat brain are essentially similar to those that have been reported for other isolated OGC (Poduslo and Norton, 1972 ; Farooq et al., 1981; Macala et al., 1983). Although there are small differences, they must be ascribed either to differences in the procedures used for the isolation of OGC or in the analytical methods employed. It is noticeable that the PL composition of the oligodendroglial perikaryon appears to be much closer to that of any other mammalian cells (Norton and Poduslo, 1971, Kameyama et aL, 1987, Leray et al., 1987), than to that described for myelin.

The major developmental changes in the PL com- position of isolated OGC were found in the content of PE, PI, PS and SPH which increased markedly

between 18 and 30 days of age. The increase in content of PC was smaller than for the other PLs, in spite of the fact that it is the most abundant phospholipid and that together with cholesterol, it exerts its impact on glial cell differentiation by lowering membrane fluidity (Volpe et al., 1986).

It is well established that during the first three weeks of life, Mn interferes with rat brain maturation. Den- dritic proliferation is impaired and migration of glial cells is delayed (Bass, 1971). Pasquini et al. (1981) found that in Mn rats the amount of myelin was markedly reduced and that all the components of isolated 20 day old rat myelin were also significantly diminished. The abnormal myelinogenesis could be the consequence of a reduction in the number of OGC and/or of a decrease in their capacity to generate myelin lamellae. Several studies appear to indicate that the number of OGC in the brain of nutritionally deprived developing rats is unchanged or slightly increased (Wiggins, 1982) and our data tend to sup- port these conclusions. In previous studies we have ibund that OGC from Mn rat brain behave normally regarding the pattern of proliferation (Larocca et al.,

1985). Although histological studies of OGC diam- eters in Mn rat brain do not reveal significant differ- ences with those of normals (Wiggins, 1982), our data indicate that the perikarya could be smaller, since both lipid and protein content per cell is significantly decreased.

Thyroid hormones have been found to play an important role in OGC differentiation (Bhat et al.,

1981). Previous studies by Walters and Morell (1981) and by Adamo et al. (1990) revealed that thyroid hormones accelerate the formation of CNS myelin producing precocious myelination. We have found that in OGC isolated from Ht rat brain, the PL con- tent remained constant while the protein content increased markedly. We have no explanation for this unusual finding.

Oligodendroglial cell preparations obtained from the brains of animals of different ages have been shown to have a similar degree of purity (Berti Mat- tera et al., 1984). However, in animals submitted to different treatments, it is possible that preparations of variable purity, or slightly contaminated with myelin, could be obtained. Although our electrophoretic stud- ies of OGC total particulates do not support such assumption, the possibility should be borne in mind.

Although the results presented in this study reflect the composition of a cell containing different types of subcellular membranes, we assume that they are the expression of changes occurring mainly in the plasma membrane of the OGC and that they would provide

Phospholipids in oligodendroglial cells 291

fur ther in format ion a b o u t the metabol ic activity of the myelin m e m b r a n e itself.

Acknowledgements--This work was supported by grants from Consejo Nacional de Investigaciones Cientificas y Trcnicas (CONICET), Universidad de Buenos Aires and Fundacirn Antorchas, Argentina.

REFERENCES

Adamo A. M., Aloise P. A., Soto E. F. and Pasquini J. M. (1990) Neonatal hiperthyroidism in the rat produce an increase in the activity of microperoxisomal marker enzymes coincident with biochemical signs of accelerated myelination. J. Neurosci. Res. 25, 353-359.

Bass N. H. (1971) Influence of neonatal undernutrition on the development of rat cerebral cortex : a microchemical study. In : Chemistry and Brain Development. (Paoletti R. and Davison A. N., eds), pp. 413-424. Plenum Press, New York.

Berti Mattera L. N., Larocca J. N., De Iraldi A. P., Pasquini J. M. and Soto E. F. (1984) Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient. Neurochem. Int. 6, 41-50.

Bhat N. R., Subha Rao G. and Pieringer R. A. (1981) Inves- tigation on myelination in vitro regulation of sulfolipid synthesis by thyroid hormone in cultures of dissociated brain cells from embryonic mice. J. biol. Chem. 256, 1167 1171.

Chalvardjian A. and Rudnicki E. (1970) Determination of lipid phosphorus in the nanomolar range. Analyt. Biochem. 36, 225-226.

Cocks J. A., Balazs R., Johnson A. L. and Eayrs J. T. (1970) Effect of thyroid hormone on the biochemical maturation of rat brain: conversion of glucose-carbon into amino acids. J. Neurochem. 17, 1275-1285.

Farooq M., Cammer W., Snyder D. S., Raine C. S. and Norton W. T. (1981) Properties of bovine oligodendroglia isolated by a new procedure using physiologic conditions. J. Neurochem. 36, 431-440.

Fewster M. E., Blackstone S. C. and Ihrig T. J. (1973) The preparation and characterization of isolated oli- godendroglia from bovine white matter. Brain Res. 63, 263 271.

Folch Pi J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 499-509.

Kameyama Y., Yashiro K., Mizuno M., Okada A., Tak- ahashi K. and Yokota Y. (1987) Comparison of membrane phospholipid and its fatty acid compositions in developing rat salivary glands. Comp. Biochem. Physiol. 87B, 741- 746.

Larocca J. N., Sato C., Berti Mattera L., Pasquini J. M. and Soto E. F. (1985) Incorporation of [3H]Thyamidine into DNA and of [35S]Sulfate into sulfatides of oli- godendroglial cells during develoment: effect of mal- nutrition. Neurochem. Res. 10, 89-98.

Leray C., Pelletier X., Hemmendinger S. and Cazenave J. P. (1987) Thin-layer chromatography of human platelet phospholipids with fatty acid analysis. J. Chromatogr. 420, 411-416.

Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.

Macala L. J., Yu R. K. and Ando S. (1983) Analysis of brain lipids by high performance thin-layer chromatography and densitometry. J. Lipid Res. 24, 1243-1250.

Norton W. T. and Poduslo S. E. (1971) Neuronal perikarya and astroglia of rat brain ; chemical composition during myelination. J. Lipid Res. 12, 84-90.

Pasquini J. M., Faryna de Reveglia I. A., Capitman N. and Soto E. F. (1981) Neonatal Hypothyroidism and early undernutrition in the rat: defective maturation of struc- tural membrane components in the Central Nervous System. Neurochem. Res. 6, 979-991.

Poduslo S. E. and Norton W. T. (1972) Isolation and some chemical properties of oligodendroglia from calf brain. J. Neurochem. 19, 772-736.

Shatton J. B., Ward C., Williams A. and Weinhouse S. (1983) A micro colorimetric assay of inorganic pyrophosphatase. Analyt. Biochem. 1311, 114 119.

Snyder D. S., Raine C. S., Farooq M. and Norton W. (1980) The bulk isolation of oligodendroglia from whole rat fore- brain : a new procedure using physiologic media. J. Neuro- chem. 34, 1614 1621.

Szuchet S., Arnason B. G. and Polak P. E. (1980) Separation of oligodendrocytes into distinct bands on a linear sucrose density gradient. J. Neurosci. Meth. 3, 7-19.

Van Veldhoven P. V. and Mannaerts G. P. (1987) Inorganic and organic phosphate measurements in the nanomolar range. Analyt. Biochem. 161, 45-48.

Volpe J. J., Iimori Y., Haven G. G. and Goldberg R. I. (1986) Relation of cellular phospholipid composition to oligodendroglial differentiation in C-6 glial cells. J. Neuro- chem. 46, 475 482.

Waiters S. and Morell P. (1981) Effects of altered thyroid states on myelinogenesis. J. Neurochem. 36, 1792-1801.

Weiss S. J., McKinney J. S. and Putney J. W. (1982) Regu- lation of phosphatidate synthesis by secretagogues in par- otid acinar cells. Biochem. J. 204, 587 592.

Widdowson E. M. and McCance R. A. (1960) Some effects of accelerating growth. I. General somatic development. Proc. R. Soc. Biol. 152, 188-206.

Wiggins R. (1982) Myelin development and nutritional insufficiency. Brain Res. Rev. 4, 151-175.

Wood P. and Bunge R. P. (1984) The biology of the oli- godendrocyte. In: Oliyodendroylia. (Norton W. T., ed), pp. 1-46. Plenum Press, New York.