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BIOCHEMICAL SOCIETY TRANSACTIONS
Myelin-like membrane isolated from subcultures of rat brain cells
JILL CHAPMAN, NANCY R. WILSON and
MARTIN G. RUMSBY
Department of Biology, University of York, York
YOI 5 D D , U . K .
Rat brain cell primary cultures described by us previously
(Walker et al., 1984) have been used as a starting system to
produce subcultures of brain cells from which the bulk of
the type 1 protoplasmic astrocytes (GFA+, A2B5-) have
been eliminated. The subcultures prepared by shaking
multiprocessed cells off the underlying protoplamic astrocyte layer, consist mainly of oligodendrocytes (GC+ ), type
2 astrocytes (GFA' , A2B5+ ), and neuron ( N F + ). Subcultures developed steadily (Walker et al., 1985); cells are
sparsely distributed initially but then begin to expand and
interact. Most noticeable are specific interactions occurring
between oligodendrocytes and neurons. Oligodendrocytes
appear to migrate to areas of culture flasks where the relatively few neurons are present: aggregates of interacting cells
form and fibres extend from one aggregate of cells to
another. In such aggregates a myelin-like membrane is
produced. On examination by transmission electron microscopy the myelin-like membrane in subcultures appears
as loosely associated whorls of membrane showing compaction in only a few areas. In these compaction sites main
dense lines and intraperiod dense lines are apparent. The
whorls of membrane often surround axons but in other
cases are elaborated in a more random way. In appearance
the material generally resembles that seen in the Shiverer
mouse (Privat et al., 1979) or the M L D mouse (Ginalski
et al., 1980). We have now isolated this myelin-like membrane and analyses are reported here.
Myelin-like membrane in subcultures was recovered by
density gradient centrifugation. Normally myelin floats on
0.85 M-SUCrOSe after density gradient centrifugation to equilibrium. This recovery criteria was applied to separate the
myelin-like membrane from subcultures. Subcultures, prepared as previously described (Walker et al., 1985) in
25cm2 Nunc culture flasks, were taken 4 weeks after seeding. Cells were rinsed free of medium with Tris/saline and
distilled water (1 ml) was added per flask. Cells were scraped
out and homogenized. The homogenate was layered onto a
0.32 ~ - / 0 . 8 M-sucrose
5
gradient which was then centrifuged
overnight at 40 OOOg. Membrane at the 0.32 ~ - / 0 . 8 5Msucrose interface was recovered, diluted with distilled water
and pelleted. This myelin-like membrane was washed once
with water by homogenization and repelleted to remove
sucrose before being homogenized in water and made to
volume for analysis.
Polypeptide analysis by SDS/polyacrylamide-gel electrophoresis on 6 3 0 % gradients revealed that the myelin-like
membrane partially resembled normal myelin. Polypeptide
bands characteristic of myelin basic protein, proteolipid
protein and Wolfgram components were present but at
much reduced levels relative to normal myelin. The myelinlike membrane contained many more polypeptides compared with normal myelin, mostly of a higher M,. Myelinassociated glycoprotein was not a significant component
of the myelin-like membrane; lectin binding studies on
myelin-like membrane polypeptides transferred to nitrocellulose showed that there was no concanavalin A-binding
component in the normal myelin-associated glycoprotein
position.
Sufficient myelin-like membrane was collected for lipid
analysis by thin-layer chromatography. The major lipid
groups of myelin, cholesterol, cerebroside and phospholipid
were present in the myelin-like membrane but in modified
proportions. Cerebroside in the myelin-like membrane was
reduced compared with normal myelin and in the cerebroside doublet resolved by t.1.c. the hydroxy fatty acidcontaining cerebroside component was reduced compared
with normal myelin. Analysis of phospholipid classes
showed that choline phosphoglycerides were the major class
in the myelin-like membrane compared with ethanolamine
phosphoglycerides in normal myelin.
The myelin-like mebrane showed evidence of 2',3'-cyclic
nucleotide 3'-phosphohydrolase activity. Wolfgram components were identified after separation of polypeptides and
immunoblotting (undertaken by Dr. P. Brophy, University
of Stirling). Several polypeptide components of varying M ,
in the myelin-like membrane, including myelin basic protein
and a component in the Wolfgram region, were hosphorylated when the membrane was incubated with [ 'PIATP, in
the absence of cyclic AMP (undertaken by Dr. R. Thompson,
University of Cambridge).
The results of the analyses showed that the myelinlike membrane elaborated in subcultures showing oligodendrocyte-neuron interactions is different from normal
myelin in several respects even though it has the same
density characteristics on gradient. Especially noticeable are
reduced proportions of some major myelin proteins myelin
basic protein, proteolipid protein and myelin-associated
glycoprotein, as well as abnormalities in the lipids. In this
respect this myelin-like membrane resembles that isolated
and characterized from other culture systems in vitro showing myelin formation, including explants (Bradbury, 1977)
and aggregating systems (Matthieu et al., 1978; Sarlieve
et al., 1983). Though signals between neurons and oligodendrocytes exist in the subcultures and perhaps are necessary
for initial elaboration of extra membrane by oligodendrocytes other signals to ensure the correct composition of
the membrane are lacking and remain to be defined. Factors
such as (a) the ratio of oligodendrocytes to neurons in the
system, (b) components of the culture medium which
include foetal calf serum, and (c) the role of the astrocytes
in myelin elaboration, are being explored.
P
This work was supported by the Multiple Sclerosis Society of Great
Britain and Northern Ireland and the Multiple Sclerosis Research Trust
Limited.
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Ginalski, H., Friede, R. L., Cohen, S. R. & Matthieu, J.-M., (1980) in
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(1984) J. Neuroimmunol. 7, 1--20
Walker, A. G., Chapman, J. A. & Rumsby, M . G. (1985) J . Neuroimmunol. 9, 159- 177
1986