SUBCELLULAR DISTRIBUTION AND STRUCTURAL POLYMORPHISM OF MYELIN BASIC PROTEIN IN NORMAL AND JIMPY MOUSE BRAIN E. BARBARESE,' J. H. CARSON* and P. E. BRAUN Department of Biochemistry. McGill Universitj. Montreal. Quebec. Canada (Rt*ceiwd 24 Jufj 1978. .4tccpred 6 Duccwih~,i1978) Abstrac!- Brains from 20 day old normal and 20 day old Jimp! mice were Jractionatrd b j ii modifcat i o n of the procedure described by EICHBEKG('t af. (1964). Each of the fractions obtained was subjected to radioimmunoassay (RIA) for myelin basic protein (MBP). From both the normal brain and the Jimpy brain MBP was recovered in three separate membrane fractions designated PIA. P2A. and P3A. which differed in their sedimentation properties but which had similar densities (less than 1.08 g'ml). In the Jimpy brain compared to normal brain the amounts of PIA and P2A w r c great11 reduced. but the amount of P3A was increased. During development in the normal brain the amount of MBP in the PIA fraction increased in parallel with the accumulation of myelin. The amount of MBP in P2A increased gradually during active myelination and decreased slightly in the adult. The amount of MBP in P3A increased sharply during the period of most active myelination. and decreased approx 10-fold as the rate of myelination in the brain declined. Electron microscopic examination revealed that the PIA and P2A fractions from normal brain contained myelin fragments. while the PIA and P2A fractions from Jimpy brain contained numerous vesicular membranous structures a i t h little if any identifiable myelin. The P3A fraction from both normal and Jimpy brain contained small vesicles of uniform size, some with polyribosomes attached. Each of the fractions was analyzed by a technique combining sodium dodecyl sulfate polyacrylamide gel electrophoresis with R I A for MBP in order to identify and quantitate the four different forms of MBP with molecular weights of 21.5 K. 18.5 K. 17 K and 14 K dalton. The proportions of the four MBPs were characteristic for each fraction. The relative proportions of the four proteins were 14 K > 18.5 K > 17 K > 21.5 K daltons in all the fractions except PIA Jimpy in which 21.5 K dalton protein was the predominant form of MBP present.
The cellular origin of the MBP containing fractions from normal and Jimpy brain is discussed.
FORMATION of the myelin membrane in the central nervous system (CNS) of the mouse can be studied at the subcellular and molecular levels by taking advantage of certain properties of this system. Firstly, one of the major components of the membrane is the myelin basic protein (MBP), which is found exclusively in myelin and which can be wed as a marker for myelin-related structures. Secondly, myelin accumulates rapidly in all regions of the brain between 10 and 30 days after birth (UZMAN& RUMLEY. 1958). Thirdly, there is a mutant (Jimpy) in which the process of myelination is blocked (SIDMAN er d., 1964: M E t E R & BISCHOFF,1974, 1975) but in which the synthesis of the MBP occurs at a normal rate (CARSON et af., 1975). Fourthly, M B P exhibits structural polymorphism BAR BARES^ et d.. 1977). and the propor-
' Present address: Department of Microbiology, University of Connecticut Health Center. Farmington. CT 06032. U.S.A. ' Present address: Department of Biochemistq, University of Connecticut Health Center, Farmington. CT 06032. U.S.A. Abhreriations used: MBP. myelin basic protein; PAGE. polyacrylamide gel electrophoresis; RIA. radioimmunoassay; SDS. sodium dodecyl sulfate.
tions of the various molecular forms change during development (BARBARESE et a/., 1978) so that the ontogenic relationships among MBP-containing structures can be established. The present work was undertaken to identify stages in the process of assembly of MBP into myelin. If myelinogenesis is a multistep process. then in the normal mouse brain intermediates may accumulate during the period when the rate of myelin formation is most active (15-20 days after birth) and disappear when the rate of myelin formation declines in the adult. Likewise, if the process is blocked at a particular step in the Jimpy brain. then intermediates preceding the blocked step may accumulate. Therefore, b j analyzing the subcellular distribution of MBP at various times during development in the normal brain and in the Jimpy brain, one might det-ect subcellular fractions that are derived from intermediates in myelin biogenesis. The cellular origin of these fractions can be deduced by correlating their morphology and protein composition with the structures observed during myelin morphogenesis in the normal brain and with the pathological structures observed in the Jimpy brain. The ontogenic relationship among MBP-containing fractions can be determined from
1437
1438
E BARBARFSF. J H CARSOVand P E BRA^^
the proportions of the four forms of MBP (21.5K. 18.5K. 1 7 K and 1 4 K daltons). The proportions of the four MBPs change from 1 :5:2:10, respectively. in myelin from ~ O U I animals I ~ (15-30 days old) to 1 : 10:3.5:35, respectively, in myelin from adults (BARBARESE et a/.. 1978). Since the proteins in myelin turn over extremel! slowly (FISCHER & MORELL,1974). this change in the proportions of the four M B P s during development presumabl! reflects the accretion of newly formed myelin with a changing MBP composition. This provides a criterion for distinguishing between 'old' myelin. 'new' myelin. and intermediates in myelin biogenesis. MATERIALS AND METHODS .4uiriiul.\. Normal brown mice (C57BLj6J-A' ') and heteroz!gous females (Tajp + I(C57BLi6J-A"') carrying the closel? linked Tabb! and Jimp) mutations were obtained from Bar Harbor Laboratories (Bar Harbor, ME, U.S.A.). Thc normal mice used in this study were (C57BL/6J-A"') males. Jimp! mice were produced by crossing the Tajp + + females with the normal males. Male mice carrying the Jimp! mutation were identified by the onset of clinical symptoms (hind leg tremor. ataxia) at approx I2 da)s after birth. The Jimp) mouse brain showed a deficit of MBP (as measured bq R I A ) relative to normal mouse brain. Environmental. nutritional. and breeding conditions Here a s recommended by Bar Harbor Laboratories. Suhtcllirlur ,fiuctiotiutiori.The mice were killed by cervical dislocation and the brains were removed and freed from cranial nerkes. olfactory lobes. and cerebellum. All subsequent steps were performed at 44'C. The brains were homogenized in 0.25 ?rl-sucrose to a final concentration of lo",, b) 10 up-and-doRn strokes in a Teflon-glass homogenizer. The homogenate was fractionated according to the method of EICHBERG ct d. (1964) with the following modifications. The P3. or microsomal, fraction was resuspended in 0.25 wsucrose and layered over 0.5 M-sucrose. The resulting discontinuous gradient was centrifuged for 2 h at 100.o00y. after which the material at the interface between the 0.25 hf-sucrose and the 0.5 M-SUCTOSC was collected and designated P3A. and the pellet was collected and designated P3B. It was necessary to use 0.5 M rather than 0.8 M-sucrose as in the gradients for fractionating P1 and P2 because some non-MBP containing ma!erial in P3 had a densit) less than that of 0.8 M-sucrose. Each of the fractions was precipitated with cold trichloroacetic acid (final concentration of 20",). The precipitated material was washed 3 times with cold ether-ethanol (3:2) and once with ether. The pellets were dried. resuspended in H,O. and stored at -2O'C. Protriri rleterriibitrrioti. Protein was determined by the method of L O W R or ~ a / . (1951) with bovine serum albumin as a standard. Each sample was dissolved in 0.1", sodium dodecyl sulfate (SDS) prior to analysis. SDS polyucrylnmide ye1 electrophoresis ( P A G E ) . SDS PAGE was performed as described by SWANK & MUNKRES (1971) in gels containing IO", acrylamide and 0.50,; bisacrylamide. Proteins were stained with Coomassie Blue as described b) FAIRBANKS V I ul. (1971). Radioirfiriiuriou.\.rcr,.( R I A ) . R I A of total MBP was performed as described by B A R B A R ~ctSa/. E (1977. 1978) with '251-labelled 14 K dalton protein and antiserum against purified 14K dalton protein. Each sample was dissolved
in T3 buffer (0.2 M-Tris-acetate. pH 7.2. lo, Triton X-100. O.lO, Trasylol), and each determination was performed in
duplicate. R I A of individual MBPs after separation on SDS PAGE was performed as described by BARRARESL P I 01. (1977). Electroil microscopy. Small aliquots of each fraction were fixed at 4'C overnight with an equal volume of half strength Karnovsky fixative (KARNOVSKY. 1965) and pelleted in Beem capsules (Better Equipment for Electron Microscopy. Inc., New York, NY. U.S.A.). according to COTMAN & FLASSBI'RG (1970). The pellets were postfixed for 1 h in 1.33",, osmium buffered with collidine and block stained for 1 ' 2 h in saturated aqueous uranyl acetate ( 0 s - U L treatment). The tissue was then dehydrated in graded ethanol and embedded in Vestopal W (KCRTZ. 1961). Ultrathin sections Here cut with glass knives on an LKB Ultratome 111. Sections were mounted on copper grids with carbon coated support lilm. The sections were . stained with Reynold's lead citrate for I min ( P ~ A s L1964). All grids were examined in a Philips 300 electron microscope operated as 60 kV. A 20 or 30pm objective aperture was used. Micrographs were recorded on Kodak electron image plates and magnification was calibrated with an E.F. Fullani carbon grating replica of 28,ooO lines per inch or 54.864 lines per inch. cat. no. loo0 and 1002. respectivel). Clierniculs. All chemicals were obtained from Fisher Scientific Co. (Montreal. Canada) except for the following: Triton X-100 and Coomassie Blue R250 from Sigma (St. Louis, MO, U.S.A.). acrylamide and bis-acrylamide from BioRad Laboratories (Richmond. CA. U.S.A.). and Vestopal W from M. Jaeger (Geneva. Switzerland).
RESULTS
Subcellular distrihufioti of M B P dtrritiy t r c f i w
ttiwlitiu-
tioti Brains from normal and Jimpy mice at 20 days after birth were fractionated by a modification of the method of EICHBERG ~t a/. (1964). This procedure separates 8 morphologically distinct subcellular fractions identified as: large myelin (PIA), small myelin contaminated by a few microsomes and synaptosomes (P2A), nuclei and mitochondria (PIB), synaptosomes (P2B).tissue and cell debris and red blood cells (PIC). mitochondria (P2C). microsomes (P3). and cell sap (S) (EICHBERGet a/., 1964). The amount of MBP present in each fraction was determined by RIA. Although most of the MBP was recovered in PIA and P2A, there was a significant amount of MBP in the P3 fraction. The microsomal fraction (P3) was analyzed on a continuous sucrose density gradient, and each fraction of the gradient was subjected t o RIA for MBP. T h e MBP was associated with a particulate component with a density between 1.03 and 1.06g/ml. In order to separate the MBP-containing component from the rest of the microsomal material, the P3 fraction was subjected to a discontinuous sucrose density gradient step. This step generated 2 additional fractions: the material at the interface between 0.25 M and 0.5 M-sucrose (P3A) and the material in the pellet (P3B).
Myelin basic proti:ins in mouse brain TABLE1. SUBCELLULAR DISTRIBUTION OF MBP AND PROTEIN IN 20 day OLD NORMAL AND JIMPSMOUSE Su bcellular fraction
PIA P2A P3A PI B P2B P3B PIC P2C S Homogenate
MBP (nmol/brain) Normal Jimpy 8.5 1.8 0.01
0.06 0.17 0.08
-
-
~
~
~
10.3
~
~
-
0.32
TOTAL BRAIN
Total protein (mgbrain) Normal Jimpy 0.5 1.8 0.02 0.6 4. I 0.L 1.2 0.2 4.0 16
0.2 0.2 0.1 1.1
4.8 0.2 I .2 0.1 2.8 13
Twenty day old normal and Jimpy mouse brains were fractionated as described in Materials and Methods. Each fractionation‘ was carried out with at least 3 brains. The total protein ‘content of each fraction was determined by the method of LOWRS er a/. (1951). The MBP content of each fraction was determined by RIA. Each value represents the mean of at least 3 separate fractionations. The variation was always less than loo;. (-): MBP was not detectable.
The analytical data (Table I ) show that at 20 days after birth the normal mouse brain contained approx 10 nmol of MBP distributed in the following manner: 820;, in PIA, 18% in P2A and 0.1% in P3A. At the same age the Jimpy mouse brain contained approx 0.3 nmol of MBP, of which 20% was recovered in PlA, 537; in P2A and 277; in P3A. In terms of absolute amounts the P3A fraction from the Jimpy brain contained 8 times as much MBP and 5 times as much protein as the same fraction from normal brain. None of the other fractions from either normal or Jimpy brain had detectable amounts of MBP.
1439
pared to P1A but did not change significantly during development. Morphologj, of MBP-containiny fracrions fioni noritid arid Jinipj. brains
Electron micrographs of PlA, PZA, and P3A from 20 day old normal and 20 day old Jimpy brain are shown in Figs. 2-4. The PIA fraction from normal brain (Fig. 2A) contained large multilamellar membranous sheets with occasional cross sections of intact myelinated axons. The axonal material seemed to be the only component other than myelin which was present. The corresponding fraction isolated from Jimpy brain (Fig. 2B) appeared as a heterogeneous population of unilamellar membranous vesicles. No morphologically identifiable myelin membranes were observed. The P2A fraction from normal brain (Fig. 3A) contained multilamellar membranes similar to those observed in PlA, but of overall smaller size. It also had a considerable proportion of unilamellar vesicles which could be microsomal or synaptosomal in origin. The P2A fraction from the Jimpy brain (Fig. 3B) appeared as a heterogeneous population of membranous structures. No typical myelin membranes were found, although some multilamellar membranes were occasionally observed. The P3A fraction from both normal (Fig. 4A) and Jimpy (Fig. 4B) brains consisted of unilamellar vesicles which
Accumulation of [he MBP-containing fractions during derelopmmr
Brains from normal mice at different ages were fractionated, and the amount of total protein and MBP present in PIA, P2A and P3A was determined (Fig. 1). At 10 days after birth MBP was detectable in all 3 fractions. In PIA it accumulated at an approximately linear rate between 10 and 30 days after birth and at a reduced rate between 30 and 60 days after birth. This fraction contained greater than 75% of the total brain MBP at every age after 10 days, and the ratio of M B P to total protein (20 nmol/mg) was constant. In P2A MBP accumulated gradually between 10 and 30 days, after which the amount of MBP declined. The ratio of MBP to total protein in this fraction varied during development, increasing up to a maximum (2.5nmol/mg) at 30 days after birth and then decreasing. The amount of MBP in P3A increased sharply between 10 and 15 days after birth and then decreased so that the amount at 60 days was approx 10% of the amount at 15 days after birth. The ratio of MBP to total protein in P3A was low (0.5 nmol/mg) com3.c. 3 2 , 5 - ~
0.05
I t
.“r ;‘I
/ I /
OO
O
lo
‘s. ..
--- - - - -- - --
P3A
-0-
;o
20 40 Days after birth
50
___D
$0
FIG. 1. Accumulation of MBP in PIA, P2A and P3A from normal brain during development. The brains of normal mice at 5, 10, 15, 20, 30 and 60 days after birth were fractionated as described in Materials and Methods. Five brains were used at each age. The results are expressed as mol MBP per brain. The values are the mean of 3 separate experiments. The variation was lo”/, or less.
E.
1440
~
[
BARBARESt,
J. H. CARSON and P. E. BRAUN
PIA NORMAL
I P2A
P l A JlMPY
1
I
P 3 A NORMAL
1
P J A JlMPY
NORMAL
P 2 A JlMPY
30
I
i
I
20
30
SLICE NUMBER
FIG. 5. RIA profiles of PIA, P2A and P3A fractions from normal and Jimpy mouse brain at 20 days after birth. The PlA, P2A and P3A fractions from normal and Jimpy brains were run on SDS polyacrylamide gel for 14 h at 3 mA/gel. Approximately 5-120 p g of protein was applied to the gel depending on the concentration of MBP in the sample. Routinely, 2 different protein concentrations were tested for each fraction. After staining, the gels were processed for R I A (BAKBARESE et nl., 1977). Only the gel region that contains MBP is shown. No cross-reacting material other than the 21.5 K, 18.5 K, 17 K and 14 K dalton species was found. Each RIA sample was done in duplicate. The effective range of the assay is between 2 and 15 x mol. The RIA profiles obtained in a typical experiment are shown. Similar profiles were obtained in 4 separate experiments.
were quite homogenous in size (150 nm average dia.). Numerous polyribosomes were also present, often with one end in close apposition to the vesicles. Analysis of subcellular fractions by SDS PAGE and RIA The 9 subcellular fractions from 20 day old normal and 20 day old Jimpy brains were analyzed by SDS PAGE. Each of the fractions had a characteristic protein profile. The gels of the non MBP-containing fractions (PlB, PlC, P2B, P2C, P3B, S ) were identical for both normal and Jimpy. The protein profile of PIA normal was the same as reported previously by BARBARESE et al. (1978). The protein profiles for P1A and P2A Jimpy were the same as for the corresponding fractions from normal brain, except that the 4 MPBs were not detected by staining. The protein profiles for P3A from normal and Jimpy were identical but the small amount of MBP was obscured by other bands. The gels of the MBP-containing fractions (PlA, P2A, P3A) from both normal and Jimpy brains were further analyzed by RIA to determine the relative proportions.of each of the 4 MBPs in each fraction. The radioimmunoassay profiles of the gels are shown in Fig. 5. The different molecular forms of MBP were detected in characteristic proportions in each of the MBP-containing fractions. In the fractions from normal brain the relative proportion of 1 4 K dalton MBP tended to decrease from P3A t o P2A t o P l A . The proportions of the 4 proteins in P2A and P3A
from Jimpy brain were similar to the proportions in the corresponding fractions from normal brain. In P l A Jimpy, the 21.5 K dalton protein, which was the least abundant form in all the other fractions, was the predominant species of MBP. DISCUSSI0N
The work described in this paper shows that in normal and Jimpy mouse brain there are 3 distinct subcellular fractions that contain MBP. The fractions, designated PlA, P2A, and P3A, all have densities less than 1.08 g/ml but can be separated on the basis of their sedimentation properties. In order to determine from which cellular structure each fraction was derived, their morphology, overall protein composition, and developmental pattern of accumulation were analyzed. In order to determine the ontogenic relationships among the different fractions the proportions of the 21.5K, 18.5K,1 7 K and 1 4 K daltons MBPs in each fraction were compared. The P1A fraction from normal brain is almost certainly derived from the mature myelin sheath. By electron microscopy the fraction appears as sheets of multilamellar compacted membranes with occasional cross sections of myelinated axons. The axonal material may contribute to some of the high molecular weight proteins present on the SDS gel of this fraction. The overall protein composition and the ratio of MBP t o total protein are similar to those observed in myelin isolated from adult mouse brain by conven-
FIG.
2. Electron micrographs of P1A from normal (A) and Jimpy (B) brains at 20 days after birth. x 58,500.
1441
FIG.3. Electron micrographs of P2A from normal (A) and Jimpy (B) brains at 20 days after birth. x 58,500.
1442
FIG.4. Electron micrographs of P3A from normal (A) and Jimpy (B) brains at 20 days after birth x 58.500.
1443
Myelin basic proteins in mouse brain tional methods (BARBARESE et ul., 1977). The pattern of accumulation of P i A during development parallels the accumulation of myelin in the mouse brain (UZMAN & RUMLEY, 1958). The ratio of MBP to total protein (20 nmol/mg) is constant throughout development. All these factors are indications that P I A is a relatively pure preparation of fragments of the mature myelin membrane. Although the P2A fraction from normal brain is a mixture of membranous structures, the MBP-containing component is probably derived from newly formed or immature myelin. Multilamellar membranes similar to those in PIA are present in the fraction, indicating that it contains myelin. The protein profile of the fraction shows typical myelin components on a background of non-myelin proteins. Two observations suggest that P2A from normal brain contains newly formed myelin rather than a subfraction of mature myelin. Firstly, it has the developmental pattern of accumulation that is cxpected for newly formed myelin. At 10 days after birth, when most of the myelin in the brain is newly formed, greater than 50% of the total MBP is recovered in P2A. Between 15 and 30 days after birth, when the rate of myelin formation is maximum, the amount of MBP in P2A increases. At 60 days, when the rate of myelin formation is slower, the amount of MBP in P2A is decreased. Secondly, the proportions of the four MBPs in P2A are distinct from the proportions in mature myelin. P2A contains a greater proportion of 1 4 K dalton MBP than PlA, and the proportion of this form of MBP in myelin increases during development. This suggests that P2A represents ‘new’ myelin while P1A represents ‘old’ myelin. There are several possible explanations for the presence of MBP in the P3A fraction. One possibility is that when the amount of myelin in the homogenate is very small (as in the Jimpy brain) it fractionates in an anomalous fashion. This explanation is unlikely because when 20 day old Jimpy or newborn normal brains were homogenized in the presence of trace amounts of 3H-labelled myelin isolated from 20 day old normal brain, the radioactivity was recovered primarily in P1A and P2A with very little in P3A (BARBARESE, unpublished observation). A second possibility is that the MBP in P3A is due to adventitious binding of soluble MBP to some microsomal component during the fractionation procedure. This is also unlikely, since when brains were homogenized in the presence of soluble ‘Z51-labelled 18.5 K dalton MBP and fractionated, negligible radioactivity was recovered in the P3A fraction (BARBARESE, unpublished observation). The third possibility is that P3A is derived from an MBP-containing subcellular component which is distinct from PlA or P2A. The fractionation procedure is designed to isolate enriched fractions, and biochemical characterization would be needed to assess the purity of the P3A fraction. However, the fact that P3A from normal brain has a constant MBP to total protein ratio throughout develop-
1445
ment and the fact that the fraction accumulates dramatically during active myelination suggests that a large proportion of the membranes in the fraction are involved in myelin formation. The proportions of the four MBPs support the possibility that P3A is an intermediate in myelin assembly. P3A contains a higher proportion of 14 K dalton MBP than either P2A or PlA, suggesting that it is derived from a ‘newer’ structure than either of these fractions. The P3A fraction from the Jimpy brain appears identical in every respect to P3A from normal brain and is probably derived from the same type of membranous intermediates in myelin biogenesis. The fact that this fraction accumulates in the Jimpy brain to a greater extent than the normal brain at a comparable age suggests that in the Jimpy brain intermediates in myelin assembly are formed but are not converted into myelin. Morphological studies of oligodendroglial cells in Jimpy mice (MEIER& BISCHOFF,1974, 1975) show that the perikaryon and processes are filled with vesicles and other membranous structures, some of which may represent the intracellular intermediates from which P3A Jimpy is derived. The nature of the P2A fraction from the Jimpy brain is less certain. Since the bulk of the material in this fraction is probably derived from components that are unrelated to myelin, the overall morphology and protein composition of the fraction is uninformative. The proportions of the four MBPs in this fraction are similar to those in PIA and P2A from normal brain, suggesting that the MBP-containing material in P2A Jimpy could be derived from myelinlike structures such as the membranous tubes in the perikaryon of the Jimpy oligodendroglial cell or the small amount of loose myelin that accumulates in the Jimpy brain (MEIER& BISCHOFF,1974). The origin of the P I A fraction from Jimpy brain is probably the initial wrapping of oligodendroglial plasma membrane around the axon. Since this fraction is isolated by sedimentation at low speed (1000 g for 10 min), it is presumably derived from large subcellular structures. However, by electron microscopy it appears as an heterogeneous population of small vesicles. This may mean that the original structure vesicularized during the isolation procedure. The protein profile of the P1A fraction from 20 day old Jimpy brain is similar to the profile of the P l A fraction from 5 or 10 day old normal brain in which myelination is just beginning (BARBARESE, unpublished observation). MEIER& BISCHOFF(1975) have reported that in the Jimpy CNS each axon is surrounded by a thin tongue of oligodendroglial cytoplasm which presumably represents one of the early stages in myelin morphogenesis. The Jimpy mutation blocks the extension of this initial wrapping to form the multiple layers of the myelin sheath. It seems likely that the MBP containing component in P1A Jimpy is derived from this single wrapping of the oligodendroglial process around the axon. The results presented in this paper have important
1446
E. BARBARESE, J. H. CARSON and P. E. BRAUN
implications concerning both the molecular and the cellular mechanisms in myelination in normal and Jimpy mouse brain. Firstly, the characteristic polymorphism of MBP in the different subcellular fractions from normal and Jimpy brain, particularly the predominant 21.5 K dalton MBP in the P1A Jimpy, can best be explained by the hypothesis that the mode of MBP gene expression changes during myelin morphogenesis, from predominantly 21.5 K dalton MBP in the initial stages to predominantly 14K dalton MBP in the latter stages. This hypothesis is consistent with the earlier observations that the proportions of the four MBPs in myelin change during mouse brain development (BARBARESE et a!., 1978) and that the rate of synthesis of 14K dalton MBP relative to 18.5K dalton MBP increases during brain development (CAMPAGNONI et a/., 1978). Secondly, the properties of the P3A fraction suggest that it represents an intracellular intermediate in -myelin formation. The role of this intermediate may be to translocate MBP from its site of synthesis to the site of myelin assembly. In this regard, CULLEN& WEBSTER(1977) have reported that when myelination in the optic nerve of Xenopus tadpole is blocked by low temperature, membrane vesicles accumulate in the perikarya and inner tongue process of the oligodendroglial cells. These vesicles may correspond to the intraceliular precursors of myelin from which P3A is derived. The accumulation of the P3A fraction in Jimpy brain suggests that the Jimpy mutation also blocks the conversion of the precursor membranes into myelin. Acknow/edgements- We thank J. ABRAMSfor useful discussion and for his help in preparing the samples for electron for taking the electron micromicroscopy, L. GULUZIAN graphs and photographs, and M. SCHINDLER for her excellent technical assistance. This work was supported by grants from the Medical Research Council of Canada and the Multiple Sclerosis Society of Canada.
REFERENCES E., BRAUN P. E. & CARSON J. H. (1977) IdentifiBARBARESE cation of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins. Proc. natn. Acad. Sci., U.S.A. 74, 3360-3364. BARBAKESE E., CARSONJ. H. & BRAUNP. E. (1978) Ac-
cumulation of the four myelin basic proteins in mouse brain during development. J . Neurochem. 31, 779-782. CAMPAGNONI C. W., CAREYG. D. & CAMPAGNONI A. T. (1978) Synthesis of myelin basic proteins in the developing mouse brain. Archs Biochem. Biophys. 190, 118-125. CARSON J. H., HERSCHKOWITZ N. N. & BRAUNP. E. (1975) Synthesis and degradation of myelin basic protein in normal and Jimpy mouse brain. Trans. Am. SOC.Neurochem. 6 , 207. COTMAN C . W. & FLANSBURG D. A. (1970) An analytical micro-method for electron microscopic study of the compositipn and sedimentation properties of subcellular fractions. Brain Res. 22, 152-156. CULLENM. J. & WEBSTER H. DE F. (1977) The effects of low temperature on myelin formation in optic nerves of Xenopus tadpoles. Tissue and Cell 9, 1-10. EICHBFRG J., WHITTAKER U. P. & DAWSONR. M. C. (1964) Distribution of lipids in subcellular particles of guinea pig brain. Biochem. J . 90, 91- 100. FAIRBANKS G., STECKT. L. & WALLACH D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606 -2617. FISCHERC. A. & MORELLP. (1974) Turnover of proteins in myelin and myelin-like material in mouse brain. Brain Res. 74, 5165. KARNOVSKY M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. 1. Cell. Biol. 27, 137A-138A. KURTZS. M. (1961) A new method for embedding tissue in Vestopal W. J . Ultrastruct. Res. 5, 468-477. LDWRY 0. H., ROSEBROUGHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J . biol. Chem. 193, 265-275. MEIERC. & BISCHOFF A. (1974) Dysmyelination in ‘Jimpy’ mouse. Electron microscopic study. J . Neuropath. exp. Neurol. 33, 343-353. A. (1975) Oligodendroglial cell develMEIERC. & BISCHOFF opment in Jirnpy mice and controls. An electron-microscopic study in the optic nerve. J . Neurol. Sci. 26, 5 17-528. PEASE D. C. (1964) in Histological Techniques f o r Electron Microscopy. 381 pp. Academic Press, New York. SIDMAN R. L., DICKIEM. M. & APPEL S. H. (1964) Mutant mice (quaking and Jimpy) with deficient myelination in the central nervous system. Science 144, 309-31 1. SWANK R. T. & MUNKRES K. D. (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Analyt. Biochem. 39, 462-477. UZMANL. L. & RUMLEY M. K. (1958) Changes in the composition of the developing mouse brain during early myelination. J . Neurochem. 3, 170-184.
The cellular origin of the MBP containing fractions from normal and Jimpy brain is discussed.
FORMATION of the myelin membrane in the central nervous system (CNS) of the mouse can be studied at the subcellular and molecular levels by taking advantage of certain properties of this system. Firstly, one of the major components of the membrane is the myelin basic protein (MBP), which is found exclusively in myelin and which can be wed as a marker for myelin-related structures. Secondly, myelin accumulates rapidly in all regions of the brain between 10 and 30 days after birth (UZMAN& RUMLEY. 1958). Thirdly, there is a mutant (Jimpy) in which the process of myelination is blocked (SIDMAN er d., 1964: M E t E R & BISCHOFF,1974, 1975) but in which the synthesis of the MBP occurs at a normal rate (CARSON et af., 1975). Fourthly, M B P exhibits structural polymorphism BAR BARES^ et d.. 1977). and the propor-
' Present address: Department of Microbiology, University of Connecticut Health Center. Farmington. CT 06032. U.S.A. ' Present address: Department of Biochemistq, University of Connecticut Health Center, Farmington. CT 06032. U.S.A. Abhreriations used: MBP. myelin basic protein; PAGE. polyacrylamide gel electrophoresis; RIA. radioimmunoassay; SDS. sodium dodecyl sulfate.
tions of the various molecular forms change during development (BARBARESE et a/., 1978) so that the ontogenic relationships among MBP-containing structures can be established. The present work was undertaken to identify stages in the process of assembly of MBP into myelin. If myelinogenesis is a multistep process. then in the normal mouse brain intermediates may accumulate during the period when the rate of myelin formation is most active (15-20 days after birth) and disappear when the rate of myelin formation declines in the adult. Likewise, if the process is blocked at a particular step in the Jimpy brain. then intermediates preceding the blocked step may accumulate. Therefore, b j analyzing the subcellular distribution of MBP at various times during development in the normal brain and in the Jimpy brain, one might det-ect subcellular fractions that are derived from intermediates in myelin biogenesis. The cellular origin of these fractions can be deduced by correlating their morphology and protein composition with the structures observed during myelin morphogenesis in the normal brain and with the pathological structures observed in the Jimpy brain. The ontogenic relationship among MBP-containing fractions can be determined from
1437
1438
E BARBARFSF. J H CARSOVand P E BRA^^
the proportions of the four forms of MBP (21.5K. 18.5K. 1 7 K and 1 4 K daltons). The proportions of the four MBPs change from 1 :5:2:10, respectively. in myelin from ~ O U I animals I ~ (15-30 days old) to 1 : 10:3.5:35, respectively, in myelin from adults (BARBARESE et a/.. 1978). Since the proteins in myelin turn over extremel! slowly (FISCHER & MORELL,1974). this change in the proportions of the four M B P s during development presumabl! reflects the accretion of newly formed myelin with a changing MBP composition. This provides a criterion for distinguishing between 'old' myelin. 'new' myelin. and intermediates in myelin biogenesis. MATERIALS AND METHODS .4uiriiul.\. Normal brown mice (C57BLj6J-A' ') and heteroz!gous females (Tajp + I(C57BLi6J-A"') carrying the closel? linked Tabb! and Jimp) mutations were obtained from Bar Harbor Laboratories (Bar Harbor, ME, U.S.A.). Thc normal mice used in this study were (C57BL/6J-A"') males. Jimp! mice were produced by crossing the Tajp + + females with the normal males. Male mice carrying the Jimp! mutation were identified by the onset of clinical symptoms (hind leg tremor. ataxia) at approx I2 da)s after birth. The Jimp) mouse brain showed a deficit of MBP (as measured bq R I A ) relative to normal mouse brain. Environmental. nutritional. and breeding conditions Here a s recommended by Bar Harbor Laboratories. Suhtcllirlur ,fiuctiotiutiori.The mice were killed by cervical dislocation and the brains were removed and freed from cranial nerkes. olfactory lobes. and cerebellum. All subsequent steps were performed at 44'C. The brains were homogenized in 0.25 ?rl-sucrose to a final concentration of lo",, b) 10 up-and-doRn strokes in a Teflon-glass homogenizer. The homogenate was fractionated according to the method of EICHBERG ct d. (1964) with the following modifications. The P3. or microsomal, fraction was resuspended in 0.25 wsucrose and layered over 0.5 M-sucrose. The resulting discontinuous gradient was centrifuged for 2 h at 100.o00y. after which the material at the interface between the 0.25 hf-sucrose and the 0.5 M-SUCTOSC was collected and designated P3A. and the pellet was collected and designated P3B. It was necessary to use 0.5 M rather than 0.8 M-sucrose as in the gradients for fractionating P1 and P2 because some non-MBP containing ma!erial in P3 had a densit) less than that of 0.8 M-sucrose. Each of the fractions was precipitated with cold trichloroacetic acid (final concentration of 20",). The precipitated material was washed 3 times with cold ether-ethanol (3:2) and once with ether. The pellets were dried. resuspended in H,O. and stored at -2O'C. Protriri rleterriibitrrioti. Protein was determined by the method of L O W R or ~ a / . (1951) with bovine serum albumin as a standard. Each sample was dissolved in 0.1", sodium dodecyl sulfate (SDS) prior to analysis. SDS polyucrylnmide ye1 electrophoresis ( P A G E ) . SDS PAGE was performed as described by SWANK & MUNKRES (1971) in gels containing IO", acrylamide and 0.50,; bisacrylamide. Proteins were stained with Coomassie Blue as described b) FAIRBANKS V I ul. (1971). Radioirfiriiuriou.\.rcr,.( R I A ) . R I A of total MBP was performed as described by B A R B A R ~ctSa/. E (1977. 1978) with '251-labelled 14 K dalton protein and antiserum against purified 14K dalton protein. Each sample was dissolved
in T3 buffer (0.2 M-Tris-acetate. pH 7.2. lo, Triton X-100. O.lO, Trasylol), and each determination was performed in
duplicate. R I A of individual MBPs after separation on SDS PAGE was performed as described by BARRARESL P I 01. (1977). Electroil microscopy. Small aliquots of each fraction were fixed at 4'C overnight with an equal volume of half strength Karnovsky fixative (KARNOVSKY. 1965) and pelleted in Beem capsules (Better Equipment for Electron Microscopy. Inc., New York, NY. U.S.A.). according to COTMAN & FLASSBI'RG (1970). The pellets were postfixed for 1 h in 1.33",, osmium buffered with collidine and block stained for 1 ' 2 h in saturated aqueous uranyl acetate ( 0 s - U L treatment). The tissue was then dehydrated in graded ethanol and embedded in Vestopal W (KCRTZ. 1961). Ultrathin sections Here cut with glass knives on an LKB Ultratome 111. Sections were mounted on copper grids with carbon coated support lilm. The sections were . stained with Reynold's lead citrate for I min ( P ~ A s L1964). All grids were examined in a Philips 300 electron microscope operated as 60 kV. A 20 or 30pm objective aperture was used. Micrographs were recorded on Kodak electron image plates and magnification was calibrated with an E.F. Fullani carbon grating replica of 28,ooO lines per inch or 54.864 lines per inch. cat. no. loo0 and 1002. respectivel). Clierniculs. All chemicals were obtained from Fisher Scientific Co. (Montreal. Canada) except for the following: Triton X-100 and Coomassie Blue R250 from Sigma (St. Louis, MO, U.S.A.). acrylamide and bis-acrylamide from BioRad Laboratories (Richmond. CA. U.S.A.). and Vestopal W from M. Jaeger (Geneva. Switzerland).
RESULTS
Subcellular distrihufioti of M B P dtrritiy t r c f i w
ttiwlitiu-
tioti Brains from normal and Jimpy mice at 20 days after birth were fractionated by a modification of the method of EICHBERG ~t a/. (1964). This procedure separates 8 morphologically distinct subcellular fractions identified as: large myelin (PIA), small myelin contaminated by a few microsomes and synaptosomes (P2A), nuclei and mitochondria (PIB), synaptosomes (P2B).tissue and cell debris and red blood cells (PIC). mitochondria (P2C). microsomes (P3). and cell sap (S) (EICHBERGet a/., 1964). The amount of MBP present in each fraction was determined by RIA. Although most of the MBP was recovered in PIA and P2A, there was a significant amount of MBP in the P3 fraction. The microsomal fraction (P3) was analyzed on a continuous sucrose density gradient, and each fraction of the gradient was subjected t o RIA for MBP. T h e MBP was associated with a particulate component with a density between 1.03 and 1.06g/ml. In order to separate the MBP-containing component from the rest of the microsomal material, the P3 fraction was subjected to a discontinuous sucrose density gradient step. This step generated 2 additional fractions: the material at the interface between 0.25 M and 0.5 M-sucrose (P3A) and the material in the pellet (P3B).
Myelin basic proti:ins in mouse brain TABLE1. SUBCELLULAR DISTRIBUTION OF MBP AND PROTEIN IN 20 day OLD NORMAL AND JIMPSMOUSE Su bcellular fraction
PIA P2A P3A PI B P2B P3B PIC P2C S Homogenate
MBP (nmol/brain) Normal Jimpy 8.5 1.8 0.01
0.06 0.17 0.08
-
-
~
~
~
10.3
~
~
-
0.32
TOTAL BRAIN
Total protein (mgbrain) Normal Jimpy 0.5 1.8 0.02 0.6 4. I 0.L 1.2 0.2 4.0 16
0.2 0.2 0.1 1.1
4.8 0.2 I .2 0.1 2.8 13
Twenty day old normal and Jimpy mouse brains were fractionated as described in Materials and Methods. Each fractionation‘ was carried out with at least 3 brains. The total protein ‘content of each fraction was determined by the method of LOWRS er a/. (1951). The MBP content of each fraction was determined by RIA. Each value represents the mean of at least 3 separate fractionations. The variation was always less than loo;. (-): MBP was not detectable.
The analytical data (Table I ) show that at 20 days after birth the normal mouse brain contained approx 10 nmol of MBP distributed in the following manner: 820;, in PIA, 18% in P2A and 0.1% in P3A. At the same age the Jimpy mouse brain contained approx 0.3 nmol of MBP, of which 20% was recovered in PlA, 537; in P2A and 277; in P3A. In terms of absolute amounts the P3A fraction from the Jimpy brain contained 8 times as much MBP and 5 times as much protein as the same fraction from normal brain. None of the other fractions from either normal or Jimpy brain had detectable amounts of MBP.
1439
pared to P1A but did not change significantly during development. Morphologj, of MBP-containiny fracrions fioni noritid arid Jinipj. brains
Electron micrographs of PlA, PZA, and P3A from 20 day old normal and 20 day old Jimpy brain are shown in Figs. 2-4. The PIA fraction from normal brain (Fig. 2A) contained large multilamellar membranous sheets with occasional cross sections of intact myelinated axons. The axonal material seemed to be the only component other than myelin which was present. The corresponding fraction isolated from Jimpy brain (Fig. 2B) appeared as a heterogeneous population of unilamellar membranous vesicles. No morphologically identifiable myelin membranes were observed. The P2A fraction from normal brain (Fig. 3A) contained multilamellar membranes similar to those observed in PlA, but of overall smaller size. It also had a considerable proportion of unilamellar vesicles which could be microsomal or synaptosomal in origin. The P2A fraction from the Jimpy brain (Fig. 3B) appeared as a heterogeneous population of membranous structures. No typical myelin membranes were found, although some multilamellar membranes were occasionally observed. The P3A fraction from both normal (Fig. 4A) and Jimpy (Fig. 4B) brains consisted of unilamellar vesicles which
Accumulation of [he MBP-containing fractions during derelopmmr
Brains from normal mice at different ages were fractionated, and the amount of total protein and MBP present in PIA, P2A and P3A was determined (Fig. 1). At 10 days after birth MBP was detectable in all 3 fractions. In PIA it accumulated at an approximately linear rate between 10 and 30 days after birth and at a reduced rate between 30 and 60 days after birth. This fraction contained greater than 75% of the total brain MBP at every age after 10 days, and the ratio of M B P to total protein (20 nmol/mg) was constant. In P2A MBP accumulated gradually between 10 and 30 days, after which the amount of MBP declined. The ratio of MBP to total protein in this fraction varied during development, increasing up to a maximum (2.5nmol/mg) at 30 days after birth and then decreasing. The amount of MBP in P3A increased sharply between 10 and 15 days after birth and then decreased so that the amount at 60 days was approx 10% of the amount at 15 days after birth. The ratio of MBP to total protein in P3A was low (0.5 nmol/mg) com3.c. 3 2 , 5 - ~
0.05
I t
.“r ;‘I
/ I /
OO
O
lo
‘s. ..
--- - - - -- - --
P3A
-0-
;o
20 40 Days after birth
50
___D
$0
FIG. 1. Accumulation of MBP in PIA, P2A and P3A from normal brain during development. The brains of normal mice at 5, 10, 15, 20, 30 and 60 days after birth were fractionated as described in Materials and Methods. Five brains were used at each age. The results are expressed as mol MBP per brain. The values are the mean of 3 separate experiments. The variation was lo”/, or less.
E.
1440
~
[
BARBARESt,
J. H. CARSON and P. E. BRAUN
PIA NORMAL
I P2A
P l A JlMPY
1
I
P 3 A NORMAL
1
P J A JlMPY
NORMAL
P 2 A JlMPY
30
I
i
I
20
30
SLICE NUMBER
FIG. 5. RIA profiles of PIA, P2A and P3A fractions from normal and Jimpy mouse brain at 20 days after birth. The PlA, P2A and P3A fractions from normal and Jimpy brains were run on SDS polyacrylamide gel for 14 h at 3 mA/gel. Approximately 5-120 p g of protein was applied to the gel depending on the concentration of MBP in the sample. Routinely, 2 different protein concentrations were tested for each fraction. After staining, the gels were processed for R I A (BAKBARESE et nl., 1977). Only the gel region that contains MBP is shown. No cross-reacting material other than the 21.5 K, 18.5 K, 17 K and 14 K dalton species was found. Each RIA sample was done in duplicate. The effective range of the assay is between 2 and 15 x mol. The RIA profiles obtained in a typical experiment are shown. Similar profiles were obtained in 4 separate experiments.
were quite homogenous in size (150 nm average dia.). Numerous polyribosomes were also present, often with one end in close apposition to the vesicles. Analysis of subcellular fractions by SDS PAGE and RIA The 9 subcellular fractions from 20 day old normal and 20 day old Jimpy brains were analyzed by SDS PAGE. Each of the fractions had a characteristic protein profile. The gels of the non MBP-containing fractions (PlB, PlC, P2B, P2C, P3B, S ) were identical for both normal and Jimpy. The protein profile of PIA normal was the same as reported previously by BARBARESE et al. (1978). The protein profiles for P1A and P2A Jimpy were the same as for the corresponding fractions from normal brain, except that the 4 MPBs were not detected by staining. The protein profiles for P3A from normal and Jimpy were identical but the small amount of MBP was obscured by other bands. The gels of the MBP-containing fractions (PlA, P2A, P3A) from both normal and Jimpy brains were further analyzed by RIA to determine the relative proportions.of each of the 4 MBPs in each fraction. The radioimmunoassay profiles of the gels are shown in Fig. 5. The different molecular forms of MBP were detected in characteristic proportions in each of the MBP-containing fractions. In the fractions from normal brain the relative proportion of 1 4 K dalton MBP tended to decrease from P3A t o P2A t o P l A . The proportions of the 4 proteins in P2A and P3A
from Jimpy brain were similar to the proportions in the corresponding fractions from normal brain. In P l A Jimpy, the 21.5 K dalton protein, which was the least abundant form in all the other fractions, was the predominant species of MBP. DISCUSSI0N
The work described in this paper shows that in normal and Jimpy mouse brain there are 3 distinct subcellular fractions that contain MBP. The fractions, designated PlA, P2A, and P3A, all have densities less than 1.08 g/ml but can be separated on the basis of their sedimentation properties. In order to determine from which cellular structure each fraction was derived, their morphology, overall protein composition, and developmental pattern of accumulation were analyzed. In order to determine the ontogenic relationships among the different fractions the proportions of the 21.5K, 18.5K,1 7 K and 1 4 K daltons MBPs in each fraction were compared. The P1A fraction from normal brain is almost certainly derived from the mature myelin sheath. By electron microscopy the fraction appears as sheets of multilamellar compacted membranes with occasional cross sections of myelinated axons. The axonal material may contribute to some of the high molecular weight proteins present on the SDS gel of this fraction. The overall protein composition and the ratio of MBP t o total protein are similar to those observed in myelin isolated from adult mouse brain by conven-
FIG.
2. Electron micrographs of P1A from normal (A) and Jimpy (B) brains at 20 days after birth. x 58,500.
1441
FIG.3. Electron micrographs of P2A from normal (A) and Jimpy (B) brains at 20 days after birth. x 58,500.
1442
FIG.4. Electron micrographs of P3A from normal (A) and Jimpy (B) brains at 20 days after birth x 58.500.
1443
Myelin basic proteins in mouse brain tional methods (BARBARESE et ul., 1977). The pattern of accumulation of P i A during development parallels the accumulation of myelin in the mouse brain (UZMAN & RUMLEY, 1958). The ratio of MBP to total protein (20 nmol/mg) is constant throughout development. All these factors are indications that P I A is a relatively pure preparation of fragments of the mature myelin membrane. Although the P2A fraction from normal brain is a mixture of membranous structures, the MBP-containing component is probably derived from newly formed or immature myelin. Multilamellar membranes similar to those in PIA are present in the fraction, indicating that it contains myelin. The protein profile of the fraction shows typical myelin components on a background of non-myelin proteins. Two observations suggest that P2A from normal brain contains newly formed myelin rather than a subfraction of mature myelin. Firstly, it has the developmental pattern of accumulation that is cxpected for newly formed myelin. At 10 days after birth, when most of the myelin in the brain is newly formed, greater than 50% of the total MBP is recovered in P2A. Between 15 and 30 days after birth, when the rate of myelin formation is maximum, the amount of MBP in P2A increases. At 60 days, when the rate of myelin formation is slower, the amount of MBP in P2A is decreased. Secondly, the proportions of the four MBPs in P2A are distinct from the proportions in mature myelin. P2A contains a greater proportion of 1 4 K dalton MBP than PlA, and the proportion of this form of MBP in myelin increases during development. This suggests that P2A represents ‘new’ myelin while P1A represents ‘old’ myelin. There are several possible explanations for the presence of MBP in the P3A fraction. One possibility is that when the amount of myelin in the homogenate is very small (as in the Jimpy brain) it fractionates in an anomalous fashion. This explanation is unlikely because when 20 day old Jimpy or newborn normal brains were homogenized in the presence of trace amounts of 3H-labelled myelin isolated from 20 day old normal brain, the radioactivity was recovered primarily in P1A and P2A with very little in P3A (BARBARESE, unpublished observation). A second possibility is that the MBP in P3A is due to adventitious binding of soluble MBP to some microsomal component during the fractionation procedure. This is also unlikely, since when brains were homogenized in the presence of soluble ‘Z51-labelled 18.5 K dalton MBP and fractionated, negligible radioactivity was recovered in the P3A fraction (BARBARESE, unpublished observation). The third possibility is that P3A is derived from an MBP-containing subcellular component which is distinct from PlA or P2A. The fractionation procedure is designed to isolate enriched fractions, and biochemical characterization would be needed to assess the purity of the P3A fraction. However, the fact that P3A from normal brain has a constant MBP to total protein ratio throughout develop-
1445
ment and the fact that the fraction accumulates dramatically during active myelination suggests that a large proportion of the membranes in the fraction are involved in myelin formation. The proportions of the four MBPs support the possibility that P3A is an intermediate in myelin assembly. P3A contains a higher proportion of 14 K dalton MBP than either P2A or PlA, suggesting that it is derived from a ‘newer’ structure than either of these fractions. The P3A fraction from the Jimpy brain appears identical in every respect to P3A from normal brain and is probably derived from the same type of membranous intermediates in myelin biogenesis. The fact that this fraction accumulates in the Jimpy brain to a greater extent than the normal brain at a comparable age suggests that in the Jimpy brain intermediates in myelin assembly are formed but are not converted into myelin. Morphological studies of oligodendroglial cells in Jimpy mice (MEIER& BISCHOFF,1974, 1975) show that the perikaryon and processes are filled with vesicles and other membranous structures, some of which may represent the intracellular intermediates from which P3A Jimpy is derived. The nature of the P2A fraction from the Jimpy brain is less certain. Since the bulk of the material in this fraction is probably derived from components that are unrelated to myelin, the overall morphology and protein composition of the fraction is uninformative. The proportions of the four MBPs in this fraction are similar to those in PIA and P2A from normal brain, suggesting that the MBP-containing material in P2A Jimpy could be derived from myelinlike structures such as the membranous tubes in the perikaryon of the Jimpy oligodendroglial cell or the small amount of loose myelin that accumulates in the Jimpy brain (MEIER& BISCHOFF,1974). The origin of the P I A fraction from Jimpy brain is probably the initial wrapping of oligodendroglial plasma membrane around the axon. Since this fraction is isolated by sedimentation at low speed (1000 g for 10 min), it is presumably derived from large subcellular structures. However, by electron microscopy it appears as an heterogeneous population of small vesicles. This may mean that the original structure vesicularized during the isolation procedure. The protein profile of the P1A fraction from 20 day old Jimpy brain is similar to the profile of the P l A fraction from 5 or 10 day old normal brain in which myelination is just beginning (BARBARESE, unpublished observation). MEIER& BISCHOFF(1975) have reported that in the Jimpy CNS each axon is surrounded by a thin tongue of oligodendroglial cytoplasm which presumably represents one of the early stages in myelin morphogenesis. The Jimpy mutation blocks the extension of this initial wrapping to form the multiple layers of the myelin sheath. It seems likely that the MBP containing component in P1A Jimpy is derived from this single wrapping of the oligodendroglial process around the axon. The results presented in this paper have important
1446
E. BARBARESE, J. H. CARSON and P. E. BRAUN
implications concerning both the molecular and the cellular mechanisms in myelination in normal and Jimpy mouse brain. Firstly, the characteristic polymorphism of MBP in the different subcellular fractions from normal and Jimpy brain, particularly the predominant 21.5 K dalton MBP in the P1A Jimpy, can best be explained by the hypothesis that the mode of MBP gene expression changes during myelin morphogenesis, from predominantly 21.5 K dalton MBP in the initial stages to predominantly 14K dalton MBP in the latter stages. This hypothesis is consistent with the earlier observations that the proportions of the four MBPs in myelin change during mouse brain development (BARBARESE et a!., 1978) and that the rate of synthesis of 14K dalton MBP relative to 18.5K dalton MBP increases during brain development (CAMPAGNONI et a/., 1978). Secondly, the properties of the P3A fraction suggest that it represents an intracellular intermediate in -myelin formation. The role of this intermediate may be to translocate MBP from its site of synthesis to the site of myelin assembly. In this regard, CULLEN& WEBSTER(1977) have reported that when myelination in the optic nerve of Xenopus tadpole is blocked by low temperature, membrane vesicles accumulate in the perikarya and inner tongue process of the oligodendroglial cells. These vesicles may correspond to the intraceliular precursors of myelin from which P3A is derived. The accumulation of the P3A fraction in Jimpy brain suggests that the Jimpy mutation also blocks the conversion of the precursor membranes into myelin. Acknow/edgements- We thank J. ABRAMSfor useful discussion and for his help in preparing the samples for electron for taking the electron micromicroscopy, L. GULUZIAN graphs and photographs, and M. SCHINDLER for her excellent technical assistance. This work was supported by grants from the Medical Research Council of Canada and the Multiple Sclerosis Society of Canada.
REFERENCES E., BRAUN P. E. & CARSON J. H. (1977) IdentifiBARBARESE cation of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins. Proc. natn. Acad. Sci., U.S.A. 74, 3360-3364. BARBAKESE E., CARSONJ. H. & BRAUNP. E. (1978) Ac-
cumulation of the four myelin basic proteins in mouse brain during development. J . Neurochem. 31, 779-782. CAMPAGNONI C. W., CAREYG. D. & CAMPAGNONI A. T. (1978) Synthesis of myelin basic proteins in the developing mouse brain. Archs Biochem. Biophys. 190, 118-125. CARSON J. H., HERSCHKOWITZ N. N. & BRAUNP. E. (1975) Synthesis and degradation of myelin basic protein in normal and Jimpy mouse brain. Trans. Am. SOC.Neurochem. 6 , 207. COTMAN C . W. & FLANSBURG D. A. (1970) An analytical micro-method for electron microscopic study of the compositipn and sedimentation properties of subcellular fractions. Brain Res. 22, 152-156. CULLENM. J. & WEBSTER H. DE F. (1977) The effects of low temperature on myelin formation in optic nerves of Xenopus tadpoles. Tissue and Cell 9, 1-10. EICHBFRG J., WHITTAKER U. P. & DAWSONR. M. C. (1964) Distribution of lipids in subcellular particles of guinea pig brain. Biochem. J . 90, 91- 100. FAIRBANKS G., STECKT. L. & WALLACH D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606 -2617. FISCHERC. A. & MORELLP. (1974) Turnover of proteins in myelin and myelin-like material in mouse brain. Brain Res. 74, 5165. KARNOVSKY M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. 1. Cell. Biol. 27, 137A-138A. KURTZS. M. (1961) A new method for embedding tissue in Vestopal W. J . Ultrastruct. Res. 5, 468-477. LDWRY 0. H., ROSEBROUGHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J . biol. Chem. 193, 265-275. MEIERC. & BISCHOFF A. (1974) Dysmyelination in ‘Jimpy’ mouse. Electron microscopic study. J . Neuropath. exp. Neurol. 33, 343-353. A. (1975) Oligodendroglial cell develMEIERC. & BISCHOFF opment in Jirnpy mice and controls. An electron-microscopic study in the optic nerve. J . Neurol. Sci. 26, 5 17-528. PEASE D. C. (1964) in Histological Techniques f o r Electron Microscopy. 381 pp. Academic Press, New York. SIDMAN R. L., DICKIEM. M. & APPEL S. H. (1964) Mutant mice (quaking and Jimpy) with deficient myelination in the central nervous system. Science 144, 309-31 1. SWANK R. T. & MUNKRES K. D. (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Analyt. Biochem. 39, 462-477. UZMANL. L. & RUMLEY M. K. (1958) Changes in the composition of the developing mouse brain during early myelination. J . Neurochem. 3, 170-184.
