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Composition and Metabolism of Myelin Phosphoglycerides During Maturation and Aging
Composition and Metabolism of Myelin Phosphoglycerides During Maturation and Aging LLOYD A. HORROCKS Department of Physiological Chemistry, College of Medicine, The Ohio State University, Columbus, Ohio, 43210 (U.S.A.)
Morphological studies’ have established that the myelin sheath in the central nervous system is continuous with the plasma membrane of the oligodendroglial cells and myelin constitutes the major portion of these cells. Myelin fractions can be isolated by a number of methods2 for subcellular fractionation of dispersions of CNS tissues from adult mammals. These myelin fractions have the morphological appearance of myelin, are nearly completely soluble in chloroform-methanol (2 : 1, v/v), form a band on sucrose density gradients at a density less than that of 0.8 M sucrose, and have a characteristic lipid3-’ and protein2 composition. Contamination of isolated myelin fractions by mitochondria and axoplasm is almost completely removed by
z
o Horrocks4 Sun and Somorajski” 0 Rawlins and Smith” Norton e l al.“
AGE, Days (Logarithmic Scale1
Fig. 1. The amount of myelin isolated from the brains of rats and mice as a function of age. For the mice, the amount of myelin is expressed as the amount of myelin lipid phosphorus in order to correct for differences in lipid composition during early development. The bars represent the S.E. The value for 730-day-old mice is based on myelin isolations from 21 individual mouse brains. 0 and 0, mouse; 0 and B, rat. Abbreviations: CDP, cytidine diphosphate; CNS, central nervous system; CPG, choline phosphoglycerides; EPG, ethanolamine phosphoglycerides; GPC, sn-glycero-3-phosphorylcholine;GPE, snglycero-3-phosphorylethanolamine; GPS, sn-glycero-3-phosphorylserine;IPG, inositol phosphoglycerides; SPG, serine phosphoglycerides; SRA, specific radioactivity. References p . 393-395
384
L. A. HORROCKS
TABLE 1 LIPID COMPOSITION OF SUBCELLULAR FRACTIONS FROM BRAINS OF
2-YEAR-OLD MICE
Values are expressed as the mean & S.E. For lipid-P, cholesterol, and galactolipids, n = 21; for myelin phospholipids, n = 9; for microsomal phospholipids, n = 7; and for mitochondria1 phospholipids, n = 5. The acid-stable EPG include diacyl-GPE, alkylacyl-GPE, and cardiolipin (mitochondrial fraction only).
~
Cholesterol Galactolipids Alk-1 -enylacyl-GPE Acid-stable EPG CPG Sphingom yelin SPG plus IPG
Mjielin ,fraction
1.33 0.03 0.65 0.02 0.331 3Z 0.006 0.146 i 0.007 0.251 0.007 0.053 0.004 0.215 0.012
**
Mole ratio, component: lipid-P - -Microsomal fraction 0.78 0.20 0.177 0.174 0.403 0.047 0.197
5 0.02 0.02 k 0.007 5 0.002 0.010 & 0.008 & 0.009
+
Mitochondria1 fraction 0.64 0.04 0.105 0.296 0.410 0.043 0.147
0.09
k 0.02
0.004 & 0.015 0.008 5 0.009 f 0.008
osmotic shock followed by reisolation of the myelin. Contamination by fragments of endoplasmic reticulum may be a problem. Miller and Dawson’ found a substantial amount of NADPH-cytochrome c reductase, apparently a microsomal enzyme, in crude isolated myelin, but Jungalwala and Dawson’ could not detect this enzyme in osmotically shocked and purified myelin. Further studies of microsomal enzymes in isolated myelin fractions during purification are required. Relatively few studies on isolated myelin have been extended to ages beyond maturity’, ‘O-’’ . In the mouse and rat, each study has shown that the amount of myelin in the brain continues to increase throughout the life span of the animals (Fig. 1). Although the rate of increasc: of the amount of myelin decreases with age, the net deposition of myelin does not stop. Thus “period of maximum rate of myelination” is a more precise term than “period of myelination”. The characteristic lipid composition of myzlin is illustrated in Table 1. To date, similar studies have not been made with brains from larger animals. In man, measurements have been made of the content of lipids such as cerebrosides and ethanolamine plasmalogens which are concentrated in the myelin sheath13. Since these lipids decrease in amount after the age of 30, the amount of myelin in the human brain may also decrease during aging. I n 1965 we reported that mouse brain myelin changes composition during development14. As a result of skepticism by several members of the audience, the experiment was replicated with myelin isolated by several different methods from the brains of mice4, rats’, l o , ‘’-I7 ,rabbits”, and man’’, 92. Regardlelessofthemodeofexpression of the lipid composition of the isolated myelin, all investigators agree that the relative proportions of galactolipids, cholesterol, and ethanolamine plasmalogens increase and the relative proportion of choline phosphoglycerides decreases during maturation (Figs. 2 and 3). The increases in the relative proportions of galactolipids and cholesterol are nearly equal on a molar basis (Fig. 2). Cuzner and DavisonI6
MYELIN COMPOSITION AND METABOLISM
8 P
385
80-
H
::
2
60
.
40
-
-
E
0
7
In
-
0
- loo\P
d
P
E
v)
2o
m,
W 4
.
GALACTOLIPIOS
0,O
~
0 CHOLESTEROL
802
5
-
p
i
i
0
10
20
40
100
200
400
60
800
AGLdoyr (logarithmic scale)
Fig. 2. The amounts of galactolipids and cholesterol relative to phospholipids in myelin as a function of age. 0 and 0, mouse4; 0 and W, rat2. 40
r
a 1 10
0 , O CHOLINE
20
PHOSPHOGLYCERIOES
40
100
200
AGE, doys ( logarithmic scole)
400
800
.,
Fig. 3. The proportions of ethanolamine plasmalogens and choline phosphoglycerides in myelin mouse4; 0 and rat2. phospholipids as a function of age. 0 and 0,
described an even more pronounced increase in these lipids that was probably due to the inclusion of the myelin-like fraction in the early myelin2. The attainment of the myelin composition characteristic of the mature animal is much more rapid in the mouse than in the rat. The cholesterol and galactolipid content of rat brain increases substantially until at least 275 days of age2’. The molar ratio of the net increase of cholesterol to galactolipid is about 2 which is the ratio found in myelin isolated from adult rats. The proportion of ethanolamine plasmalogens also increases during maturation (Fig. 3). In myelin from the brains of adult mammals, the ethanolamine plasmalogens account for 31 to 37% of the phospholipid?’. The proportion of ethanolamine plasmalogens in human myelin increases slightly from maturity to old age2’. The adult level is reached between 14 and 24 days of age in the mouse but not until after 30 days of age in the rat. In contrast, the proportion of choline phosphoglycerides declines during maturation at the same rate in the myelin from the mouse and rat. References p . 393-395
386
L. A. HORROCKS
TABLE 2 ALKENYL AND ACYL GROUP COMPOSITIONS OF ETHANOLAMINE PLASMALOGENS FROM ISOLATED HUMAN MYELIN (n = 5)
Weight
Alkenyl groups
16:O 17:O 18:O 18:l
Acyl groups
16:O 18:O 18:l 20: 1 20:2 20:3 (n - 6) 20:4 (n - 6) 22:3 22:4 (n - 6) 2 2 ~ 6(n - 3) 2 4 ~ 4(n - 6)
%
Age (years): 36-50
57-77
25.6 1.1 16.4 56.8
26.9 1 .o 15.6 56.5
3.2 3.2 45.1 8.1 1.8 0.9 9.7 1.8 20.2 2.1 1.9
3.8 3.7 46.9 10.3 1.6 0.9 7.3 1.3 18.9 2.0 1.2
The lipid composition of myelin is quite constant during aging with the possible exception of galactolipids (Fig. 2). The major phosphoglycerides of myelin are ethanolamine plasmalogens, phosphatidylcholine and phosphatidylserine. The alkenyl groups of the ethanolamine plasmalogens from human myelin are about 57 % 18 :1 , 26 % 16 :0 and 16 % 18 :0 with no apparent change in composition during aging. The content of monounsaturated alkenyl groups in ethanolamine plasmalogens is much greater in myelin than in gray matter. The acyl groups from ethanolamine plasmalogens are almost exclusively unsaturated as expected for acyl groups from the 2-position of phosphoglycerides. When compared to gray matter ethanolamine plasmalogens, the acyl groups from myelin ethanolamine plasmalogens have relatively large proportions of 18 :1 , 20 : 1 and 22 :4, about the same proportion of 20 :4, but a much lower proportion of 22 :6 acyl groups. With increasing age, the proportions of 18 :1 and 20 : 1 acyl groups increased and 20 :4 and 22 :4 acyl groups decreased significantly” (Table 2). Similar changes have been observed in the total EPG from whole brainz3. The phosphatidylserine from human myelin contains nearly equal proportions of 18 :O and 18 : 1 acyl groups with very small proportions of 16 : O and polyunsaturated acyl groups (Table 3). With increasing age, there is a sniall increase in 18:O and a small decrease in 18 : 1 acyl groups. In gray matter, polyunsaturated acyl groups account for most of the unsaturated acyl groups. The phosphatidylcholine from human brain contains small proportions of polyunsaturated acyl groups, even in gray matter. In myelin, the phosphatidylcholine has lower proportions of 16 :O and poly-
387
MYELIN COMPOSITION AND METABOLISM
TABLE 3 ACYL GROUP COMPOSITIONS OF PHOSPHATIDYLCHOLINEAND PHOSPHATIDYLSERINE FROM ISOLATED HUMAN MYELIN
(n = 5)
Weight Phosphatidyleholine 57-77 Age (years): 36-50 Aeyl group 16:O 16:l 18:O 18:l 20:l 20:4 (n - 6) 22~4 (n - 6)
28.9 2.4 13.6 47.7 1.3 2.6 0.9
% Phosphatidylserine 36-50 57-77
30.8 2.0 12.4 49.5 1.6 2.0 0.4
2.3
1.3
42.6 48.9 3.5 0.5 0.6
44.2 46.2 4.4 1.o 1.1
TABLE 4 ACYL GROUP COMPOSITIONS OF CEREBROSIDES FROM ISOLATED HUMAN MYELIN
(n
=
4)
Methyl ester derivatives of unsubstituted acyl groups and methyl ester and trimethyl silyl ether derivatives of 2-hydroxyacyl groups were subjected to gas-liquid chromatography. A hydrogen-flame detector was used. Area Age (years): Acyl group 16:O 18:O 18:l 20:o 22:o 22:l 23 :O 23:l 24:O 24:l 25 :O 25 : 1 26:O 26:l
Ratio 24:1/24:0
Unsubstituted acyl groups 36-50 57-77
0.8 7.7 0.8 0.5 3.0 0.4 5.4 0.6 19.8 40.7 7.1 8.5 0.6 4.8
1.6 10.8 1.4 0.6 2.4 0.4 3.8 0.3 13.4 49.7 4.2 7.6 0.7 4.3
2.1
3.7
% 2-Hydroxy aeyl groups 36-50 57-77
0.9
1.3
0.2 4.6
0.1 6.0
15.4
19.3
37.4 27.1 9.6 2.2 0.7 2.3
31.0 29.3 6.9 2.7
0.72
0.4
2.6
0.95
unsaturated acyl groups with a much higher proportion of 18 :I acyl groups. These differences are further accentuated during aging (Table 3). Thus, the non-polar groups from human myelin phosphoglycerides differ from the non-polar groups of neuronal phosphoglycerides by having higher proportions of monounsaturated (18 :1 and 20 :1) acyl groups and lower proportions of saturated (16 :0 and 18 :0) and n-3 polyReferences p . 393-395
388
L. A. HORROCKS
unsaturated (22 :6) acyl groups and these differences tend to become larger during aging2’. Cerebrosides, which are also major components of myelin, are characterized by very long-chain acyl groups such as unsubstituted and 2-hydroxy 24 : 0 and 24 : 1 acyl groups. Both types of 24:O acyl groups were found in decreased proportions in cerebrosides from human myelin from older brains (Table 4). Increased proportions of unsubstituted 24 : 1 and 2-hydroxy 23 :0 were found instead. Relatively large changes in acyl group compositions of myelin lipids might be expected during maturation because lipid synthesis is most active and the lipid class composition of the myelin changes most at that time. No results are available. In addition, the lipid class and acyl group compositions of the presumed myelin precursors, the myelin-like fraction and the oligodendroglial plasma membrane, should be determined during maturation. “Early myelin” (myelin isolated from very young animals) has a composition that is similar to that of the myelin from quaking mice (a mutant with arrested myelination). Myelin from the quaking mouse, when compared to myelin from littermate controls, has more CPG but is deficient in cerebrosides, ethanolamine plasmalogens, and the apolar p r ~ t e i n ’ ~ - ~ In ’ . addition, compared to controls quaking mouse brain myelin had much lower proportions of monounsaturated alkenyl and acyl groups in the EPG and 24 :1 hydroxy and unsubstituted acyl groups in the c e r e b r ~ s i d e sThese ~~. results suggest that the apolar protein has relatively more binding sites for cerebrosides and ethanolamine plasmalogens and relatively fewer binding sites for CPG than the other myelin proteins. The “early myelin” may represent a transition form between the oligodendroglial plasma membrane and mature myelin’*. Davison’s group has studied the purification of crude “early myelin”. Banik and DavisonZ9 reported that a myelin-like fraction could be separated from compact myelin from young rat brains. Further studies by Agrawal et have shown that the myelin-like fraction resembles myelin in enzyme activity but the myelin-like fraction does not contain the basic protein or cerebrosides. They suggested that the myelin-like fraction represents a transition between oligodendroglial plasma membrane and compact myelin and that the differences observed in early myelin are due to a larger proportion of the myelin-like fraction in a mixture of compact mydin and myelin-like fraction. On the basis of 2’,3’-cyclic nucleotide phosphohydrolase activities, Zanetta et aL3’ suggested that myelin-like and nerveending fractions may both contain fragments of oligodendroglial plasma membrane. Th- identity of the myelin-like fraction with the oligodendroglial plasma membrane is supported by the report that a myelin-like fraction from quaking mice has neither the basic protein nor the apolar protein but only the high molecular weight myelin proteins’’. Similar results were reported by Savolainen and pal^^^ for myelin-like fractions, fetal myelin, and glial plasma membranes from human brains. Unfortunately, the glia were probably astrocytes and lipids and enzymes were not assayed. Phosphoglyceride biosynthesis is an essential part of myelin deposition. In the brain, the diacyl-GPC and diacyl-GPE are formed from CDP-choline or CDPethanolamine and 1,2-diacylgly~erols~~~ 34. The diacyl-GPE and diacyl-GPS may
MYELIN COMPOSITION A N D METABOLISM
389
undergo exchange reactions with free ethanolamine or serine3', 3 6 . The alkyl groups of brain phosphoglycerides are formed from fatty alcohols37- 4 3 . Phosphoglycerides with alkyl groups are involved in the biosynthesis of ethanolamine plasmalogens as follows: (a) l-Alkyl-2-acyl-sn-glycero-3-phosphates -+ 1-alkyl-2-acyl-sn-glycerols + Pi (b) 1-Alkyl-2-acyl-sn-glycerols CDP-ethanolamine .+ l-alkyl-2-acyl-sn-glycero-3phosphorylethanolamines CMP -+ 1-alkenyl-2-acyl-sn-gly(c) l-Alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines cero-3-phosphorylethanolamines+ 2[H]
+
+
Reaction (a) was demonstrated by Snyder et ~ 1 . Ethanolamine ~ ~ . phosphotransferase (EC 2.7.8.1) catalyzes Reaction (b)45. Direct evidence for Reaction (c) in cell-free systems from brain tissue has been provided by Horrocks and Rad~minska-Pyrek~' Since the products of these three reactions were all quite radioand by Blank et active at short times after intracerebral injections of ['4C]hexadecano141, this pathway is operative in vivo. Indirect evidence for this pathway had been reported previously4'. The activity of the fatty acid Fatty acid synthetase is present in the synthetase gradually decreases during development when the activity is expressed in terms of tissue weight5'. Fatty acids can be elongated in the mitochondrias2*53, and microsomess4. Fatty acid desaturase activity is present in brain microsomes from very young rats but is virtually absent by 11 days of age55. The utilization of these pathways has been established by the uptake of radioactivity from labeled acetate or glucose into alkenyl and acyl groups'" 'O, 56-60. Brain fatty acids may also be taken up from the free fatty acids in the circulating blood. Dhopeshwarkar and Mead6' - 6 3 and Dhopeshwarkar et have established that small amounts of 16:0, 18:l and 19:2 fatty acids administered orally or intraarterially to rats can be recovered in brain tissue. In this laboratory, we have found that the same fatty acids are taken up in significant amounts by monkey brain after the infusion of ['4C]fatty acid-albumin complexes into the internal carotid artery. The perfused rabbit brain takes up ['4C]palmitate from the perfusate into the reported that linoleic acid was taken up by phosphog1ycerides6'. Bernsohn et brain stem and spinal cord from the cerebral spinal fluid (CSF). These experiments demonstrate that exogenous fatty acids are taken up by the brain (see review by D ' A d a m ~ and ~ ~ )used in part of the synthesis of phospholipids. Labeled fatty acids are incorporated into intracellular phospholipids by slices6' and ex plant^^^. 7 0 of rat cerebrum. The choroid plexus incorporates palmitic acid from the ventricles into lipids7'. Intraczrebral injections of [14C]fatty acids have been used to determine the fate of free fatty acids in brain tissue. In mice, about one-fourth of the injected radioactivity from [14C]palmitic acid was recovered in the brain lipids7', 7 3 . At times of more than 12 hours after injection, about 85 % of the recovered radioactivity was found in the phospholipid^^^ with relative specific radioactivities of the phosphoglycerides that were nearly identical to those reported by Winkelmann References P . 393-395
390
L. A. HORROCKS
and G e r ~ k e n The ~ ~ . incorporation into phosphoglycerides increases during the first hour after injection73. An intermediate in phosphoglyceride biosynthesis, the 1,2diacylglycerols, reached its highest SRA between 6 and 10 minutes after injection. The flux of radioactivity through the diacylglycerol pool was sufficient to account for all of the choline and ethanolamine phosphoglyceride turnover with estimated half-lives of 9 minutes for the diacylglycerols and 7 days for the phosphoglycerides. Sun74has found that the half-life of the diacylglycerols in mouse brains was the same at 3, 10 and 28 months of age, but the uptake of palmitic acid into the diacylglycerols decreased markedly with age. The synthesis of myelin lipids takes place in the oligodendroglial endoplasmic reticulum8. The myelin lipids can then be transported to newly synthesized myelin or exchanged with existing myelin lipids75. The exchange process is presumably mediated by a cytosol protein as shown for cerebroside sulfates by Herschkowitz et and Pleasure and P r ~ c k o pand ~ ~for mitochondria1 phosphoglycerides by Miller and Dawson7’. In several studies, with different radioactive lipid precurs0rs9, 78-80 , about 7 days are required before the SRA of myelin phosphoglycerides are equal to the SRA of microsomal phosphoglycerides. In one of these studies”, the SRA of phosphoglycerides from the crude myelin fraction was much higher than the SRA of the purified myelin phosphoglycerides during the first few days after injection. The material with a high SRA that was lost during purification may include the myelin-like fraction that was described earlier in this section. The metabolism and characterization of this “nascent” myelin deserve further study. The exchange of lipids between mature myelin and the cytosol is difficult to visualize, but we must remember that a portion of each lamella is in contact with oligodendroglial cytosol at the nodes of Ranvier. Since membrane lipids are fluid and can diffuse laterally, all of the myelin lipids can equilibrate with cytosol transport lipoproteins. Irregardless of the extent of contact of cytosol with myelin, the rate of this equilibration and subsequent metabolic processes will determine the rate of myelin turnover. The turnover of brain lipids is usually measured from graphs of the SRA against time. In addition to problems with the reproducibility of injections and the heterogeneity of pools. the interpretation of turnover studies is also dependent on the degree of reutilization of catabolic products. This is illustrated by the results of Abdel-Latif and Smith” for the metabolism of rat brain CPG. The half-lives of the CPG were 2.9 days with [‘4C]glycerol as the precursor, 11.7 with [‘4C]choline, and 18.6 days with [32P]phosphate. Lapetina et reported half-lives of 5.1 days with [‘“Clglycerol, 9.2 days with [3’P]phosphate, and 38 days with [14C]acetate for the total lipids from rat cerebral cortex. These differences in the half-lives reflect the differences in reutilization of the products from phosphoglyceride catabolism. An extensive reutilization of palmitic acid was found in the mouse brain”. At 30 days after injection, only 22% of the radioactivity in the phosphoglyceride acyl groups was in the 16:0 acyl groups. Kishimoto et ~ 1found . that ~ ~16 :O acyl groups from phosphoglycerides were the source of the palmitic acid used for elongation. These findings help to explain the half-life of 38 days for rat brain lipids after [I4C]acetate injections8’ and the half-life of 7 months for the EPG from rat brain myelin after intraperitoneally
39 1
MYELIN COMPOSITION AND METABOLISM
injected ['4C]acetate*4. In neither case was the SRA of the 16 :0 acyl groups measured. In the latter case, the very low content of 16:O acyl groups in the myelin EPG was not considered. It is necessary to distinguish between the apparent turnover which includes reutilization and the true turnover. Another form of turnover is the exchange of intact lipids which are not catabolized as shown for cholesterol by Banik and Davison", 86.The exchange phenomenon that was described previously is supported TABLE 5 SPECIFIC RADIOACTIVITIES OF THE
EPG FROM SUBCELLULAR
FRACTIONS OF MOUSE BRAINS
C57 BL/lO mice at 150 days of age were given intracerebral injections of [14C]ethanolamine as described by H o r r o ~ k s Lipids ~ ~ . were isolated according to Horrocks and Sungo.
2
SRA (dpm/pmole) 4
6
Diacyl-GPE Myelin Microsomes Mitochondria
2170 3830 1590
1320 1880 1280
530 1070 350
Alk-I-enylacyl-GPE Myelin Microsomes Mitochondria
1550 2250 3600
950 1440 2620
630 630 1110
Alkylacyl-GPE Myelin Microsomes Mitochondria
1780 4480 5160
980 1240 2720
310 240 1180
Days after injection:
TABLE 6 SPECIFIC RADIOACTIVITIES OF THE
EPG IN
BRAIN SUBCELLULAR FRACTIONS FROM 2-YEAR-OLD MICE
The specific radioactivity of the EPG were measured after intracerebral injections of [14C]ethanolamine as described by H o r r o c k ~ ~ ~ . Days after injection:
SRA (dpm/pmole) 7
_________.--
4
I4
.
Acid-stable EPG Myelin Microsomes Mitochondria
2140 3520 1730
1650 2080 1490
1090 1280 720
Alk-1 -enylacyl-GPE Myelin Microsomes Mitochondria
1110 2100 3100
930 1650 1600
780 1070 1060
References P . 393-395
392
L. A. HORROCKS TABLE 7
THE HALF-LIVES OF
EPG
FROM SUBCELLULAR FRACTIONS OF BRAINS OF MICE DURING DEVELOPMENT AND AGING
-
-~
~~
~
t ~ (days) p
Age at injection (days):
40
150
Mitochondria Diacyl-GPE Alk-1 -enylacyl-GPE Alkylacyl-GPE
-
-
1.9 2.4 2.3
1.9 6.3 -
Microsomes Diacyl-GPE Alk-1-enylacyl-GPE Alkylacyl-GPE
1.3 1.3 -
1.9 2.4 1.3
6.9 10.3 -
Myelin Diacyl-GPE Alk-1 -enylacyl-GPE Alkylacyl-GPE
1.7 1.6 -
3.0 3.2 2.0
10.3 20.3 -
730
by the finding that the lipids from different brain membranes, including myelin, have The turnover rates for specific lipids are probably similar turnover rates', determined by the magnitude of the forces binding them to membranes and by the activity of catabolic enzymes. Since membrane proteins have a multitude of binding sites, their turnover rate should be much slower than membrane lipids. In fact, halflives of 21 days for the myelin basic proteins7, and 35 days for the myelin apolar proteins" have been reported. These values agree with those reported for brain mitochondrial proteins''. In the latter report, no difference was found in the turnover rate of mitochondrial proteins from 1-year-old and 2-year-old rats. Intracerebral injections of ['4C]ethanolamine label the brain EPG quite rapidly7' with very little recycling of the radioactivity. Experiments of this type have been done with maturing mice7', adult mice (Table 5), and aged mice (Table 6). The halflives of the myelin EPG in mice (Table 7) are 1-2 days in maturing mice, 2-3 days in adult mice, 6-20 days in aged mice and 5-15 days in rats33, 7 8 . Similar values have been obtained with palmitic acid as the precursor". Jungalwala and Dawson' repeated the ethanolamine experiment of Ansell and Spanner7' with nearly identical results. Slices of rat brains and spinal cords were incubated with labeled acetate and leucine. The amounts of incorporation into myelin lipids and proteins decreased rapidly with age up to 6 months, but the incorporation at 6 months, 12 months and 18 months were about the same". These results indicate that the rate of myelin biosynthesis probably slows down markedly during maturation, then plateaus during adulthood. As shown in Table 7, the turnover rate for the myelin EPG is much lower in 2-year-old mice. Because the amount of myelinin the mouse brain increases throughout the first 2 years of life (Fig. 1) the rate of myelin biosynthesis must always exceed the rate of myelin catabolism. "3
MYELIN COMPOSITION AND METABOLISM
393
SUMMARY
In the CNS, myelin is an extension of the plasma membrane of the oligodendroglial cell. Compact myelin from adult brains can be isolated easily and has a characteristic composition. During early maturation, myelin isolation techniques yield “early myelin” which has a different composition, perhaps because it represents a transitional form between the oligodendroglial plasma membrane and compact myelin. In addition, a “myelin-like” fraction can be isolated. The latter fraction may be fragments of oligodendroglial plasma membrane or a transitional form. During aging, the amounts of myelin in mouse and rat brains increase until at least 2 years of age. The lipid composition changes very little during aging, but some differences are seen in the fatty acid composition of myelin phosphoglycerides and cerebrosides from human brains. Myelin lipids are synthesized by the endoplasmic reticulum. A cytosol lipoprotein is presumably involved in the equilibration process which may require a week or more due to the time required for lateral diffusion of myelin lipids to a portion of the myelin that is in contact with the cytosol. Nevertheless, the equilibration process is effective because myelin lipids turnover at rates that are comparable with the turnover rates for microsomal lipids. In rats, the biosynthetic rates for myelin lipids and proteins decrease markedly during maturation. In mice, the turnover of ethanolamine phosphoglycerides slows down with increasing age. The half-lives were about 50 % higher for 5-month-old mice (1.3-3.2 days) than for 40-day-old mice (1.3-1.7 days) and were much higher for 2-year-old mice (6.3-20.3 days).
ACKNOWLEDGMENTS
I am indebted to Dr. Grace Y. Sun, Cleveland Psychiatric Institute, for discussions, the National Institutes of Health, U.S. Public Health Service, for support through research grants NS-05510, NS-08291, and GRS-5409, and the National Multiple Sclerosis Society for support from the Benjamin Miller Memorial Grant for Research on Multiple Sclerosis.
REFERENCES 1 A. PETERS AND J. E. VAUGHAN, in A. N . DAVISON AND A. PETERS (eds.), Myelination, Charles C Thomas, Springfield Ill., 1970, pp. 3-79. 2 W. T. NORTON, in R. PAOLETTI AND A. N. DAVISON (eds.), Chemistry and Brain Development, Plenum Press, New York, N.Y., 1971, pp. 327-337. 3 L. A. HORROCKS, J . Lipid Res., 8 (1967) 569. 4 L. A. HORROCKS, J. Neurochem., 15 (1968) 483. 5 M. E. SMITH,Adv. Lipid Res., 5 (1967) 241. 6 J. EICHBERG, G . HAUSERAND M. L. KARNOVSKY, in G. H. BOURNE(ed.), The Structure and Function of Nervous Tissue, Vol. 3, Biochemistry and Disease, Academic Press, New York, N.Y., 1969, pp. 185-287.
394
L. A. HORROCKS
7 L. C. MOKRASCH, in A. LAJTHA(ed.), Handbook of Neurochemistry, Vol. I, Plenum Press, New York, N.Y.. 1969, pp. 171-193. Biochem. J., 126 (1972) 805. 8 E. K. MILLERAND R. M. C. DAWSON, 9 F. B. JUNGALWALA AND R. M. C. DAWSON, Biochem. J., 123 (1971) 683. S. E. PODUSLO AND K. SUZUKI, Abstr. Int. SOC.Neurochem., Strasbourg, (1967) 10 W. T. NORTON, 161-162. AND M. E. SMITH,J. Neurochem., 18 (1971) 1861. 11 F. A. RAWLINS 12 G . Y. SUNAND T. SAMORAJSKI, J. Gerontol., 27 (1972) 10. 13 G. ROUSER,A. YAMAMOTO AND G. KRITCHEVSKY, in R. PAOLETTI AND A. N. DAVISON (cds.), Chemistry and Brain Development, Plenum Press, New York, N.Y., 1971, pp. 91-109. 14 L. A. HORROCKS, R. J. MECKLER AND R. L. COI.LINS, in G. B. ANSELL(ed.), Variations in the Chemical Composition of the Nervous System as Determined by Development and Genetic Factors, Pergamon Press, Oxford, 1966, p. 46. 15 A. N. DAVISON, M. L. CUZNER, N. L. BANIKAND J. M. OXBERRY, Nature, 212 (1966) 1373. 16 M. L. CUZNER AND A. N. DAVISON, Biochem J., 106 (1968) 29. 17 L. F. ENGAND E. P. NOBLE,Lipids, 3 (1968) 157. 18 K. B. DALALAND E. R. EINSTEIN, Bruin Res., 16 (1969) 441. D. PRATTAND M. G. TAVASTSTJERNA, Biochemistry, 7 (1968) 19 L. F. ENG,F. C. CHAO,B. GERSTL, 4455. 20 Y. KISHIMOTO, W. E. DAVIES AND N. S. RADIN,J. Lipid Res., 6 (1965) 532. 21 L. A. HORROCKS, J. Lipid Res., 9 (1968) 469. 22 G. Y. SUNAND L. A. HORROCKS, Fed. Proc., 30 (1971) 1248 Abs. 23 H. B. WHITE,JR., C. GALLIAND R. PAOLETTI, J. Neurochem., 18 (1971) 1337. 24 H. SINGH,N. SPRITZAND B. GEYER, J. Lipid Res., 12 (1971) 473. W. T. NORTONAND P. MORELL, J. Neurochem., 18 (1971) 2119. 25 S. GREENFIELD, 26 N. A. GREGSON AND J. M. OXBERRY, J. Neurochem., 19 (1972) 1065. 27 C. M. JACQUE, C. F. LOUIS,C. GUEDEAND N. A. BAUMANN, C.R. Acad. Sci., Paris, Ser. D , 274 (1972) 126. 28 S. E. PODUSLO AND W. T. NORTON, J. Neurochem., 19 (1972) 727. 29 N. L. BANIKAND A. N. DAVISON, Biochem. J., 115 (1969) 1051. 30 H. C. AGRAWAL, N. L. BANIK,A. H. BONE,A. N. DAVISON, R. F. MITCHELL AND M. SPOHN, Biochem. J., 120 (1970) 635. P. BENDA,G. GOMBOS AND I. G. MORGAN, J. Neurochem., 19 (1972) 881. 31 J. P. ZANETTA, 32 H. SAVOLAINEN AND J. PALO,FEBS Lett., 20 (1972) 71. 33 G. B. ANSELL, in R. PAOLETTI AND A. N. DAVISON (eds.), Chemistry and Bruin Development, Plenum Press, New York, N.Y., 1971, pp. 63-73. AND K. P. STRICKLAND, in A. LAJTHA (ed.), Handbook of Neurochemistry, Vol. 111, 34 R. J. ROSSITER Plenum Press, New York, N.Y., 1970, pp. 467489. 35 G. PORCELLATI AND F. DIJESO(eds.), Membrane Bound Enzymes, Plenum Press, New York, N.Y., 1971, pp. 111-134. 36 G. PORCELLATI, G. ARIENTI,M. PIROTTA AND D. GIORGINI, J. Neurochem., 18 (1971) 1395. 37 A. K. HAJRA,Biochem. Biophys. Res. Commun., 39 (1970) 1037. 38 H. H. 0. SCHMID AND T. TAKAHASHI, J. Lipid Res., 11 (1970) 412. Biochim. Biophys. Acta, 244 (1971) 217. 39 R. H. LUMBAND F. SNYDER, 40 W. STOFFEL AND D. LEKIM,Hoppe Seyler’s 2.Physiol. Chem., 352 (1971) 501. in J. DOMONKOS, A. FONYO,I. H U S Z ~AND K J. SZENTAGOTHAI (eds.), 3rd In41 L. A. HORROCKS, ternational Meeting of the International Society for Neurochemistry, Academiai Kiado, Budapest, 1971, p. 312. in F. SNYDER (ed.), Ether Lipids: Chemistry and Biology, Academic Press, New 42 L. A. HORROCKS, York, N.Y., 1972, pp. 177-272. 43 F. SNYDER (ed.), Ether Lipids: Chemistry and Biology, Academic Press, New York, N.Y., 1972, pp. 121-157. 44 F. SNYDER, M. L. BLANKAND B. MALONE, J. Biol. Chem., 245 (1970) 4016. 45 A. RADOMINSKA-PYREK AND L. A. HORROCKS, J. Lipid Res., 13 (1972) 580. 46 L. A. HORROCKS AND A. RADOMINSKA-PYREK, FEBS Lett., 22 (1972) 190. Biochem. Biophys. Res. Commun., 47 (1972) 1203. 47 M. L. BLANK,R. L. WYKLEAND F. SNYDER, 48 L. A. HORROCKS AND G. B. ANSELL, Lipids, 2 (1967) 329.
MYELIN COMPOSITION AND METABOLISM
395
49 J. M. BOURRE,S. POLLET,0. DAUDU AND N. BAUMANN, C.R. Acad. Sci., Paris, Ser. D , 273 (1971) 1534. 50 J. GRIPPOAND J. H. MENKES, Pediatr. Res., 5 (1971) 466. J. Neurochem., 19 (1972) 737. 51 J. J. VOLPEAND Y . KISHIMOTO, 52 S. C. BOONEAND s. J. WAKIL,Biochemistry, 9 (1970) 1470. 53 F. YATSUAND s. Moss, J. Newrochem., 18 (1971) 1895. 54 E. AEBERHARD, J. GRIPPOAND J. H. MENKES, Pediatr. Res., 3 (1969) 590. AND M. PASCAUD, Biochimie, 53 (1971) 699. 55 C. STROUV~-VALLET 56 S. JOFFE,J. Neurochem., 16 (1969) 715. 57 A. ETZRODT AND H. DEBUCH, Hoppe Seyler's 2. Physiol. Chem., 351 (1970) 603. 58 M. E. SMITH,J. Newrochem., 16 (1969) 83. 59 W. E. STONE,J. K. TEWS,K. E. WHISLERAND D. J. BROWN,J. Neurochem., 19 (1972) 321. 60 G. A. DHOPESHWARKAR, C. SUBRAMANIAN AND J. F. MEAD, Biorhim. Biophys. Acta, 248 (1971)41. 61 G . A. DHOPESHWARKAR AND J. F. MEAD,Biorhim. Biophys. Acta, 187 (1969) 461. 62 G. A. DHOPESHWARKAR A N D J. F. MEAD,Biochim. Biophys. Acta, 210 (1970) 250. AND J. F. MEAD,Biochim. Biophys. Arta, 231 (1971) 8. 63 C . A. DHOPESHWARKAR C. SUBRAMANIAN, D. H. MCCONNELL AND J. F. MEAD,Biochim. 64 G . A. DHOPESHWARKAR, Biophys. A d a , 255 (1972) 572. AND G. GERCKEN, Eur. J. Physiol., 312 (1969) R124. 65 G. WINKELMANN L. M. STEPHANIDES AND H. NORGELLO, Bruin Res., 28 (1971) 327. 66 J . BERNSOHN, 67 A. F. D'ADAMO,JR., in A. LAJTHA(ed.), Handbook of Newrochemistry, Vol. 111, Plenum Press, New York, N.Y., 1970, pp. 525-546. 68 C . R. WEBSTER, Biochem. J., 102 (1967) 373. 69 J. H. MENKES, J. Neurochem., 18 (1971) 1433. Lz)ids, 7 (1972) 135. 70 J. H. MENKES, 71 G. V. MARINETTI, A. WEINDL AND J. KELLY,J. Neurochem., 18 (1971) 2003. 72 G. Y. SUNAND L. A. HORROCKS, J. Neurochem., 16 (1969) 181. J. Neurochem., 18 (1971) 1963. 73 G. Y. SUNAND L. A. HORROCKS, 74 G. Y. SUN,Neurobiology, 1 (1971) 232. 75 E. K. MILLERAND R. M. C. DAWSON, Biochem. J., 126 (1972) 823. 76 N. HERSCHKOWITZ, G. M. MCKHANN, S. SAXENA AND E. M. SHOOTER, J. Neurochem., 15 (1968) 1181. AND D. J. PROCKOP, J. Neurochem., 19 (1972) 283. 77 D. E. PLEASURE 78 G. B. ANSELLAND S. SPANNER, J. Neurorhem., 15 (1968) 1371. 79 L. A. HORROCKS, J. Neurochem., 16 (1969) 13. 80 G. Y. SUNAND L. A. HORROCKS, J. Lipid Res., 14 (1973) 206. 81 A. A. ABDEL-LATIF AND J. P. SMITH,Biorhim. Biophys. Acta, 218 (1970) 134. 82 E. G. LAPETINA, G. RODRIGUEZ DE LORES ARNAIZAND E. DE ROBERTIS, Biochim. Biophys. Acta, 176 (1969) 643. W. E. DAVIES AND N. S. RADIN,J. Lipid Res., 6 (1965) 525. 83 Y. KISHIMOTO, 84 M. E. SMITHAND L. F. ENG, J. Am. Oil Chem. Soc., 42 (1965) 1013. 85 N. L. BANIKAND A. N. DAVISON, Biochem. J., 122 (1971) 751. 86 N. L. BANIF. AND A. N. DAVISON, Biochem. J., 122 (1971) 26 P. 87 J. G . WOODAND N. KING, Nature, 229 (1971) 56. 88 M. E. SMITH,Biochim. Biophys. Actu, 164 (1968) 285. AND P. H. GOLD,J. Biol. Chem., 246 (1971) 2425. 89 R. A. MENZIES 90 L. A. HORROCKS AND G. Y. SUN,in R. RODNIGHT AND N. MARKS (eds.), Research Methods in Newrochemistry,Plenum Press, New York, N.Y., 1972, pp. 223-231. 91 B. GERSTL,L. C. RUBINSTEIN, L. F. ENGAND M. G. TAVASTSTJERNA, Arch. Neuvol., 15 (1966) 603. Abstr. Int. SOC.Neurochem., 92 L. F. ENG, B. GERSTL,D. V. PRATTAND M. G. TAVASTSTJERNA, Strusbourg, (1 967) 62.
Morphological studies’ have established that the myelin sheath in the central nervous system is continuous with the plasma membrane of the oligodendroglial cells and myelin constitutes the major portion of these cells. Myelin fractions can be isolated by a number of methods2 for subcellular fractionation of dispersions of CNS tissues from adult mammals. These myelin fractions have the morphological appearance of myelin, are nearly completely soluble in chloroform-methanol (2 : 1, v/v), form a band on sucrose density gradients at a density less than that of 0.8 M sucrose, and have a characteristic lipid3-’ and protein2 composition. Contamination of isolated myelin fractions by mitochondria and axoplasm is almost completely removed by
z
o Horrocks4 Sun and Somorajski” 0 Rawlins and Smith” Norton e l al.“
AGE, Days (Logarithmic Scale1
Fig. 1. The amount of myelin isolated from the brains of rats and mice as a function of age. For the mice, the amount of myelin is expressed as the amount of myelin lipid phosphorus in order to correct for differences in lipid composition during early development. The bars represent the S.E. The value for 730-day-old mice is based on myelin isolations from 21 individual mouse brains. 0 and 0, mouse; 0 and B, rat. Abbreviations: CDP, cytidine diphosphate; CNS, central nervous system; CPG, choline phosphoglycerides; EPG, ethanolamine phosphoglycerides; GPC, sn-glycero-3-phosphorylcholine;GPE, snglycero-3-phosphorylethanolamine; GPS, sn-glycero-3-phosphorylserine;IPG, inositol phosphoglycerides; SPG, serine phosphoglycerides; SRA, specific radioactivity. References p . 393-395
384
L. A. HORROCKS
TABLE 1 LIPID COMPOSITION OF SUBCELLULAR FRACTIONS FROM BRAINS OF
2-YEAR-OLD MICE
Values are expressed as the mean & S.E. For lipid-P, cholesterol, and galactolipids, n = 21; for myelin phospholipids, n = 9; for microsomal phospholipids, n = 7; and for mitochondria1 phospholipids, n = 5. The acid-stable EPG include diacyl-GPE, alkylacyl-GPE, and cardiolipin (mitochondrial fraction only).
~
Cholesterol Galactolipids Alk-1 -enylacyl-GPE Acid-stable EPG CPG Sphingom yelin SPG plus IPG
Mjielin ,fraction
1.33 0.03 0.65 0.02 0.331 3Z 0.006 0.146 i 0.007 0.251 0.007 0.053 0.004 0.215 0.012
**
Mole ratio, component: lipid-P - -Microsomal fraction 0.78 0.20 0.177 0.174 0.403 0.047 0.197
5 0.02 0.02 k 0.007 5 0.002 0.010 & 0.008 & 0.009
+
Mitochondria1 fraction 0.64 0.04 0.105 0.296 0.410 0.043 0.147
0.09
k 0.02
0.004 & 0.015 0.008 5 0.009 f 0.008
osmotic shock followed by reisolation of the myelin. Contamination by fragments of endoplasmic reticulum may be a problem. Miller and Dawson’ found a substantial amount of NADPH-cytochrome c reductase, apparently a microsomal enzyme, in crude isolated myelin, but Jungalwala and Dawson’ could not detect this enzyme in osmotically shocked and purified myelin. Further studies of microsomal enzymes in isolated myelin fractions during purification are required. Relatively few studies on isolated myelin have been extended to ages beyond maturity’, ‘O-’’ . In the mouse and rat, each study has shown that the amount of myelin in the brain continues to increase throughout the life span of the animals (Fig. 1). Although the rate of increasc: of the amount of myelin decreases with age, the net deposition of myelin does not stop. Thus “period of maximum rate of myelination” is a more precise term than “period of myelination”. The characteristic lipid composition of myzlin is illustrated in Table 1. To date, similar studies have not been made with brains from larger animals. In man, measurements have been made of the content of lipids such as cerebrosides and ethanolamine plasmalogens which are concentrated in the myelin sheath13. Since these lipids decrease in amount after the age of 30, the amount of myelin in the human brain may also decrease during aging. I n 1965 we reported that mouse brain myelin changes composition during development14. As a result of skepticism by several members of the audience, the experiment was replicated with myelin isolated by several different methods from the brains of mice4, rats’, l o , ‘’-I7 ,rabbits”, and man’’, 92. Regardlelessofthemodeofexpression of the lipid composition of the isolated myelin, all investigators agree that the relative proportions of galactolipids, cholesterol, and ethanolamine plasmalogens increase and the relative proportion of choline phosphoglycerides decreases during maturation (Figs. 2 and 3). The increases in the relative proportions of galactolipids and cholesterol are nearly equal on a molar basis (Fig. 2). Cuzner and DavisonI6
MYELIN COMPOSITION AND METABOLISM
8 P
385
80-
H
::
2
60
.
40
-
-
E
0
7
In
-
0
- loo\P
d
P
E
v)
2o
m,
W 4
.
GALACTOLIPIOS
0,O
~
0 CHOLESTEROL
802
5
-
p
i
i
0
10
20
40
100
200
400
60
800
AGLdoyr (logarithmic scale)
Fig. 2. The amounts of galactolipids and cholesterol relative to phospholipids in myelin as a function of age. 0 and 0, mouse4; 0 and W, rat2. 40
r
a 1 10
0 , O CHOLINE
20
PHOSPHOGLYCERIOES
40
100
200
AGE, doys ( logarithmic scole)
400
800
.,
Fig. 3. The proportions of ethanolamine plasmalogens and choline phosphoglycerides in myelin mouse4; 0 and rat2. phospholipids as a function of age. 0 and 0,
described an even more pronounced increase in these lipids that was probably due to the inclusion of the myelin-like fraction in the early myelin2. The attainment of the myelin composition characteristic of the mature animal is much more rapid in the mouse than in the rat. The cholesterol and galactolipid content of rat brain increases substantially until at least 275 days of age2’. The molar ratio of the net increase of cholesterol to galactolipid is about 2 which is the ratio found in myelin isolated from adult rats. The proportion of ethanolamine plasmalogens also increases during maturation (Fig. 3). In myelin from the brains of adult mammals, the ethanolamine plasmalogens account for 31 to 37% of the phospholipid?’. The proportion of ethanolamine plasmalogens in human myelin increases slightly from maturity to old age2’. The adult level is reached between 14 and 24 days of age in the mouse but not until after 30 days of age in the rat. In contrast, the proportion of choline phosphoglycerides declines during maturation at the same rate in the myelin from the mouse and rat. References p . 393-395
386
L. A. HORROCKS
TABLE 2 ALKENYL AND ACYL GROUP COMPOSITIONS OF ETHANOLAMINE PLASMALOGENS FROM ISOLATED HUMAN MYELIN (n = 5)
Weight
Alkenyl groups
16:O 17:O 18:O 18:l
Acyl groups
16:O 18:O 18:l 20: 1 20:2 20:3 (n - 6) 20:4 (n - 6) 22:3 22:4 (n - 6) 2 2 ~ 6(n - 3) 2 4 ~ 4(n - 6)
%
Age (years): 36-50
57-77
25.6 1.1 16.4 56.8
26.9 1 .o 15.6 56.5
3.2 3.2 45.1 8.1 1.8 0.9 9.7 1.8 20.2 2.1 1.9
3.8 3.7 46.9 10.3 1.6 0.9 7.3 1.3 18.9 2.0 1.2
The lipid composition of myelin is quite constant during aging with the possible exception of galactolipids (Fig. 2). The major phosphoglycerides of myelin are ethanolamine plasmalogens, phosphatidylcholine and phosphatidylserine. The alkenyl groups of the ethanolamine plasmalogens from human myelin are about 57 % 18 :1 , 26 % 16 :0 and 16 % 18 :0 with no apparent change in composition during aging. The content of monounsaturated alkenyl groups in ethanolamine plasmalogens is much greater in myelin than in gray matter. The acyl groups from ethanolamine plasmalogens are almost exclusively unsaturated as expected for acyl groups from the 2-position of phosphoglycerides. When compared to gray matter ethanolamine plasmalogens, the acyl groups from myelin ethanolamine plasmalogens have relatively large proportions of 18 :1 , 20 : 1 and 22 :4, about the same proportion of 20 :4, but a much lower proportion of 22 :6 acyl groups. With increasing age, the proportions of 18 :1 and 20 : 1 acyl groups increased and 20 :4 and 22 :4 acyl groups decreased significantly” (Table 2). Similar changes have been observed in the total EPG from whole brainz3. The phosphatidylserine from human myelin contains nearly equal proportions of 18 :O and 18 : 1 acyl groups with very small proportions of 16 : O and polyunsaturated acyl groups (Table 3). With increasing age, there is a sniall increase in 18:O and a small decrease in 18 : 1 acyl groups. In gray matter, polyunsaturated acyl groups account for most of the unsaturated acyl groups. The phosphatidylcholine from human brain contains small proportions of polyunsaturated acyl groups, even in gray matter. In myelin, the phosphatidylcholine has lower proportions of 16 :O and poly-
387
MYELIN COMPOSITION AND METABOLISM
TABLE 3 ACYL GROUP COMPOSITIONS OF PHOSPHATIDYLCHOLINEAND PHOSPHATIDYLSERINE FROM ISOLATED HUMAN MYELIN
(n = 5)
Weight Phosphatidyleholine 57-77 Age (years): 36-50 Aeyl group 16:O 16:l 18:O 18:l 20:l 20:4 (n - 6) 22~4 (n - 6)
28.9 2.4 13.6 47.7 1.3 2.6 0.9
% Phosphatidylserine 36-50 57-77
30.8 2.0 12.4 49.5 1.6 2.0 0.4
2.3
1.3
42.6 48.9 3.5 0.5 0.6
44.2 46.2 4.4 1.o 1.1
TABLE 4 ACYL GROUP COMPOSITIONS OF CEREBROSIDES FROM ISOLATED HUMAN MYELIN
(n
=
4)
Methyl ester derivatives of unsubstituted acyl groups and methyl ester and trimethyl silyl ether derivatives of 2-hydroxyacyl groups were subjected to gas-liquid chromatography. A hydrogen-flame detector was used. Area Age (years): Acyl group 16:O 18:O 18:l 20:o 22:o 22:l 23 :O 23:l 24:O 24:l 25 :O 25 : 1 26:O 26:l
Ratio 24:1/24:0
Unsubstituted acyl groups 36-50 57-77
0.8 7.7 0.8 0.5 3.0 0.4 5.4 0.6 19.8 40.7 7.1 8.5 0.6 4.8
1.6 10.8 1.4 0.6 2.4 0.4 3.8 0.3 13.4 49.7 4.2 7.6 0.7 4.3
2.1
3.7
% 2-Hydroxy aeyl groups 36-50 57-77
0.9
1.3
0.2 4.6
0.1 6.0
15.4
19.3
37.4 27.1 9.6 2.2 0.7 2.3
31.0 29.3 6.9 2.7
0.72
0.4
2.6
0.95
unsaturated acyl groups with a much higher proportion of 18 :I acyl groups. These differences are further accentuated during aging (Table 3). Thus, the non-polar groups from human myelin phosphoglycerides differ from the non-polar groups of neuronal phosphoglycerides by having higher proportions of monounsaturated (18 :1 and 20 :1) acyl groups and lower proportions of saturated (16 :0 and 18 :0) and n-3 polyReferences p . 393-395
388
L. A. HORROCKS
unsaturated (22 :6) acyl groups and these differences tend to become larger during aging2’. Cerebrosides, which are also major components of myelin, are characterized by very long-chain acyl groups such as unsubstituted and 2-hydroxy 24 : 0 and 24 : 1 acyl groups. Both types of 24:O acyl groups were found in decreased proportions in cerebrosides from human myelin from older brains (Table 4). Increased proportions of unsubstituted 24 : 1 and 2-hydroxy 23 :0 were found instead. Relatively large changes in acyl group compositions of myelin lipids might be expected during maturation because lipid synthesis is most active and the lipid class composition of the myelin changes most at that time. No results are available. In addition, the lipid class and acyl group compositions of the presumed myelin precursors, the myelin-like fraction and the oligodendroglial plasma membrane, should be determined during maturation. “Early myelin” (myelin isolated from very young animals) has a composition that is similar to that of the myelin from quaking mice (a mutant with arrested myelination). Myelin from the quaking mouse, when compared to myelin from littermate controls, has more CPG but is deficient in cerebrosides, ethanolamine plasmalogens, and the apolar p r ~ t e i n ’ ~ - ~ In ’ . addition, compared to controls quaking mouse brain myelin had much lower proportions of monounsaturated alkenyl and acyl groups in the EPG and 24 :1 hydroxy and unsubstituted acyl groups in the c e r e b r ~ s i d e sThese ~~. results suggest that the apolar protein has relatively more binding sites for cerebrosides and ethanolamine plasmalogens and relatively fewer binding sites for CPG than the other myelin proteins. The “early myelin” may represent a transition form between the oligodendroglial plasma membrane and mature myelin’*. Davison’s group has studied the purification of crude “early myelin”. Banik and DavisonZ9 reported that a myelin-like fraction could be separated from compact myelin from young rat brains. Further studies by Agrawal et have shown that the myelin-like fraction resembles myelin in enzyme activity but the myelin-like fraction does not contain the basic protein or cerebrosides. They suggested that the myelin-like fraction represents a transition between oligodendroglial plasma membrane and compact myelin and that the differences observed in early myelin are due to a larger proportion of the myelin-like fraction in a mixture of compact mydin and myelin-like fraction. On the basis of 2’,3’-cyclic nucleotide phosphohydrolase activities, Zanetta et aL3’ suggested that myelin-like and nerveending fractions may both contain fragments of oligodendroglial plasma membrane. Th- identity of the myelin-like fraction with the oligodendroglial plasma membrane is supported by the report that a myelin-like fraction from quaking mice has neither the basic protein nor the apolar protein but only the high molecular weight myelin proteins’’. Similar results were reported by Savolainen and pal^^^ for myelin-like fractions, fetal myelin, and glial plasma membranes from human brains. Unfortunately, the glia were probably astrocytes and lipids and enzymes were not assayed. Phosphoglyceride biosynthesis is an essential part of myelin deposition. In the brain, the diacyl-GPC and diacyl-GPE are formed from CDP-choline or CDPethanolamine and 1,2-diacylgly~erols~~~ 34. The diacyl-GPE and diacyl-GPS may
MYELIN COMPOSITION A N D METABOLISM
389
undergo exchange reactions with free ethanolamine or serine3', 3 6 . The alkyl groups of brain phosphoglycerides are formed from fatty alcohols37- 4 3 . Phosphoglycerides with alkyl groups are involved in the biosynthesis of ethanolamine plasmalogens as follows: (a) l-Alkyl-2-acyl-sn-glycero-3-phosphates -+ 1-alkyl-2-acyl-sn-glycerols + Pi (b) 1-Alkyl-2-acyl-sn-glycerols CDP-ethanolamine .+ l-alkyl-2-acyl-sn-glycero-3phosphorylethanolamines CMP -+ 1-alkenyl-2-acyl-sn-gly(c) l-Alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines cero-3-phosphorylethanolamines+ 2[H]
+
+
Reaction (a) was demonstrated by Snyder et ~ 1 . Ethanolamine ~ ~ . phosphotransferase (EC 2.7.8.1) catalyzes Reaction (b)45. Direct evidence for Reaction (c) in cell-free systems from brain tissue has been provided by Horrocks and Rad~minska-Pyrek~' Since the products of these three reactions were all quite radioand by Blank et active at short times after intracerebral injections of ['4C]hexadecano141, this pathway is operative in vivo. Indirect evidence for this pathway had been reported previously4'. The activity of the fatty acid Fatty acid synthetase is present in the synthetase gradually decreases during development when the activity is expressed in terms of tissue weight5'. Fatty acids can be elongated in the mitochondrias2*53, and microsomess4. Fatty acid desaturase activity is present in brain microsomes from very young rats but is virtually absent by 11 days of age55. The utilization of these pathways has been established by the uptake of radioactivity from labeled acetate or glucose into alkenyl and acyl groups'" 'O, 56-60. Brain fatty acids may also be taken up from the free fatty acids in the circulating blood. Dhopeshwarkar and Mead6' - 6 3 and Dhopeshwarkar et have established that small amounts of 16:0, 18:l and 19:2 fatty acids administered orally or intraarterially to rats can be recovered in brain tissue. In this laboratory, we have found that the same fatty acids are taken up in significant amounts by monkey brain after the infusion of ['4C]fatty acid-albumin complexes into the internal carotid artery. The perfused rabbit brain takes up ['4C]palmitate from the perfusate into the reported that linoleic acid was taken up by phosphog1ycerides6'. Bernsohn et brain stem and spinal cord from the cerebral spinal fluid (CSF). These experiments demonstrate that exogenous fatty acids are taken up by the brain (see review by D ' A d a m ~ and ~ ~ )used in part of the synthesis of phospholipids. Labeled fatty acids are incorporated into intracellular phospholipids by slices6' and ex plant^^^. 7 0 of rat cerebrum. The choroid plexus incorporates palmitic acid from the ventricles into lipids7'. Intraczrebral injections of [14C]fatty acids have been used to determine the fate of free fatty acids in brain tissue. In mice, about one-fourth of the injected radioactivity from [14C]palmitic acid was recovered in the brain lipids7', 7 3 . At times of more than 12 hours after injection, about 85 % of the recovered radioactivity was found in the phospholipid^^^ with relative specific radioactivities of the phosphoglycerides that were nearly identical to those reported by Winkelmann References P . 393-395
390
L. A. HORROCKS
and G e r ~ k e n The ~ ~ . incorporation into phosphoglycerides increases during the first hour after injection73. An intermediate in phosphoglyceride biosynthesis, the 1,2diacylglycerols, reached its highest SRA between 6 and 10 minutes after injection. The flux of radioactivity through the diacylglycerol pool was sufficient to account for all of the choline and ethanolamine phosphoglyceride turnover with estimated half-lives of 9 minutes for the diacylglycerols and 7 days for the phosphoglycerides. Sun74has found that the half-life of the diacylglycerols in mouse brains was the same at 3, 10 and 28 months of age, but the uptake of palmitic acid into the diacylglycerols decreased markedly with age. The synthesis of myelin lipids takes place in the oligodendroglial endoplasmic reticulum8. The myelin lipids can then be transported to newly synthesized myelin or exchanged with existing myelin lipids75. The exchange process is presumably mediated by a cytosol protein as shown for cerebroside sulfates by Herschkowitz et and Pleasure and P r ~ c k o pand ~ ~for mitochondria1 phosphoglycerides by Miller and Dawson7’. In several studies, with different radioactive lipid precurs0rs9, 78-80 , about 7 days are required before the SRA of myelin phosphoglycerides are equal to the SRA of microsomal phosphoglycerides. In one of these studies”, the SRA of phosphoglycerides from the crude myelin fraction was much higher than the SRA of the purified myelin phosphoglycerides during the first few days after injection. The material with a high SRA that was lost during purification may include the myelin-like fraction that was described earlier in this section. The metabolism and characterization of this “nascent” myelin deserve further study. The exchange of lipids between mature myelin and the cytosol is difficult to visualize, but we must remember that a portion of each lamella is in contact with oligodendroglial cytosol at the nodes of Ranvier. Since membrane lipids are fluid and can diffuse laterally, all of the myelin lipids can equilibrate with cytosol transport lipoproteins. Irregardless of the extent of contact of cytosol with myelin, the rate of this equilibration and subsequent metabolic processes will determine the rate of myelin turnover. The turnover of brain lipids is usually measured from graphs of the SRA against time. In addition to problems with the reproducibility of injections and the heterogeneity of pools. the interpretation of turnover studies is also dependent on the degree of reutilization of catabolic products. This is illustrated by the results of Abdel-Latif and Smith” for the metabolism of rat brain CPG. The half-lives of the CPG were 2.9 days with [‘4C]glycerol as the precursor, 11.7 with [‘4C]choline, and 18.6 days with [32P]phosphate. Lapetina et reported half-lives of 5.1 days with [‘“Clglycerol, 9.2 days with [3’P]phosphate, and 38 days with [14C]acetate for the total lipids from rat cerebral cortex. These differences in the half-lives reflect the differences in reutilization of the products from phosphoglyceride catabolism. An extensive reutilization of palmitic acid was found in the mouse brain”. At 30 days after injection, only 22% of the radioactivity in the phosphoglyceride acyl groups was in the 16:0 acyl groups. Kishimoto et ~ 1found . that ~ ~16 :O acyl groups from phosphoglycerides were the source of the palmitic acid used for elongation. These findings help to explain the half-life of 38 days for rat brain lipids after [I4C]acetate injections8’ and the half-life of 7 months for the EPG from rat brain myelin after intraperitoneally
39 1
MYELIN COMPOSITION AND METABOLISM
injected ['4C]acetate*4. In neither case was the SRA of the 16 :0 acyl groups measured. In the latter case, the very low content of 16:O acyl groups in the myelin EPG was not considered. It is necessary to distinguish between the apparent turnover which includes reutilization and the true turnover. Another form of turnover is the exchange of intact lipids which are not catabolized as shown for cholesterol by Banik and Davison", 86.The exchange phenomenon that was described previously is supported TABLE 5 SPECIFIC RADIOACTIVITIES OF THE
EPG FROM SUBCELLULAR
FRACTIONS OF MOUSE BRAINS
C57 BL/lO mice at 150 days of age were given intracerebral injections of [14C]ethanolamine as described by H o r r o ~ k s Lipids ~ ~ . were isolated according to Horrocks and Sungo.
2
SRA (dpm/pmole) 4
6
Diacyl-GPE Myelin Microsomes Mitochondria
2170 3830 1590
1320 1880 1280
530 1070 350
Alk-I-enylacyl-GPE Myelin Microsomes Mitochondria
1550 2250 3600
950 1440 2620
630 630 1110
Alkylacyl-GPE Myelin Microsomes Mitochondria
1780 4480 5160
980 1240 2720
310 240 1180
Days after injection:
TABLE 6 SPECIFIC RADIOACTIVITIES OF THE
EPG IN
BRAIN SUBCELLULAR FRACTIONS FROM 2-YEAR-OLD MICE
The specific radioactivity of the EPG were measured after intracerebral injections of [14C]ethanolamine as described by H o r r o c k ~ ~ ~ . Days after injection:
SRA (dpm/pmole) 7
_________.--
4
I4
.
Acid-stable EPG Myelin Microsomes Mitochondria
2140 3520 1730
1650 2080 1490
1090 1280 720
Alk-1 -enylacyl-GPE Myelin Microsomes Mitochondria
1110 2100 3100
930 1650 1600
780 1070 1060
References P . 393-395
392
L. A. HORROCKS TABLE 7
THE HALF-LIVES OF
EPG
FROM SUBCELLULAR FRACTIONS OF BRAINS OF MICE DURING DEVELOPMENT AND AGING
-
-~
~~
~
t ~ (days) p
Age at injection (days):
40
150
Mitochondria Diacyl-GPE Alk-1 -enylacyl-GPE Alkylacyl-GPE
-
-
1.9 2.4 2.3
1.9 6.3 -
Microsomes Diacyl-GPE Alk-1-enylacyl-GPE Alkylacyl-GPE
1.3 1.3 -
1.9 2.4 1.3
6.9 10.3 -
Myelin Diacyl-GPE Alk-1 -enylacyl-GPE Alkylacyl-GPE
1.7 1.6 -
3.0 3.2 2.0
10.3 20.3 -
730
by the finding that the lipids from different brain membranes, including myelin, have The turnover rates for specific lipids are probably similar turnover rates', determined by the magnitude of the forces binding them to membranes and by the activity of catabolic enzymes. Since membrane proteins have a multitude of binding sites, their turnover rate should be much slower than membrane lipids. In fact, halflives of 21 days for the myelin basic proteins7, and 35 days for the myelin apolar proteins" have been reported. These values agree with those reported for brain mitochondrial proteins''. In the latter report, no difference was found in the turnover rate of mitochondrial proteins from 1-year-old and 2-year-old rats. Intracerebral injections of ['4C]ethanolamine label the brain EPG quite rapidly7' with very little recycling of the radioactivity. Experiments of this type have been done with maturing mice7', adult mice (Table 5), and aged mice (Table 6). The halflives of the myelin EPG in mice (Table 7) are 1-2 days in maturing mice, 2-3 days in adult mice, 6-20 days in aged mice and 5-15 days in rats33, 7 8 . Similar values have been obtained with palmitic acid as the precursor". Jungalwala and Dawson' repeated the ethanolamine experiment of Ansell and Spanner7' with nearly identical results. Slices of rat brains and spinal cords were incubated with labeled acetate and leucine. The amounts of incorporation into myelin lipids and proteins decreased rapidly with age up to 6 months, but the incorporation at 6 months, 12 months and 18 months were about the same". These results indicate that the rate of myelin biosynthesis probably slows down markedly during maturation, then plateaus during adulthood. As shown in Table 7, the turnover rate for the myelin EPG is much lower in 2-year-old mice. Because the amount of myelinin the mouse brain increases throughout the first 2 years of life (Fig. 1) the rate of myelin biosynthesis must always exceed the rate of myelin catabolism. "3
MYELIN COMPOSITION AND METABOLISM
393
SUMMARY
In the CNS, myelin is an extension of the plasma membrane of the oligodendroglial cell. Compact myelin from adult brains can be isolated easily and has a characteristic composition. During early maturation, myelin isolation techniques yield “early myelin” which has a different composition, perhaps because it represents a transitional form between the oligodendroglial plasma membrane and compact myelin. In addition, a “myelin-like” fraction can be isolated. The latter fraction may be fragments of oligodendroglial plasma membrane or a transitional form. During aging, the amounts of myelin in mouse and rat brains increase until at least 2 years of age. The lipid composition changes very little during aging, but some differences are seen in the fatty acid composition of myelin phosphoglycerides and cerebrosides from human brains. Myelin lipids are synthesized by the endoplasmic reticulum. A cytosol lipoprotein is presumably involved in the equilibration process which may require a week or more due to the time required for lateral diffusion of myelin lipids to a portion of the myelin that is in contact with the cytosol. Nevertheless, the equilibration process is effective because myelin lipids turnover at rates that are comparable with the turnover rates for microsomal lipids. In rats, the biosynthetic rates for myelin lipids and proteins decrease markedly during maturation. In mice, the turnover of ethanolamine phosphoglycerides slows down with increasing age. The half-lives were about 50 % higher for 5-month-old mice (1.3-3.2 days) than for 40-day-old mice (1.3-1.7 days) and were much higher for 2-year-old mice (6.3-20.3 days).
ACKNOWLEDGMENTS
I am indebted to Dr. Grace Y. Sun, Cleveland Psychiatric Institute, for discussions, the National Institutes of Health, U.S. Public Health Service, for support through research grants NS-05510, NS-08291, and GRS-5409, and the National Multiple Sclerosis Society for support from the Benjamin Miller Memorial Grant for Research on Multiple Sclerosis.
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