Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all of the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55 % and proteolipid protein for 46 % of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F. and Prensky, A. L. (1972) J. Neurochem. 19, 2083–2089) exhibited a developmental “switch” between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

Elevated specific activity of 5′-nucleotidase in a spinal cord myelin fraction from shiverer mice comparison with other myelin-associated enzymes and myelin proteins

Meylin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5′-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2′,3′-cyclic nucleotide-3′-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiveres, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hhypothetically, the 5′-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.     

Hypotheses regarding myelination derived from comparisons of myelin subfractions.

Patterns of myelin-associated proteins and glycoproteins in subfractions of central nervous system myelin and changes in these during development have been reviewed. Several hypotheses are put forward regarding classification of these proteins as structural components of myelin, as components of the membranes from which myelin is derived or as molecules present to play a role in myelin formation rather than as true components of compact myelin. 2′, 3′-Cyclic nucleotide 3′-phosphohydrolase is considered to fit into this last category and two different hypothesis are proposed for its role. The major myelin glycoprotein is postulated to serve a function in recognition of axons by oligodendrocytes, a function not needed in the peripheral nervous system where Schwann cells and axons develop in close contact.     

Protein and Glycoprotein Composition of Myelin Subfractions from the Developing Rat Optic Nerve and Tract

Abstract: The rat optic nerve and tract (representing a relatively homogeneous part of the CNS) were utilised for a detailed examination of the protein and glycoprotein composition of developing myelin membranes. Animals aged from 5 days through to adulthood were used. Myelin fractions could first be isolated from the nerve 8 days after birth and the yield increased until 60 days of age, before declining slightly to the adult level; a similar (but possibly slightly delayed) pattern was apparent for the optic tract. The homogeneity of optic nerve myelin (compared with that from brain and spinal cord) was demonstrated by zonal centrifugation on continuous sucrose-density gradients; myelin from both 20-day and adult animals exhibited narrow, Gaussian-like distributions, with 19–22% of the total myelin at the population modes. During development, the myelin density profile was shifted to a denser region of the sucrose gradients. Micro-polyacrylamide gel electrophoretic analyses of "light" and "heavy" myelin subfractions from both optic nerve and tract indicated that the gross developmental changes in protein composition were similar to those previously described for myelin prepared from larger CNS areas, particularly the forebrain. The glycoprotein components of the myelin fractions were stained directly on micro-gels using fluorescein isothiocyanate-labelled concanavalin A. The relative proportion of the major high-molecular-weight glycoprotein decreased rapidly during the early phases of myelination. A number of lower-molecular-weight glycoproteins were also apparent; the proportions of these varied during development and in light and heavy myelin subfractions, but definitive data are not available to determine whether they are components of the myelin sheath or of contaminating membranes.     

Metabolic studies on myelin. Evidence for a precursor role of a myelin subfraction

Myelin prepared from brain tissue of the developing rat (15 days post partum) can be separated into several subfractions. These are (1) ;myelin-like' and ;purified myelin', by the technique of Davison and co-workers (Agrawal et al., 1970b) and (2) ;membrane fraction,' ;light myelin' and ;heavy myelin' by the discontinuous-sucrose-gradient procedure described in the present paper. ;Myelin-like' and ;membrane-fraction' subfractions appear to be similar in chemical properties, but different in detailed morphology by electron microscopy. Both fractions are related to myelin, on the basis of demonstrable myelin basic protein by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate and the presence of a myelin-marker enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase. These two fractions have a low lipid content (17% for ;myelin-like' and 40% for ;membrane-fraction' subfractions) compared with myelin (67-72%). No cerebroside was detected in these two fractions, whereas cerebrosides are a major component of myelin itself. The administration of [2,3-(3)H]tryptophan to young rats results in more rapid incorporation into proteins of the ;myelin-like' and ;membrane-fraction' subfractions when compared with incorporation into myelin. Data are presented which are consistent with a precursor-product relationship for conversion of ;myelin-like' and ;membrane-fraction' subfractions into myelin.     

Localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes

The location of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes was determined. This was accomplished by comparing the enzyme's accessibility with that of glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic surface marker) and acetylcholinesterase (external marker) in sealed and unsealed ghosts and normal and inverted membrane vesicles. The results showed that 2′,3′-cyclic nucleotide 3′-phosphodiesterase, like glyceraldehyde-3-phosphate dehydrogenase, meets several criteria for an inner (cytoplasmic) membrane location: (1) the enzyme was accessible to substrate in unsealed ghosts and inside-out vesicles but not in sealed or right-side-out vesicles, (2) latent activity in sealed ghosts could be exposed with detergent (Triton X-100), (3) activity in unsealed ghosts was gradually sequestered during resealing and could be re-exposed with detergent, and (4) the enzyme was susceptible to trypsin proteolysis only in unsealed ghosts. These results demonstrate that the active site of 2′,3′-cyclic nucleotide 3′-phosphodiesterase faces the cytoplasm of erythrocytes and that the enzyme may not span the lipid bilayer of the membrane. The localization of the phosphodiesterase on the inner membrane surface of erythrocytes suggests that the similar enzyme of myelin may be embedded within the major dense line of the compact lamellae.     

PROTEINS AND GLYCOPROTEINS IN MYELIN PURIFIED FROM THE DEVELOPING BOVINE AND HUMAN CENTRAL NERVOUS SYSTEMS

Myclin was purified from bovine and human midbrain at various stages of prenatal and postnatal development. Basic protein and proteolipid proteins were the major individual proteins at all stages. The specific activity of 2′3′-cyclic nucleotide 3′-phosphohydrolase remained constant in the bovine myclin during development but decreased slightly in human myelin. A high molecular weight glycoprotein with electrophoretic mobility similar to that previously reported in rodent myelin (QUARLES et al., 1973) was present in both bovine and human myelin at all stages of development. The intensity of staining of this glycoprotein with periodic acid-Schiff reagents per mg total myelin protein was less in mature bovine and human myelin than in rat myelin.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain.

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2',3'-cyclic nucleotide-3'-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55% and proteolipid protein for 46% of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H.C., Burton, R. M., Fishman, M.A., Mitchell, R.F. and Prensky, A.L. (1972) J. Neurochem. 19, 2083-2089) exhibited a developmental "switch" between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development.

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.     

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.     

Developmental profiles of glial enzymes in the chick embryo: In vivo and in culture

The activities of two glial cell enzymes, glutamine synthetase (a marker for astrocytes) and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (a marker for oligodendrocytes and myelination) were studied in the developing chick embryo brain in vivo and in cultures derived from chick embryos. The in vivo findings showed that the activities of both enzymes parallel the patterns of gliogenesis and myelination. Glutamine synthetase follows similar patterns in culture and in vivo, whereas the developmental profile of 2′,3′-cyclic nucleotide 3′-phosphohydrolase appears to be affected by the culture conditions.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P₀ protein and a basic protein of slightly larger mol wt. than the P₁ protein of rat myelin.     

Myelin changes induced by incubation of brain slices with serum

Forebrain and brain stem slices prepared from adult rats were incubated with pooled normal human serum. Following the incubation, the tissue was homogenized and the fraction floating on 0.32 M sucrose as well as two myelin subfractions (light and heavy) were isolated. Addition of serum into the incubation medium increased generation of the floating fraction by the cerebral slices. Changes in the myelin membrane were also observed. Thus, myelin isolated from forebrain slices revealed pronounced increase in the buoyant density of its particles and loss of basic protein. Furthermore, in spite of the intensive washing employed during the isolation procedure, some serum proteins were found firmly attached to the membraneous fractions. The demonstration of the myelin alterations in the living cerebral tissue exposed to serum during incubation may contribute to understanding the pathogenesis of multiple sclerosis.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Biochemical Changes in Central Nervous System Membranes in Experimental Allergic Encephalomyelitis

Biochemical and morphological studies of myelin subfractions were undertaken on Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE). Myelin subfractions, obtained by sucrose density gradient centrifugation at 10 days post-induction, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed for 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Aliquots were processed for electron microscopic analysis. When comparing the myelin subfractions of EAE-affected animals with those of controls, differences were observed only in the light fractions, i.e., a decrease in the specific activity of CNPase and in the percentage of basic proteins relative to the total proteins of the fraction. This decrease was also evident in the basic protein/proteolipid protein ratio which is frequently used in the literature. In addition, electron microscopic observations demonstrated strong differences in the morphology of the same fraction. These findings suggest that the light fraction is the most sensitive in the early stages of the disease and must play a key role in demyelinating processes.     

Rapid Incorporation In Vivo of Intracerebrally Injected 32Pi into Polyphosphoinositides of Three Subfractions of Rat Brain Myelin

Abstract: At intervals ranging from 1 to 10 min after injection of ³²Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphospho-inositides (PPI) was then determined. There was rapid incorporation of ³²Pi into PPI, which contained 50–70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated ³²Pi into total recovered PPI in the order of medium > heavy > light fraction: however, the order of relative specific radioactivities was heavy > light > medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phos-phatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di- (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged ³²Pi at rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.     

THE BIOSYNTHESIS AND CONCENTRATION OF GALACTOSYL DIGLYCERIDE IN GLIAL AND NEURONAL ENRICHED FRACTIONS OF ACTIVELY MYELINATING RAT BRAIN

—In continuation of our studies on the association of the galactosyl diglycerides of brain with myelination, we have measured the biosynthesis and concentration of these glyceride glycolipids, in oligodendroglial, astroglial, neuronal, and myelin enriched fractions from brains of rats of postnatal age 16, 19 and 29 days. The relative purity of cell fractions and myelin derived from 50 to 60 brains of each age-group was checked by phase contrast microscopy and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity. The relative purity was comparable to that reported by other investigators for cell fractions from bovine brain. Of the three cell types, the oligodendroglia had the highest and the neurons had the lowest capacity to enzymatically synthesize and to accumulate monogalactosyl diglyceride. The amount of monogalactosyl diglyceride found in myelin compared to that found in oligodendroglial fraction greatly increased during development between 16 and 29 days of age. The biosynthesis of galactosyl ceramide but not glucosyl ceramide was highest in oligodendroglial enriched cell fraction. However, ceramide glucosyl-transferase activity, which was greatly affected by the method used for cellular separation, was highest in a microsomal fraction derived from grey matter. Our results support the contention that the oligodendroglial cells are the site of synthesis of myelin constituents of the central nervous system, and that there is a temporal relationship between this site of synthesis and the site of deposition (myelin).