COMPOSITION OF CEREBRAL LIPIDS IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

The composition of sphingolipids and phospholipids of mouse brain during myelination was determined in the Quaking mutant, which manifests a genetic disorder of myelin formation, and in littermate controls. The biochemical changes during myelination in the brains of the controls corresponded quantitatively with previous findings in a different strain of mice. The Quaking mutant exhibited concentrations of sphingolipids and phospholipids in brain which were comparable to those of controls in the early stage of myelination but the tissue content failed to increase with maturation. The greatest differences occurred in the cerebrosides which at 65 days of postnatal age were only 10 per cent of control levels. During development the pattern of cerebral levels of sphingomyelin, plasmalogen and total phospholipid in the mutants tended to resemble that of the cerebrosides. The defect in the Quaking mutant is compatible with a failure in maturation of myelin. These findings have been compared with those in the Jimpy mutant, a different genetic disorder of myelin in the mouse previously studied in a similar fashion. The Jimpy mutant is characterized by a quantitatively more pronounced deficiency of myelin lipids and a decline in cerebrosides during brain development.     

A STUDY OF LIPID COMPONENTS IN BRAIN OF THE 'JIMPY' MOUSE, A MUTANT WITH MYELIN DEFICIENCY

(1) Brain composition and developmental changes were investigated in a mutant (‘Jimpy’) mouse characterized by a severe myelin deficiency. (2) Significantly lower cholesterol, phospholipid and galactolipid values were observed, and the accumulation of these lipids during the myelination period was markedly reduced or absent. (3) The most remarkable feature of ‘Jimpy’ brain was a very small galactolipid content. In 29-day-old mutants the concentration of galactolipids was 0-18 μ moles/g wet wt., representing a 46-fold decrease when compared to values determined in normal mice. (4) There was no such striking change in the distribution of different phospholipids. However, lowered relative amounts of some phospholipids, e.g. ethanolamine plasmalogen, sphingomyelin and phosphatidylserine, were observed in ‘Jimpy’ brain. (5) Protein content was also lower in mutant brains and showed an absolute decrease after 23 days of life. (6) These data support the statement that the process of myelination is disturbed at an early stage, resulting in a deficiency of mature myelin sheaths and leading probably to the breakdown of primitive myelin structures.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

Jimpy Mice: Quantitation of Myelin-Associated Glycoprotein and Other Proteins

Myelin-associated glycoprotein (MAG), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity, myelin basic protein (BP), and proteolipid protein (PLP) were quantitated in the brains of 20-day-old Jimpy and control mice. The levels of MAG, CNPase, and BP in Jimpy brains were 5.3%, 9.7%, and 1.9% of those in control brains, respectively. Immunoblotting analysis did not reveal an increased apparent Mr for MAG in the Jimpy mouse, as has been observed in some other hypomyelinating murine mutants. PLP was reduced more than the other proteins, as it was not detected by an immunoblotting technique that was capable of detecting 0.5% of the control level.     

ANOMALIES OF MYELIN-ASSOCIATED GLYCOPROTEINS IN''QUAKING MICE

Abstract— Proteins and glycoproteins in a myelin fraction isolated from Quaking mutant mice were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and stained with Fast Green or with periodic acid-Schiff reagents. Double labelling experiments with [³H]fucose and [¹⁴C]fucose were also used to compare glycoproteins in myelin from the mutant mice with those from control mice. In the myelin fraction from the Quaking mice the basic proteins and proteolipid protein were decreased relative to the high molecular weight proteins. Some glycoproteins which are present in small amounts in myelin from normal mice were increased relative to the major glycoprotein in the myelin fraction of the Quaking mice. Furthermore, the major myelin-associated glycoprotein was shifted toward higher apparent molecular weight in comparison with controls of the same age or even with 9-day-old controls. The abnormal glycoproteins in the mutant myelin fraction could be a factor in the impairment of myelination.     

2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE IN BRAINS OF MUTANT MICE WITH DEFICIENT MYELINATION

—The brains of Jimpy and Quaking mice were compared with those of the corresponding normal controls during the course of development. The activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was found to be markedly reduced in the affected animals. The reduction in the Jimpy mice was greater than in the Quaking mice. The activity of CNP seems to be proportional to that of myelin in the mutant mice. A similar reduction was found in spinal cords of the mutant mice, but there was no difference in CNP activity between the sciatic nerves of the mutant mice and those of the corresponding normal controls.     

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.     

THE FATTY ACID COMPOSITION OF PHOSPHOLIPIDS AND GLYCOLIPIDS IN JIMPY MOUSE BRAIN

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin-layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas-liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono-unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long-chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non-hydroxy fatty acids was reduced to a greater extent than in sphingomyelin. The distribution of fatty acids in sphingolipids from the myelin and microsomal fractions was also investigated in both types of mice. Cerebrosides were characterized by a high content of long-chain fatty acids in myelin as well as in microsomes. Sulphatides and sphingomyelin, on the other hand, showed a higher content of medium-chain fatty acids in microsomes than in myelin. In the mutant brain, the amount of long-chain fatty acids was reduced in both subcellular fractions. The deviation from normal in the pattern of fatty acid distribution in Jimpy brain is discussed in relation to the current concepts of glycolipid biosynthesis.     

ENZYMIC SYNTHESIS OF PSYCHOSINE SULPHATE

—An enzyme which catalyses the synthesis of psychosine sulphate by the transfer of [³⁵S]sulphate from 3′-phosphoadenosine 5′-phosphosulphate to galactosyl-sphingosine has been demonstrated in the brain of the young mouse. The enzyme activity appears to be bound to a microsomal fraction which is spun down with the synaptosomes. The product of the incubation mixture has been characterized as psychosine sulphate by a variety of TLC separations and other chemical procedures. Several parameters (detergent, cations, substrate and 3′-phosphoadenosine 5′-phosphosulphate concentrations, pH and incubation time), affecting the 3′-phosphoadenosine 5′-phosphosulphate-psychosine sulphotrans-ferase activity, have also been investigated. In the normal mouse brain there is a maximum enzyme activity at 17–19 days after birth, which is the period of most rapid myelin formation. In the brains of Jimpy mice, mutants with myelin deficiency, the activity is reduced and reaches a maximum around the 13th day. The lower activity correlates with the small amounts of sulphatides in Jimpy mouse brains. The results are discussed and related to present knowledge of galactolipid biosynthesis.     

SUBCELLULAR DISTRIBUTION AND STRUCTURAL POLYMORPHISM OF MYELIN BASIC PROTEIN IN NORMAL AND JIMPY MOUSE BRAIN

Abstract— Brains from 20 day old normal and 20 day old Jimpy mice were fractionated by a modification of the procedure described by Eichberg et al. (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 P1A. 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 P1A and P2A were greatly 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 P1A and P2A fractions from normal brain contained myelin fragments while the P1A and P2A fractions from Jimpy brain contained numerous vesicular membranous structures with 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 RIA 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 P1A 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.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

Myelin-Associated Glycoprotein and Other Proteins in Trembler Mice

The myelin-associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20-day-old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/micrograms of total protein in the controls to 1.4 ng/micrograms protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20-day-old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age-matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt-Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous system (PNS) of the Trembler mice may contribute to the pathology in this mutant.     

CHANGES IN THE PROTEIN COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Abstract— Myelin was isolated from the brains of mice at various ages by a procedure involving a final purification on a continuous CsCl gradient. Myelin protein accumulated throughout development, increasing from 0.25 mg of protein/brain at 8 days of postnatal age to 3.5 mg of protein/brain at 300 days, although the rate of accumulation was greatest at about 21 days of age. Quantitative studies of the protein composition of these samples were carried out, utilizing discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Mouse brain myelin, contained (in order of increasing molecular weight) two basic proteins, an uncharacterized doublet, proteolipid protein, and a group of high molecular weight proteins. There were marked changes in the quantitative distribution of these proteins with increasing postnatal age. The basic protein fraction of total myelin protein increased from about 18 per cent at 8 days to 30 per cent at 300 days of age. Proteolipid protein increased even more dramatically, from 7 to 27 per cent in the same time interval. These chemical studies were correlated with ultrastructural investigations, both of the developing myelin sheath in situ and the isolated myelin obtained from mice of various ages. A hypothesis, relating the observed changes in protein composition of myelin during development to its mode of formation, is developed. Another subcellular fraction, separated from myelin, by virtue of its greater density in a CsCl gradient, was also studied. It was a vesicular, membranous fraction present at a level of 0.35 mg of protein/brain at all ages and was related to myelin in terms of protein composition.     

BIOSYNTHESIS OF PSYCHOSINE AND LEVELS OF CEREBROSIDES IN THE CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF QUAKING MICE

Abstract— The levels of cerebrosides in neural tissues of adult mice were determined by densitometry of cerebroside spots on charred thin-layer chromatograms of washed total lipid extracts. Values for brain, spinal cord and peripheral nerves were 9·2, 33·0 and 36·9 mg/g of tissue, respectively. In adult Quaking mice these values were 6·4, 24 and 35 % of normal, respectively. Normal levels in brain, spinal cord and peripheral nerve of 21-day-old mice were 3·10, 13·5 and 17·8 mg/g, respectively. In 21-day-old Quaking mice the levels were reduced to 16,21 and 57% of normal, respectively. Biosynthesis of psychosine (galacto-sylsphingosine) by homogenates of Quaking brain, spinal cord and peripheral nerve, respectively, was 18, 24 and 42% of the normal rates at 21 days after birth and 16, 66 and 60% of the normal rates at 94 days. Our results suggest a quantitative relationship between the rate of formation of psychosine in vitro and the rate of accumulation of cerebrosides. in vivo. Biosynthesis of lactosylceramide was not reduced in homogenates of brain and spinal cord from Quaking mice. Cerebroside levels in normal and Quaking spinal cord and in normal brain increased 2- to 3-fold after 21 days of age, but in Quaking brain there was little or no increase.     

LIPID COMPOSITION OF OPTIC NERVE MYELIN

Abstract— Myelin was isolated from bovine optic nerves by differential ultracentrifugation and its lipid composition was analysed. Optic nerve myelin contained 76·3 per cent lipid. The major lipids were cholesterol, ethanolamine glycerophosphatides (EGP) and cerebroside. Serine glycerophosphatides (SGP), sphingomyelin and cerebroside sulphate were present in smaller proportions. EGP and SGP contained 34·6 and 0·5 per cent aldehydes. The major fatty aldehydes were palmitaldehyde, stearaldehyde and octadecenaldehyde. The fatty acids of EGP, SGP and choline glycerophosphatides (CGP) were chiefly 16:0, 18:0 and 18:1, with small proportions of 20 and 22 carbon polyunsaturates. The sphingolipids contained predominantly saturated and monounsaturated fatty acids of chain lengths of 20–26 carbon atoms. Optic nerve myelin and white matter myelin resembled one another closely in overall lipid composition and in the fatty acid compositions of their constituent lipids. Optic nerve myelin and white matter myelin are chemically similar membranes, but both of these differ in their lipid composition from spinal root myelin.     

COMPOSITION OF CEREBRAL LIPIDS IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

Abstract— The composition of sphingolipids and phospholipids of mouse brain during myelination was determined in normal animals and in mice with a genetically-determined disorder of myelin formation. Myelination was normally characterized by a two-fold increase in total phospholipids of brain, a four-fold increase in total sphingolipids, and a six-fold increase in cerebrosides. The Jimpy mutant, with defective formation of myelin in the central nervous system, demonstrated a marked deficiency of cerebrosides and a significantly lower content of total sphingolipids, without alteration of the composition of phospholipids. The increasing content of cerebrosides in the brains of the leucodystrophic mutant at the time in development when myelination is most active and the subsequent relative deficit suggest that the failure of myelin formation is not the result of a defect in biosynthesis of cerebrosides.     

THE COMPOSITION OF MYELIN FROM THE MUTANT MOUSE ''QUAKING''

Abstract— Myelin was isolated from the brains of adult Quaking mice, a mutant showing a deficiency of myelin in the central nervous system, and normal controls. The mutant myelin was found to have a higher flotation density than that of the control and showed marked differences in lipid composition. The myelin from Quaking mice was found to be deficient in cerebroside and ethanolamine phospholipid. Acrylamide gel electrophoresis of total myelin protein demonstrated a pronounced deficiency of proteolipid protein. The activity of cyclic 2',3'-AMP phosphohydrolase was normal.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

BIOSYNTHESIS OF FREE AMINO ACIDS IN THE BRAIN OF JIMPY MICE

—The metabolism of free amino acids: γ-aminobutyric acid (GABA), glutamine, glycine and glutathione has been studied. The labelling of these free amino acids in normal and in myelin-deficient brains of Jimpy mice was followed after intraperitoneal injection of ¹⁴C-labelled glucose precursor. The quantitative distribution of these amino acids in the two kinds of mouse brain has been compared. A higher level of GABA and a faster labelling of the amino acids in Jimpy than in normal mouse brain was observed.     

METABOLISM IN VIVO OF BRAIN GALACTOLIPIDS: THE JIMPY MUTANT

Abstract— The incorporation in vivo of [U-¹⁴C]glucose into the galactolipids of the brain of control and Jimpy mutant mice was examined. Over a 24-h period of incorporation there was no indication of an increased rate of turnover of brain galactolipids in the mutant. The Jimpy mutant was identified at ages prior to and at the inception of myelination (7–10 days post partum) with a coat marker (Tabby). There was similar total radioactivity in galactolipids of the Jimpy at these ages but a reduction to 13 per Cent of control at 13 days and to 6 per cent at 16 days of postnatal age. This devetopmental pattern of galactolipid synthesis in Jimpy brain is not in accord with a primary defect in the biosynthesis of cerebrosides and sulphatides.