Meylin types with different protein components in the same species

Abstract— Myelin was isolated from bovine optic nerve, cerebral white matter, spinal cord white matter and peripheral nerve (intradural spinal roots). The freeze-dried myelin completely dissolved in phenol-formic acid-water (14:3:3, w/v/v), and acrylamide gel electrophoresis of the myelin proteins was performed with this solvent. Qualitative and quantitative differences were observed in the myelin proteins from the various regions of the CNS. Myelin of peripheral nerve contained proteins that are apparently unique to it and which are not found in the myelin of the CNS.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Central Nervous System Oligosaccharide Storage in Caprine β-Mannosidosis

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.     

Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2 3 cyclic nucleotide 3 phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.     

CHANGES OCCURRING IN THE CHEMICAL COMPOSITION OF THE CENTRAL NERVOUS SYSTEM DURING FOETAL AND POST-NATAL DEVELOPMENT OF THE SHEEP

(1) The chemical composition of the CNS (separated into cerebrum, cerebellum, brain stem and spinal cord) was determined in sheep during foetal and post-natal development and in adults. (2) The spinal cord differed from the remainder of the CNS in growing more after the period studied (50-day-old foetuses to 5-week-old lambs) than before it. This was largely attributable to lipid accumulation. (3) Chemical growth (accumulation of DNA, protein and lipid) proceeded linearly in spinal cord, logarithmically in cerebrum and cerebellum while in brain stem growth was described by a sigmoid function. (4) Fat-free dry matter, protein, total lipid, cholesterol and phospholipid concentrations increased progressively in all parts of the CNS but DNA concentrations changed little. In the cerebrum alone there was an increase in DNA concentration during maturation suggesting an increased cell population. Cholesterol was present predominantly in the free form but esters were detected in foetal tissues from 70 up to 120 days gestation. (5) Cerebroside, the characteristic lipid of myelin, increased in concentration soon after 85 days of gestation, up to which point very low values were recorded, the rate varying according to the region of the CNS examined. Rates of increase in total regional cerebroside content were used to identify periods of myelination and the results suggest that there are two periods of peak activity, one about 20 days before birth and the other at 10-20 days after birth. (6) The composition of lipids added during the two phases of myelination and during maturation were characteristically different. In the spinal cord, lipid analyses best reflect changes in myelin composition.     

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

REGIONAL DIFFERENCES IN LIPID COMPOSITION IN RABBIT NERVOUS TISSUE

Lipid composition was determined for different regions of rabbit nervous tissue. In the white matter of adult rabbit, the ratio between cholesterol, phospholipids and sphingolipids was quite constant. Among the subclasses of phospholipids, phosphatidylcholine and sphingomyelin tended to compensate for each other as constituents of myelin, as did galactosphingolipids and sphingomyelin amongst the sphingolipids. The brain was rich in phosphatidylcholine and gakactosphingolipids, while peripheral nerves (PN) were rich in sphingomyelin. The spinal cord showed a composition intermediate between the brain and PN. The sphingolipid to phosphatidylcholine ratio seems to be useful as a myelin maturation index applicable to both CNS and PN. The rostral part of the CNS showed a high ratio of molecular species of cerebroside with α-hydroxy fatty acids to those with unsubstituted fatty acids (CH/CN). The caudal part of the CNS had a high concentration of cerebrosides with C24-monoenoic fatty acids, so that there was an inverse relationship between CH/CN and C24:1/C24:0 for different regions of CNS. The present data show that the lipid composition as well as the fatty acid composition of myelin-specific lipids are influenced by neural differentiation and development or by neuroglial relationships.     

Kinetic properties of the 5 alpha-reductase of testosterone in the purified myelin, in the subcortical white matter and in the cerebral cortex of the male rat brain

The 5 alpha-reductase, the enzyme which converts testosterone into dihydrotestosterone (DHT), is present in several CNS structures of the rat. Recent reports from this laboratory indicate that the subcortical white matter and the myelin possess a 5 alpha-reductase activity several times higher than that present in the cerebral cortex. Moreover, previous ontogenetic observations indicate that in all cerebral tissues examined (including the myelin) the 5 alpha-reductase has a higher activity in immature animals. This study was performed in order to verify whether the differences in the 5 alpha-reductase activity on the various brain components might be due to the presence of different concentrations of the same enzyme or to different isoenzymes. To this purpose, the kinetic properties Km and Vmax were measured in the purified myelin as well as in homogenates of the subcortical white matter and of the cerebral cortex, obtained from the brain of adult (60-90-day-old), immature (23-day-old), and aged (greater than 20-month-old) male rats. The results indicate that the enzymes present in the myelin, in the subcortical white matter and in the cerebral cortex of adult male rats possess a very similar apparent Km (1.93 +/- 0.2, 2.72 +/- 0.73 and 3.83 +/- 0.49 microM respectively). On the contrary, the Vmax values obtained in the myelin (34.40 +/- 5.54), in the white matter (19.57 +/- 2.36) and in the cerebral cortex (6.47 +/- 1.03 ng/h/mg protein) of adult animals have been found to be consistently different. Very similar Km values were found in the myelin obtained from the brain of immature and very old rats (2.14 +/- 0.11 and 3.39 +/- 0.75 microM respectively). The Vmax measured in the myelin purified from the immature rat brain (62.25 +/- 4.52) showed a value which was much higher than that found in the myelin of adult animals (34.40 +/- 5.54); a Vmax (34.31 +/- 9.41) almost identical to that of adult animals was found in the myelin prepared from the brain of aged rats.     

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.     

N-Acetylsuccinimidylglutamate, a Cyclic Imide Form of the Peptide N-Acetylaspartylglutamate, Is Present in Low Micromolar Concentrations in Murine and Bovine CNS

Abstract: N -Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N -acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the α- and β-carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 ± 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 ± 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amounts in the superior colliculus and lateral genicutale nucleus (1.17 ± 0.05 and 0.82 ± 0.13 pmol/mg wet weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions ( r = 0.74, p < 0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)-NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.     

The composition of the myelin proteins of the central nervous system

Abstract— The amino acid composition of human, monkey and bovine centrum ovale myelin, of bovine optic nerve myelin, and of bovine spinal cord white matter myelin has been determined. In general, the amino acid patterns of the centrum ovale myelin of these species and the optic nerve myelin are identical. Differences are noted when these are compared to the spinal cord white matter myelin. It is shown that the amino acid composition of myelin cannot be duplicated by any combination of the Folch–Lees proteolipid protein and the basic protein fraction of myelin. It is necessary to postulate the existence of a third protein fraction that is rich in dicarboxylic amino acids.     

Codistribution of neurite growth inhibitors and oligodendrocytes in rat CNS: appearance follows nerve fiber growth and precedes myelination

Higher vertebrate CNS myelin and oligodendrocytes in vitro contain membrane-bound surface proteins of 35 and 250 kDa with marked inhibitory properties for neurite growth and for fibroblast spreading. The inhibitory activity is neutralized by monoclonal antibody IN-1, which binds to the inhibitory proteins. IN-1 also neutralizes the nonpermissive substrate properties of adult rat optic nerve explants and spinal cord white matter in vitro, thus suggesting a crucial involvement of these inhibitors in the nonpermissive nature of the adult CNS of higher vertebrates. We have determined time of appearance and distribution of the IN-1-sensitive inhibitory activity in the rat. In the optic nerve, inhibitors appear after the period of axonal growth and before myelination. A similar schedule was found for the spinal cord and for the cerebellum. No IN-1-sensitive inhibitory activity was found outside the CNS or in oligodendrocyte-free regions of the CNS. Where investigated, the distribution of inhibitory oligodendrocytes and of IN-1-sensitive inhibitory activity correlated well. Our data suggest that IN-1-sensitive inhibitory activity in vivo might be an oligodendrocyte-specific property.     

Lysosulfatide (Sulfogalactosylsphingosine) Accumulation in Tissues from Patients with Metachromatic Leukodystrophy

We describe here a sensitive assay method for lysosulfatide (sulfogalactosylsphingosine) in human tissues using HPLC. The method involves extraction of lipids, saponification, isolation using a C18 Sep-Pak column, derivatization with o-phthalaldehyde, and detection of the fluorescent lysosulfatide using HPLC. In control subjects, a small amount of lysosulfatide was detected in the cerebral white matter (9-35 pmol/mg of protein), spinal cord (35 pmol/mg of protein), sciatic nerve (14 pmol/mg of protein), and kidney (approximately 2 pmol/mg of protein) but not in the cerebral gray matter and liver. A marked accumulation of the lipid was noted in tissues from six patients with metachromatic leukodystrophy (MLD). The concentration of lysosulfatide was high in the cerebral white matter, spinal cord, and sciatic nerve (223-1,172 pmol/mg of protein). Even in the cerebral gray matter, kidney, and liver, where lysosulfatide was hardly detected in the control sample, a considerable amount (3-45 pmol/mg of protein) accumulated in MLD patients. The concentration and distribution pattern of lysosulfatide were similar to those of galactosylsphingosine (psychosine) accumulated in patients with Krabbe disease. Therefore, the accumulation of lysosulfatide may explain the demyelination in patients with MLD, as is the case with Krabbe disease.     

METABOLIC ACTIVITY OF CNS PROTEINS IN RATS AND MONKEYS WITH EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)

—The metabolic activity of proteins from myelin and non-myelin fractions of slices of lesions in monkey brains and in spinal cords of Lewis rats with acute experimental allergic encephalomyelitis was investigated using [1-¹⁴C]leucine as a protein precursor. The uptake in vitro of [1-¹⁴C]leucine into the monkey EAE lesions was greatly increased in both the myelin and non-myelin fractions. Similar findings were made in spinal cord slices of the EAE rat with an average specific activity 341 per cent of control measured in proteins of purified myelin and 415 per cent of control in the non-myelin protein. The increased uptake appeared with the onset of paralytic symptoms 10–14 days after injection. The increased uptake did not appear to be a result of an increased amino acid pool size as measured with uniformly labelled l -leucine, valine, arginine and phenylalanine. The increase in specific activity of the myelin protein of the EAE rats was shown to be associated with the peaks characteristic of myelin protein when separated on polyacrylamide gels and the serial slices counted. Most of the radioactivity of both the control and EAE myelin protein migrated with the high molecular weight fraction, and the largest increase in radioactivity in myelin protein appeared in this fraction. Some increase in specific activity was also found in the basic and proteolipid proteins. Four different guinea-pig antigens were used to induce EAE: whole spinal cord, purified basic protein, purified myelin and basic protein + cerebroside. All caused paralytic symptoms and greatly increased incorporation in vitro of [1-¹⁴C]leucine into spinal cord proteins. The incorporation of [1-¹⁴C]leucine into slices of the inguinal and popliteal lymph nodes of the EAE and Freund's adjuvant control rats were measured and compared with the incorporation into the spinal cord non-myelin fractions. The specific activity of lymph node proteins was of the order of 10 × that of the non-myelin protein of the control spinal cord. Invasion of a moderate number of cells of the order of activity of these lymph nodes could account for the large increase in rate of protein synthesis in the EAE nervous tissue. It is concluded that much of the increased protein synthesis could be due to the inflammatory cells, although a small amount of the total increase appears to be associated with myelin protein. Other changes in metabolism of the CNS tissue of the EAE rat include a lower rate of lipid synthesis and a decreased activity of the tricarboxylic acid cycle.     

Accumulation of lysosulfatide (sulfogalactosylsphingosine) in tissues of a boy with metachromatic leukodystrophy

Abnormal accumulation of lysosulfatide (sulfogalactosylsphingosine) was evident in autopsied tissues from a boy with late-infantile metachromatic leukodystrophy. The concentration was high in the cerebral white matter, spinal cord and sciatic nerve (116-787 pmol/mg protein) and low in the cerebral gray matter, kidney and liver (4-40 pmol/mg protein). As is the case with galactosylsphingosine, lysosulfatide inhibited cytochrome c oxidase activity, in a dose-dependent manner. Judging from the tissue distribution of the accumulated lysosulfatide and because of the cytotoxicity, the lysosulfatide presumably explains the demyelination seen in the nervous tissues of patients with metachromatic leukodystrophy.     

A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase

Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production.     

Regional studies of myelin proteins in human brain and spinal cord

The myelin specific proteins, myelin basic protein (MBP) and myelin proteolipid protein (PLP) were quantitated by radioimmunoassay (RIA) and the activity of the enzyme 23-cyclic 3 phosphohydrolase (CNP) measured, in 27 regions of normal brain and spinal cord. Varying regional concentrations for each protein and regional variations for protein ratios were noted, supporting the concept of a varying chemical composition for myelin throughout the central nervous system (CNS). Variation was also noted among myelin subfractions from a single region. Regions with special sensitivity to the multiple sclerosis process had relatively lower proportions of CNP in several, but not all cases.     

MYELIN PROTEINS FROM DIFFERENT REGIONS OF THE CENTRAL NERVOUS SYSTEM

—The protein composition of myelin prepared from specific anatomical regions of the bovine brain and spinal cord was studied by a modification of the method of Gonzalez -Sastre (1970). Spinal cord myelin contained lesser amounts of chloroform-methanol soluble protein and proteolipid protein and had a lower activity of the enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase than did myelin from subcortical white matter. There was no difference, however, in the protein composition of myelin from the various levels of the spinal cord. The amino acid composition of both proteolipid and basic protein showed no significant regional differences. Myelin preparations from both brain and spinal cord contained DM-20 protein.     

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.