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.     

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.     

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.     

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.     

PROTEIN COMPOSITION OF AXONS and MYELIN FROM RAT and HUMAN PERIPHERAL NERVES

Abstract— Proteins of rat and human peripheral nerves were studied in whole nerve homogenates and in purified myelin and axonal preparations of peripheral nerve. Both myelin and axonal fractions were obtained from desheathed and minced nerve segments by flotation and sedimentation, respectively, in 0.85 m -sucrose following hypotonic treatment. The purity of myelin and axonal preparations was confirmed by electron microscopic examination of pelleted material. Nerve proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3 and 7.4. Major protein bands of fresh whole nerve homogenates corresponded to polypeptide bands of either the purified myelin or axon preparations. The most prominent electrophoretic band in peripheral nerve was identified as a myelin glycoprotein with molecular weight of 27,000. The major polypeptides of axon preparations had molecular weights of 200,000, 150,000, 69,000, 55,000 and 27,000. The latter two proteins were believed to represent tubulin and residual major myelin protein, respectively. The three largest axonal polypeptides were believed to be derived from neurofilaments, which represented the predominant organelle of the purified axons. Collagen was also seen in whole nerve homogenates and in purified axons but could be distinguished by its metachromatic staining with Coomassie blue.     

Zonal centrifuge profiles of rat brain homogenates: Instability in sucrose,stability in iso-osmotic Ficoll-sucrose

By substituting iso-osmotic Ficoll-sucrose for hyperosmotic sucrose between the densities of 1.043 and 1.088 in sucrose density gradients in the B-XV rotor of an Anderson-NIH-AEC zonal centrifuge system, it was possible to stabilize the zonal centrifuge absorbancy profiles of adult rat brain homogenates. The reason for the instability in ordinary sucrose gradients was found to be the interaction of myelin with other brain structures in hyperosmotic sucrose. No such interaction occurred in isoosmotic sucrose (0.32 M) with or without Ficoll. In Ficoll-sucrose, myelin was separated at three reproducible densities of 1.054, 1.060, and 1.066 gm/ml. No myelin appeared at a density if 1.094 gm/ml, which represented the main collection point in ordinary sucrose. Synaptosomes were separated at peak densities of 1.072 and 1.152 gm/ml. Mitochondria were obtained at a density of 1.176 gm/ml. Areas under zonal centrifuge absorbancy profiles of rat brain homogenates were found to be constant regardless of the values of ω²t that were reached.     

Effects of Rumpshaker Mutation on CNS Myelin Composition and Structure

Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jp ^rsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jp ^rsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jp ^rsh myelin, further substantiating that PLP is not a component of this junctional complex. X-raydiffraction measurements on unfixed optic nerves showed that the jp ^rsh period is 5–10 Å larger than normal. Moreover, jp ^rsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jp ^rsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jp ^rsh mutant correlate with a wider periodicity and less stable packing of the myelin.     

Evidence for aerobic ATP synthesis in isolated myelin vesicles

Even though brain represents only 2–3% of the body weight, it consumes 20% of total body oxygen, and 25% of total body glucose. This sounds surprising, in that mitochondrial density in brain is low, while mitochondria are thought to be the sole site of aerobic energy supply. These data would suggest that structures other than mitochondria are involved in aerobic ATP production. Considering that a sustained aerobic metabolism needs a great surface extension and that the oxygen solubility is higher in neutral lipids, we have focused our attention on myelin sheath, the multilayered membrane produced by oligodendrocytes, hypothesizing it to be an ATP production site. Myelin has long been supposed to augment the speed of conduction, however, there is growing evidence that it exerts an as yet unexplained neuro-trophic role. In this work, by biochemical assays, Western Blot analysis, confocal laser microscopy, we present evidence that isolated myelin vesicles (IMV) are able to consume O₂ and produce ATP through the operation of a proton gradient across their membranes. Living optic nerve sections were exposed to MitoTracker, a classical mitochondrial dye, by a technique that we have developed and it was found that structures closely resembling nerve axons were stained. By immunohystochemistry we show that ATP synthase and myelin basic protein colocalize on both IMV and optic nerves. The complex of data suggests that myelin sheath may be the site of oxygen absorption and aerobic metabolism for the axons.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency ($\text{msd}$), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking > immature > trembler ? normal adult; and in sciatic nerves: $\text{trembler > immature > quaking}\text{msd ?}\text{normal}$ adult. Repeat periods about 3 Å less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190–200 Å period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is ≈ 2 Å thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is ≈ 5 Å thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Biochemical Analysis of Myelin Proteins in a Novel Neurological Mutant: The Taiep Rat

Abstract: Hemispheres, spinal cords, and sciatic nerves were taken from taiep, carrier, and control rats at ages ranging from 1 day to 16 months. Absolute myelin yields from CNS taiep tissues peaked at ～2 months and then decreased until they reached a low but stable level. Myelin yield from the affected hemispheres expressed as a percentage of age-matched controls decreased continuously from 2 weeks until it reached a stable level of ～10–15%. The same was true for the spinal cords, but here the myelin yield reached a plateau at a slightly higher percentage of 20–25%. In comparison with control rats, isolated CNS myelin fractions from the affected rats had a greater content of high molecular weight proteins. Western blot analyses of CNS homogenates revealed that myelin basic protein (MBP), proteolipid protein, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase were all present but decreased to levels generally consistent with the deficiencies of myelin. However myelin-associated glycoprotein (MAG) levels always were reduced much more than those of the other three myelin proteins, and at younger ages the apparent molecular weight for MAG was increased in the mutants. Western blot analyses of sciatic nerve homogenates showed that the levels of MBP, MAG, and P₀ were not significantly different in control and mutant animals. These results suggested an early hypomyelination of the CNS, with peak levels of myelin at 2 months, followed by a prolonged period of myelin loss, until a very low but stable myelin level was reached. The consistently greater loss of MAG, in comparison with other CNS myelin proteins, is different from most other hypomyelinating mutants in which MAG is relatively preserved in comparison with the proteins of compact myelin. This might be due to microtubular abnormalities in the taiep mutant interfering with transport of myelin proteins and having the greatest effect on MAG because of its most distal location in the periaxonal oligodendroglial membranes.     

Rapid transport of protein in the optic system of the goldfish

Abstract— Several amino acids, particularly [³H]proline and [³H]asparagine specifically and efficiently labelled rapidly transported proteins in the goldfish optic nerve and tectum after intraocular injection. Studies with these amino acids showed that the rapidly transported proteins moved as a discrete band at a rate which was temperature-dependent, and was equal to 70-100 mm per day at 20°C. Transported protein in the optic tectum was 80 per cent particulate and was found in synaptosomal, mitochondrial, and myelin fractions, but not in purified nuclei or ribosomes.     

Functional Recovery Occurs Even After Partial Remyelination of Axon-Meshed Median and Ulnar Nerves in Mice

Upper limb nerve injuries are common, and their treatment poses a challenge for physicians and surgeons. Experimental models help in minimum exploration of the functional characteristics of peripheral nerve injuries of forelimbs. This study was conducted to characterize the functional recovery (1, 3, 7, 10, 14, and 21 days) after median and ulnar nerve crush in mice and analyze the histological and biochemical markers of nerve regeneration (after 21 days). Sensory–functional impairments appeared after 1 day. The peripheral nerve morphology, the nerve structure, and the density of myelin proteins [myelin protein zero (P0) and peripheral myelin protein 22 (PMP22)] were analyzed after 21 days. Cold allodynia and fine motor coordination recovery occurred on the 10th day, and grip strength recovery was observed on the 14th day after injury. After 21 days, there was partial myelin sheath recovery. PMP22 recovery was complete, whereas P0 recovery was not. Results suggest that there is complete functional recovery even with partial remyelination of median and ulnar nerves in mice.

     

Myelin Proteins, Glycoproteins, and Myelin-Related Enzymes in Experimental Demyelination of the Rabbit Optic Nerve: Sequence of Events

Wallerian degeneration of the rabbit optic nerve was investigated by the technique of retinal ablation which precludes edema, hemorrhage, or macrophage infiltration. After 8 days of degeneration, marked degradation of axons and some myelin abnormalities appeared in the optic nerve, optic chiasma, and optic tract. Myelin lesions were maximal 32 days after retinal destruction. The amount of material stained with a myelin dye decreased drastically between 32 and 90 days after the operation. Biochemical parameters gave the following sequence of events. The concentration of the major periodic acid--Schiff staining glycoproteins was decreased after 2 days, and 6 days later the presence of cholesterol esters was detected in the optic tissue. After 16 days of Wallerian degeneration, the specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase not associated with myelin decreased, indicating a possible de-differentiation of oligodendrocytes. Degradation of myelin basic protein became significant at 32 days and the amount of myelin isolated decreased later. The loss of myelin basic protein coincided with a reduction of myelin periodicity as measured in purified fractions by electron microscopy. These results show that secondary myelin destruction in the absence of edema, hemorrhage, or macrophages is a very slow process, and in this situation myelin undergoes a selective and sequential loss of its constituents.     

Effect of inorganic lead exposure on myelination in the rat

The effect of defined lead burdens on myelination of the central and peripheral nervous systems was studied in neonatal Long-Evans rats. Pups were exposed to inorganic lead (100 or 400 mg Pb as lead acetate/kg body wt/day by gastric intubation) from day 2 following birth to 30 days of age. Accumulation of myelin in forebrain was not affected by the 100-mg dosage, but at the 400 mg/kg dosage level, myelin accumulation was reduced by approximately 42% on a per gram forebrain basis relative to vehicle-intubated animals. The deficit was over 50% on a per forebrain basis, since there was also a slight reduction in brain weight. This lead effect was observed at both 15 and 30 days of age. Accumulation of myelin in optic nerve (determined on the basis of proteolipid protein concentration) was also reduced by 30% relative to controls by this dosage level. However, myelination in sciatic nerve (determined on the basis of P₀ protein concentration) was not affected by this exposure regimen. Myelin deficits were greater than could be accounted for by undernutrition arising secondary to lead exposure and were not due to a developmental delay in the onset of myelination.     

Simultaneous determination of olanzapine,clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography

An automated method for simultaneous routine quantification of the antipsychotic drugs clozapine, olanzapine and their demethylated metabolites is described. The method included adsorption on a cyanopropyl (CPS) coated clean-up column (10 μm; 10×2.0 mm I.D.), washing off interfering serum constituents to waste, and separation on C18 ODS Hypersil reversed phase material (5 μm; 250×4.6 mm I.D.) using acetonitrile–water–tetramethylethylenediamine (37:62.6:0.4, v/v/v) adjusted to pH 6.5 with concentrated acetic acid. UV-detection was performed at 254 nm. The limit of quantification was 10–20 ng/ml. Relative day to day standard variations ranged between 4.5 and 13.5%. The method is suitable for routine monitoring of olanzapine and clozapine including their demethylated metabolites.     

Biochemical studies of myelin in Wallerian degeneration of rat optic nerve

Abstract— Wallerian degeneration of the optic nerves of the rat was induced by removal of the eyes. After 54, 66, 76 or 90 days of degeneration a myelin fraction of the nerves was obtained by the procedure of Laatsch et al. (1962). The yield of myelin from the degenerated nerves was decreased, but the isolated myelin appeared to be morphologically normal. The proportion of cholesterol in the myelin lipids was slightly increased, whereas that of the ethanolamineglycerophosphatides was decreased and galactolipids were normal. After one‘cycle’of myelin purification, the high-molecular-weight fraction formed a much greater percentage of the total protein in myelin isolated from degenerated optic nerves. After 2–3‘cycles’of purification, the distribution of protein in myelin isolated from degenerated and normal optic nerves was similar, an observation suggesting that the high-molecular-weight fraction in‘1-cycle myelin’from degenerated optic nerves may have been partly attributable to contamination. With the possible exception of ethanolamineglycerophosphatides, our data suggest that there was no preferential breakdown of myelin lipid constituents nor of protein constituents during Wallerian degeneration of rat optic nerve. As assessed by SDS-gel electrophoresis of the water-insoluble particulate fraction, the percentage of myelin protein was markedly decreased after 76 days of degeneration. However, the major myelin protein constituents in this fraction (the two basic proteins and proteolipid protein) appeared to decrease in the same relative proportions.     

Myelin-Associated Glycoprotein: Electron Microscopic Immunocytochemical Localization in Compact Developing and Adult Central Nervous System Myelin

Light microscopic immunocytochemical studies have shown that myelin-associated glycoprotein (MAG) is localized in myelin of the developing CNS; but in the adult, MAG appears to be restricted to periaxonal regions of myelinated fibers. To extend these observations, we embedded optic nerves of 15-day-old rats, adult rats, and an adult human in epon after aldehyde and osmium tetroxide fixation. After 5% H2O2 pretreatment, thin sections were immunostained with 1:250-1:5,000 rabbit antiserum to rat CNS MAG according to the avidin-biotin-peroxidase complex (ABC) method. Dense deposits of reaction product covered compact myelin in both developing and adult optic nerves. When we used 1:500, 1:1,000, and 1:2,000 anti-MAG, less intense immunostaining of myelin was found. We also obtained the same localization in compact myelin with the peroxidase-antiperoxidase (PAP) method. With 1:250 anti-MAG, dense deposits of reaction product were not observed on axolemmal membranes or on oligodendroglial membranes located periaxonally and paranodally. In thin sections of adult human optic nerve, anti-MAG also stained compact myelin intensely. When thin sections of rat and human optic nerves were treated with preimmune or absorbed serum, no immunostaining was observed. Immunoblot tests showed that our MAG antisera did not react with any non-MAG myelin proteins. In contrast with earlier light microscopic data, this study shows that MAG localization does not change during CNS development; both developing and adult compact myelin sheaths contain MAG. As many biochemical studies also show that MAG is present in compact myelin, we suggest that this 100,000 dalton glycoprotein now be called myelin glycoprotein (MGP) instead of MAG.     

THE EFFECTS OF UNDERNUTRITION ON THE PROTEINS OF OPTIC AND SCIATIC NERVES DURING DEVELOPMENT

Abstract— Polyacrylamide gel electrophoresis has been used to assess the appearance of some optic and sciatic nerve proteins in normal developing rats and in undernourished rats. Of the myelin proteins, the'Wolfgram'proteolipid is already present about the time myelination begins. The basic myelin proteins appear later, first in sciatic and then in optic nerve. A non-myelin basic protein, assumed to be a histone, is present at high levels in both nerves before myelination begins. There is no apparent effect of undernutrition on the appearance and amount of myelin proteins at 12, 16 and 22 days of age. The'histone'protein is reduced in optic and sciatic nerves at times corresponding roughly to the transition periods from cellular proliferation to myelin formation. The possibilities are discussed that myelin basic proteins are synthesized as compact myelin formation occurs, and that there may be retarded cellular proliferation in nerves of undernourished rats.