PROTEIN AND GLYCOPROTEIN COMPOSITION OF MYELIN AND MYELIN SUBFRACTIONS FROM BRAINS OF ''QUAKING'' MICE

Abstract— Quaking mutants in mice are known to be affected by an arrest of myelinogenesis and to have a purified myelin which is more dense than that of controls. Their myelin has been shown to demonstrate a striking decrease in proteolipid protein, a lesser decrease in the small myelin basic protein and changes in glycoproteins comprising reduction in the major peak and shift of this peak towards a higher apparent molecular weight. The possibility that these findings might reflect merely contamination of myelin with other membranes was tested by subfractionation. Light myelin (floats on 0.62 m -sucrose) is generally accepted as more compact and mature than the heavier subfraction (floating on 0.85 m -sucrose). The changes previously found were present in both subfractions and even more marked in the light myelin. These results indicate that the anomalies of myelin proteins and glycoproteins were not caused by contaminants and are present in compact myelin as well as in membranes which are transitional between the glial plasma membrane and the myelin sheath. Therefore, we suggest that the Quaking mutation results in dysmyelination rather than hypomyelination.     

White Matter Rafting––Membrane Microdomains in Myelin

The myelin membrane comprises a plethora of regions that are compositionally, ultrastructurally, and functionally distinct. Biochemical dissection of oligodendrocytes, Schwann cells, and central and peripheral nervous system myelin by means such as cold-detergent extraction and differential fractionation has led to the identification of a variety of detergent-resistant membrane assemblies, some of which represent putative signalling platforms. We review here the different microdomains that have hitherto been identified in the myelin membrane, particularly lipid rafts, caveolae, and cellular junctions such as the tight junctions that are found in the radial component of the CNS myelin sheath. This article is dedicated to Drs. Anthony and Celia Campagnoni.     

Binding of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase to Myelin: An In Vitro Study

Abstract: The binding of 2′,3′-cyclic nucleotide 3′-phosphodiesterase isoform 1 (CNP1) to myelin and its association with cytoskeletal elements of the sheath have been characterized with in vitro synthesized polypeptides and purified myelin. We have previously shown that the cysteine residue present in the carboxy-terminal CXXX box of CNP1 is isoprenylated, and that both C₁₅ farnesyl and C₂₀ geranylgeranyl isoprenoids can serve as substrates for the modification. Here, we have mutated the CXXX box to obtain selectively farnesylated CNP1 or geranyl-geranylated CNP1 and found that these two modified forms of CNP1 behave identically in all of the assays performed. Isoprenylation is essential but not sufficient for the binding of in vitro synthesized CNP1 to purified myelin, because a control nonmyelin protein is isoprenylated, yet unable to bind to myelin. In our assay, membrane-bound CNP1 partitions quantitatively into the non-ionic detergent-insoluble phase of myelin, suggesting that CNP1 binds to cytoskeletal elements within myelin. However, isoprenylated CNP1 fails to bind to the cytoskeletal matrix isolated from myelin by detergent treatment, implying that both detergent-soluble and insoluble myelin components are involved in the binding of CNP1. A model for the interactions between CNP1 and myelin is presented, consistent with models proposed for other isoprenylated proteins.     

Membrane structure in isolated and intact myelins.

The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions.     

Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.     

FROG SCIATIC NERVE MYELIN: A CHEMICAL CHARACTERIZATION

Abstract— The lipid and protein compositions of PNS myelin from two species of frogs, Xenopus laevis and Rana catesbiana , are compared with each other and with those of the rat. The relative proportions of PNS myelin lipids in the two frog species and rat are very similar except for a somewhat higher phosphatidyl choline content in frog myelin. The protein compositions of frog PNS myelin of the two species are also much alike, but a protein smaller than P₂ is found in Xenopus , which is not seen in Rana . The results are discussed in terms of results found by others in other species and the compositions are compared with those of the corresponding CNS myelin.     

Demyelinating Antibodies to Myelin Oligodendrocyte Glycoprotein and Galactocerebroside Induce Degradation of Myelin Basic Protein in Isolated Human Myelin

Abstract: Although the specificity of multiple sclerosis (MS) brain immunoglobulins (lgs) remains unknown, the incubation of these lgs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.     

Identification of Plasmolipin as a Major Constituent of White Matter Clathrin-Coated Vesicles

We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.     

Myelin consists of a continuum of particles of different density with varying lipid composition: major differences are found between normal mice and quaking mutants

Mouse brain myelin consists of a continuum of particles of different densities, as shown by sucrose density gradient centrifugation. In normal animals most of the material (65 per cent) is concentrated between 0.6 and 0.7 M sucrose (the maximum being found at 0.66 M sucrose, corresponding to 23 per cent). The density differences among various myelin fractions are related to their protein/lipid ratios, as lighter fractions contain less protein and more lipid. Lipid analysis shows a decrease in the amount of every lipid from the lightest to the heaviest fraction: the light fraction is richer in phosphatidyl-ethanolamine, phosphatidyl-serine and cerebrosides. The distribution is highly abnormal in purified myelin from Quaking mutant ; very low quantities of myelin with normal density are found, but unexpected large amount of high density particles are found, possibly related to a "pre-myelin" material (oligodendrogial) processes which are not maturing into normal myelin).     

On the spontaneous adherence of myelin basic protein to T lymphocytes.

We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation.     

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).     

The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin-Deficient (mld) Mutant Mice

The myelin-deficient (mld) mutation is autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mld. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin-associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low-molecular-mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.     

Protein and Lipid Composition of Radial Component-Enriched CNS Myelin

Abstract: The radial component is a junctional complex that is believed to stabilize the apposition of myelin membranes in the internode of CNS myelin. Based on our previous finding that the radial component of compact myelin retains its structure in tissue treated with the detergent Triton X-100, we have attempted to isolate the junctional complex from spinal cord myelin treated with this detergent. Using 0.5% Triton X-100, our procedures yielded a fraction of isolated myelin that was enriched in well-preserved radial component. This fraction that contained morphologically well-defined radial component was examined by sodium dodecyl sulfate-polyacrylamide gel electropho-resis and immunoblotting, and TLC, and was found to be significantly and consistently enriched in the 21.5-kDa and 17-kDa isoforms of myelin basic protein, and in cerebro-sides, hydroxy sulfatide, and sphingomyelin. In addition, the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase, tubulin, and actin tended to be resistant to Triton extraction. The fraction of isolated myelin that contained radial component was deficient in proteolipid protein and DM-20, the 18.5-and 14-kDa isoforms of myelin basic proteins, and in the major phospholipids, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. Our data indicate that the radial component can be isolated and that certain myelin and cytoskeletal proteins and lipids are closely associated with it.     

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.     

The habenular nuclei of the elasmobranch "Scyllium stellare": myelinated perikarya.

Some habenular cells of the elasmobranch, Scyllium stellare, have a perikaryon completely enveloped by myelin of the compact type. They are found only in the left habenula and not in the right. The myelin sheath accompanies the proximal segment of the process arising from the perikaryon, in a node of Ranvier-like pattern. The identity of these myelinated cells and the functional significance of their myelination are unknown.     

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.     

OCCURRENCE OF ALKANES IN BRAIN MYELIN. COMPARISON BETWEEN NORMAL AND QUAKING MOUSE

Alkanes, a new class of neurolipid, were found in mouse brain, the level being reduced in the Quaking mutant. These hydrocarbons are concentrated in myelin; minor amounts being found in microsomes, mitochondria and synaptosomes. The average recovery is 7.1 μg/mg in normal myelin, 2.2 in the Quaking myelin. The distribution pattern of these alkanes was determined by gas liquid chromatography and was found to differ in normal and Quaking myelin; the hydrocarbons consist mainly of n-alkanes ranging from C₂₁ to C₃₂ with even and odd aliphatic chains.     

Biochemical Characterization of Myelin Isolated from the Central Nervous System of Xenopus Tadpoles

Abstract: Myelin isolated from the central nervous system of Xenopus tadpoles was characterized biochemically and compared with Xenopus frog and mammalian myelins. Xenopus tadpole myelin contains the characteristic protein and lipid components of mammalian myelin, although quantitative differences exist. The biochemical composition of Xenopus tadpole myelin suggests that it is an immature form of XePnopus frog myelin. Basic protein and proteolipid protein are prominent components of Xenopus myelin, but isolated tadpole myelin contains a greater proportion of higher molecular weight proteins than Xenopus frog or mature mammalian myelin. The basic protein has a higher apparent molecular weight than mammalian myelin basic protein. The levels of 2',3'-cyclic nucleotide 3'-phosphodiesterase are significantly higher in whole tadpole brain homogenate and purified myelin than in similar mammalian preparations. Tadpole myelin lipids contain a higher proportion of phospholipids and less galactolipid than mammalian myelin. Tadpole myelin galactolipids include a high (16%) percentage of monogalactosyl diglyceride, a component found in only trace quantities (0.9%) in bovine myelin.     

Myosin II has distinct functions in PNS and CNS myelin sheath formation

The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.