Cell lineage analysis of Schwann cells in the PNS determined by shiverer-normal mouse chimeras

The myelin of the peripheral nervous system from the shiverer mutant mice is characterized by the absence of myelin basic protein, while the other myelin protein components are present at normal levels. Myelin lamella formation is normal in the shiverer mutant. Therefore, by using antiserum against myelin basic protein, we can distinguish the shiverer from the wild-type control myelin immunohistochemically. To study the cell lineage of Schwann cells, chimeras produced by the aggregation of eight-cell embryos from wild-type mice and shiverer mice have been used. Using myelin basic protein as a marker, it was observed that Schwann cells in the sciatic nerve existed as patches of cells with like-genotype. The patches occurred in a linear array along the axons with some intermingling of Schwann cells. Complete randomization by intermingling of Schwann cells was not observed and clones of Schwann cells may persist as contiguous groups throughout peripheral nerve development.     

Progressive segregation of unmyelinated axons in peripheral nerves, myelin alterations in the CNS, and cyst formation in the kidneys of myelin and lymphocyte protein-overexpressing mice

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.     

Molecular biology of myelin basic protein: gene rearrangement and expression of anti-sense RNA in myelin-deficient mutants.

1. Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system (PNS). 2. Myelin basic protein (MBP) is a major protein in CNS myelin. 3. MBP is expressed specifically in the nervous system. 4. The MBP gene has been cloned and characterized. 5. Two mutant mice, Shiverer (shi) and myelin-deficient (mld. shimid), are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the central nervous system. 6. It was found that rearrangement within the MBP gene results in low expression of the gene. 7. In Shiverer, the MBP gene is partially deleted (from exons 3 to 7), and in mld, the gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. 8. In mld, anti-sense RNA complementary to exons 3-7, which correspond to the inverted segment, was detected by RNase protection studies, and presumed to be responsible for the reduced expressions of MBP. 9. The mechanism of gene rearrangement in MBP was also characterized. 10. This article reviews the recent progress in the study of the MBP gene, especially the rearrangement of the gene and its expression in mutant mice.     

FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin

Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were approximately 5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.     

A novel fluorescent probe that is brain permeable and selectively binds to myelin.

Myelin is a multilayered glial cell membrane that forms segmented sheaths around large-caliber axons of both the central nervous system (CNS) and peripheral nervous system (PNS). Myelin covering insures rapid and efficient transmission of nerve impulses. Direct visual assessment of local changes of myelin content in vivo could greatly facilitate diagnosis and therapeutic treatments of myelin-related diseases. Current histologic probes for the visualization of myelin are based on antibodies or charged histochemical reagents that do not enter the brain. We have developed a series of chemical compounds including (E,E)-1,4-bis(4'-aminostyryl)-2-dimethoxy-benzene termed BDB and the subject of this report, which readily penetrates the blood-brain barrier and selectively binds to the myelin sheath in brain. BDB selectively stains intact myelinated regions in wild-type mouse brain, which allows for delineation of cuprizone-induced demyelinating lesions in mouse brain. BDB can be injected IV into the brain and selectively detect demyelinating lesions in cuprizone-treated mice in situ. These studies justified further investigation of BDB as a potential myelin-imaging probe to monitor myelin pathology in vivo.     

Wrapping it up: the cell biology of myelination

During nervous system development, oligodendroglia in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) synthesise large amounts of specific proteins and lipids to generate myelin, a specialised membrane that spirally ensheathes axons and facilitates fast conduction of the action potential. Myelination is initiated after glial processes have attached to the axon and polarisation of the plasma membrane has been triggered. Myelin assembly is a multi-step process that occurs in spatially distinct regions of the cell. We propose that assembly of myelin proteins and lipids starts during their transport through the biosynthetic pathway and continues at the plasma membrane aided by myelin-basic protein (MBP). These sequential processes create the special lipid and protein composition necessary for myelin to perform its insulating function during nerve conduction.     

Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles

Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis.     

Myelin Basic Protein Citrullination in Multiple Sclerosis: A Potential Therapeutic Target for the Pathology

Multiple sclerosis (MS) is a multifactorial demyelinating disease characterized by neurodegenerative events and autoimmune response against myelin component. Citrullination or deimination, a post-translational modification of protein-bound arginine into citrulline, catalyzed by Ca²⁺ dependent peptidylarginine deiminase enzyme (PAD), plays an essential role in physiological processes include gene expression regulation, apoptosis and the plasticity of the central nervous system, while aberrant citrullination can generate new epitopes, thus involving in the initiation and/or progression of autoimmune disorder like MS. Myelin basic protein (MBP) is the major myelin protein and is generally considered to maintain the stability of the myelin sheath. This review describes the MBP citrullination and its consequence, as well as offering further support for the “inside-out” hypothesis that MS is primarily a neurodegenerative disease with secondary inflammatory demyelination. In addition, it discusses the role of MBP citrullination in the immune inflammation and explores the potential of inhibition of PAD enzymes as a therapeutic strategy for the disease.     

The Nodes of Ranvier: Molecular Assembly and Maintenance

Action potential (AP) propagation in myelinated nerves requires clustered voltage gated sodium and potassium channels. These channels must be specifically localized to nodes of Ranvier where the AP is regenerated. Several mechanisms have evolved to facilitate and ensure the correct assembly and stabilization of these essential axonal domains. This review highlights the current understanding of the axon intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the peripheral nervous system (PNS) and central nervous system (CNS).Axons conduct electrical signals, called action potentials (APs), among neurons in a circuit in response to sensory input, and between motor neurons and muscles. In mammals and other vertebrates, many axons are myelinated. Myelin, made by Schwann cells and oligodendrocytes in the peripheral nervous system (PNS) and central nervous system (CNS), respectively, is a multilamellar sheet of glial membrane that wraps around axons to increase transmembrane resistance and decrease membrane capacitance. Although myelin is traditionally viewed as a passive contributor to nervous system function, it is now recognized that myelinating glia also play many active roles including regulation of axon diameter, axonal energy metabolism, and the clustering of ion channels at gaps in the myelin sheath called nodes of Ranvier. Together, the active and passive properties conferred on axons by myelin, result in axons with high AP conduction velocities, low metabolic demands, and reduced space requirements as compared with unmyelinated axons. Thus, myelin and the clustering of ion channels in axons permitted the evolution of the complex nervous systems found in vertebrates. This review highlights the current understanding of the axonal intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the PNS and CNS.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

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.     

Phylogenetic development of myelin glycosphingolipids

Myelin is a highly specialized membrane, which enwraps axons and facilitates saltatory nerve conduction in vertebrates. Galactocerebroside and its sulfate ester, sulfatide, are highly localized in myelin. To understand the role played by these galactosphingolipids we investigated the changes of these myelin-specific compounds during the course of the evolution of myelin. We found that urodele nerve myelin lacks alpha-hydroxy fatty acid-containing galactosphingolipids. Our morphological and physiological studies of urodele nerves indicated that these hydroxy fatty acid-containing galactosphingolipids probably contribute to fast nerve conduction. Also it is suspected that they are involved in the regulation of the thickness of myelin in relation to the size of the axon. In another study, we discovered that glucocerebroside, which has glucose instead of galactose as its carbohydrate component, is abundantly present in the myelin-like sheath membrane of crustacean nerves. Subsequently, the phylogenetic study indicated that galactocerebrosides were limited to the nervous system of deuterostomes, while all protostome nerves contain glucocerebrosides. The role of glucocerebrosides in multilayered membranes and in the conduction velocity of the protostome nervous system is discussed.     

Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes

Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin.     

Immunoreactive myelin basic proteins are not detected when shiverer mutant Schwann cells and fibroblasts are co-cultured with normal neurons

Shiverer (shi) is an autosomal recessive mutation in mice that results in hypomyelination in the central nervous system (CNS) but normal myelination in the peripheral nervous system (PNS). Myelin basic proteins (MBPs) are virtually absent in both PNS and CNS. It is not known whether the cellular target in the PNS is the myelin-forming Schwann cell or another cell type which secondarily affects the Schwann cell. To determine the cellular target of the shi gene, we have adapted tissue culture techniques that allow co-culture of pure populations of mouse sensory neurons of one genotype with Schwann cells and fibroblasts of another genotype under conditions that permit myelin formation. These cultures were stained immunocytochemically as whole mounts to determine whether MBPs were expressed under various in vitro conditions. In single-genotype cultures, presence or absence of MBPs was consistent with earlier in vivo results: +/+ cultures were MBP-positive and shi/shi cultures were MBP-negative. In mixed-genotype cultures, visualization of MBPs in myelin accorded with the genotype of the non-neuronal Schwann cells and fibroblasts and not with the neurons--those cultures that contained +/+ non-neuronal cells were MBP-positive and those with shi/shi non-neuronal cells were MBP-negative, independent of the neuronal genotype. These results rule out neurons or circulating substances as mediators of the influence of the shi genetic locus on MBP synthesis and deposition in peripheral myelin.     

The reaction of myelin phospholipids with phospholipase C and D

Phospholipase C and D hydrolyze the membrane-bound phospholipids of isolated, untreated myelin. When the membrane is treated with detergents or solvents which disrupt the membrane structure, the activity of the enzymes against the membrane-bound lipids increases. Myelin in the central nervous system is derived from the cell membrane of the oligodendroglial cell. Because the phospholipids in erythrocyte cell membranes are strikingly resistent to phospholipase C and D hydrolysis the question is raised of whether myelin in situ, as opposed to isolated myelin, is susceptible to phospholipase hydrolysis.     

Hypothesis of an Energetic Function for Myelin

Nervous system is a great oxygen consumer, but the site of oxygen absorption has remained elusive. Four proteomic studies have shown that the respiratory complexes I to V may be expressed in isolated myelin. Myelin is an outgrowth of glial cells, surrounding many axons in multiple spires both in peripheral and central nervous system. Recent quantitative analyses strongly support the daring hypothesis that myelin is functional in aerobic ATP production, to supply the neuron with chemical energy. A vision of myelin sheath as a structure devoted to the oxygen absorbance for glucose combustion in nervous system thank to its enormous surface, would be also supported by an impressive series of characteristics and properties of myelin that do not presently find an explanation, all of which are herein examined.     

Composition of myelin from peripheral and central nervous systems of the squirrel monkey

Myelin was prepared from the brachial plexus and cervical spinal cord of adult squirrel monkeys (Saimiri sciureus). Brachial plexus myelin contained a larger amount of sphingomyelin and smaller amounts of cholesterol, lipid galactose, ethanolamine phosphoglyceride, choline phosphoglyceride, and alk-1-enyl ether than spinal cord myelin when compared as ratios to total lipid phosphorus. The peripheral nervous system myelin had a higher proportion of protein. All of these differences were statistically significant. Thus peripheral nervous system myelin and central nervous system myelin differ in protein content and lipid composition in this subhuman primate.     

PCDHα在髓鞘形成和少突胶质细胞发育中的作用

该文通过免疫组化及蛋白免疫印迹的方法分别对Pcdhα基因敲除和对照组小鼠的中枢神经系统内的髓鞘碱性蛋白表达以及少突胶质细胞的发育进行了测定。结果表明:1)Pcdhα基因缺失小鼠中枢神经系统中的髓鞘碱性蛋白较对照组小鼠明显减少;2)Pcdhα基因敲除可导致少突胶质细胞发育异常:在小脑中,处于成熟期的少突胶质细胞减少,而处于前体细胞阶段的少突胶质细胞增多。上述结果提示Pcdhα可以通过调控少突胶质细胞的成熟过程进而影响髓鞘的形成。