Human Central Nervous System Myelin Research Articles

Conservation and divergence of myelin proteome and oligodendrocyte transcriptome profiles between humans and mice.

Human myelin disorders are commonly studied in mouse models. Since both clades evolutionarily diverged approximately 85 million years ago, it is critical to know to what extent the myelin protein composition has remained similar. Here, we use quantitative proteomics to analyze myelin purified from human white matter and find that the relative abundance of the structural myelin proteins PLP, MBP, CNP, and SEPTIN8 correlates well with that in C57Bl/6N mice. Conversely, multiple other proteins were identified exclusively or predominantly in human or mouse myelin. This is exemplified by peripheral myelin protein 2 (PMP2), which was specific to human central nervous system myelin, while tetraspanin-2 (TSPAN2) and connexin-29 (CX29/GJC3) were confined to mouse myelin. Assessing published scRNA-seq-datasets, human and mouse oligodendrocytes display well-correlating transcriptome profiles but divergent expression of distinct genes, including Pmp2, Tspan2, and Gjc3. A searchable web interface is accessible via www.mpinat.mpg.de/myelin. Species-dependent diversity of oligodendroglial mRNA expression and myelin protein composition can be informative when translating from mouse models to humans.

Bad wrap: Myelin and myelin plasticity in health and disease.

Human central nervous system myelin development extends well into the fourth decade of life, and this protracted period underscores the potential for experience to modulate myelination. The concept of myelin plasticity implies adaptability in myelin structure and function in response to experiences during development and beyond. Mounting evidence supports this concept of neuronal activity-regulated changes in myelin-forming cells, including oligodendrocyte precursor cell proliferation, oligodendrogenesis and modulation of myelin microstructure. In healthy individuals, myelin plasticity in associative white matter structures of the brain is implicated in learning and motor function in both rodents and humans. Activity-dependent changes in myelin-forming cells may influence the function of neural networks that depend on the convergence of numerous neural signals on both a temporal and spatial scale. However, dysregulation of myelin plasticity can disadvantageously alter myelin microstructure and result in aberrant circuit function or contribute to pathological cell proliferation. Emerging roles for myelin plasticity in normal neurological function and in disease are discussed. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 123-135, 2018.

SDSL-EPR Study of a C-terminal Segment of Myelin Basic Protein in a Myelin Mimetic Environment

Myelin basic protein (MBP) is a peripheral membrane protein and a major component of human central nervous system myelin. It is a multifunctional, highly positively charged, intrinsically disordered protein that undergoes extensive post-translational modifications. Its multifunctionality includes among others, binding of cytoskeletal proteins to a membrane surface and polymerization and bundling of cytoskeletal proteins. These interactions are modulated by interaction of MBP with Ca2+-calmodulin. We report here the use of site-directed spin labeling and continuous wave power saturation electron paramagnetic resonance spectroscopy (SDSL-EPR) to examine the conformation of segment A141-L154 of a recombinant 18.5 kDa murine isoform of MBP in a reconstituted membrane environment. This segment overlaps the primary calmodulin binding region (T147-D158) of MBP and is predicted, on the basis of helical wheel constructs, to form an amphipathic alpha helix. Solution NMR investigations also showed this region to exist as a transient alpha helix in 30% TFE. Our measurements using SDSL-EPR reveal that this region forms an extended helix with a period of ∼3.8 residues per turn and with a tilt angle of ∼4.3 degrees with respect to the plane of the lipid bilayer. The N-terminal end of the helix appears to be buried more deeply in the membrane bilayer than the C-terminal end. The C-terminal region of the segment bears two Lys residues whose side chains interact with the lipid head groups causing this end to be more exposed. The accessibility of the C-terminal region of A141-L154, which forms a part of the primary calmodulin binding segment, could facilitate interaction of this region with cytosolic proteins viz., calmodulin and modulate cytoskeletal binding to the oligodendrocyte plasma membrane.(Supported by CIHR, NSERC and MS Society of Canada)

Differentiation of blood monocytes to dendritic cells following reverse transmigration across the blood-brain barrier (94.6)

Abstract Dendritic cells (DC) are absent in the normal human central nervous system (CNS) but their presence has been documented in neuroinflammation. The origin of CNS DC is still controversial. The objective of this study was to determine whether monocytes that cross the blood-brain barrier (BBB) differentiate into DC. Primary human brain microvessel endothelial cells (HBMEC) were cultured on collagen gels. Purified human CD14+ monocytes were incubated with confluent HBMEC for 48h and the reverse transmigrated (RT) cells were characterized by FACS analysis. Approximately 50% of the monocytes reverse transmigrated across HBMEC monolayers and 70% of the RT cells expressed DC-SIGN. Monocytes incubated on gels in the absence of HBMEC retained their phenotype. The percentage of RT cells expressing CD83 increased from 25% to 70% when human CNS myelin was incorporated into the gels, and there was significant increase in CCR7 expression. Analysis of culture supernatants showed high levels of IL-6, TNF-α, PGE2 and IL-1β when HBMEC were cultured on myelin gels suggesting that these cytokines may contribute to the differentiation of monocytes to mature DC. There was an associated increase of MIP-3β, MCP-1, MIP-1α, and MIP-1β in the supernatants. Mixed lymphocyte reactions by co-culturing CFSE-labeled T cells and RT cells showed that RT cells were capable of stimulating allogeneic T cell proliferation. In conclusion, the results suggest that monocytes which reverse-transmigrate across HBMEC differentiate into mostly mature DC capable of inducing T cell proliferative responses. (Supported by MSS, Canada)

Backbone Dynamics of the 18.5 kDa Isoform of Myelin Basic Protein Reveals Transient α-Helices and a Calmodulin-Binding Site

The 18.5kDa isoform of myelin basic protein (MBP) is the predominant form in adult human central nervous system myelin. It is an intrinsically disordered protein that functions both in membrane adhesion, and as a linker connecting the oligodendrocyte membrane to the underlying cytoskeleton; its specific interactions with calmodulin and SH3-domain containing proteins suggest further multifunctionality in signaling. Here, we have used multidimensional heteronuclear nuclear magnetic resonance spectroscopy to study the conformational dependence on environment of the protein in aqueous solution (100mM KCl) and in a membrane-mimetic solvent (30% TFE-d2), particularly to analyze its secondary structure using chemical shift indexing, and to investigate its backbone dynamics using 15N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity toward α-helicity that was stabilized by membrane-mimetic conditions: T33-D46, V83-T92, and T142-L154 (murine 18.5kDa sequence numbering). All of these regions corresponded with bioinformatics predictions of ordered secondary structure. The V83-T92 region comprises a primary immunodominant epitope that had previously been shown by site-directed spin labeling and electron paramagnetic resonance spectroscopy to be α-helical in membrane-reconstituted systems. The T142-L154 segment overlapped with a predicted calmodulin-binding site. Chemical shift perturbation experiments using labeled MBP and unlabeled calmodulin demonstrated a dramatic conformational change in MBP upon association of the two proteins, and were consistent with the C-terminal segment of MBP being the primary binding site for calmodulin.

Pathogen Specificity and Autoimmunity Are Distinct Features of Antigen-Driven Immune Responses in Neuroborreliosis

Neuroborreliosis (NB) is a chronic infectious disease of the central nervous system (CNS) caused by a tick-borne spirochete, Borrelia burgdorferi. In addition to direct effects of the causative infectious agent, additional immunity-mediated mechanisms are thought to play a role in the CNS pathology of NB. In order to further understand the involvement of humoral immune mechanisms in NB, we dissected the intrathecal antibody responses down to the single-plasma-cell level. Starting with single-cell reverse transcription-PCR of fluorescence-activated cell sorter-sorted cerebrospinal fluid plasma cells from an NB patient, we identified expanded clones and resurrected the antigen specificity of their secreted antibodies through recombinant expression of the correctly paired immunoglobulin heavy- and light-chain genes as monoclonal antibodies (MAbs). As expected, we found specificity for the causative infectious agent, B. burgdorferi, among the clonally expanded plasma cell (cePC)-derived MAbs. However, from an independent cePC of the same patient, we could derive MAbs specific for human CNS myelin, without detectable cross-reactivity with B. burgdorferi antigens. While reactivity against B. burgdorferi is a known feature of humoral immune responses in NB, we show (i) that immune responses specific for self antigens may be a distinct feature of CNS infections independent of pathogen reactivity and (ii) that humoral autoimmunity in NB (since found in cePC) is the result of a truly antigen-driven immune response. Our findings indicate that in NB mechanisms may be at play that induce distinct immune responses specific for pathogen and self antigens independent from "molecular mimicry."

Characterization of myelin-associated glycoprotein (MAG) proteolysis in the human central nervous system.

Purified human central nervous system myelin contains an endogenous cysteine protease which degrades the 100-kDa myelin-associated glycoprotein into a slightly smaller 90-kDa derivative called dMAG, and which has been implicated in demyelinating diseases. The native proteolytic site in human MAG was determined in order to characterize this cysteine protease in humans further. This was accomplished by identifying the carboxy-terminus of purified dMAG. The results of these experiments, in conjunction with peptidolysis assays of myelin, demonstrated that the enzyme which proteolyses MAG is extracellular and has cathepsin L-like specificity. Furthermore, it was shown that this cathepsin L-like activity potentially was regulated by the endogenous extracellular inhibitor cystatin C.

Implantation of C6 astrocytoma spheroid into collagen type I gels: invasive, proliferative, and enzymatic characterizations.

A three-dimensional model has been developed in which C6 astrocytoma spheroids of defined sizes are embedded into collagen type I gels. The authors have monitored cell invasive behavior; obtained quantitative data on cell invasion, proliferation, and enzymatic activity; assessed cell-cell interactions by altering the spheroid size used; and studied cell-matrix interactions by modifying the matrix components. Their results show that C6 astrocytoma cells detach from the spheroid surface and invade the gel as single cells by means of a system that appears to be dependent on metalloprotease function. These invasive cells have a low proliferative index. Larger spheroids with central hypoxic microregions possess cells that invade the gel at faster rates; this could be correlated with the release of increased collagen type I degrading activity. Extracellular matrix proteins, such as laminin, fibronectin, and collagen type IV have no significant influence on invasive activity, whereas hyaluronic acid decreases and human central nervous system myelin increases invasion. New strategies directed at the treatment of malignant gliomas must take into account the subpopulation of malignant cells located long distances from the major tumor mass. The spheroid invasion model may provide specific insights into the behavior of these invasive cells.

High molecular weight protein of human central nervous system myelin inhibits neurite outgrowth: an effect which can be neutralized by the monoclonal antibody IN-1.

Neurite outgrowth of PC12 cells in the presence of nerve growth factor and the spreading of 3T3 fibroblasts were inhibited by human myelin proteins from different areas of the central nervous system (CNS) in a dose-dependent manner. Application of liposomes containing human CNS myelin proteins induced rapid collapse of PC12 growth cones. When 3T3 fibroblasts were plated on a human CNS myelin protein-coated substrate the cells remained round, and spreading was inhibited. All these inhibitory effects could be neutralized by the monoclonal antibody IN-1, which was raised against a 250 kDa neurite growth-inhibiting protein (NI-250) of rat CNS myelin. Comparison of the inhibitory properties of human and bovine CNS myelin on PC12 neurite outgrowth showed that human CNS myelin was slightly more inhibitory per unit of myelin protein. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that in human myelin, as in rat and bovine myelin, a high molecular weight protein is responsible for the inhibitory activities on neurite outgrowth and fibroblast spreading.

Human central nervous system myelin inhibits neurite outgrowth

In vitro and animal studies have identified molecules in mammalian CNS myelin which inhibit neuritic extension and which may be responsible, at least in part, for the lack of axonal regeneration after injury in the injured brain, optic nerve and spinal cord. To determine whether such inhibitory activity may be present in human CNS myelin, we used a bioassay to characterize neurite outgrowth on this substrate. Human CNS myelin strongly inhibited neuritic outgrowth from newborn rat dorsal root ganglion neurons and NG-108-15 cells, a neuroblastoma-glioma hybrid cell line. Similar but less potent inhibitory activity was identified in human gray matter. The CNS myelin inhibition of neuritic outgrowth appeared to be dependent on direct contact between the myelin substrate and neuntes. The inhibitory activity in human CNS myelin closely resembled that described in adult rodents. Inhibition of neurite growth by human CNS myelin in this in vitro bioassay mirrors the lack of regeneration in vivo and can be used as a model to develop strategies designed to enhance axonal regeneration and neural recovery.

Migration of Human Glioma Cells on Myelin

Histoanatomically invading astrocytoma cells appear to migrate along distinct structures within the brain. Astrocytoma invasion may occur along extracellular matrix (ECM) protein-containing structures, such as blood vessels, but most frequently occurs along tracts of myelinated fibers. This behavior most likely is a consequence of the use of constitutive extracellular ligands expressed along the pathways of preferred dissemination. Enzymatic modification of the extracellular space or deposition of ECM by the tumor cells may also create a more permissive environment. Established human glioma cell lines and two preparations of primary cells isolated from glioblastoma biopsies were studied with the use of cell adhesion and monolayer migration assays to investigate whether crude human central nervous system myelin extracts present specific cell adhesion ligands that promote glioma attachment and cell migration. Two cell lines showed high levels of adhesion and migration on central nervous system myelin similar to levels of migration on the ECM protein merosin, which has previously been shown to be a highly permissive substrate for cultured astrocytoma cells. Two other cell lines showed lower but specific migratory response; one cell line did not attach or specifically migrate on crude myelin extracts. For both glioblastoma primary cell preparations, myelin and merosin were the most permissive substrates for attachment and migration. Other ECM proteins (collagen type IV, fibronectin, and vitronectin) were moderate or nonpermissive substrates. Our findings indicated that astrocytoma cells may be able to use oligodendrocyte membrane-associated ligands as well as ECM proteins of the basement membranes for invasion of normal brain.

A phosphatidylinositol-linked peanut agglutinin-binding glycoprotein in central nervous system myelin and on oligodendrocytes.

Here we report the isolation and initial biochemical characterization of a 120-kD peanut agglutinin-binding glycoprotein from the adult human central nervous system (CNS), which is anchored to membranes through a phosphatidylinositol linkage. Myelin incubated with phosphatidylinositol-specific phospholipase C released the protein as a soluble polypeptide of 105 kD, which was isolated with peanut agglutinin-agarose affinity chromatography. The protein was found to be highly glycosylated. The protein appears to be confined to the CNS, where its developmental expression is region specific and parallels myelination. It is in greater quantity in white matter than in gray matter and it is in isolated human CNS myelin. Furthermore, ovine oligodendrocytes in culture contain the protein on their surfaces and release it into the supernatant as a soluble 105-kD form. We call this protein the oligodendrocyte-myelin protein.

The species distribution of nervous system antigens that react with anti-myelin-associated glycoprotein antibodies

The reactivity of monoclonal and polyclonal antibodies directed against human central nervous system (CNS) myelin-associated glycoprotein (MAG) was investigated in a number of animal species. The antibodies included mouse monoclonal antibodies obtained by immunization with human MAG; HNK-1, a mouse monoclonal antibody raised against a human lymphoblastoma and used to identify a subset of lymphocytes with natural killer function; human IgM paraproteins associated with neuropathy; and polyclonal antibodies obtained from rabbits immunized with rat or human MAG. Following polyacrylamide gel electrophoresis of CNS and peripheral nervous system (PNS) tissue from human, bovine, cat, rabbit, guinea pig, rat, mouse, frog, gold fish and chicken, proteins were electrophoretically transferred onto nitrocellulose. The immune-staining of electroblots showed distinct interspecies variation in the reactivity of the antibodies with MAG. In addition, the species distribution of several low molecular weight glycoproteins present in PNS tissue that cross-react with anti-MAG antibodies was determined. These low molecular weight antigens are not present in CNS homogenates or in purified human CNS myelin. It was also shown that IgM from a patient with peripheral neuropathy and paraproteinemia associated with anti-MAG antibodies recognized these low molecular weight antigens. The results suggest that IgM paraproteins, HNK-1 and some mouse monoclonal antibodies react with carbohydrate determinants shared by MAG and several lower molecular weight glycoproteins present only in human, bovine, cat and chicken PNS. Rabbit polyclonal anti-rat MAG antisera and mouse monoclonal antibodies reacting with peptide epitopes of MAG are much more specific for detecting MAG than antibodies reacting with carbohydrate epitopes of human MAG. The results are discussed in relation to human demyelinating peripheral neuropathy associated with IgM paraproteinemia.

Changes in the composition of two molecular species of ethanolamine plasmalogen in normal human myelin during development

Changes in the ratio of two molecular species of ethanolamine plasmalogen, Pl-LE-1 and Pl-PE-2, of human central nervous system myelin during development were measured by a TLC procedure. The ratio was found to decrease sharply with age up to 6 months as a result of an increase in the amount of Pl-PE-2, believed to be a unique myelin lipid with 18:1 chains in both positions of the glycerol. The ratio continued to decrease gradually with age and did not reach the adult level until an age of 17 years.

Thermotropic phase transitions in normal human myelin as observed in a sensitive microcalorimeter

Using a differential heat capacity calorimeter, phase transitions have been observed in normal human central nervous system myelin. Two large endothermic transitions at 33 and 77°C, respectively, and a smaller transition at 61–62°C, were routinely observed. The relatively simple protein composition of myelin has allowed us to assign the lower transition (33°C) to proteolipid-lipid interactions and the higher temperature transition (72°C) to basic protein-lipid interactions. The small transition at 61–62°C was not assigned, but is probably due to lipid. The abolition of the high temperature transition, after extraction of basic protein from myelin, the lack of phase transitions when a total lipid extract or the isolated proteins were studied, enabled us to assign the high temperature transition. Because the only other major protein was the proteolipid fraction, the low temperature transition (33°C) was assigned to proteolipid-protein interaction.

Plasma cell dyscrasia and peripheral neuropathy: identification of the myelin antigens that react with human paraproteins.

In some cases of polyneuropathy and plasma cell dyscrasia, the monclonal antibodies react with human peripheral nerve myelin. To identify the myelin antigens involved, we separated the proteins of human central and peripheral nerve myelin by polyacrylamide gel electrophoresis, transferred the proteins onto nitrocellulose sheets, and used an immunoenzymatic technique to detect the reactive antigens. Serum IgM but not IgG from three patients with neuropathy and complement-fixing anti-human myelin IgM paraproteins immunostained a protein of approximately 100,000 daltons in human peripheral nerve myelin and a protein or closely migrating proteins of similar size in human central nervous system myelin. In a fourth patient, both IgM and IgG immunostained the antigen. Immunostaining was specific for the paraprotein light chain type, and absorption of the patients' sera with human peripheral nerve myelin eliminated the reaction with the central nervous system proteins. No reaction was seen with rabbit peripheral nerve myelin or with membranes prepared from human myotubes, human T cells, or human fibroblasts. Control sera from six patients with neuropathy and IgM paraproteins that did not react with myelin, from four patients with IgM paraproteins but no neuropathy, and from three normal subjects did not immunostain myelin.

Phosphoprotein phosphatase of human central nervous system myelin: Purification to apparent homogeneity of a low Mr phosphatase and characterization of the high Mr phosphatase

Phosphoprotein phosphatase of human central nervous system myelin: Purification to apparent homogeneity of a low Mr phosphatase and characterization of the high Mr phosphatase

Interaction of multiple sclerosis serum with liposomes containing ganglioside GM1

Multiple sclerosis serum contains a factor or factors capable of releasing sequestered [14C]glucose from liposomes containing GM1, the major ganglioside present in human central nervous system myelin. Although some control sera can also release large quantities of liposomal glucose, multiple sclerosis and control sera differ in the mechanism of this release. Liposomal damage by multiple sclerosis serum does not appear to be complement mediated, but shares certain releasing characteristics of cholera toxin.

Effect of basic protein from human central nervous system myelin on lipid bilayer structure

The effect of myelin basic protein from normal human central nervous system on lipid organization has been investigated by studying model membranes containing the protein by differential scanning calorimetry or electron spin resonance spectroscopy. Basic protein was found to decrease the phase transition temperature of dipalmitoyl phosphatidylglycerol, phosphatidic acid, and phosphatidylserine. The protein had a greater effect on the freezing temperature, measured from the cooling scan, than on the melting temperature, measured from the heating scan. These results are consistent with partial penetration of parts of the protein into the hydrocarbon region of the bilayer in the liquid crystalline state and partial freezing out when the lipid has been cooled below its phase transition temperature. The effect of the protein on fatty acid chain packing was investigated by using a series of fatty acid spin labels with the nitroxide group located at different positions along the chain. If the protein has not yet penetrated, it increases the order throughout the bilayer in the gel phase, probably by decreasing the repulsion between the lipid polar head groups. Above the phase transition temperature, when parts of it are able to pentrate, it decreases the motion of the lipid fatty acid chains greatly near the polar head group region, but has little or no effect near the interior of the bilayer. Upon cooling again the protein still decreases the motion near the polar head group region but increases it greatly in the interior. Thus, the protein penetrates partway into the bilayer, distorts the packing of the lipid fatty acid chains, and prevents recrystallization, thus decreasing the phase transition temperature. The magnitude of the effect varied with the lipid and was greatest for phosphatidic acid and phosphatidylglycerol. It could be reversed upon cooling for phosphatidylglycerol but not phosphatidic acid. The protein was only observed to decrease the phase transition temperature of phosphatidylserine upon cooling. It had only a small effect on phosphatidylethanolamine and no effect on phosphatidylcholine. Thus, the protein may penetrate to a different extent into different lipids even if it binds to the polar head group region by electrostatic interactions.

Circular dichroism studies on lipid-protein complexes of a hydrophobic myelin protein.

Circular dichroism spectra of lipophilin (a hydrophobic protein purified from human central nervous system myelin) were analyzed by the method of Chen et al. (1974) to obtain information on its secondary structure in aqueous and lipid environments. When introduced into phosphatidylcholine vesicles by dialysis from 2-chloroethanol, the protein possessed about 75% alpha helix. A new water-soluble form of lipophilin also containing over 70% alpha helix was obtained by a similar dialysis in the absence of lipid. This product had a higher helical content than two other water-soluble preparations derived by dialysis from phenol-acetic acid-urea. Interaction of all three aqueous forms of the protein with lysolecithin micelles resulted in increases in total helical content or in the average length of helical segments. The amount of beta sheet was at a minimum for lipophilin incorporated into vesicles, where the presence of lipid also provided some protection against thermal denaturation.