Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.     

The QKI-6 and QKI-7 RNA Binding Proteins Block Proliferation and Promote Schwann Cell Myelination

Background

The quaking viable (qk^v) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk^v mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.

Methodology/Principal Findings

To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27^KIP1 and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27^KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR.

Conclusions/Significance

Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.     

Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures

Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J. B. and Braun, P. E. (1984) Dev. Neurosci. 6, 345-355). In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes. Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture. Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope. The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments. However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period. The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied. The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of [32P] phosphate in the myelin basic proteins. These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.     

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

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

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Glioblastoma infiltration into central nervous system tissue in vitro: involvement of a metalloprotease

Differentiated oligodendrocytes and central nervous system (CNS) myelin are nonpermissive substrates for neurite growth and for cell attachment and spreading. This property is due to the presence of membrane-bound inhibitory proteins of 35 and 250 kD and is specifically neutralized by monoclonal antibody IN-1 (Caroni, P., and M. E. Schwab. 1988. Neuron. 1:85-96). Using rat optic nerve explants, CNS frozen sections, cultured oligodendrocytes or CNS myelin, we show here that highly invasive CNS tumor line (C6 glioblastoma) was not inhibited by these myelin- associated inhibitory components. Lack of inhibition was due to a specific mechanism as the metalloenzyme blocker 1,10-phenanthroline and two synthetic dipeptides containing metalloprotease-blocking sequences (gly-phe, tyr-tyr) specifically impaired C6 cell spreading on CNS myelin. In the presence of these inhibitors, C6 cells were affected by the IN-1-sensitive inhibitors in the same manner as control cells, e.g., 3T3 fibroblasts or B16 melanomas. Specific blockers of the serine, cysteine, and aspartyl protease classes had no effect. C6 cell spreading on inhibitor-free substrates such as CNS gray matter, peripheral nervous system myelin, glass, or poly-D-lysine was not sensitive to 1,10-phenanthroline. The nonpermissive substrate properties of CNS myelin were strongly reduced by incubation with a plasma membrane fraction prepared from C6 cells. This reduction was sensitive to the same inhibitors of metalloproteases. In our in vitro model for CNS white matter invasion, cell infiltration of optic nerve explants, which occurred with C6 cells but not with 3T3 fibroblasts or B16 melanomas, was impaired by the presence of the metalloprotease blockers. These results suggest that C6 cell infiltrative behavior in CNS white matter in vitro occurs by means of a metalloproteolytic activity, which probably acts on the myelin-associated inhibitory substrates.     

Glia unglued: how signals from the extracellular matrix regulate the development of myelinating glia

The health and function of the nervous system relies on glial cells that ensheath neuronal axons with a specialized plasma membrane termed myelin. The molecular mechanisms by which glial cells target and enwrap axons with myelin are only beginning to be elucidated, yet several studies have implicated extracellular matrix proteins and their receptors as being important extrinsic regulators. This review provides an overview of the extracellular matrix proteins and their receptors that regulate multiple steps in the cellular development of Schwann cells and oligodendrocytes, the myelinating glia of the PNS and CNS, respectively, as well as in the construction and maintenance of the myelin sheath itself. The first part describes the relevant cellular events that are influenced by particular extracellular matrix proteins and receptors, including laminins, collagens, integrins, and dystroglycan. The second part describes the signaling pathways and effector molecules that have been demonstrated to be downstream of Schwann cell and oligodendroglial extracellular matrix receptors, including FAK, small Rho GTPases, ILK, and the PI3K/Akt pathway, and the roles that have been ascribed to these signaling mediators. Throughout, we emphasize the concept of extracellular matrix proteins as environmental sensors that act to integrate, or match, cellular responses, in particular to those downstream of growth factors, to appropriate matrix attachment.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

Tumor necrosis factor stimulates multiple serine/threonine protein kinases in Swiss 3T3 and L929 cells. Implication of casein kinase-2 and extracellular signal-regulated kinases in the tumor necrosis factor signal transduction pathway.

Incubation of Swiss 3T3 or L929 cells with tumor necrosis factor (TNF) leads to the rapid stimulation of several cytosolic Ser/Thr kinases active toward myelin basic protein, the S6 peptide (RRLSSLR), the G peptide (SPQPSRRGSESSEE), and Kemptide (LRRASLG). This confirms the hypothesis that kinases other than protein kinases A and C may be involved in the TNF signal transduction. Chromatography on Mono Q resolved multiple kinase peaks with each substrate tested and moreover revealed a TNF-mediated casein kinase-2 activation in both cell lines, measurable with the specific RRREEESEEE peptide or with the G peptide. The TNF-stimulated myelin basic protein kinases-1 and -2 were identified as extracellular signal-regulated kinases-2 and -1, respectively, based on their elution pattern on Mono Q chromatography, their inactivation by protein phosphatase action, their reaction with phosphothreonine and phosphotyrosine antibodies, and by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 42- and 44-kDa proteins recognized by anti-extracellular signal-regulated kinase antibodies.     

PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: Involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways

PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24 h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 μM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 μM) but not the MAPKK inhibitor (PD98059, 50 μM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.     

Hypotheses regarding myelination derived from comparisons of myelin subfractions.

Patterns of myelin-associated proteins and glycoproteins in subfractions of central nervous system myelin and changes in these during development have been reviewed. Several hypotheses are put forward regarding classification of these proteins as structural components of myelin, as components of the membranes from which myelin is derived or as molecules present to play a role in myelin formation rather than as true components of compact myelin. 2′, 3′-Cyclic nucleotide 3′-phosphohydrolase is considered to fit into this last category and two different hypothesis are proposed for its role. The major myelin glycoprotein is postulated to serve a function in recognition of axons by oligodendrocytes, a function not needed in the peripheral nervous system where Schwann cells and axons develop in close contact.     

Isoprenylated Proteins in Myelin

Abstract: Incubation of rat brainstem slices with [³H]- mevalonate ([³H]MVA) in the presence of lovastatin resulted in the incorporation of label into three groups of myelin-associated proteins with molecular masses of 47, 21–27, and 8 kDa, as revealed on sodium dodecyl sulfate- polyacrylamide rod gel electrophoresis. Although the gel patterns of [³H]MVA-derived prenylated proteins were similar, the relative level of ³H incorporated into each protein species differed between myelin and the brainstem homogenate. Immunoprecipitation studies identified the 47-kDa prenylated protein as a 2′-3′-cyclic nucleotide phospho- diesterase, whereas the 8-kDa protein proved to be the γ subunit of membrane-associated guanine nucleotide regulatory protein. The ³H-labeled 21–27-kDa group in myelin corresponds to the molecular mass of the extensive Ras- like family of monomeric GTP-binding proteins known to be prenylated in other tissues. Increase in lovastatin concentration resulted in reduced levels of [³H]MVA-labeled species in myelin and concomitantly increased levels in the cytosol. A cold MVA chase restored to normality the appearance of [³H]MVA-labeled proteins in myelin. Furthermore, a high lovastatin concentration in the brainstem slice incubation mixture altered the appearance of newly synthesized nonprenylated myelin proteins, including proteolipid protein and the 17-kDa subspecies of myelin basic protein. Because other myelin proteins were unaffected by the high lovastatin concentration, restricting the availability of MVA in myelin-forming cells may selectively alter processes required for myelinogenesis. Although the molecular basis for the” different MVA requirements in myelin- forming cells remains undefined, it may involve an alteration in the biological activity of certain proteins that require prenylation to be functionally active, and that are responsible for promoting insertion of specific proteins into the myelin membrane.     

Separation of the major proteins of central and peripheral nervous system myelin using reversed-phase high-performance liquid chromatography

A general method to separate the major proteins of rat central and peripheral nervous system myelin has been developed. The key step is the initial quantitative removal of the lipids under conditions where the proteins retain their solubility in HPLC solvents. Lipids are removed by a combination of solvent extraction and column chromatography on Sephadex LH-60 in 2-chloroethanol:10 mM HCl (9:1). Proteins are then separated by reversed-phase (RP) HPLC. Samples are applied to a wide pore reversed-phase C-3 column and eluted with a linear gradient of 10-70% 1-propanol in 0.1% trifluoroacetic acid (0-100% B) over a 60-min period. Myelin basic proteins elute between 25 and 30% B, Wolfgram and other high molecular weight proteins at 35-50% B, proteolipid protein at 65-80% B, and P0 glycoprotein at 55-65% B. This elution pattern is consistent with the known relative hydrophobicity of these proteins. Protein recovery for the entire procedure is greater than 74%. Proteolipid and P0 proteins isolated by HPLC contain 2.3 and 1.1 mol of covalently bound fatty acids, respectively. This fatty acid composition is similar to that previously reported using different isolation procedures. The analysis of central and peripheral nervous system myelin proteins by RP-HPLC permits the isolation of purified proteins for structural and metabolic experiments.     

The QKI-6 RNA Binding Protein Localizes with the MBP mRNAs in Stress Granules of Glial Cells

Background

The quaking viable (qk^v) mouse has several developmental defects that result in rapid tremors in the hind limbs. The qkI gene expresses three major alternatively spliced mRNAs (5, 6 and 7 kb) that encode the QKI-5, QKI-6 and QKI-7 RNA binding proteins that differ in their C-terminal 30 amino acids. The QKI isoforms are known to regulate RNA metabolism within oligodendrocytes, however, little is known about their roles during cellular stress.

Methodology/Principal Findings

In this study, we report an interaction between the QKI-6 isoform and a component of the RNA induced silencing complex (RISC), argonaute 2 (Ago2). We show in glial cells that QKI-6 co-localizes with Ago2 and the myelin basic protein mRNA in cytoplasmic stress granules.

Conclusions

Our findings define the QKI isoforms as Ago2-interacting proteins. We also identify the QKI-6 isoform as a new component of stress granules in glial cells.     

IGF binding protein alterations on periplaque oligodendrocytes in multiple sclerosis: implications for remyelination

Why myelin repair greatly fails in multiple sclerosis (MS) is unclear. The insulin-like growth factor (IGF) system plays vital roles in oligodendrocyte development, survival, and myelin synthesis. We used immunohistochemistry to study IGF-I, IGF-I receptors and IGF binding proteins (IGFBPs) 1-6 on oligodendrocytes at the edges of chronic demyelinated plaques and normal appearing white matter of MS, and in cerebral white matter of controls without neurological disease. Oligodendrocytes in all conditions were immunoreactive for IGF-I, IGF-I receptors and IGFBPs-1-5. Oligodendrocytes at the edges of demyelinated plaques displayed enhanced immunoreactivity for IGF-I, IGF-I receptors, IGFBPs-1 and -6. Because increased expression of IGFBPs-1 and -6 has been associated with impaired synthesis of myelin proteins in oligodendrocyte lineage cells, pharmacological approaches to reduce their expression might be useful for promoting remyelination of axons in MS lesions.