Unique responses of differentiating neuronal growth cones to inhibitory cues presented by oligodendrocytes.

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice.

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.

A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord.

Axon growth inhibitors associated with myelin play an important role in the failure of axon regeneration in the adult mammalian central nervous system (CNS). Several inhibitors are present in the mature CNS. We now present a novel therapeutic vaccine approach in which the animals' own immune system is stimulated to produce polyclonal antibodies that block myelin-associated inhibitors without producing any detrimental cellular inflammatory responses. Adult mice immunized in this manner showed extensive regeneration of large numbers of axons of the corticospinal tracts after dorsal hemisection of the spinal cord. The anatomical regeneration led to recovery of certain hind limb motor functions. Furthermore, antisera from immunized mice were able to block myelin-derived inhibitors and promote neurite growth on myelin in vitro.

Differential regulation of the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene during oligodendrocyte development.

The two major isoforms of 2',3'-cyclic nucleotide phosphodiesterase (CNP), 48 and 46 kDa, have recently been shown to be produced from a single gene by alternative splicing. In addition, messenger RNA encoding the larger isoform is transcribed from a separate promoter, approximately 1 kb upstream from that encoding the smaller isoform. We have investigated the expression of these two CNP isoforms and have found that they are differentially expressed during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only the mRNA encoding the larger protein is found. At the time of oligodendrocyte differentiation, however, both CNP mRNAs are induced. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation.

Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth.

Contact-dependent axon growth inhibitory activity is present in CNS myelin, but the inhibitory proteins have not been fully characterized. We report here that at least two peaks of inhibitory activity can be separated by fractionating solubilized CNS myelin proteins by DEAE chromatography. A major peak of inhibitory activity corresponded to the elution profile of myelin-associated glycoprotein (MAG). Immunodepletion of MAG from these inhibitory fractions removed neurite growth inhibition, whereas recombinant MAG (ectodomain) was a potent inhibitor of neurite outgrowth. Immunodepletion of MAG from total extracts of CNS myelin restored neurite growth up to 63% of control levels. These results establish that MAG is a significant, and possibly the major, inhibitor in CNS myelin; this has broad implications for axonal regeneration in the injured mammalian CNS.

Myelin-associated glycoprotein, a cell adhesion molecule of oligodendrocytes, is phosphorylated in brain.

Myelin-associated glycoprotein (MAG) has been implicated in the mediation of interactions between oligodendrocytes and neurons during the development of the myelin sheath. Here we show that MAG is phosphorylated in intact myelinating mouse brain primarily at serine residues and to a lesser extent at threonine and tyrosine residues. In vivo, only the larger of the two developmentally regulated MAG isoforms is phosphorylated. MAG can be phosphorylated at tyrosine by the v-fps and v-src protein-tyrosine kinases in vitro and by a kinase endogenous to myelin membrane preparations. MAG phosphorylated in myelin membranes in vitro also contains phosphoserine and phosphothreonine. These observations suggest that phosphorylation of MAG is physiologically significant in regulating oligodendrocyte-neuron interactions.

Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter.

Previous studies have suggested the persistence of oligodendrocyte progenitor cells in the adult mammalian subcortical white matter. To identify oligodendrocyte progenitors in the adult human subcortical white matter, we transfected dissociates of capsular white matter with plasmid DNA bearing the gene for green fluorescence protein (hGFP), placed under the control of the human early promoter (P2) for the oligodendrocytic protein cyclic nucleotide phosphodiesterase (P/hCNP2). Within 4 d after transfection with P/hCNP2:hGFP, a discrete population of small, bipolar cells were noted to express GFP. These cells were A2B5-positive (A2B5(+)), incorporated bromodeoxyuridine in vitro, and constituted <0.5% of all cells. Using fluorescence-activated cell sorting (FACS), the P/hCNP2-driven GFP(+) cells were then isolated and enriched to near-purity. In the weeks after FACS, most P/hCNP2:hGFP-sorted cells matured as morphologically and antigenically characteristic oligodendrocytes. Thus, the human subcortical white matter harbors mitotically competent progenitor cells, which give rise primarily to oligodendrocytes in vitro. By using fluorescent transgenes of GFP expressed under the control of an early oligodendrocytic promoter, these oligodendrocyte progenitor cells may be extracted and purified from adult human white matter in sufficient numbers for implantation and cell-based therapy.

Molecular characteristics of the epitope in myelin-associated glycoprotein that is recognized by a monoclonal IgM in human neuropathy patients.

The antigen for the IgM monoclonal antibody from patients with IgM paraproteinemia and peripheral neuropathy is the myelin-associated glycoprotein (MAG), a minor protein component of both human PNS myelin and human CNS myelin. Sera from five patients were found to react with identical proteolytically derived fragments of MAG indicating that the monoclonal IgM from these patients is recognizing a common epitope. Furthermore, the lectin concanavalin A reacts with these fragments and deglycosylation of isolated MAG abolishes the recognition of MAG by the patient monoclonal IgM. Therefore, it appears that the monoclonal IgM from these five patients recognizes a common epitope which contains carbohydrate moieties. These data are consistent with the idea that the peripheral myelin sheath is involved in an autoimmune response directed against MAG.

The characterization and cellular distribution of a family of antigens related to myelin associated glycoprotein in the developing nervous system.

The antigenic epitope detected on myelin associated glycoprotein (MAG) by the monoclonal antibody HNK-1 (Leu 7) was sensitive to degradation by trifluoromethane-sulfonic acid (TFMS) and is therefore probably carbohydrate in nature. This antigen was found to be widely distributed within the rat and chicken embryonic nervous system and was present on cultured central and peripheral neurons (100%), oligodendrocytes (100%) and astrocytes (70-80%) as detected by double marker immunofluorescence. The antigen could be removed from cultured neurons by trypsinization and its resynthesis was blocked by cycloheximide, suggesting that the carbohydrate epitope detected by HNK-1 was attached to a de novo synthesized protein. Several molecular species were detected on Western blots of detergent extracts from 13-15d rat embryonic brain and neuron-enriched cultures from chick spinal cord and dorsal root ganglia. Protein components with molecular weights in the ranges of 90-100 kd to 280 kd were observed and comprise a family of glycoproteins containing the HNK-1 reactive carbohydrate epitope present on MAG. These glycoproteins could play a role in intercellular interactions within the developing nervous system.

Myelin-associated glycoprotein (MAG) distribution in human central nervous tissue studied immunocytochemically with monoclonal antibody.

Recent biochemical data show that myelin-associated glycoprotein (MAG) is the antigen for a monoclonal antibody found in sera of patients with IgM paraproteinemia and neuropathy (Braun et al. 1982). Immunoreactivity of this antibody with CNS has not been described. To study this, monoclonal anti-MAG was used in the avidin-biotin-peroxidase complex method (Hsu et al. 1981) to immunostain paraffin and epon sections of human CNS. Well characterized polyclonal MAG antiserum (Quarles et al. 1981) was employed in comparison tests. In paraffin sections of developing CNS, both monoclonal and polyclonal MAG antisera stained oligodendroglia and myelin. In adult CNS, periaxonal regions of myelin sheaths were immunostained in paraffin sections and semithin epon sections treated with monoclonal and polyclonal anti-MAG. In electron-microscopic experiments that included milder pretreatment of epon thin sections and more precise reaction product localization, entire thickness of myelin sheaths were immunostained. Thus, in electron micrographs, monoclonal and polyclonal anti-MAG immunoreactivity also have the same localization. In other electron-microscopic experiments, the same reaction product localization was observed with antiserum to myelin basic protein (MBP), a known constituent of compact myelin. Thus, results with this monoclonal anti-MAG provide important new evidence to support the localization of MAG in compact CNS myelin. Our data also suggest that monoclonal antibodies against MAG will be useful in studies of the pathogenesis of multiple sclerosis and other demyelinating diseases.

Chloroform markedly stimulates the phosphorylation of myelin basic proteins.

We have investigated the effect of chloroform on the phosphorylation of myelin basic proteins because tumor-promoting agents such as phorbol esters and chloroform are known to enhance the activity of protein kinase C. We report that the presence of chloroform, at a concentration known to enhance protein kinase C activity, stimulated the phosphorylation of myelin basic proteins 15-17 fold over control conditions. The phosphorylation of a 50 kiloDalton myelin protein was also stimulated but to a lesser extent. The concentration of chloroform required for the maximal phosphorylation of myelin basic proteins and the 50 kiloDalton protein was approximately 2% (v/v).

Abnormal expression of the myelin-associated glycoprotein in the central nervous system of dysmyelinating mutant mice.

Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin-associated glycoprotein (MAG)-related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal littermates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five- to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypeptides. The quantity of MAG synthesized by 15-day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthesized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated developmental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.

Release of proteins from the surface of bovine central nervous system myelin by salts and phospholipases.

Incubation of bovine CNS myelin with phospholipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholipase C from Clostridium perfringens, phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9-12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.

Gene expression of the central and peripheral nervous system myelin membrane 2', 3'-cyclic nucleotide 3'-phosphodiesterase in development.

Determination of gene expression for the 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) during development shows that the amount of translatable RNA encoding this enzyme increases in parallel with myelination in the mouse brain. The two isoforms of this enzyme are developmentally regulated in parallel, in contrast to several other myelin proteins. By comparison, the amount of translatable RNA encoding CNP in the peripheral nervous system (rat sciatic nerve) remains constant during development. The two isoforms that are expressed are identical in apparent molecular weight to rat brain CNP. Moreover, no apparent differences were detected by proteolytic digestion (Cleveland mapping) of CNP 1 from the central or peripheral nervous systems.

Acylation in vitro of the myelin proteolipid protein and comparison with acylation in vivo: acylation of a cysteine occurs nonenzymatically.

Characteristics of fatty acylation of myelin proteolipid protein (PLP) in vitro were compared with the corresponding process in vivo. Rapid and efficient separation of labelled PLP from other proteins and lipids was effected by extraction into chloroform/methanol/0.1 N HCl (10/10/1) and chromatography on Sephadex LH-60 in the same solvent. Covalent linkage of [3H]-palmitate to PLP was demonstrated by repetitive chromatography on LH-60, thin layer chromatography, and polyacrylamide gel electrophoresis. Reductive cleavage with sodium borohydride of PLP acylated in vitro or in vivo yielded [3H]-hexadecanol, identifying at least one of the acyl linkages as a thiolester bond. When PLP was acylated with acyl-CoA as the fatty acid donor, the reaction occurred non-enzymatically as supported by the following observations: 1) acylation activity increased with increasing pH above pH 7.5, 2) acylation activity was heat stable, 3) acylation activity was not removed from PLP during purification in organic solvents or in Triton X-100-containing buffers, and 4) acylation of tryptic fragments occurred in the absence of an exogenously added enzyme source. The relevance of in vitro fatty acylation of PLP to that in vivo was confirmed by comparison of proteolytically derived peptide maps that showed that likely the same domain of PLP was acylated in vitro and in vivo.

Cross-linking studies on the conformation and dimerization of myelin basic protein in solution.

Myelin basic protein was isolated from both cat and bovine central nervous system. Cat and bovine myelin basic protein, which are shown to be similar by tryptic mapping, exhibit identical behavior when cross-linked with the bifunctional reagent difluorodinitrobenzene. Myelin basic protein is cross-linked into only a dimer under certain conditions in the presence of sodium dodecyl sulfate. In contrast, many oligomers are formed when myelin basic protein is cross-linked in the absence of detergent. The formation of cross-linked dimers in the absence of other oligomer formation suggests that the protein is at least partly dimeric in the presence of sodium dodecyl sulfate. The conformation of them myelin basic protein monomer in sodium dodecyl sulfate was also studied. N-Bromosuccinimide and cyanogen bromide cleavage reactions were used to demonstrate that difluorodinitrobenzene had introduced intramolecular cross-links between the two peptides resulting from each of the cleavage ractions. However, these types of intramolecular cross-links cannot be detected under conditions in which only dimers have formed. Some of the lysine residues which are modified by difluorodinitrobenzene were identified by tryptic mapping. In several respects, the conformation of myelin basic protein in a sodium dodecyl sulfate solution appears to be similar to the conformation of the protein in the membrane.