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Several cell adhesion molecules of the immunoglobulin superfamily (IGSF) are expressed differently during the development and myelination of the peripheral nervous system. To examine the relationship between the expression of IGSF molecules and Schwann cell differentiation, we established a useful system for myelin formation in vitro on collagen gel using primary neuron/Schwann cell co-cultures from neonatal dorsal root ganglions (DRG). At 10 days in vitro (DIV), many Schwann cells were found in the areas surrounding aggregates of DRG neurons. After 20 DIV, Schwann cells positioned next to axons and elongated their processes along the axons. Some of them started loosely elaborating a large axon. Under electron microscopy, compact myelin was shown to be formed at 30 DIV. Thus the speed of myelination was much slower in vitro than in vivo. In co-cultures, L1 and neural cell adhesion molecule (NCAM) were detected at the premyelinating stage, L1 was precisely expressed earlier than NCAM. Expression of myelin associated glycoprotein (MAG) was transiently up-regulated at the early stage of myelination, and then P0 expression was finally increased as myelination proceeded. The change of expression pattern of these molecules in co-cultures was quite similar to that observed in the development in vivo. When Schwann cell proliferation was blocked by low serum culture condition, L1 and NCAM expressions were up-regulated. In contrast, the presence of cholera toxin in low serum media markedly increased expressions of P0 and MAG, but decreased the levels of both L1 and NCAM. These results suggest that both L1 and NCAM play roles in the contact and/or recognition between axons and Schwann cells at an early stage of myelination. On the other hand, MAG and P0 are important for axon ensheathment and myelin compaction.

The reaction of oligodendrocyte progenitor cells (OPCs) after spinal cord injury (SCI) is poorly understood. In this study, we examined oligodendroglial reactions after contusion SCI in adult rats by immunohistochemistry. OPCs were identified by staining with monoclonal antibodies (mAbs) A2B5 and O4. Each of the A2B5-, O4-positive OPCs and galactocerebroside-positive oligodendrocytes dramatically increased in the lesion of the dorsal posterior funiculus. Bromodeoxyuridine (BrdU) incorporation studies showed that most O4-positive cells in the lesion were labeled with BrdU, suggesting that these OPCs were proliferative. In contrast, the expression of myelin basic protein was decreased in the lesion compared with controls that received laminectomy only. From the injured cord, OPCs were isolated by immunopanning with mAb A2B5. We observed an increased number of OPCs from the injured spinal cords compared with those isolated from controls and unoperated animals. After several days in culture, the OPCs from the lesion expressed galactocerebroside. These results suggest that OPCs are induced and can differentiate following SCI in the adult rat., (Copyright 2001 Wiley-Liss, Inc.)

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.

PASII/PMP22 protein (mol. wt 22 kDa, pI 8.8) is an abundant and extremely hydrophobic glycoprotein of PNS myelin which is solubilized effectively by SDS. In humans, this protein is involved in hereditary neuropathies. Here we describe a simple method for purification of PASII/PMP22, suitable for crystallization trials. We usually obtained 10-20 mg PASII/PMP22 from 10 g bovine spinal roots. It is notable, that the original protocol was designated for purification of P0 myelin glycoprotein, and purification of PASII/PMP22 is a bonus. Extensive crystallization trials are in progress.

At the premyelinating stage, the Schwann cells of peripheral nerves are able to recognize the axon, to arrange themselves along it in a nonoverlapping manner, and finally to establish a one-to-one cell-axon relationship. The mechanism that regulates these processes is not known in detail. We found the existence of a significant Schwann cell apoptosis in vivo of rat postnatal sciatic nerve, peaking around postnatal day 3. More than 50% of the neonatal Schwann cells cultured in axon-free medium undergo a rapid apoptosis. The apoptosis can be suppressed by addition of survival factors such as Neu differentiation factors or by increasing the adhesion of Schwann cells to substratum. We suggest that in neonatal nerves in vivo, Schwann cells are highly susceptible to apoptosis, but they are saved from death by contact with axons. The dramatic increase in number of Schwann cells between postnatal day 0 and 3 overcomes the number of axons available for them. Consequently the Schwann cells that fail to contact an axon undergo apoptosis. In conclusion, the number of Schwann cells in the developing nerves is regulated by the apoptosis and clearly depends on the survival signals from axons.

We demonstrated the cell lineage of oligodendrocytes from the glial precursor cells to mature oligodendrocytes forming myelin sheath around the axon. There are several different stages of oligodendrocyte development in vito. So far there are no precise data about their morphological changes during oligodendrocyte development, but by the analysis using SEM and immunostaining, the characteristic morphological changes with serial expression of cell markers were observed in each developmental steps of oligodendrocyte. We have also clearly demonstrated how oligodendrocytes wrap around the axon by using video time-lapse movies. These results will be useful for understanding the exact cellular mechanism of myelination in the CNS.

Oligodendrocyte precursor cells that develop into myelin-forming cells of the central nervous system (CNS) were cultured from newborn rat brain to study how they proliferate and differentiate in normal conditioning medium, and their cell development was characterized by scanning electron microscopy (SEM) observation and immunocytochemical studies. We have identified A2B5-negative pre-O2A progenitor cells (so-called "type-1" oligodendrocytes) in the secondary cultures on the astrocyte feeder layer. These cells are very small (diameter: 3.5 microns), round, and glossy, and develop into the process-bearing O2A progenitor cells (called "type-2" oligodendrocytes), which also express myelin basic protein (MBP) both in the cell body and in their cell processes. Finally, they develop into mature oligodendrocytes (called "type-3" oligodendrocytes). After MBP expression is elicited in these cells and MBP accumulates in the cell process in the area in contact with the axon, these cells are capable of forming the myelin sheath. Therefore, we examined the mechanism of myelin-sheath formation of "type-3" oligodendrocytes using video time-lapse movies, and demonstrated that these cells initially sent out processes to search for axons several times before the onset of myelination. Then thick filopodia extended towards the axon, and at the same time, the axonal part of neuron moved forward. Finally the ruffling lamellipodial parts wrapped up the axon similarly to a transverse wave with the secured thick filopodial process on the axon acting as scaffolding. These results suggest that our experimental systems are useful in studying normal oligodendrocyte development and their cellular biochemistry, as well as investigating the mechanism of myelin formation by oligodendrocytes.

Oligodendrocyte precursor cells were cultured from newborn rat brain and studied their differentiation and proliferation. They have identified type-1, type-2, and type-3 oligodendrocytes based on the expression of characteristic marker molecules that frequently used to stage oligodendrocyte development. The type-3 oligodendrocytes were observed to send but tentative that locate axons prior to myelination. These processes terminate in lamellipodia, which eventually enwrap the axon and begin the myelination process with several steps. At the first stage, ruffling is immediately induced at the lamellipodia with filopodia made of oligodendrocyte processes, and the axon is contacted several times; then process retraction occurs to reform the filopodial and lamellipodial parts prior to the onset of the myelination. Second, after filopodial movements and lamellipodial ruffling occur again, their morphology is dramatically changed to become three thick filopodia that anchor to the axon. Finally, lamellipodial ruffling parts ripple, the angle between the position of the resting filopodium and the axon change, depending on the start of axonal movement, and the lamellipodia turn around the axon like a transverse wave with one stroke of the brush, as observed on the video screen, and their rolling membrane changes to the bursting form within minutes in real time.

A full length cDNA of PMP-22 (PAS-II/SR13/Gas-3) of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 1823 base pairs (bp) in length and contains a 480 bp open reading frame encoding a polypeptide of 160 residues. The deduced amino acid sequence is highly homologous to PMP-22 from bovine (PAS-II), rat (SR13) and mouse (Gas-3).

Myelin proteins in mammalian peripheral nerve were studied comparatively. 1. While each content of P1 and P2 in the myelin varied among species, additional content of P1 and P2 are relatively constant. 2. The antigenic determinants of P2 for induction of experimental allergic neuritis were reported. 3. Amino acid sequence analysis of P0 revealed that P0 is conserved across species and belongs to the immunoglobulin superfamily. 4. The characteristic carbohydrate chain of P0 containing sulfate and sialic acid showed a positive reaction to the molecule-related immunity and adhesion. 5. Molecular architecture of the myelin is discussed.

The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid-dependent protein kinase (protein kinase C), calcium/calmodulin-dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.

Membrane preparations from chick peripheral nervous system (PNS) catalyzed the transfer of [3H]glucose from UDP-[3H]glucose into glucosylphosphoryl dolichol. The initial rate of glucosylphophoryl dolichol formation in a non-myelin membrane fraction from actively myelinating chick PNS was 11 fold higher than that from adult. Exogenous dolichyl monophosphate stimulated glucosylphosphoryl dolichol synthesis in both fractions. The higher level of glucosylphosphoryl dolichol synthesis corresponded to the onset of myelination in chick PNS. Exogenous dolichyl monophosphate also stimulated the labeling of glucosylated oligosaccharide lipids and glycoproteins in the fraction. On SDS polyacrylamide gel electrophoresis, the relative mobility of the major and minor radioactive glycoprotein corresponded with that of the P0 and PASII glycoprotein in PNS myelin, respectively. The results suggest that myelin glycoproteins in PNS are glycosylated via lipid intermediates.

Experimental allergic neuritis (EAN) is an experimentally produced demyelinating disease of peripheral nervous system. Several peptides of bovine P2 protein were tested for neuritogenic activity in Lewis rats. The hexacosapeptide CiT4 (residues 53--78 of bovine P2 protein) showed the highest neuritogenic activity among the peptides tested. The nonapeptide (residues 70--78) and the tridecapeptide (residues 66--78) were synthesized using the liquid phase peptide synthesis technique. The tridecapeptide showed mild, but definite activity in inducing EAN in the rats, while the nonapeptide was inactive. The localization of the neuritogenic determinant of bovine P2 protein in Lewis rats is discussed.

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins, could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2- terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.

1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide.