Forms Of Myelin Basic Protein Research Articles

Pediatric multiple sclerosis: an integrated outlook at the interplay between genetics, environment and brain-gut dysbiosis.

Multiple sclerosis (MS) is a debilitating autoimmune condition caused by demyelination, neurodegeneration and persistent inflammation of the central nervous system. Pediatric multiple sclerosis (PMS) is a relatively rare form of the disease that affects a significant number of individuals with MS. Environmental exposures, such as viral infections and smoking, can interact with MS-associated human leukocyte antigens (HLA) risk alleles and influence the immune response. Upregulation of immune response results in the disruption of immune balance leading to cascade of inflammatory events. It has also been established that gut microbiome dysbiosis poses a higher risk for pro-inflammation, and it is essentially argued to be the greatest environmental risk factor for MS. Dysbiosis can cause an unusual response from the adaptive immune system and significantly contribute to the development of disease in the host by activating pro-inflammatory pathways that cause immune-mediated disorders such as PMS, rendering the body more vulnerable to foreign attacks due to a weakened immune response. All these dynamic interactions between biological, environmental and genetic factors based on epigenetic study has further revealed that upregulation or downregulation of some genes/enzyme in the central nervous system white matter of MS patients produces a less stable form of myelin basic protein and ultimately leads to the loss of immune tolerance. The diagnostic criteria and treatment options for PMS are constantly evolving, making it crucial to have a better understanding of the disease burden on a global and regional scale. The findings from this review will aid in deepening the understanding of the interplay between genetic and environmental risk factors, as well as the role of the gut microbiome in the development of pediatric multiple sclerosis. As a result, healthcare professionals will be kept abreast of the early diagnostic criteria, accurately delineating other conditions that can mimic pediatric MS and to provide comprehensive care to individuals with PMS based on the knowledge gained from this research.

The identification of phosphorylated forms of myelin basic protein associated with mitochondria

Current data demonstrate that neurodegenerative processes are associated with mitochondrial dysfunction. One of the causes of mitochondrial dysfunction is increased permeability of the inner membrane and the formation of the so-called non-selective mitochondrial permeability transition pore (mPTP), which is recognized as an early stage of programmed cell death. One of the factors of neurodegeneration is disruption of the interaction between glial cells and axons. We recently demonstrated that the myelin protein 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), which is localized in mitochondria, is involved in the regulation of the mPTP. Here, we show that two phosphoproteins with molecular weights of 21.5 and 17 kDa increase their phosphorylation state after pore opening. Using two-dimensional electrophoresis followed by mass spectrometry, we identified these proteins as phosphorylated isoforms of the myelin basic protein (MBP). We found that association of the 21.5 and 17 kDa isoforms of MBP increased under conditions of an opened pore. Thus, one more myelin protein is probably involved in the regulation of the mPTP.

Interleukin 17-producing T-helper cells and autoimmune diseases: Time for a paradigm shift?

Introduction For almost 20 years, the pathogenic role of helper T cells has been intensively studied in autoimmune diseases. In multiple sclerosis (MS), an autoimmune disorder affecting the brain and spinal cord, overwhelming evidence has indicated that interferon (IFN)–secreting T-helper type 1 (Th1) cells are involved in the inflammatory process leading to the destruction of myelin sheath [1]. Adoptive transfer of Th1 cells reactive to myelin antigen cause an MS-like disease called experimental autoimmune encephalomyelitis (EAE), supporting this postulate. Notably, the role of Th1 cells in MS has been emphasized not only by experiments with laboratory animals but also by unwanted results of clinical trials. In past clinical trials, administration of IFNpromoted exacerbations of MS, whereas recombinant IFNhas been shown to suppress them [2]. In a more recent study, an altered form of myelin basic protein (MBP) peptide was evaluated for its therapeutic potential. However, it caused serious relapses and massive expansion of MBP-reactive Th1 cells [3]. The deleterious effects of IFNand the MBP analogue peptide have been regarded as evidence for the role of Th1 cells in the pathogenesis of MS. On the other hand, T-helper 2 (Th2) cells that secrete interleukin (IL)-4, IL-5, and IL-10 were shown to counterregulate Th1 cell functions via secretion of Th2 cytokines. This finding led to speculation that autoimmune diseases could be cured by shifting the functional balance between Th1 cells and Th2 cells toward Th2. The relevance of this idea has been verified vigorously in numerous studies, which have actually demonstrated that Th2-biasing agents are effective for downmodulating clinical and pathologic manifestations of EAE. Given such theoretic, empiric, and experimental evidence, our understanding of the pathogenesis of autoimmune diseases has been heavily influenced by the Th1/Th2 paradigm [4]. However, contradictory to the “Th1 disease” dogma, EAE and collagen-induced arthritis (CIA) became worse in mice deficient in those molecules that are critical for Th1 cell development (eg, IL-12 receptor 2 and IFN) [5]. Thus, the question has arisen as to whether Th1 cells are truly needed for induction of autoimmune diseases. The recent discovery of a new T-helper cell subset characterized by production of IL-17 has clarified that the IL-17–producing T-helper cells (Th17) would mediate autoimmune disease models including EAE, CIA, and experimentally induced colitis without help from Th1 cells [6–9]. Very recent papers have identified that a combination of proinflammatory cytokine (IL-6) and anti-inflammatory cytokine (transforming growth factor [TGF]) would cause differentiation of Th17 cells [10–12], unveiling a new principle governing the maintenance and disruption of immunologic tolerance.

The effect of a circulating monoclonal antibody (mAb) recognizing a native form of myelin basic protein (MBP) on clinical and histopathological expression of EAE in DA rats

The effect of a circulating monoclonal antibody (mAb) recognizing a native form of myelin basic protein (MBP) on clinical and histopathological expression of EAE in DA rats

HLA-DR-restricted presentation of purified myelin basic protein is independent of intracellular processing.

Antigen presentation to CD4+ T cells involves intracellular antigen processing and loading of peptides onto newly synthesized major histocompatibility complex (MHC)-class II molecules. Some antigens, such as the lipid-bound, native form of myelin basic protein (LB-MBP) can also be presented by recycling of cell surface MHC class II molecules. The data reported here demonstrate that a preparation of highly purified, delipidated MBP (HP-MBP) follows yet another presentation pathway. Similar to LB-MBP, presentation of HP-MBP to HLA-DR1-restricted T cells was independent of HLA-DM, of newly synthesized proteins, and of invariant chain expression. However, in contrast to LB-MBP, presentation of HP-MBP was also independent of internalization of surface HLA-DR molecules. The different requirements for the presentation of the two molecular forms of MBP were further confirmed by use of the protease inhibitor E64: presentation of LB-MBP but not of HP-MBP was inhibited after treatment of target cells with E64. Furthermore, intact HP-MPB bound to isolated HLA-DR molecules in vitro with an association rate that was considerably faster than that of short peptides. These results show that presentation of HP-MBP is independent of intracellular processing and suggest that it may be presented to T cells by direct binding to surface HLA-DR molecules.

Localization and partial characterization of a 60 kDa citrulline-containing transport form of myelin basic protein from MO3-13 cells and human white matter.

The localization of myelin basic proteins (MBPs) in an immortalized human-human hybrid cell line (MO3-13) formed by fusion of rhabdomyosarcoma TE671-TG6 with primary human oligodendrocytes, cultured from surgical specimens, demonstrated an intracellular localization in vesicles and vacuoles with an intricate internal membranous network and to the external surface of the cell by immunogold electron microscopy. The availability of antibodies to one of the components of MBP, i.e., the citrulline containing component ("C-8"), permitted us to localize this component of MBP to intracellular vacuoles and also on the external surface of the MO3-13 cells. Since the apposition of the external surfaces of the oligodendrocyte is responsible for the intraperiod line of the myelin sheath, localization of C-8 to the external surface of non-permeabilized cells by immunogold scanning electron microscopy is consistent with our observations that C-8 is localized to the intraperiod line of myelin (McLaurin et al.: J Neurosci Res 35:618-628, 1993). Western blots of isolated MBP from MO3-13 cells, probed with an antibody reactive with residues 130-137 of MBP, recognized a protein in the 60 kDa range. No immunoreactivity was found in the 18.5 kDa range. This 60 kDa protein also reacted with a monoclonal antibody raised with residues 70-84 of MBP, 2 different polyclonals raised with whole bovine MBP, an antibody to human MBP raised in monkeys, and the anti-citrulline antibody. These data strongly suggested that the 60 kDa protein contained MBP sequences within its primary structure. A similar protein has been isolated from human myelin-containing fractions but not from compact myelin demonstrating that the 60 kDa protein from MO3-13 cells was not an artefact related to fusion. Sequence determination of peptides obtained from enzymic and chemical cleavages revealed that the 60 kDa protein contained MBP sequences and peptides with 55-60% homology with dynamin, a protein involved in intracellular transport. These data suggest that the externalization of MBP in this cell involves transport by fusion of MBP with another protein. By sequestering MBP in a larger protein, the possibility of inducing autoimmune disease by MBP released, due to cell death, is minimized.

Antibodies specific for the lipid-bound form of myelin basic protein during experimental autoimmune encephalomyelitis

On the hypothesis that myelin basic protein isolated with surrounding lipids may constitute an autoantigen in demyelinating diseases we studied the antibody response to the lipid-free and lipid-bound form of myelin basic protein during the course of experimental autoimmune encephalomyelitis induced in rats with either form of protein. Immunization with the lipid-bound form of myelin basic protein induced high titres of antibodies directed to the protein, accompanied by no antibodies to cerebroside 30 days after immunization. Antibodies specifically directed to the lipid-bound form of myelin basic protein were revealed after removal of antibodies recognizing the delipidated myelin basic protein. Anti lipid-bound myelin basic protein antibodies could already be detected at day 10 post-immunization, reaching a maximum at day 20 post-immunization. Demonstrations of antibodies entirely specific for the lipid-bound form of myelin basic protein suggests that this molecule may present epitopes not to be found in its already extensively studied primary structure, possibly the result of conformational changes following lipid binding.

Characterization of a cell line derived from a human oligodendroglioma.

A novel clonal cell line derived from a human glioma (HOG) was found to express some oligodendrocyte-specific proteins including a 15-kDa form of myelin basic protein (MBP) and high 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Expression of the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide) was low. HOG cells did not express the characteristic astrocyte markers glial fibrillary acidic protein (GFAP) or significant glutamine synthetase (GS) activity. After initial plating, HOG cells were flat and epitheloid and thus showed a limited oligodendrocyte-like morphology. However, after cells became more confluent, some cells were phase-bright and elaborated short processes. Receptor types expressed by HOG cells included A2-adenosine, prostaglandin E1 (PGE1), and beta 2-adrenergic receptors (beta-ARs) linked to stimulation of adenylate cyclase, and muscarinic cholinergic and H1-histamine coupled to phosphatidyinositol turnover (Post and Dawson, 1991). HOG cells should therefore provide a useful model for studying the extracellular regulation and phosphorylation of oligodendrocyte-specific proteins.

Detection of cerebrospinal fluid antibodies against myelin basic protein in patients with AIDS dementia complex

We evaluated cerebrospinal fluid (CSF) antibody levels against a lipid-free, denatured form of myelin basic protein (LF-MBP) in 11 patients with AIDS dementia complex (ADC) by using an enzyme-linked immunosorbent assay (ELISA). In 9 out of 11 patients, anti-LF-MBP antibody levels were significantly higher than those observed both in 15 human immunodeficiency virus (HIV)-infected patients without neurological disorders and in 9 anti-HIV-negative subjects affected by other neurological diseases. Furthermore, we followed up anti-MBP levels in 5 out of the 11 ADC patients and detected a strict relationship with the encephalopathy progression. At the same time, with the aim to detect early demyelinating events we investigated CSF antibody levels against a lipid-bound, native-like form of MBP (LB-MBP). Results did not show any significant difference between LF-MBP and LB-MBP in terms of antibody reactivity. The detection of anti-MBP antibodies in CSF may provide the opportunity to assess a diagnostic tool for discovering demyelinating lesions in ADC patients.

Metalloendoprotease Cleavage of 18.2‐ and 14.1‐Kilodalton Basic Proteins Dissociating from Rodent Myelin Membranes Generates 10.0‐ and 5.9‐Kilodalton G‐Terminal Fragments

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37 degrees C; pH 7.4). After 1-3 h, electrophoresis of the membranes revealed loss of 50% of 18.2- and 14.1-kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane-associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4-kDa and minor 10.0- and 5.9-kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2- and 14.1-kDa forms of MBP were cleaved near residue 73 to produce a 10.0- and 5.9-kDa C-terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5-20% using leupeptin (20 microM) but up to 50% by dithiothreitol mM), phenylmethylsulphonyl fluoride (1 mM), and phosphoramidon (50 microM) but up to 50% by dithiothreitol (DDT, 10 mM). Only DDT and 1,10-phenanthroline substantially blocked the formation of the characteristic 10.0-and 5.9-kDa C-terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N-ethylmaleimide/leupeptin-sensitive calpains and phosphoramidon-sensitive endopeptidase 24.11.

Equilibrium binding of myristoyllysophosphatidylcholine to bovine myelin basic protein: an example of ligand-mediated acceptor association

The interaction of myristoyllysophosphatidylcholine with bovine myelin basic protein at pH 7.4 and 4.5, I = 0.48, has been investigated by a recycling partition equilibrium technique with Bio-Gel P-2 as the gel phase. Important points to emerge from this direct binding study are that it is a monomeric (not micellar) amphiphile that binds to myelin basic protein, that the amphiphile binds preferentially to the monomeric form of myelin basic protein, that this binding to monomer is highly cooperative, that the similarity of binding behavior in the two environments tested is consistent with the dominance of a hydrophobic contribution to the protein-amphiphile interaction, and that the self-association of myelin basic protein in the presence of phospholipid [Smith, R. (1982) Biochemistry 21, 2697-2701] must reflect the aggregation of a protein-amphiphile complex(es) coupled with concomitant release of some lipid. These findings are then related to earlier nuclear magnetic resonance and circular dichroism studies in which the results were interpreted on the basis that myelin basic protein bound preferentially to micellar phospholipid.

Identification of a cDNA coding for a fifth form of myelin basic protein in mouse.

The primary sequences of four molecular mass variants (14, 17, 18.5, and 21.5 kDa) of the mouse myelin basic protein (MBP) have recently been determined through analysis of cDNA clones of their mRNAs. The mRNAs coding for the four MBP variants are thought to arise by differential splicing of two exons (exons 2 and 6) from a single gene. In contrast, exons 2 and 5 may be spliced out in the posttranscriptional processing of the human MBP gene. To investigate the possibility that a third exon (exon 5) may also be differentially spliced out in the processing of the mouse MBP gene transcript, a mouse cDNA library was screened to search for cDNAs missing exon 5. A MBP cDNA was isolated whose coding region specified a fifth mouse MBP variant with a molecular mass of approximately equal to 17 kDa. The mass of this variant (17,257 Da) is so close to that of the other 17-kDa mouse MBP (17,224 Da) that the two would be indistinguishable on NaDodSO4/polyacrylamide gels. Analysis of the sequence of the cDNA clone indicates that excision of exons 2 and 5 of the mouse MBP gene would produce the mRNA encoding this newly described 17-kDa MBP, whereas excision of exon 6 would produce the mRNA for the other 17-kDa MBP variant. Thus, the "17-kDa" mouse MBP consists of at least two molecular forms with very similar molecular masses but markedly different primary sequences. Of five full-length or near full-length cDNAs representing 17-kDa MBPs, one was missing exons 2 and 5 and four were missing exon 6.

A New Form of Myelin Basic Protein Found in Human Brain

Human myelin basic protein was subjected to ion-exchange chromatography at high pH to separate the differently charged components. Polyacrylamide gel electrophoretic patterns of the fractions showed that the less basic fractions 3, 4, and 5 contained significant amounts of a protein somewhat smaller than the more common 18.5-kDa form. Fraction 3 consisted of approximately equal amounts of this smaller polypeptide and component 3, the 18.5-kDa form found in other mammalian myelin basic protein preparations. The two proteins in fraction 3 were separated by fast protein liquid chromatography. Both have blocked N termini and identical C termini (-Met-Ala-Arg-Arg). When the tryptic digests of the two proteins were fractionated by HPLC, the elution profiles were similar, except that four peaks found in the chromatogram of the larger protein were missing from the chromatogram of the smaller one. In addition, an extra peak was found in the elution pattern of the latter chromatogram. Amino acid analysis of the individual tryptic peptides indicated that the smaller protein lacked residues 106-116 (-Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-). The deleted portion corresponds exactly to the amino acid sequence encoded by exon 5 of the mouse basic protein gene. This new form of myelin basic protein has a molecular weight of 17,200, calculated from its amino acid composition.

Recombination within the myelin basic protein gene created the dysmyelinating shiverer mouse mutation.

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint of the deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. The recombination junction shares several features with the junctions characterized by Anderson et al. [Anderson, R., Kato, S. & Camerini-Otero, D. (1984) Proc. Natl. Acad. Sci. USA 81, 206-210] in mouse L cells and is consistent with their model for a partially homologous recombination event. The structure of the shi recombination junction suggests that the donor DNA molecules were aligned in a partially homologous region before staggered cutting and joining occurred.

Alternative splicing accounts for the four forms of myelin basic protein

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

Spectroscopic assessment of secondary and tertiary structure in myelin basic protein

Myelin basic protein conformation and hydrophobicity, along with the protein's behavior in the presence of the fluorescent probe 6-(p-toluidino)-2-naphthalenesulfonate, have been studied by using Fourier transform infrared (FT-IR) and Raman spectroscopy. The FT-IR and Raman spectra provided compelling evidence for the presence of a small amount of beta structure, ca. 25%, in the aqueous solution and solid-state forms of myelin basic protein. The enhanced fluorescence and shift in the emission maximum of 6-(p-toluidino)-2-naphthalenesulfonate when bound to myelin basic protein are consistent with the presence of at least one hydrophobic region in the molecule. Loss of the fluorescence enhancement in the presence of denaturing agents indicates that native myelin basic protein has a folded structure in solution. All of the results provide support for conformational predictions derived from the application of Edmundson wheels to the primary structure.

The induction of liposome aggregation by myelin basic protein

Myelin basic protein derived from bovine spinal cord has been interacted with liposomes of varying brain lipid compositions. The effects of salt and protein concentration on liposome cross linking has been investigated. It appears that myelin basic protein cannot link liposomes composed of brain-derived phosphatidyl choline. Myelin basic protein can link liposomes composed of phosphatidyl serine; phosphatidyl serine + cholesterol; phosphatidyl serine + cholesterol + cerebroside sulphate. Linking of liposomes occurs at protein concentrations lower than those required for myelin basic protein dimers to be formed. Therefore, it seems that the monomeric form of myelin basic protein links lipid bilayers. The presence of cholesterol in the bilayer increases the ability of myelin basic protein to aggregate such liposomes compared with the linking ability of the polycationic polypeptide, poly-l-lysine.

Characterization of mouse myelin basic protein messenger RNAs with a myelin basic protein cDNA clone.

Using a family of synthetic tetradecamer oligonucleotides as a primer for cDNA synthesis and a second family of tetradecamers as a hybridization probe, we have prepared and isolated a cDNA clone of mouse myelin basic protein (MBP). The clone, pNZ111, corresponds to the region of the mRNA that codes for an amino acid sequence present in all four major forms of MBP. The relative abundance of MBP mRNA, estimated by dot blot hybridization, increased with the age of the mouse to a maximum at 18 days, then decreased to about one-fourth of that amount at later ages. Mouse MBP mRNAs, selected by their ability to hybridize to the clone, translate into the four forms of myelin basic protein. In RNA blot analyses, pNZ111 hybridized to multiple species of mouse mRNA. The predominant hybridization is to a broad band of RNAs ranging in length from 2,350 to 2,100 bases. These mRNA species are extremely long, considering that the largest MBP could be encoded by approximately 600 bases. In addition to these, there are also minor bands that hybridize with pNZ111, including a band of 4,100 bases and smaller ones of 1,900, 1,500, and 1,200 bases.

The effect of the shiverer mutation on myelin basic protein expression in homozygous and heterozygous mouse brain.

We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co-dominantly with the wild-type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wild-type amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild-type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +/shi animals. Third, the small amount of myelin basic protein-related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild-type brain. The pattern of myelin basic protein-related polypeptides in +/shi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein-related polypeptides qualitatively similar to that encoded by wild-type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein-related translation products are spared. The pattern of myelin basic protein-related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis-acting regulatory element controlling that gene (or genes).

Developmental regulation of myelin basic protein in dispersed cultures.

The expression of myelin basic protein, a major component of the myelin membrane, was studied in the absence of myelin formation in a unique situation in which these two processes have been uncoupled. The oligodendrocytes that contained myelin basic protein were identified by immunofluorescence in primary dispersed cultures derived from 20-day-old fetal rat brain. Their number increased 20-fold between 15 and 34 days in culture. Morphologically identifiable myelin was never observed. The oligodendrocytes elaborated a complex network of processes and membranous sheets resembling "unfurled" myelin. Myelin basic protein appeared concomitantly in the perikaryon, processes, and membranous sheets of the oligodendrocytes and remained distributed in these compartments throughout the culture period. The oligodendrocytes synthesized the four forms of myelin basic protein found in rodent brain with molecular weights of 21,500, 18,500, 17,000, and 14,000 and modulated their expression with time in culture. The onset of rapid myelin basic protein accumulation, as measured by radioimmunoassay, took place after 25 days in culture. Myelin basic protein accumulated at the rate of 0.2 fmol per oligodendrocyte per day and reached a level of 300 pmol/mg of protein by 34 days. By 60 days, the amount of myelin basic protein had declined to 100 pmol/mg of protein, a level maintained up until at least 120 days. When the amount of myelin basic protein was correlated with the number of oligodendrocytes, it was estimated that each induced cell contained on average 1 fmol of this protein at the three time points (15, 28, and 34 days in culture) at which cells were counted. Our results indicate that the accumulation, modulation of the molecular forms, and insertion of myelin basic protein into the membrane can occur in the absence of myelin formation, but that continued metabolic stability, subcellular sequestration, and fine control of the relative proportions of the different forms of myelin basic protein may be dependent on myelin morphogenesis.