myelin-basic-protein and galactocerebroside

During postnatal development of the higher vertebrate CNS, large populations of oligodendroglia are generated from precursor cells in a very dependable way. In adult lesioned CNS tissues, local populations of oligodendroglia are replenished by proliferation of this replenishment varies from one species to another and also from one lesion type another. Studies on the developmental generation of oligodendroglia are reviewed here, delineating what is known of the early relationships between the CNS glial lineages and of what regulates this development. Contributions from recent cell biological work are considered against the background of morphological and radioautographic results. The quiescent condition of extremely slow turnover in the normal adult CNS is noted, and the dramatic effects of lesions on the neural cell environment are considered. Lesions can trigger proliferation at a much greater rate in the mature oligodendroglial population, as observed both in situ and in tissue culture; in addition to persisting stem cells, the mature cells participate in replenishing the local oligodendroglial population. This regeneration from cells already committed to the oligodendroglial lineage may minimise such disturbing effects of the lesion environment as might distort replenishment of the population from precursor cells.

Topics: Animals; Blood-Brain Barrier; Cell Differentiation; Cell Separation; Cell Survival; Cells, Cultured; Central Nervous System; Demyelinating Diseases; Galactosylceramides; Glial Fibrillary Acidic Protein; Glucose; Humans; Insulin; Mice; Mice, Neurologic Mutants; Mitosis; Myelin Basic Protein; Myelin Proteins; Myelin Sheath; Neuroglia; Oligodendroglia; Rats

We have established and efficient system to specify NG2/PDGF-Rα/OLIG2+ oligodendrocyte precursor cells (OPCs) from human embryonic stem cells (hESCs) at low, physiological (3%) oxygen levels. This was achieved via both forebrain and spinal cord origins, with up to 98% of cells expressing NG2. Developmental insights reveal a critical role for fibroblast growth factor 2 (FGF-2) in OLIG2 induction via ventral forebrain pathways. The OPCs mature in vitro to express O4 (46%) and subsequently become galactocerebroside (GALC), O1, and myelin basic protein-positive (MBP+) multibranching oligodendrocytes. These were cultured alongside hESC-derived neurons. The electrophysiological properties of human OPCs are similar to those of rat OPCs, with large voltage-gated sodium currents and the ability to fire action potentials. Exposure to a selective retinoid X receptor agonist increased the proportion of O4+ oligodendrocytes that express MBP from 5% to 30%. Thus, we have established a developmentally engineered system to investigate the biological properties of human OPCs and test the effects of putative remyelinating agents prior to clinical application.

Topics: Action Potentials; Basic Helix-Loop-Helix Transcription Factors; Cell Lineage; Cells, Cultured; Embryonic Stem Cells; Fibroblast Growth Factor 2; Galactosylceramides; Humans; Myelin Basic Protein; Nerve Tissue Proteins; Neural Stem Cells; Neurogenesis; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Oxygen; Prosencephalon; Retinoid X Receptors; Sodium; Spinal Cord

To investigate age-related intrinsic regulation of the capacity of human fetal oligodendrocyte progenitor cells (OPCs) to myelinate, potential OPCs were selected from 15- to 23-gestational-week (gw) human fetal brain tissue based on the expression of gangliosides--recognized with the monoclonal antibody A2B5, which detects multipotent cells including OPCs--or platelet-derived growth factor receptor α (PDGFRα), an early marker of the oligodendroglial lineage. Cells were either cultured alone or cocultured with rat dorsal root ganglia neurons (DRGNs). When cultured alone, both the A2B5- and PDGFRα-selected cells exhibited age-dependent increases in early to mid-stage lineage markers, including sulfatides (O4 antibody) and the transcription factor Olig2, while the cell death rate correlated negatively with age. In coculture with neurons, cells also expressed the myelin components galactocerebroside (GC) and myelin basic protein (MBP), and ensheathed axons. In DRGN cocultures, A2B5+ cells derived from >19 gw produced more GC+/MBP+ cells compared with the 15-17-week cells. The number of GC+ cells making axonal contacts, and ensheathing axonal segments per cell increased proportionally to gestational age. This age-dependent difference in GC/MBP cell number and capacity to ensheath axons persisted when PDGFRα selection was used to enrich for the number of OPCs in cultures derived from younger ages. Addition of the growth factors brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) enhanced OPC differentiation under all conditions. These findings indicate that intrinsic regulatory mechanisms associated with the chronological age of the donor cells are key variables to assess when considering the myelination capacity of OPCs for cellular replacement therapy.

Topics: Age Factors; Animals; Antibodies, Monoclonal; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Brain; Brain-Derived Neurotrophic Factor; Cell Count; Cell Death; Cell Differentiation; Cell Lineage; Cells, Cultured; Coculture Techniques; Fetus; Galactosylceramides; Ganglia, Spinal; Gestational Age; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Neurons; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Rats; Receptor, Platelet-Derived Growth Factor alpha; Stem Cells

Galactocerebroside (GalC) and its sulfated derivative sulfatide (SUL) are galactosphingolipids abundantly expressed in oligodendrocytes (OLs). Despite their biological importance in OL development and function, attempts to visualize GalC/SUL in tissue sections have met with limited success. This is at least in part because permeabilization of tissue sections with detergents such as Triton X-100 results in significant degradation of GalC/SUL immunoreactivity. Here we establish a novel method that enables visualization of endogenous GalC/SUL in OLs and myelin throughout the entire depth of brain sections. We show that treating brain sections with the cholesterol-specific detergent digitonin instead of Triton X-100 or methanol leads to efficient antibody penetration into tissue sections without disrupting GalC/SUL immunoreactivity. We also determine the optimal concentrations of digitonin using confocal microscopy. With our method, the morphology and the number of GalC/SUL-expressing OLs can be visualized three-dimensionally. Furthermore, our method is applicable to double immunostaining with anti-GalC/SUL antibody and other antibodies which recognize intracellular antigens. Our simple method using digitonin should prove to be useful in enabling detailed examination of GalC/SUL expression in the brain in both physiological and pathological conditions.

Topics: Animals; Animals, Newborn; Autophagy-Related Proteins; Brain; Detergents; Digitonin; Dose-Response Relationship, Drug; Galactosylceramides; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Myelin Basic Protein; O Antigens; Octoxynol; Oligodendroglia; Phosphopyruvate Hydratase; Sulfoglycosphingolipids

Insulin-like growth factor-1 (IGF-1) interacts with the Type I receptor to activate two main signaling pathways, the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K)-Akt cascades, which mediate proliferation or survival of oligodendrocyte (OL) progenitors (OLPs). In other cellular systems, mammalian target of rapamycin (mTOR) and the p70 S6 kinase are downstream effectors that phosphorylate translation initiation factors (e.g. eIF-4E), their regulators (e.g. 4E-binding protein 1, 4E-BP1) and ribosomal protein S6 (S6). The aim of this study was to determine whether these pathways are involved in IGF-1-stimulated protein synthesis, important for growth and differentiation of OLs. Rat cultured OLPs were treated with IGF-1 with or without inhibitors of PI3K (LY294002 or Wortmannin), mTOR (rapamycin), MEK (PD98059), and Akt (III or IV), as well as an adenovirus encoding a dominant negative form of Akt. Protein synthesis, as assessed by [(35)S]-methionine incorporation, was stimulated by IGF-1 and required the upstream activation of PI3K, Akt, mTOR and MEK/ERK. Concordant with the experiments using protein kinase inhibitors, western blotting revealed that IGF-1 stimulates phosphorylation of Akt, mTOR, ERK, S6 and 4E-BP1. Activation of S6 and inactivation of 4E-BP1, necessary for protein synthesis to take place, were dependent on the upstream activation of PI3K and mTOR. Finally, IGF-1 consistently stimulated protein synthesis through mTOR in differentiating OLPs but mRNA transcription was not required at day 4, indicating a differential role of IGF-1 throughout OL development.

Topics: Animals; Animals, Newborn; Cells, Cultured; Complement C3; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Galactosylceramides; Gangliosides; Glial Fibrillary Acidic Protein; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinases; Myelin Basic Protein; Oligodendroglia; Phosphatidylinositol 3-Kinases; Protein Biosynthesis; Protein Kinases; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Stem Cells; TOR Serine-Threonine Kinases; Transfection

Mature oligodendrocytes myelinate axons in the CNS. The development of the myelin sheath is dependent on the proper maturation of oligodendrocytes from precursors cells, a spatially restricted process that is regulated by inductive and repressive cues. Several members of the bone morphogenetic protein family (BMP2 and 4) have been implicated as repressors of oligodendrocyte development in vitro by shifting oligodendrocyte precursors into the astrocyte lineage. We now report on a second role of BMPs in oligodendrocyte development, regulation of myelin protein expression in immature oligodendrocytes. Purified immature rodent oligodendrocytes treated with BMP4 maintained galactocerebroside (GalC) expression, whereas the expression of three key myelin proteins, proteolipid protein (PLP), myelin basic protein (MBP), and 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP), was severely decreased. Paradoxically, BMP-treated oligodendrocytes show increased process extension and complexity, normally a feature of oligodendrocyte maturation. We also investigated whether BMP4 could inhibit myelin protein expression in an E 12.5 mouse explant culture of cervical spinal cord and hindbrain that maintains the in vivo cellular relationships and architecture. Beads soaked in BMP protein implanted into these explants inhibited the expression of myelin proteins, proteolipid protein, and myelin-associated glycoprotein (MAG), in the local area surrounding the bead. Since these explants also contained precursors cells, expression of galactocerebroside and O4, an oligodendrocyte marker, were also decreased by BMP treatment but to a much lesser degree than the myelin markers. Together, these data indicate that BMPs have multiple roles in oligodendrocyte development. At earlier stages, they affect cell lineage decisions and at later stages, they inhibit cell specialization.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Antigens, Differentiation; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Central Nervous System; Down-Regulation; Galactosylceramides; Growth Inhibitors; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Associated Glycoprotein; Nerve Fibers, Myelinated; Oligodendroglia; Rats; Rats, Sprague-Dawley; Stem Cells

To probe the effects of possible inhibitors or enhancers of in vivo myelination, we have modified a technique widely used in studies of the developing neuromuscular system that involves incorporation of test compounds into a silicon rubber solution, which solidifies on contact with air. U-shaped rubber implants are inserted around the sciatic nerve of 1-day-old rats and left in place for 24-48 h. Sections from the region of the nerve lying within the implant, with or without the test compound, are then immunolabeled, examined with in situ hybridization or electron microscopy. Application of EDTA (440 microg/implant) in this way strongly suppressed the levels of the myelin-associated molecules protein P0, myelin basic protein (MBP), and galactocerebroside (Galc). mRNA levels for P0 and the myelin-related transcription factor Krox-20 were also reduced, further supporting association of the EDTA-induced effect with the myelinating Schwann cells. In contrast, no obvious differences were observed in either neurofilament (NF) protein or glial fibrillary acidic protein (GFAP) expression, suggesting absence of influence on axons or nonmyelinating Schwann cells. Despite the severely altered molecular composition of myelin in the presence of EDTA, examination in the electron microscope did not reveal any apparent ultrastructural changes in the myelin sheaths or nerve development. This work introduces a novel method for studying nerve development and shows that EDTA, which chelates divalent cations such as Ca(2+) and Mg(2+), strongly and selectively reduces levels of molecules, which, on postnatal days 1-4, are expressed in myelinating cells at much higher levels than in cells not engaged in myelination.

Topics: Animals; Animals, Newborn; Chelating Agents; Disease Models, Animal; DNA-Binding Proteins; Down-Regulation; Drug Carriers; Early Growth Response Protein 2; Edetic Acid; Galactosylceramides; Growth Inhibitors; Implants, Experimental; Microscopy, Electron, Transmission; Myelin Basic Protein; Myelin P0 Protein; Myelin Proteins; Myelin Sheath; Nerve Regeneration; Neurosurgical Procedures; Rats; RNA, Messenger; Schwann Cells; Sciatic Nerve; Silicone Elastomers; Transcription Factors

Neurological deficit in experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) is probably a consequence of synergy between T and B cell responses to CNS antigens. During the demyelinating phase of chronic relapsing EAE in ABH mice, anti-myelin oligodendrocyte glycoprotein (MOG) responses were increased compared to the inflammatory acute phase, but such levels did not correlate with the severity of clinical disease. The pathogenicity of antibodies (Ab) to MOG, myelin basic protein (MBP), proteolipid protein (PLP) and galactocerebroside (GalC) was investigated in vivo following injection at the onset of EAE. An IgG2a monoclonal Ab (mAb), clone Z12, directed to MOG augmented clinical disease and demyelination in ABH and C57BL/6 mice, but not MOG knock-out mice. No effect was observed with F(ab(2))' fragments of Z12 or with the anti-MOG IgG1 mAbs, clones Y10 or 8-18C5. Cobra venom factor partially reduced the augmenting effect of mAb Z12 suggesting a role for complement. The pathogenic effect of anti-myelin Abs was not restricted to MOG since an anti-GalC mAb exacerbated inflammation in the CNS while an MBP mAb (clone 22) reduced clinical disease. Taken together, these data provide further evidence that myelin-reactive Abs generated during autoimmune neurological disease may play an important role not only in the pathogenesis of disease but also the regulation of myelin-targeted autoimmune disease.

Topics: Animals; Autoantibodies; Complement System Proteins; Demyelinating Diseases; Disease Models, Animal; Elapid Venoms; Encephalomyelitis, Autoimmune, Experimental; Female; Galactosylceramides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Myelin Proteins; Myelin Proteolipid Protein; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Recurrence; Spinal Cord

Evidence has been accumulated showing that neurosteroids, particularly progesterone (PROG) and its metabolites, may participate in myelination and remyelination in the peripheral nervous system, but very few studies have been undertaken in the central nervous system (CNS). The aim of this work was to investigate the capacities of synthesis and metabolism of PROG at three important stages of the oligodendroglial lineage: oligodendrocyte pre-progenitors (OPP), oligodendrocyte progenitors (OP), and fully differentiated oligodendrocytes (OL). Experiments have been conducted in vitro using highly purified primary cell cultures from rat brain. Cells were incubated with (3)H-pregnenolone ((3)H-PREG), the immediate precursor of PROG, or with (3)H-PROG, and steroids metabolites were then identified by thin layer chromatography and high-performance liquid chromatography (HPLC). mRNA expression of key steroidogenic enzymes was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that only OPP and OP, but not OL, expressed 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA and were able to synthesize PROG from PREG. In the three cell types studied, PROG was metabolized by the type 1 isoform of 5 alpha-reductase into 5 alpha-dihydroprogesterone (5 alpha-DHPROG). This enzyme exhibited a 5-fold higher activity in OL than in OPP and OP. 5 alpha-DHPROG was further transformed either into 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THPROG), known as a positive allosteric modulator of the GABA(A) receptor, or into the 3 beta-isomer. The 3 alpha,5 alpha-THPROG synthesis was 10 times higher in OPP than in the other cell studied, while the 3 beta,5 alpha-THPROG production did not change with cell differentiation. PROG synthesis and metabolism and the dramatic changes in neurosteroidogenesis observed during the oligodendroglial differentiation may contribute to oligodendrocyte development or the myelination process.

Topics: Aging; Animals; Animals, Newborn; Astrocytes; Brain; Cell Differentiation; Cell Lineage; Cells, Cultured; Ectodysplasins; Fluorescent Antibody Technique; Galactosylceramides; Gangliosides; Glial Fibrillary Acidic Protein; Membrane Proteins; Microglia; Myelin Basic Protein; Neural Cell Adhesion Molecule L1; Neural Cell Adhesion Molecules; Oligodendroglia; Progesterone; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sialic Acids; Stem Cells

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.

Topics: Animals; Animals, Newborn; Cells, Cultured; Galactosylceramides; Myelin Basic Protein; Myelin P0 Protein; Myelin Proteins; Myelin Sheath; Myelin-Associated Glycoprotein; Progesterone; Rats; Schwann Cells

The N20.1 immortalized cell line has several characteristics of differentiating oligodendrocytes (OLs), including expression of the glycolipids galactocerebroside (GalC) and sulfatide, and the myelin proteins CNPase and myelin basic protein (MBP) (1,2). Addition of 1-100 microM forskolin to elevate cyclic AMP (cAMP) levels changed cell morphology from irregular and flattened to a more rounded birefringent cell with multiple branched processes. GalC and sulfatide were detected immunocytochemically after permeabilization in the untreated cells and levels appeared to increase slightly following exposure to forskolin. Further analysis showed that most of the glycolipid was internal, with virtually no detectable levels on the cell surface in untreated cells and a very slight change following treatment with forskolin. Synthesis of the two lipids as measured by [H3]galactose incorporation doubled within 24 hours of treatment with forskolin. Levels of message for UDP-galactose: ceramide galactosyl transferase (CGT), a key enzyme in the synthesis of GalC and sulfatide, were compared with those of MBP and proteolipid protein (PLP), before and after elevation of cAMP. No changes were observed in levels of mRNA for CGT and PLP after 24 hours, with a possible increase by 48 hours. In contrast, levels of MBP message dropped precipitously by 24 hours; this was accompanied by an increase in levels of message for suppressed cAMP-inducible POU (SCIP). Thus CGT transcription is regulated independently of MBP and SCIP in N20.1 cells. Analysis of MBP levels by immunocytochemistry and Western blot showed little or no change in protein levels at 24 and 48 hours, in contrast to the sharp decrease in message levels by 24 hours, indicating a relatively long half life for MBP in this cell line. Thus, the N20.1 cells are an informative model for examining regulation of expression of myelinotypic proteins and GalC, as well as the transport of this lipid to the plasma membrane.

Topics: Animals; Cell Line; Cell Size; Colforsin; Cyclic AMP; Galactosylceramides; Gene Expression Regulation; Mice; Myelin Basic Protein; Myelin Sheath; Oligodendroglia; RNA, Messenger; Sulfoglycosphingolipids

Although the specificity of multiple sclerosis (MS) brain immunoglobulins (Igs) remains unknown, the incubation of these Igs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.

Topics: Antigens, Surface; Autoantibodies; Chelating Agents; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Galactosylceramides; Humans; Immunoglobulin Fab Fragments; Myelin Basic Protein; Myelin Proteins; Myelin Sheath; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Peptide Fragments

Oligodendroglia synthesize myelin in the CNS. In vitro, oligodendroglia may be identified by the binding of monoclonal antibodies against galactocerebroside, a myelin-specific galactolipid. Oligodendroglial trophic factor is a protein mitogen for cells of the oligodendroglial lineage. When oligodendroglia in cerebral white matter cultures are treated with oligodendroglial trophic factor, galactocerebroside-positive cells undergo mitosis but fail to express the myelin structural proteins, myelin basic protein and proteolipid protein. Oligodendroglia treated with oligodendroglial trophic factor, however, do express 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein in a manner similar to oligodendroglia treated with platelet-derived growth factor. Oligodendroglial trophic factor, therefore, generates a population of somewhat 'immature' oligodendroglia, which are galactocerebroside, myelin-associated glycoprotein and 2', 3'-cyclic nucleotide 3' phosphodiesterase positive but myelin basic protein and proteolipid protein negative.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Antibodies, Monoclonal; Brain; Cells, Cultured; Galactosylceramides; Gene Expression; Mitogens; Myelin Basic Protein; Myelin Proteins; Myelin Proteolipid Protein; Myelin-Associated Glycoprotein; Nerve Growth Factors; Neuroglia; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; RNA, Messenger

Treatment of cultured oligodendrocytes with a monoclonal antibody to galactocerebroside (GalC) triggers a cascade of events including the redistribution of membrane surface GalC over internal domains of MBP and loss of microtubular structures within the sheets (Dyer and Benjamins: J Neurosci 8:4307-4318, 1988; Dyer and Benjamins: J Neurosci Res 24:212-221, 1989). In this report, wild type and myelin basic protein (MBP)-deficient shiverer oligodendrocytes were used to study the possible relationships between these events, and specifically to determine if MBP mediates signals which destabilize microtubular assemblies in cultured oligodendrocytes. We now show that MBP and GalC, which are both initially Triton X-100 soluble, become Triton X-100 insoluble following anti-GalC binding and anti-GalC:GalC complex redistribution, suggesting that the surface anti-GalC: GalC complexes become associated with cytoplasmic MBP. Mediation of the signaling event by MBP is further demonstrated by 1) a decreased phosphorylation of MBP in wild type oligodendrocytes after antibody binding, and 2) the absence of responses, such as GalC redistribution and microtubule loss, in MBP-deficient shiverer oligodendrocytes treated with anti-GalC. Continuous activation of the GalC/MBP pathway for 7 days in wild type oligodendrocytes results in enlarged cell bodies and production of numerous microprocesses, a morphology that is similar to MBP-deficient shiverer oligodendrocytes. A second signaling pathway which produces an opposite effect, i.e., the stabilization and apparent up-regulation of microtubular structures in cultured oligodendrocyte membrane sheets, remains functional in shiverer oligodendrocytes. Thus, MBP appears to be important for mediating extracellular signals that cause a loss of microtubular structures in oligodendrocyte membrane sheets and abnormal morphology.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Biomarkers; Cell Membrane; Cell Size; Cells, Cultured; Cytoskeleton; Female; Galactosylceramides; Male; Mice; Mice, Neurologic Mutants; Microtubules; Myelin Basic Protein; Octoxynol; Oligodendroglia; Phosphorylation; Polymers; Protein Processing, Post-Translational; Signal Transduction; Solubility

Subacute sclerosing panencephalitis (SSPE) patients carry persistent measles virus infection in the brain. Furthermore, the blood lymphocytes contain viral RNA. Lymphocytes derived from 6 SSPE patients were stimulated with Epstein-Barr virus (EBV). Production of antibodies against measles virus of the IgG isotype was detected in the supernatants of cell cultures of all patients, regardless of the disease's activity, duration or interferon therapy. In contrast, only some of these cell cultures also produced antibodies against myelin.

Topics: Adolescent; Adult; Antibodies, Viral; Antibody Specificity; B-Lymphocytes; Cell Line, Transformed; Cells, Cultured; Child; Encephalitis; Female; Galactosylceramides; Herpesvirus 4, Human; Humans; Immunoglobulin G; Lymphocyte Activation; Male; Measles virus; Multiple Sclerosis; Myelin Basic Protein; Pregnancy; Pregnancy Complications, Infectious; Subacute Sclerosing Panencephalitis

Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.

Topics: Animals; Base Sequence; Brain; Cloning, Molecular; Demyelinating Diseases; Exons; Galactosylceramides; Gene Expression; Male; Mice; Mice, Inbred ICR; Mice, Neurologic Mutants; Molecular Sequence Data; Mutation; Myelin Basic Protein; Nucleic Acid Hybridization; Oligodendroglia; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger

There is suggestive but inconclusive evidence for a contribution of T cells and antimyelin antibodies to the pathogenesis of the Guillain-Barré polyneuropathy. We have studied the potential synergism of cellular and humoral immunity in the adoptive transfer model of EAN. EAN was induced in Lewis rats by injecting varying doses of P2 peptide (SP26)-sensitized T lymphocytes. Disease severity was dose-dependent. The addition of intravenous GC-AB to a subclinical dose of SP26-sensitized T cells resulted in overt clinical disease and markedly enhanced demyelination. Intravenous injection of antibody alone had no effect. We conclude that activated neuritogenic T cells, while entering into peripheral nerves, alter the blood-nerve barrier, which gives circulating demyelinating antibodies access to the endoneurium. The observations support the concept of a synergistic role of T-cell autoimmunity and humoral responses in the inflammatory demyelination of Lewis rat EAN.

Topics: Animals; Antibodies; Axons; Demyelinating Diseases; Edema; Female; Galactosylceramides; Ganglia, Spinal; Immunization, Passive; Lymphocyte Activation; Macrophages; Male; Motor Neurons; Myelin Basic Protein; Myelin P2 Protein; Nerve Degeneration; Neurilemma; Neuritis, Autoimmune, Experimental; Neurons, Afferent; Rabbits; Rats; Rats, Inbred Lew; Sciatic Nerve; Spinal Cord; Spinal Nerve Roots; T-Lymphocytes

O4+/A007+GalC- proligodendroblasts represent a distinct stage of development in the oligodendrocyte lineage, occurring just prior to the appearance of postmitotic GalC+ oligodendrocytes. These cells, isolated directly from postnatal rat telencephalon by an immunopanning procedure, can terminally differentiate and myelinate axons when transplanted back into an in vivo environment. Specifically, after 30 days in the brain of newborn shiverer mouse hosts, O4+GalC- oligodendrocyte progenitors produced myelin basic protein positive (MBP+) patches. These MBP+ patches, examined by both light and confocal microscopy, contained oligodendrocyte cell bodies and ensheathed host shiverer axons morphologically similar to those found in normal rat brain at an analogous age. These results suggest that isolated O4+GalC- cells can become biochemically mature oligodendrocytes with the capacity to elaborate myelin sheaths, and further define the period of development during which oligodendrocytes retain their capacity to myelinate axons when given a receptive environment.

Topics: Animals; Antigens, Surface; Brain; Cell Separation; Galactosylceramides; Mice; Mice, Neurologic Mutants; Myelin Basic Protein; Myelin Sheath; Oligodendroglia; Rats; Reference Values; Stem Cell Transplantation; Stem Cells

To test whether gangliosides (GA) might exert neuritogenic effects in vivo, experimental allergic neuritis (EAN) was studied clinically, neuropathologically, and immunologically in Lewis rats immunized with bovine peripheral nerve, P2 myelin protein, P2 myelin protein plus two different doses of GA, P2 with galactocerebroside (GC), and GA alone, each emulsified in adjuvant. All except the GA-treated group developed signs of EAN between days 11 and 14 after the injection. Rats immunized with P2 alone were the most severely affected. Rats given P2 plus GA and those given P2 plus GC displayed a significantly lower clinical score. Histological analysis revealed a comparable degree of inflammation of the peripheral nervous system and demyelination in the spinal nerve roots of bovine peripheral nerve- and P2-immunized rats. The P2 plus GA and P2 plus GC groups revealed similar degrees of pathology in the spinal nerve roots but the latter group stood apart from the rest in that it showed widespread peripheral nervous system changes extending distally into the sciatic nerve. Serological analysis demonstrated that P2 and GC, but not GA, elicited antibody (IgG) responses, but there was no correlation between antibody titer and clinical or histological involvement. The present data fail to support an enhancing role for gangliosides in the expression of EAN and, by extrapolation, in the Guillain-Barré syndrome, for which EAN serves as the laboratory model, and in which suggestions have been made that antibodies to GA may have pathogenetic significance.

Topics: Animals; Autoantibodies; Autoimmune Diseases; Cattle; Galactosylceramides; Gangliosides; Male; Myelin Basic Protein; Myelin P2 Protein; Neuritis, Autoimmune, Experimental; Rats; Rats, Inbred Lew; Sciatic Nerve

From previous studies on the induction and treatment of experimental autoimmune encephalomyelitis (EAE) in guinea pigs and mice, antibodies have been implicated during both demyelination and remyelination. In the present study, sera from guinea pigs with acute, chronic and myelin basic protein/galactocerebroside (MBP/GC)-treated chronic EAE were evaluated for the presence of anti-glial cell antibodies by immunocytochemical techniques. Antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The majority of sera from acute and chronic active EAE animals displayed intense labelling of astrocytes and only weak staining of oligodendrocytes when tested on sections of normal guinea pig brain tissue. In contrast, sera from animals with chronic EAE treated with MBP/GC gave strong labelling of oligodendrocytes and only minor staining of astrocytes. By immunoblotting, astrocyte staining was shown to be due to the presence of antiglial fibrillary acidic protein (GFAP) antibodies. The intense oligodendrocyte staining observed in sections reacted with sera from MBP/GC-treated guinea pigs corresponded well with high titers of serum anti-GC and anti-MBP antibodies measured by an ELISA. It was concluded that the presence of antibodies against astrocytes was possibly related to astrocytic antigens within the disease-inducing emulsion, at least during the initial phases of EAE, and not to their release from the central nervous system of affected animals.

Topics: Animals; Antibodies; Astrocytes; Blotting, Western; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Galactosylceramides; Guinea Pigs; Immune Sera; Immunoglobulin G; Immunohistochemistry; Myelin Basic Protein; Rabbits; Reference Values; Spinal Cord

Chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) in the Lewis rat, induced by the injection of spinal cord tissue in complete Freund's adjuvant (SC/CFA), was studied in vivo by treatment with liposomes containing central nervous tissue antigens, and in vitro by lymphocyte proliferation assays. Intracardiac administration of myelin basic protein (MBP) liposomes, galactocerebroside (GC) liposomes, or MBP + GC liposomes substantially reduced the clinical severity and/or delayed the onset of the initial phase of disease. Liposomes prepared from whole myelin provided even greater protection, and were effective at suppressing both the first disease episode and the relapses. These results indicate that while GC and MBP may play significant roles in the development of CR-EAE in the Lewis rat, immune responses to other antigens are probably also involved. Splenic and lymph node lymphocytes from MBP-GC liposome-treated rats, and splenic lymphocytes from cytochrome-GC (CYT-GC) liposome-treated rats, showed drastically reduced abilities to proliferate in response to MBP in culture. Spleen cells from both the MBP-GC- and CYT-GC-liposome-treated donors were able to actively suppress antigen-induced proliferation of MBP-primed lymphocytes. These findings suggest participation of both clonal anergy, and active suppressor cells in the liposome-mediated suppression of CR-EAE in the Lewis rat.

Topics: Animals; Antigens; Cell Division; Central Nervous System; Cytochrome c Group; Drug Carriers; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Guinea Pigs; Liposomes; Lymph Nodes; Lymphocyte Activation; Myelin Basic Protein; Nerve Tissue; Rats; Rats, Inbred Lew; Recurrence; Spinal Cord

Cholesterol esters and free cholesterol were determined in the serum of guinea pigs with experimental allergic encephalomyelitis (EAE). EAE was induced by immunization with myelin basic protein (MBP) and galactocerebroside (GC). The results showed an increased content of cholesterol arachidonate and cholesterol linoleate during the period of clinical signs of EAE in comparison with the content of cholesterol esters in EAE induced by MBP without galactocerebroside. The content of free cholesterol was significantly reduced. Histological analyses showed intensive inflammatory lesions and demyelination which were not found in EAE induced by MBP without galactocerebroside.

Topics: Animals; Central Nervous System; Cerebrosides; Drug Synergism; Encephalomyelitis, Autoimmune, Experimental; Female; Galactosylceramides; Guinea Pigs; Lipids; Male; Myelin Basic Protein; Myelin Sheath

Fc receptor-dependent myelin phagocytosis has been proposed as a possible important effector mechanism in several immune-mediated demyelinating diseases. The present study was designed to determine whether myelin is opsonizable by anti-myelin antibodies. Thioglycolate-elicited mouse peritoneal macrophages were cultured with 125I-labelled bovine central myelin pretreated with normal or immune serum. Serum opsonic activity was determined by a kinetic study comparing macrophage uptake of opsonized and untreated 125I-myelin. Heat-stable and heat-labile myelin opsonins were detected in normal rabbit serum. Myelin was also opsonized by normal rabbit gamma globulin and by heat-inactivated normal mouse, human, and guinea pig serum. Increased opsonic activity was detected in rabbit anti-myelin antiserum and the gamma globulin fraction prepared from this serum, in anti-myelin basic protein and anti-galactocerebroside antiserum but not in anti-myelin-associated glycoprotein antiserum or in serum from rabbits injected with Freund's adjuvant alone. One out of three anti-sheep red blood cell antisera tested also showed increased myelin opsonic activity. It is concluded that anti-myelin antibodies can promote opsonic phagocytosis, and that normal serum and normal serum gamma globulin also opsonize myelin.

Topics: Animals; Antibodies; Blood Physiological Phenomena; Cattle; Erythrocytes; Galactosylceramides; Immune Sera; Myelin Basic Protein; Myelin Proteins; Myelin Sheath; Myelin-Associated Glycoprotein; Opsonin Proteins; Reference Values; Sheep

Using immunofluorescence with a panel of antibodies that recognize antigens expressed by oligodendroglia, the myelin-producing cells of the CNS, at different stages of differentiation from precursor to mature cell, we have investigated the development of cells of this lineage in cryostat sections of rat cerebellum. Our results are consistent with the view that glial precursors, identified by their expression of the ganglioside GD3, arise in the subependymal layers of the 4th ventricle and migrate to their final position in the cerebellum via the superior medullary velum, and to some extent the peduncles. As the cells reach their final destination they make the transition to recognizable galactocerebroside (GC)-expressing oligodendroglia, via a GD3+/GC+ intermediate. The myelin-associated protein 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) appears at the same time as GC, whereas myelin basic protein (MBP) is expressed 2-3 days after GC and CNP, immediately prior to myelin formation. A very clear progression of oligodendroglial differentiation was observed from the SMV into the base of the cerebellum, up into the white matter (WM) tracts of the folia, and then away from this central white matter into the granule cell and Purkinje cell layers, and finally the molecular layer. The time delay between the expression of GC, CNP and MBP was the same for oligodendroglia in all of these layers, suggesting the presence of an intrinsic clock controlling the initial expression of these myelin components. The early appearance of CNP in oligodendroglia suggests a role for this protein in the early stages of myelinogenesis.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Cerebellum; Fluorescent Antibody Technique; Galactosylceramides; Immunohistochemistry; Myelin Basic Protein; Myelin Sheath; Neuroglia; Oligodendroglia; Rats; Rats, Inbred Strains

Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture. Exposure of oligodendroglia to rabbit anti-GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled. Anti-GalC exposure for 24 hr results in contracted membrane sheets. No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG. In cultures exposed to anti-GalC continuously for 4-7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets. This effect is reversible upon removal of anti-GalC from the culture media. By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions. Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions. The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction. In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures. Membrane sheets that are contracted in response to anti-GalC exposure for 6-24 hr show intense GalC staining over microtubular structures. Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti-GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures. In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.

Topics: Animals; Antibodies; Cells, Cultured; Cerebrosides; Cytological Techniques; Galactose; Galactosylceramides; Immunoglobulin G; Microscopy, Electron, Scanning; Microtubules; Myelin Basic Protein; Neuroglia; Oligodendroglia; Proteolipids; Sulfoglycosphingolipids; Time Factors; Tubulin

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cerebrosides; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Glycoproteins; Myelin Basic Protein; Neuroglia; Oligodendroglia; Rats; T-Lymphocytes

Topics: Animals; Cell Division; Central Nervous System; Chronic Disease; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Guinea Pigs; Myelin Basic Protein; Myelin Sheath; Neuroglia; Oligodendroglia

Primary cultures of neonatal mouse cerebra were maintained for up to 4 weeks in the absence of neurons. Oligodendrocytes in these cultures pass through a sequence of cytoarchitectural change and antigen expression which mimics the differentiation of oligodendrocytes in vivo. The cell bodies and processes of oligodendrocytes first express the myelin-specific antigen galactocerebroside (GC) by 2 days in vitro. Myelin basic protein (MBP) appears several days later. The majority of oligodendrocytes then proceed to elaborate large sheets of membranous material from the tips and lengths of cell processes. These membranous sheets, which contain GC and MBP, are reminiscent of unwrapped myelin profiles in vivo. As with the cell bodies and processes, GC is inserted into the sheets several days before MBP. Our results establish that oligodendrocytes cultured without neurons are able to produce extensive membranes containing myelin-specific antigens. They also suggest that oligodendrocyte shape and membrane production are, in part, regulated from within the oligodendrocyte itself.

Topics: Animals; Brain; Cell Differentiation; Cell Membrane; Female; Galactosylceramides; Histocytochemistry; Immunologic Techniques; Mice; Microscopy, Phase-Contrast; Myelin Basic Protein; Neuroglia; Neurons; Oligodendroglia; Pregnancy

The pathology of demyelination in rabbits with experimental allergic neuritis (EAN) or galactocerebroside-induced neuritis was compared to that in rabbits inoculated with either an emulsion of lipid haptens (gangliosides, lecithin and cholesterol) and Freund's complete adjuvant or Freund's complete adjuvant (FCA) alone. In rabbits inoculated with bovine peripheral myelin in FCA, perivenular demyelination associated with infiltrates of lymphocytes and macrophages occurred after 30 days, while those animals inoculated with galactocerebroside (GC) in Freund's adjuvant did not develop lesions until 60-90 days. GC rabbits had demyelination and severe nerve edema without cellular infiltrates. In rabbits inoculated with FCA alone, demyelination was restricted to ganglia and proximal nerve roots. Myelin basic protein (MBP) and GC antibodies from EAN, GC and lipid hapten-inoculated rabbits were detected by ELISA in sera at all post-inoculation time points. Appreciable P0 and P2 antibody titers were detected only in EAN animals. The results indicate that Freund's complete adjuvant alone or in combination with lipid haptens is capable of producing neuropathic effects in the rabbit independent of those produced by EAN or galactocerebroside neuritis.

Topics: Animals; Antibodies; Demyelinating Diseases; Female; Freund's Adjuvant; Galactosylceramides; Haptens; Lipids; Myelin Basic Protein; Myelin Sheath; Neuritis; Peripheral Nerves; Rabbits

Myelin basic protein (MBP) is one of the most important myelin components. Based on our previous studies, we hypothesized that neurons might have regulatory effects on the production of MBP by oligodendrocytes, and we conducted studies designed to verify this hypothesis. Oligodendroglia-rich cultures from total brain of neonatal rats or mice and pure cultures of embryonic rats or chicks were prepared. Cultures of mouse fibroblasts and astrocytes were prepared as well. We show here that MBP production by oligodendrocytes was greatly enhanced by treatment with either pure neurons, rat neuronal conditioned medium, or chick neuronal conditioned medium, while chemically defined, hormonally supplemented medium or medium conditioned by astrocytes and fibroblasts had no effect on MBP expression. We conclude that the production of MBP by oligodendrocytes is regulated by a nonspecies specific soluble neuronal factor. The conservation of this phenomenon from avian to rodent species implies its critical role in myelination and suggests its potential application as a treatment in demyelination.

Topics: Animals; Brain; Cell Survival; Cells, Cultured; Chick Embryo; Culture Media; Fluorescent Antibody Technique; Galactosylceramides; Myelin Basic Protein; Nerve Growth Factors; Neuroglia; Oligodendroglia; Rats; Staining and Labeling

Enzymatically dissociated cell suspensions from adult rat spinal cord were added at low densities (5 X 10(3) cells/culture) to cultures of pure dorsal root ganglion neurons. Oligodendrocytes, identified by immunostaining with a monoclonal antibody to galactocerebroside, began to proliferate by 4 days after their addition, forming large colonies of cells by the 14th day. Myelin formation by oligodendrocytes began 4 weeks after addition and myelin was abundant by 6 weeks. Oligodendrocyte proliferation and myelination did not require the immediate presence of astrocytes; the number of astrocytes overall remained low throughout the culture period. Preliminary studies indicated that the specific removal of galactocerebroside-positive cells from the cultures with anti-galactocerebroside antibody and complement 3 days after their addition prevented the subsequent generation of new oligodendrocytes and myelination. These preliminary results suggest that a major source of new myelinating cells in the adult central nervous system (CNS) might be already committed, galactocerebroside-positive, oligodendrocytes rather than uncommitted stem cells. The absence of cellular barriers between the myelinating cells and the medium make these cultures well suited for studies probing cellular and molecular mechanisms of myelination in the CNS.

Topics: Animals; Astrocytes; Cells, Cultured; Embryo, Mammalian; Galactosylceramides; Ganglia, Spinal; Myelin Basic Protein; Myelin Sheath; Neuroglia; Oligodendroglia; Rats

Serum antibodies to P2 protein, myelin basic protein (MBP) and galactocerebroside (GC) were examined to study the role of humoral factors in experimental allergic neuritis (EAN). Out of 19 outbred rabbits sensitized with peripheral nerve homogenate, 14 developed EAN and the remaining 5 rabbits did not develop the disease. Anti-P2 protein and anti-MBP antibodies were detected not only in the EAN rabbits but also in the nonresponders. On the other hand, anti-GC antibody was detected in 7 of 14 EAN rabbits but not in the nonresponders. This antibody appeared near the onset of the clinical signs. As 7 rabbits developed EAN in the absence of the antibody and the titer did not correlate with the clinical severity, it is unlikely that anti-GC antibody is the major factor in inducing EAN. However, this antibody, along with other factors, may play a role in EAN.

Topics: Animals; Autoantibodies; Autoimmune Diseases; Cerebrosides; Galactosylceramides; Myelin Basic Protein; Myelin P2 Protein; Neuritis, Autoimmune, Experimental; Peripheral Nerves; Rabbits; Time Factors

Cell type-specific markers are currently available for identifying major cell types in neural cell cultures. The markers considered specific for glial cells are numerous, but only galactocerebroside and myelin basic protein for oligodendrocytes, and glial fibrillary acidic protein for astrocytes are widely adopted by investigators for that purpose. Other surface and intracellular markers which are specific for oligodendrocytes or astrocytes in vitro are briefly described. The possibility of using different classes of gangliosides as cell type-specific markers in neural cell culture is discussed. The presence of "transitional" or "bipotential" glial cells that express both oligodendrocytic and astrocytic phenotypes in human glial cell cultures and the regulatory effect of cyclic AMP derivatives on these cells are reported. In addition, the presence of Ia antigens on the surface of a selected population of cultured human oligodendrocytes and astrocytes is described.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Aged; Antigens; Antigens, Surface; Astrocytes; Cells, Cultured; Cyclic AMP; Female; Fetus; Fluorescent Antibody Technique; Galactosylceramides; Gangliosides; Glial Fibrillary Acidic Protein; Histocompatibility Antigens Class II; Histocytochemistry; HLA-DR Antigens; Humans; Infant; Male; Microscopy, Phase-Contrast; Middle Aged; Myelin Basic Protein; Neuroglia; Oligodendroglia; S100 Proteins

The pathology of experimental allergic encephalomyelitis (EAE) induced by bovine myelin basic protein (MBP) has been examined in the guinea pig with a series of doses ranging from 37.5 micrograms to 600 micrograms. This was to investigate whether the previously demonstrated lack of demyelinative effect by MBP was dose-related. At all doses tested, MBP induced clinical disease. Inflammation was the major feature of lesions in all animals. However, no demyelination was seen when 75 micrograms MBP or less was given. At higher doses (150 micrograms upwards), MBP always induced intense inflammation but demyelination was encountered inconsistently. These observations support the contention that in addition to an immune response to MBP, other factors contribute to autoimmune demyelination.

Topics: Animals; Autoantibodies; Dose-Response Relationship, Immunologic; Encephalomyelitis, Autoimmune, Experimental; Female; Galactosylceramides; Guinea Pigs; Inflammation; Myelin Basic Protein; Myelin Sheath

Oligodendroglia isolated from adult bovine brain by the method of Farooq et al. could be plated on polylysine-coated plastic dishes with an efficiency of 55-80%, and maintained in culture for as long as 4 months. The addition of cytosine arabinoside to the nutrient medium resulted in cultures that were approximately 90% oligodendroglia and 10% large fibroblasts. From 50 g of white matter 100-160 X 10(6) oligodendroglia, containing approximately 6-10 mg protein, could be obtained in culture. These small round cells started to send out processes at 5 days in vitro and by 2 weeks they formed an extensive network of processes. By immunofluorescence, all cells of this morphology were positive for galactocerebroside (GC) and myelin basic protein (MBP), and negative for glial filament protein and fibronectin. Most of the large flat cells were positive for fibronectin and negative for GC, MBP and glial filament protein. As the cultures aged the oligodendroglia tended to clump and blebs formed on the surface of both perikarya and processes. By 4 months they showed evidence of degeneration and detached from the substrate. Electron microscope examination showed that the cells had the appearance typical of oligodendroglia in situ. The somata were round to elliptical, with eccentrically placed nuclei, and were larger than freshly isolated cells. They grew directly on the substrate or on the surface of the fibroblasts. In older cultures the cells formed tight nests. The somata were enveloped by sheets of oligodendrocyte cytoplasm, sometimes having a myelin-like appearance. Gap junctions and small desmosomes were seen between oligodendroglial processes and between oligodendroglia and fibroblasts. The cytoplasm was characterized by a prominent Golgi apparatus, many mitochondria and lysosomes, scattered rough endoplasmic reticulum, free ribosomes, frequent centrioles and an abundance of microtubules. In cells from older cultures large vacuoles were common, and rarely they had multilamellar walls with alternating major and minor dense lines resembling myelin.

Topics: Animals; Cattle; Cell Separation; Cells, Cultured; Fibronectins; Fluorescent Antibody Technique; Galactosylceramides; Intermediate Filament Proteins; Microscopy, Electron; Myelin Basic Protein; Neuroglia; Oligodendroglia

Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.

Topics: Animals; Brain; Cell Division; Cells, Cultured; Cerebrosides; Embryo, Mammalian; Female; Fluorescent Antibody Technique; Galactosylceramides; Immunoenzyme Techniques; Mice; Myelin Basic Protein; Neuroglia; Oligodendroglia; Pregnancy