Glial cells as targets and producers of neurotrophins.

Glial cells fulfill important tasks within the neural network of the central and peripheral nervous systems. The synthesis and secretion of various polypeptidic factors (cytokines) and a number of receptors, with which glial cells are equipped, allow them to communicate with their environment. Evidence has accumulated during recent years that neurotrophins play an important role not only for neurons but also for glial cells. This brief update of some morphological, immunocytochemical, and biochemical characteristics of glial cell lineages conveys our present knowledge about glial cells as targets and producers of neurotrophins under normal and pathological conditions. The chapter discusses the presence of neurotrophin receptors on glial cells, glial cells as producers of neurotrophins, signaling pathways downstream Trk and p75NTR, and the significance of neurotrophins and their receptors for glial cells during development, in cell death and survival, and in neurological disorders.

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture.

Oligodendrocytes were isolated from adult pig brain and cultivated for 18-24 days. [(14)C]acetate, [(3)H]galactose or [(35)S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [(14)C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [(14)C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [(14)C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [(3)H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [(35)S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.

Differential response of mature TrkA/p75(NTR) expressing human and pig oligodendrocytes: aging, does it matter?

A differential morphological response of mature oligodendrocytes (OL) isolated from human and pig brains to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and to the nerve growth factor (NGF) was observed. In both cases, OL regenerate their processes; however, the rate and the extension of the process formation of human OL were behind that of pig OL. Presumably, the advanced age of the human tissue in these experiments might have contributed to this decrease in process formation, an effect that was already observed for rat OL [Yong et al. (1991) J Neurosci Res 29:87-99]. The less effectivity of NGF via TrkA, which was immunocytochemically shown in human OL, and of TPA via the protein kinase C (PKC) pathway, may have its common focus on the mitogen-activated protein kinase (MAPK) cascade. In this context, it was noted that only a few studies on aging of mature OL are available. It is conceivable that age-related changes in the properties of OL could be an important factor for their cellular responsiveness during longer lasting demyelinating diseases such as multiple sclerosis. Hence, this review would like to provide a basis for future investigations on the aging of mature OL. The data presently available suggest a preliminary classification of mature OL into three categories.

NGF increases [Ca2+]i in regenerating mature oligodendroglial cells.

The effect of NGF on [Ca2+]i of mature regenerating oligodendroglial cells was investigated by measuring fluo-3 fluorescence. NGF caused transient increases in [Ca2+]i, which could be inhibited by anti-NGF antibody. The rise in [CA2+]i was in part due to influx of extracellular Ca2+ since it was markedly attenuated in Ca2+-free solution. It also depended on release of Ca2+ from intracellular stores as tested by prior depletion with cyclopiazonic acid. These results support a role for Ca2+ in the effects of NGF on oligodendroglial cells.

Neurochemical and morphological studies of bulk isolated rat brain cells. II. Preparation of viable cerebral neurons which retain synaptic complexes.

The bulk isolation from rat cerebral cortex of viable neurons retaining synaptic complexes is described. The basis of this procedure is to dissociate the neurons in situ from the surrounding glial cells. The glial structures that are normally adjacent to the neuronal cell body and to the proximal parts of the neuronal processes are largely destroyed by perfusion of the brain under special conditions. The most important of these conditions was found to be a hyperosmolar concentration of hexoses in the perfusion medium. In addition, the presence of collagenase and hyaluronidase in the perfusion medium and specific perfusate flow characteristics were required to produce the structural changes throughout the brain tissue. When the perfused brain was further dissociated into a cell suspension by mincing and sieving, isolated neurons were obtained, the majority of which retained the proximal parts of their processes. A novel feature of these neurons was the retention of synaptic boutons on the plasma membrane. Presynaptic terminals with mitochondria and vesicles as well as pre- and postsynaptic membranes and densities were observed on the isolated neurons. The neurons were fractionated to 90--95% purity using discontinuous Ficoll density gradient centrifugation with a liquid fluorocarbon as cushion. Highly purified, viable cerebral neurons retaining synaptic complexes are thus available in bulk for neurobiological studies.

Oligodendrocytes from postnatal cat brain in cell culture. I. Regeneration and maintenance.

Oligodendrocytes isolated in bulk from white matter of cat brain (8-12 weeks of age) employing a Percoll gradient as the final purification step, were cultured and maintained for more than 10 weeks. Different parameters, e.g. coating material and the age of the animals appeared to have some influence on attachment rate and survival of the cells. Oligodendrocytes from older animals, or oligodendrocytes seeded into poly-L-lysine coated culture dishes revealed a marked tendency to form aggregates. Of the dispersed cells, 80-99% can be classified as oligodendrocytes by transmission electron microscopy (TEM) and immunocytochemical markers. Aggregates which were re-seeded consisted of more than 90% oligodendrocytes. About one day after attachment to the supporting layer the cells start to regenerate their processes which sometimes broaden at their ends into shovel-like, membranous extensions.

[Dyscephalia-cataracta congenita-hypotrichosis (DCH) syndrome (Ullrich-Fremerey-Dohna, Hallermann-Streiff, Francois). Report of a case showing extrapyramidal hyperkinesia and dementia (author's transl)]. / Dyskephalie-Katarakt-Hypotrichose-Syndrom (Synonyma: DCH; Ullrich-Fremerey-Dohna; Hallermann-Streiff; Francois). Beobachtung mit extrapyramidaler Störung und Demenz

In a 43-year-old man dyscephalia, cataracta congenita, and hypotrichosis were the outstanding features. These signs were first described in 1953 by Ullrich and Fremerey-Dohna as a clinical entity. Since 1958 the DCH syndrome was published under the synonyms of "Francois syndrome" and of "Hallermann-Streiff syndrome". However, as these authors did not add any essential details relevant for the classification of the syndrome we prefer to retain the term "Ullrich-Fremerey-Dohna syndrome". In our case in addition to the above mentioned and well known manifestations, extrapyramidal hyperkinesia of the choreoanthetotic type and servere mental deficiency accompanied by mild cerebral atrophy (revealed by pneumencephalography) were found.

Nerve growth factor induces proliferation and enhances fiber regeneration in oligodendrocytes isolated from adult pig brain.

Mature oligodendrocytes (OL) isolated from adult pig brains start to regenerate their fibers after 4-5 days in vitro (DIV); after 14 DIV a network of OL fibers is formed. Growth factors, of which it was known that they play an important part during proliferation and differentiation of OL progenitor cells, were used to study their influence on the regeneration of mature OL. For this purpose, OL were treated at 6 DIV with different concentrations of various growth factors. At 24 h intervals the [3H]thymidine incorporation was measured and at 8 DIV the OL fiber production evaluated. None of these factors did influence the regenerative process to any significant extent except nerve growth factor (NGF). For the first time it could be shown that NGF enhanced the OL fiber regeneration considerably and induced the proliferation of a subset of OL. These results may have important implications for the remyelinating process in demyelinating diseases such as multiple sclerosis.

Myelin basic protein induces cell death of mature pig oligodendrocytes in vitro and produces demyelination in vivo.

Two methods prevail at present in producing demyelinated areas in the central nervous system. One uses the detergent-like effect of lysolecithin, the other is based on a cell killing effect of ethidium bromide plus x-irradiation. Unwanted side-effects are inherent in both methods. Based on the fact that myelin basic protein (MBP) kills adult pig oligodendrocytes but almost no astrocytes in vitro, we have used MBP for creating demyelinated areas in the centrum semiovale of the pig brain. These lesions are characterized by a loss of oligodendrocytes and myelin, a preservation of axons and astrocytes, and by the presence of macrophages. Thus, this type of lesion might present an alternative option for studying the fate of transplanted myelinating cells.

IMGT, the international ImMunoGeneTics database: a new design for immunogenetics data access.

IMGT, the international ImMunoGeneTics database is an integrated database specializing in Immunoglobulins (Ig), T-cell receptors (TcR) and MHC molecules of all vertebrate species, created by Marie-Paule Lefranc, University of Montpellier, CNRS, Montpellier, France (Nucleic Acids Research, Database issue, Vol 26, January 1998). IMGT includes three databases: LIGM-DB (for Ig and TcR), MHC/HLA-DB and IMGT/PRIMER-DB (an Ig, TcR and MHC-related primer database), the last two in development. IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 24.000 Immunoglobulin and T cell Receptor sequences from 81 different species. MHC/HLA-DB contains class I and class II Human Leucocyte Antigen alignment tables. An IMGT tool, DNAPLOT, developed for Ig, TcR and MHC sequence analysis, is also available. IMGT goals are to establish a common data access to all immunogenetics data, including nucleotide and protein sequences, oligonucleotide primers, gene maps and other genetic data of Ig, TcR and MHC molecules, from all species, and to provide a graphical user friendly data access. IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. In this paper, we describe our approach for the data modelisation, the automation of the annotation procedure and control of data quality in LIGM-DB database. IMGT is freely available on the CNUSC WWW server at Montpellier: http://imgt.cnusc.fr: 8104 (contact: Denys.Chaume@cnusc.fr) and on the EBI servers: http://www.ebi.ac.uk/imgt (contact: malik@ebi.ac.uk) and ftp.ebi.ac.uk/pub/databases/imgt. LIGM-DB users are encouraged to report errors or suggestions to giudi@ligm.crbm.cnrs-mop.fr. IMGT initiator and coordinator: Marie-Paule Lefranc, lefranc@ligm.crbm.cnrs-mop.fr. (fax: +33(0)467040231).

Neurochemical aspects of post-tetanic potentiation of monosynaptic reflexes in the cat spinal cord. II. Determination of phospholipids at maximum of potentiation.

A monosynaptic reflex pathway was used to produce a post-tetanic potentiation (PTP). in ten experiments (cats) one side was tetanized (via N. gastrocnemius) whereas the other one was taken as control. Tissue was punched out of the ventral horn area of the spinal cord (segment height L7/S1) for the analysis of the phospholipid content. The results demonstrate that PTP significantly increases phosphatidyl-inositol in the potentiated alpha-motoneurone area. Phosphatidylserine showed a trend towards a decrease; sphingomyelin, phosphatidylcholine and phosphatidylethanolamine remained almost unchanged. The effect of a different tetanizing time on the phospholipid content is discussed as is the intention of the present experiments.

Oligodendrocytes isolated from adult pig brain synthesise and release prostaglandins.

The synthesis and release of prostaglandins have been studied in oligodendrocytes isolated from adult pig brain. Radioactively labelled arachidonic acid and di-homo-y-linoleic acid were offered as precursors and their incorporation into individual phospholipids followed. Most of the labelling was incorporated into phosphatidylinositol, which may serve as a major source for precursors of the eicosanoid biosynthesis. Oligodendrocytes are capable of synthesising and releasing prostaglandins of the E- and F-series in vitro. Cyclooxygenase, the principal enzyme for prostaglandin synthesis, was localised in oligodendrocytes immunocytochemically by using a double-labelling technique which identified the oligodendrocytes via anti-galactosylcerebroside. The production and release of oligodendroglial prostaglandins indicate that oligodendrocytes themselves can modulate immune-mediated processes in the white matter of the central nervous system. Furthermore, prostaglandins may also play a role during remyelination.

Monogalactosyl diglyceride, a marker for myelination, activates oligodendroglial protein kinase C.

Protein kinase C (PKC) is activated by 1,2-sn-diacylglycerol (DAG), the source of which can either be phosphatidylinositol bisphosphate or phosphatidylcholine. Here, we show that monogalactosyl diglyceride (MGDG), a minor galactolipid present in oligodendrocytes (OLs) and myelin, which is designated as a marker for myelination, can enhance OL PKC activity. Based on different calcium and substrate requirements we conclude that MGDG and DAG activate different isoforms of PKC group A: MGDG primarily stimulates PKC-alpha, and DAG primarily activates PKC-gamma. The presence of these PKC isoforms in OLs was confirmed by western blotting, whereas PKC-beta was only weakly stained, if at all. Addition of MGDG to the culture medium provided a higher density of regenerating OL fibers, which was not observed when membrane-permeable DAG was used. These findings indicate that MGDG can modulate the OL PKC activity and that PKC-alpha is the major PKC isoform involved in OL process formation.

Bulk separation and long-term culture of oligodendrocytes from adult pig brain. II. Some biochemical data.

Oligodendroglial proteins labeled with radioactive amino acids were subjected to one- and two-dimensional polyacrylamide electrophoresis. Bands comigrating with myelin proteins, the basic protein (MBP), the proteolipid protein (PLP), and the Wolfgram protein (WP) doublet, were detected by Coomassie Blue staining and by autoradiography. The identity of the MBP and WP in the cellular material is evidenced by immunoblotting with specific antibodies. A comparative study of myelin samples from rat and pig CNS reveals that WP can be detected immunochemically in both species. Different protein patterns, however, are observed. Three protein bands are found with antibodies against the myelin-associated glycoprotein (MAG). The high-molecular-weight component prevails in pig myelin, whereas the medium-molecular-weight component is predominant in rat myelin. Moreover, two protein bands, of molecular weights 35,000 and 33,000 (Ol 1 and Ol 2), are present in high amounts in oligodendroglial particulate material but are not detectable in myelin. These oligodendroglial characteristic proteins are not species-specific, since they are found in preparations of cat oligodendrocytes as well. Activities of cerebroside sulfotransferase (EC 2.8.2.11) are low in freshly isolated cells and increase during the first week of culture. A reverse course of enzyme activities is observed with 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37). Values reach a minimum about day 5 in culture and recover their initial values. At day 10 they remain stable until the end of the third week of the culture period.

Isolation and cultivation of mature oligodendroglial cells.

CNS axons are ensheathed by myelin which is produced and maintained by oligodendrocytes. A disorder of this assembly results in functional disturbances, e.g., paralysis in multiple sclerosis. Methods are now available to isolate and cultivate oligodendrocytes in vitro. Thus, basic oligodendroglial properties can be now investigated: signals for oligodendroglial gene expression and their role in myelinogenesis and the interaction between oligodendrocytes and other neural cells by, e.g., the release of informational substances.

Bulk separation and long-term culture of oligodendrocytes from adult pig brain. I. Morphological studies.

A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long-term culture. The entire procedure can be accomplished within 2-3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro. Yields between 2.5 and 4 X 10(7) cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti-galactocerebroside (GC) antibodies as well as anti-myelin basic protein (MBP), anti-Wolfgram protein (WP), anti-glial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti-GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti-GC-. After 3 1/2 weeks in culture, about 60% of the cells can be labeled by anti-MBP. Here too differences in staining intensities are observed. The anti-WP stain is too weak to be defined as positive. The percentage of GFAP+ cells lies in the range 15-20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers.

Glycerol phosphate dehydrogenase activity of oligodendrocytes isolated from adult pig brain: its inducibility by hydrocortisone.

Developing oligodendrocytes cultured in vitro express glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) and are known to respond to glucocorticoid treatment by increased activity of GPDH. We present evidence that GPDH is enriched in white matter and oligodendrocytes of adult pig brain. Bulk-isolated oligodendrocytes maintained in culture for several weeks exhibit an almost constant level of GPDH activity. Furthermore, a 4-day stimulation with hydrocortisone induces GPDH specific activity of long-term cultured oligodendrocytes from adult pig brain.

Neurochemical changes in the cat's spinal cord due to orthodromic tetanic stimuli. I: phospholipids.

Posttetanic potentiation of monosynaptic reflexes has been used as a paradigm for neuronal plasticity. The explanation for this phenomenon is an increased responsiveness of the synaptic junctions. This would basically require chemical changes of the nervous structures involved. The ventral horn area of the spinal cord was therefore analyzed neurochemically. The determination of the phospholipids revealed an alteration of their composition. Sphingomyelin, phosphatidylcholine and phosphatidylinositide/serine behaved differently, whereas phosphatidylethanolamine and the total phospholipid content remained unchanged.

Virus-like particles in an estranuclear mutant of Neurospora crassa.

A particulate fraction not present in wild-type cells has been isolated from the cytoplasm and from lysed mitochondria of the respiratory-deficient extranuclear mutant "abnormal-1" of Neurospora crassa. The particles have a density between 1.13 and 1.2 g/ml. They appear in thin sections after OsO(4) fixation as virus-like polymorphic vesicles containing an electron-dense nucleoid of 120-170 nm in diameter. The central core is surrounded by one or two "unit membrane" envelopes of 100 A thickness. The particles contain a single-stranded 33S RNA that is converted to 7-9S RNA by heat treatment in the presence of sodium dodecyl sulfate and that differs in its base composition from mitochondrial and cytoplasmic ribosomal RNA. They contain 7% phospholipids rich in phosphatidylethanolamine and two major proteins, a lipoprotein of molecular weight 15,000 and a glycoprotein of molecular weight 95,000. It is suggested that these virus-like particles originate within mitochondria.

Protein kinase C stimulation enhances the process formation of adult oligodendrocytes and induces proliferation.

Oligodendrocytes (OL) isolated from adult pig brains regenerate their processes and form a network of fibers after 14-18 days in vitro (DIV). Stimulation of protein kinase C (Pk-C) by tumour promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) produces at day 7 in vitro a similar network after only 20 hr. H-7, an inhibitor of Pk-C, as well as amiloride, which inhibits the subsequent Na+/H+ exchange, reversibly suppress this effect. Activation of the protein kinase A or calmodulin pathway do not result in an increased OL process production. Furthermore, TPA induced proliferation in a subpopulation of OL. We conclude that the stimulation of Pk-C is of utmost importance for OL regeneration.