A Growth Factor from Neuronal Cell Lines Stimulates Myelin Protein Synthesis in Mammalian Brain
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The Journal of Neuroscience, February 1991, 7 7(2): 327-336 A Growth Factor from Neuronal Cell Lines Stimulates Myelin Protein Synthesis in Mammalian Brain Dana Giulian,’ Beena Johnson,’ Joseph F. Krebs,’ Mischca J. Tapscott,’ and Sandra Honda* Departments of ‘Neurology and *Cell Biology, Baylor College of Medicine, Houston, Texas 77030 Oligodendroglia growth factor (OGF) is a 16-kDa soluble ferentiation of oligodendroglia, as well as many other cell types protein produced by neuronal cell lines. This factor, when (McMorris et al., 1986; Han et al., 1987). The cytokine inter- incubated with brain glia in culture, selectively stimulates leukin-2 (IL-2) has been described by different laboratories as growth of oligodendroglia, the myelin-producing cells of the a growth promoter (Benveniste and Merrill, 1986) as a growth CNS. OGF infused into the cerebral cortex of the adult rat inhibitor (Saneto et al., 1986) or as having no effect upon oli- accelerates the production of myelin proteins as shown by godendroglia (Giulian and Lachman, 1985; Yong et al., 1988). increased specific activity of the myelin enzyme 2’,3’-cyclic All studies of factor effects upon oligodendroglia have been nucleotide 3’-phosphohydrolase (2’,3’-CNPase), by stimu- limited to cell-culture bioassaysand thus await in vivo con- lated synthesis of myelin basic protein, and by elevations in firmation. When attempting to characterize biologic actions of levels of myelin proteolipid protein RNA. The ability of OGF a growth factor, there is a needto usehighly stringent conditions to induce myelin protein production in viva suggests that with enriched cell populations and chemically defined culture neuron-secreted growth factors help to regulate myelin for- medium. Under these conditions, it is difficult to know if the mation within the CNS. concentrations of a factor tested, the components of the culture medium, and the composition of the cell population adequately Secretion of growth-promoting moleculeswithin the brain dur- reflect physiologic states. Goldman et al. (1986) caution that ing development or after injury is now widely recognized as one differentiation pathways observed for glia in vitro may in fact mechanismto stimulate proliferation, acceleratedifferentiation, not occur in vivo. One way to circumvent these problems is to or alter functions of CNS glia (Giulian et al., 1988b). A number infuse isolated growth factors directly into the brain (Giulian of proteins have been described as putative glial growth factors and Ingeman, 1988; Giulian et al., 1988a). basedupon observations of brain-cell behavior in culture. For We have previously described isolation of several different example, fibroblast growth factor (FGF, Prusset al., 198l), epi- glia-promoting factors (GPFs) found in brain tissue that se- dermal growth factor (EGF; Loret et al., 1989) platelet-derived lectively stimulate the growth of either cultured astroglia or growth factor (PDGF, Besnard et al., 1987), glia maturation oligodendroglia (Giulian et al., 1985, 1986). Soluble oligoden- factor (GMF, Lim et al., 1989) and interleukin-1 (IL-l; Giulian droglia-promoting proteins appear during periods of develop- and Iachman, 1985) stimulate growth of cultured astroglia,while ment or at times of axonal regeneration when peak levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) oligodendroglial growth and myelin membrane production oc- and multipotential colony-stimulating factor (multi-CSF, Giu- cur (Giulian and Young, 1986). Some of these proteins are pro- lian and Ingeman, 1988) serve as mitogens for cultured mi- duced by neurons (Giulian and Young, 1986; Bottenstein et al., croglia. FGF, PDGF, and ciliary neurotrophic factor (CNTF) 1988), suggestingthe existence of regulatory mechanismsto are also found to alter patterns of proliferation or differentiation control myelination. We have isolated one of these proteins, in glial progenitor cellsgrown in vitro (Hugheset al., 1988;Noble oligodendroglia growth factor (OGF, previously referred to as et al., 1988; Richardson et al., 1988). Some of these molecules GPF-1; seeGiulian et al., 1985), from regeneratingretinal gan- may, in fact, be CNS regulatory factors becausethey are found glion cells of the goldfish (Giulian and Young, 1986). Goldfish- within brain tissuesduring periods of elevated glial biosynthetic derived OGF has biochemical and biological properties iden- activity and becausethey demonstrate specificity of action upon tical to a growth factor found in the developing rat brain and certain classesof non-neural cells (Giulian et al., 1988a; Raff et to one secreted by a mm-me neuronal cell line (Giulian and al., 1988; Richardson et al., 1988). Young, 1986). As described in this report, highly purified OGF At the present time, there is no well-characterized molecule infused into the cerebral cortex of the adult rat elevated pro- that acts as a selective growth factor for myelin-producing oli- duction of 3 different myelin proteins. The ability of OGF to godendrogha.Cell-culture experiments show that insulin and in- stimulate myelin protein synthesisin vivo suggeststhat neurons sulin-like growth factor 1 (IGF- 1) promote proliferation and dif- secretemolecules to trigger myelination of axons. Materials and Methods Received July 17, 1989; revised June 4, 1990; accepted Aug. 17, 1990. This work was supported by NINDS Grant NS 25637 and by NE1 Grant EY- Glia cell culture. Mixed glial cell cultures were prepared as described 04915. We thank Jocelyn Chen, Neil Zeigler, Jeff E. Ingeman, and Yunnie Choe earlier (Giulian et al., 1986) from dissociated cerebral cortex of newborn for their technical ass&ance. We also thank Dr. Robert Milner, The Scripps albino rats (Holtzman, Madison, WI). Cells (1.2 x lo6 per dish) were Institute, for the proteolipid protein probe PLP- 1. placed upon poly-L-lysine-coatedglass coverslips (22 mm square) within Correspondence should be addressed to Dana Giulian at the above address. 35-mm plastic dishes containing chemically defined culture medium Copyright 0 1991 Society for Neuroscience 0270-6474/91/l 10327-10$03.00/O (Bottenstein and Sato, 1979) supplemented with 10% fetal bovine se- 328 Giulian et al. * Effects of Oligodendroglia Growth Factor in viva rum; after 48 hr, cells were transferred to chemically defined culture acrylamide gel electrophoresis (SDS-PAGE) used the method of Neville medium. All culture media contained 5 &ml insulin (Sigma, St. Louis) (197 1) with a 20% polyacrylamide separating gel and a 4% stacking gel and 5 fig/ml transferrin (Sigma). (10 x 15 x 0.15 cm). OGF samples were mixed with sample buffer To test for biologic activity, cell cultures grown in 1.5 ml chemically (30% glycerol, 2.8 M 2-mercaptoethanol, 0.25% SDS, and 1% bromo- defined medium were incubated with increasing concentrations of OGF phenol blue, in Tris-HCl buffer, pH 8.4). This mixture was placed in for 72 hr. Appearance of oligodendroglia, ameboid microglia, and as- boiling water for 5 min and centrifuged for 2 min at 15,000 x g. The troglia were monitored by scoring the number of specifically labeled samples were separated by electrophoresis at room temperature using cells in 9 randomly selected fields (0.3 14 mmZ) from each of 3 or more 16 mA constant current for 3.5 hr. The gels were fixed and stained with coverslips. The data were expressed as n-fold increase over control silver as described by Poehling and Neuhoff (198 1). cultures incubated with matching aliquots of PBS ranging from 1 to 50 Preparative isoelectric focusing.- Prenarative_ isoelectric focusing- (IEF) ~1 per ml of culture medium. involved a horizonal column technique using the Rotofor preparative Glialcellidenttjication. Indirect immunofluorescence techniques were isoelectric focusing cell (Bio Rad, Richmond, CA). Fractions recovered used to identify astroglia containing glial fibrillaty acidic protein (GFAP) from gel-filtration chromatography were pooled and dialyzed against and oligodendroglia containing galactocerebroside (GC, Raff et al., 1978; sterile deionized H,O overnight. Following dialysis, 2.5 ml Servalyt Giulian et al., 1986). Ameboid microglia were identified by fluorescence (pH, 3-10) isodalt-grade ampholyte solution (40% ampholyte w/v; Ser- microscopy using acetylated low-density lipoprotein (ac-LDL) bound va, NY) was added to the sample, and the volume was brought up to to the fluorescent probe 1,l ‘-dioctadecyl- l,-3,3,3’,3’-tetramethylindo- 50 ml with deionized H,O to give a final 2% ampholyte concentration. carbocyanine perchlorate (DiI; Giulian and Baker, 1986) or fluorescently The sample was separated by isoelectric focusing for 4-5 hr at 12 W labeled polystyrene microspheres (Covaspheres, Duke Scientific, Palo constant power at 4°C into 20 fractions. After a pH measurement, each Alto, CA). in vitro labeling was carried out in chemically defined culture fraction was sterilized by filtration and tested for biologic activity. Con- medium with 200 &ml DiI-ac-LDL (Biomedical Technoloaies Inc.. trol experiments showed no effect of recovered ampholytes upon oli- Cambridge, MA) for-12 hr at 37°C. ’ godendroglia cultures. Growth of neuroblastoma cell lines. The mouse neuronal cell line Intracerebral infusion of growth factors. To study the effects of OGF Cl300 (CCL 147) was obtained from American Type Tissue Culture in vivo, small volumes of material were injected into the cerebral cortex Company and grown in medium (Bottenstein and Sato, 1979) consisting of albino rats. Adult rats (250-300 gm; Holtzman, Madison, WI) were of 50% Ham’s F-12 nutrient mixture (Cellgro) with N-2-hydroxyethyl- anesthetized by intraperitoneal injection of a mixture of 8.5 mg/ml piperazine-N’-2-ethane sulfonic acid (HEPES, 1.19 gm/liter) and xylazine, 42 mg/ml of ketamine hydrochloride, and 1.4 mg/ml of ace- NaHCO, (7.84 ml/liter 7.5% NaHCO,), and 50% Dulbecco’s Modified promazine maleate using 0.7-0.9 cc per kg body weight. The animals Eagle Medium (Cellgro) with glucose (2.85 gm/liter), gentamycin (48 were held in a stereotaxic device (David Kopf Instruments, 900) as burr mg/liter), an antibiotic/antimycotic solution (Sigma, 10 ml/liter), L-glu- holes were spaced 2 mm apart at 4 mm lateral to the sagittal suture.