Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for immune-related molecules by central nervous system mixed glial cell cultures.

Cytokines secreted within the central nervous system (CNS) are important in the development of multiple sclerosis (MS) lesions. The balance between Th1, monocyte/macrophage (M/M) and Th2 cytokines in the CNS may be pivotal in determining the outcome of lesion development. We examined the effects of mixtures of cytokines on gene expression by CNS glial cells, as mixtures of cytokines are present in MS lesions, which in turn contain mixtures of glial cells. In this initial analysis by gene array, we examined changes at 6 hours to identify early changes in gene expression that represent primary responses to the cytokines. Rat glial cells were incubated with mixtures of Th1, M/M and Th2 cytokines for 6 hours and examined for changes in early gene expression employing microarray gene chip technology. A minimum of 814 genes were differentially regulated by one or more of the cytokine mixtures in comparison to controls, including changes in expression in a large number of genes for immune system-related proteins. Expression of the proteins for these genes likely influences development and inhibition of MS lesions as well as protective and regenerative processes. Analysing gene expression for the effects of various combinations of exogenous cytokines on glial cells in the absence of the confounding effects of inflammatory cells themselves should increase our understanding of cytokine-induced pathways in the CNS.

Role of calcium in nitric oxide-induced cytotoxicity: EGTA protects mouse oligodendrocytes.

Active nitrogen species are overproduced in inflammatory brain lesions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). NO has been shown to mediate the death of oligodendrocytes (OLs), a primary target of damage in MS. To develop strategies to protect OLs, we examined the mechanisms of cytotoxicity of two NO donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) on mature mouse OLs. Nitrosonium ion (NO+) rather than NO. mediates damage with both SNAP and SNP, as shown by significant protection with hemoglobin (HbO2), but not with the NO. scavenger PTIO. SNAP and SNP differ in time course and mechanisms of killing OLs. With SNAP, OL death is delayed for at least 6 hr, but with SNP, OL death is continuous over 18 hr with no delay. Relative to NO release, SNP is more toxic than SNAP, due to synergism of NO with cyanide released by SNP. SNAP elicits a Ca2+ influx in over half of the OLs within min. Further, OL death due to NO release from SNAP is Ca2+-dependent, because the Ca2+ chelator EGTA protects OLs from killing by SNAP, and also from killing by the NONOates NOC-9 and NOC-18, which spontaneously release NO. SNP does not elicit a Ca2+ influx, and EGTA is not protective. In comparison to the N20.1 OL cell line (Boullerne et al., [1999] J. Neurochem. 72:1050-1060), mature OLs are (1) more sensitive to SNAP, (2) much more resistant to SNP, (3) sensitive to cyanide, but not iron, and (4) exhibit a Ca2+ influx and EGTA protection in response to NO generated by SNAP.

Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1.

Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S-nitroso-N-acetyl-DL-penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S-nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase-3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.

Binding of cholera toxin B subunit: a surface marker for murine microglia but not oligodendrocytes or astrocytes.

GM1 ganglioside is a receptor for the B subunit of cholera toxin. In lymphocytes, B subunit elicits an influx of extracellular Ca++ (Dixon et al., 1987). To investigate this signaling pathway in glia, we assessed the presence of GM1 ganglioside on the surface of cultured murine central nervous system (CNS) glia by binding of fluorescein-labeled B subunit. B subunit binding was compared to binding of peanut agglutinin, wheat germ agglutinin, and Bandeiraea (Griffonia) simplicifolia lectin (BSL)I, a microglial marker. Antibodies to glial fibrillary acidic protein, A007/O4 antigens, and galactocerebroside were used to identify astrocytes, immature oligodendrocytes (OLs) and mature OLs, respectively. Binding patterns differed based on cell type and developmental stage. Wheat germ and peanut agglutinins bound to the surface of microglia, astrocytes, and immature OLs; neither lectin bound to any significant extent to the surface of membrane sheets of mature OLs, although wheat germ agglutinin was rapidly endocytosed. Cells identified as microglia by BSL I binding and morphology were the only cells to stain brightly on the surface with B subunit. Thus, surface GM1 ganglioside appears to be a highly enriched marker for microglia in these mixed glial cultures. The effects of B subunit on intracellular Ca++ were examined by laser cytometry in glial cultures loaded with Indo-1. No Ca++ responses were observed in microglia. Mature OLs were examined for Ca++ responses to B subunit before and after surface levels of GM1 ganglioside were increased by incubation with exogenous GM1 ganglioside. Again, no Ca++ responses were observed. Thus, cultured microglia and mature OLs do not have the GM1-mediated signal transduction pathway seen in lymphocytes. However, the presence of GM1 ganglioside on microglia may play a role in giving rise to antibodies to this glycolipid in some CNS inflammatory diseases.

Release of intracellular calcium stores leads to retraction of membrane sheets and cell death in mature mouse oligodendrocytes.

The ability of mature oligodendrocytes (OLs) to recover from insult is important in repair of damage following demyelination. Since regulation of Ca2+ levels within cells plays a critical role in function and survival, this study investigates the effects of changes in cytoplasmic Ca2+ on the viability of cultured mouse OLs and their ability to maintain membrane sheets. Mature OLs in culture respond rapidly to the calcium ionophore A23187 and promptly return to resting Ca2+ levels when the ionophore is removed. Longer exposure to 0.1-1.0 microM A23187 leads to microtubule disruption, membrane sheet retraction and eventual cell death; nuclear lysis occurs in many of the OLs, as reported by Scolding, et al. (1) for rat OLs. In our cultures, mature OLs were more susceptible to nuclear lysis than were immature OLs or astroglia. Release of intracellular Ca2+ stores with thapsigargin at 5-10 microM also leads to retraction of membrane sheets. Following 6 hours of continuous exposure to thapsigargin, the effects on membrane sheets are reversed over the next 12 hours. After 18 hours of continuous exposure to thapsigargin, only occasional nuclear lysis is observed, but a number of the mature OLs show signs of DNA fragmentation, indicating that apoptotic death is occurring. Our results suggest that mature OLs cannot survive a prolonged influx of extracellular calcium as readily as immature OLs and astroglia, but have mechanism to withstand similar increases in cytoplasmic Ca2+ following sustained release of intracellular stores.

Maintenance of membrane sheets by cultured oligodendrocytes requires continuous microtubule turnover and Golgi transport.

Oligodendrocytes in murine shakeoff cultures elaborate extensive membrane sheets containing networks of microtubules. Several membrane components, including proteolipid protein (PLP) and sulfatide, are transported through the Golgi en route to the plasma membrane or myelin (1,2). This transport is essential for membrane assembly, but its role in continuing maintenance of the sheets is not known. We examined the stability of the membrane sheets following microtubule stabilization with taxol or block of transport into the Golgi with brefeldin A. Within one to three hours, both agents had marked effects on the membrane sheets. While some oligodendrocytes maintained regions of normal membrane sheets, many showed retraction of the sheets, with the majority now exhibiting multiple processes rather than sheets. The distribution of sulfatide, PLP and tubulin in cell bodies, processes and sheets was altered in treated cells, as analyzed by immunocytochemical staining with antibodies to these components. The Golgi apparatus also showed reorganization in the presence of taxol, as visualized by binding of wheat germ agglutinin, a lectin with high affinity for distal Golgi vesicles. All of these effects were reversible when the agents were removed after 3 hours. Thus, maintenance of membrane sheets by oligodendrocytes in culture is a dynamic process, requiring ongoing microtubule turnover and transport of molecules through the Golgi.

Caprine beta-mannosidosis: regional differences in deficits of CNS myelin proteins.

Caprine beta-mannosidosis is an autosomal recessive disorder characterized by marked deficiency of beta-mannosidase activity, accumulation of oligosaccharides, and pathologic changes involving prominent dysmyelination. The myelin deficits show marked regional variation, with spinal cord mildly affected and many brain regions severely affected by morphologic criteria. In this study, levels of myelin basic protein (MBP) and proteolipid protein (PLP) were measured by immunoblotting in samples prepared from spinal cord, brainstem and cerebral hemispheres of normal and affected goats at 2-4 days (newborns) and 2-4 weeks of age. In affected goats, total levels of MBP in spinal cord were normal, while PLP levels were 60-70% of normal at both ages. In contrast, PLP and MBP in brainstem and cerebral hemispheres were severely decreased at both ages, with levels of PLP 10-13% and MBP 25-29% of normal in newborns, and generally more reduced at 2-4 weeks. When myelin fractions were isolated on 0.32/0.85 M sucrose gradients, yields were about 38 and 25% of normal in spinal cord at the two ages, but less then 3% of normal in brainstem. Yields of myelin-like fraction were decreased as well, but to lesser extents than yields of myelin. Myelin from spinal cord had a normal composition with regard to PLP and MBP content, while the myelin fraction from brainstem was markedly deficient in both proteins. This suggests formation of myelin with a very abnormal composition in brainstem, or inclusion of large amounts of membranes other than myelin in this fraction. The more severe deficits in brainstem and cerebral hemispheres compared to spinal cord are consistent with morphologic observations.(ABSTRACT TRUNCATED AT 250 WORDS)

Recovery of proteolipid protein in mice heterozygous for the jimpy gene.

We have measured levels and synthesis of proteolipid protein (PLP) and its transport into myelin in female mice heterozygous for the jimpy gene and in their normal female littermates. In both cord and cerebrum, jimpy carriers show deficits in PLP during development followed by compensation in adulthood. Recovery of PLP occurs earlier in cord than in brain. At 13 days levels of PLP in carriers compared to controls are reduced to 0.60 and 0.44, respectively, in cord and cerebrum. By 100 days, normal levels of PLP are attained in cord (1.13) whereas levels of PLP in cerebrum are only 0.78 of control. By 200 days full recovery occurs in cerebrum, with a ratio of 1.21, suggesting a possible over-compensation. The yield of myelin from cerebrum was reduced to 0.78 in carriers compared to controls at 17 days. In brain slices, incorporation of [3H]leucine into homogenate PLP from carriers is the same as in controls, whereas [3H]leucine incorporation into myelin PLP is reduced to 0.68 of control. These results indicate that synthesis of PLP in the carriers is normal at 17 days, but transport of PLP into myelin is reduced. Similarly, acylation of homogenate PLP is normal, whereas acylation of myelin PLP is reduced, as measured by incorporation of [3H]palmitic acid. Transport of PLP into myelin was compared to transport of MBP; transport of both proteins was equally decreased as indicated by the similar ratio of labeled PLP to MBP in myelin from carriers compared to noncarriers.(ABSTRACT TRUNCATED AT 250 WORDS)