Induction of antiphosphorylcholine antibody formation by anti-idiotypic antibodies.

Anti-idiotypic antibodies have been used to mimic antigen in the mouse antiphosphorylcholine response in order to investigate the induction of precursors of antibody-forming cells. We have shown that interaction of anti-idiotype antibody with receptor antibody molecules induces the formation of antibodies that are specific for phosphorylcholine and carry the idiotypic determinants. This induction is dependent on the recognition of carrier determinants on the anti-idiotype antibody by helper T cells. We conclude that receptor antibody molecules on the surface of the precursors of antibody-forming cells deliver the antigenic signal for the induction of these cells.

The use of bacterial lipopolysaccharides to show that two signals are required for the induction of antibody synthesis.

Evidence is presented that antigen-sensitive cells require two signals for induction. Normally these two signals are delivered to the cell via the recognition of two determinants on the immunogen: the first the receptor on the antigen-sensitive cell, and the second by the cooperating cell system. The special experimental situation described here depends upon the observation that bacterial lipopolysaccharides (LPS) render immunogenic a variety of haptens. When monovalent haptens (TNP-amino acids) are added to spleen cultures, specific antihapten responses are induced in the presence of LPS. After analyzing competing interpretations of this phenomenon, we propose that the antigenic signal is delivered as the consequence of a conformational change in the receptor upon interacting with antigen, and the second signal is delivered directly via the interaction of LPS with the membrane on the antigen-sensitive cell receiving the antigenic signal, or indirectly via the interaction of LPS with the cooperating cell population. These data imply LPS is not itself a mitogen, but merely completes an inductive stimulus to B cells. The experimental results from these and other studies indicate how these two signals may participate in inductive, suppressive, and paralytic stimuli to antigen-sensitive cells.

Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation.

A microwell culture system was developed for analysis of cell movements and interactions during nervous system histogenesis. Cells from trypsinized 7-day-old mouse cerebellum reaggregated within hours into clusters which later developed interconnections consisting of either sheets of migrating cells and cell processes or cables of fiber bundles with cells migrating along their surfaces. Granule cells in several stages of differentiation, basket and/or stellate neurons, some larger neurons, and two types of neuroglial cells were identified in reproducible, nonrandom patterns by scanning and transmission electron microscopy. Axonal and dendritic processes, both with growth cones, and numerous synapses were generated in vitro.

Growth factors and taurine protect against excitotoxicity by stabilizing calcium homeostasis and energy metabolism.

Taurine, brain derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) are known to control the development of early postnatal cerebellar granule cells. This study attempted to investigate possible mechanisms of this control by determining neuronal survival, calcium homeostasis, and related calcium-mediated functions, as well as the site of action during glutamate-induced excitotoxicity in cultures of cerebellar granule cells. We report that stimulation of glutamate receptors induced a rapid increase in intracellular calcium concentrations ([Ca(2+)](i)) and a decrease in mitochondrial energy metabolism. These effects of glutamate were time- and concentration-dependent and could be specifically blocked by glutamate receptor antagonists. Taurine and bFGF but not BDNF differently regulated [Ca(2+)](i), and preserved the mitochondrial energy metabolism in the presence of glutamate. The regulation of [Ca(2+)](i) by bFGF and taurine required pretreatment of cells with these factors. Confocal microscope analysis of [Ca(2+)](i) and (45)Ca(2+) uptake studies showed that bFGF reduced the magnitude of glutamate-induced calcium uptake with no apparent regulation thereafter. Taurine, on the other hand, did not affect the level of calcium uptake induced by glutamate but rather the duration of the maximal response; this maximal response was transient and returned to basal levels approximately 10 min after glutamate receptor stimulation. We conclude from these data that bFGF and taurine prevent glutamate excitotoxicity through regulation of [Ca(2+)](i) and mitochondrial energy metabolism. Furthermore, the neuroprotective role of taurine and bFGF was enhanced by their collaboration.

The regulation of phosphorylation of tau in SY5Y neuroblastoma cells: the role of protein phosphatases.

In Alzheimer disease brain the microtubule associated protein (MAP) tau is abnormally hyperphosphorylated. The role of protein phosphatases (PP) in the regulation of phosphorylation of tau was studied in undifferentiated SY5Y cells. In cells treated with 10 nM okadaic acid (OA), a PP-2A/PP-1 inhibitor, the PP-1 and -2A activities decreased by 60% and 100% respectively and the activities of MAPKs, cdc2 kinase and cdk5, but not of GSK-3, increased. OA increased the phosphorylation of tau at Thr-231/Ser-235 and Ser-3961404, but not at Ser-262/356 or Ser-199/202. An increase in tyrosinated/detyrosinated tubulin ratio, a decrease in the microtubule binding activities of tau, MAP1b and MAP2, and cell death were observed. Treatment with 1 microm taxol partially inhibited the cell death. These data suggest (1) that OA induced hyperphosphorylation of tau is probably the result of activated MAPK and cdks in addition to decreased PP-2A and PP-1 activities and (2) that in SY5Y cells the OA induced cell death is associated with a decrease in stable microtubules.

Abnormally phosphorylated tau in SY5Y human neuroblastoma cells.

In Alzheimer disease (AD) the microtubule associated protein (MAP) tau is hyperphosphorylated at several sites. In the present study, like AD tau, tau in the human neuroblastoma SH-SY5Y was found to be hyperphosphorylated, at Ser-199/202, Thr-231, Ser-396 and Ser-404. However, in contrast to AD, the tau in SY5Y cells was not hyperphosphorylated at Ser-235 and there was only one tau isoform. Quantitative analysis revealed that approximately 80% of the SY5Y-tau was phosphorylated at Ser-199/202. The phosphorylated tau was deposited in perikarya and processes of the cells whereas most of the unphosphorylated (at Ser-199/202) tau was localized in the nucleus. Tau from the cell lysates did not bind to taxol-stabilized microtubules. In contrast, MAP1b and MAP2 from cell lysates bound to stabilized microtubules in vitro and were associated to the microtubule network in situ. Phosphorylation of tau at high levels, its inactivity with microtubules and its accumulation in SY5Y cells provide for the first time a cell model of cytoskeletal changes seen in AD.

Regional differences in cytoarchitecture of the weaver cerebellum suggest a new model for weaver gene action.

This paper examines the structure and cytoarchitecture of the cerebellum of the weaver mutant mouse with particular emphasis on regional differences along the mediolateral and anterior-posterior axes. We have uncovered several, previously undescribed features of the weaver cerebellar phenotype. Perhaps the most dramatic example of our findings is the severe disruption of the folial structure of the hemispheres of the weaver cerebellum. A dorsal overgrowth of tissue occurs in the hemispheres that forms a finger-like projection superficial to an atrophic but structurally more normal cerebellar mass underneath. While this folial abnormality is most evident in the homozygote (wv/wv) the antecedents of its appearance are already apparent in the heterozygote (+/wv). At the level of the cytoarchitectonics of the mutant brain, we find substantial variation in the positioning, numbers and density of both Purkinje and granule cells. As a whole, Purkinje plus Golgi II cell numbers are down by over 40%, but this reduction occurs almost exclusively in the medial half of the cerebellum. The hemispheric region contains a nearly normal number of cells per sagittal section (although their positions are predominantly incorrect). The granule cells also show numerical variation; they are nearly absent at the midline, but a substantial number of them survive in the lateral cerebellar cortex. In the paraflocculus, for example, granule cells can be observed in a modest internal granule cell layer as late as 38 postnatal days. These results are discussed in terms of a model of wv gene action in which we propose that the effect of the mutation is a general disruption of cellular distribution in the cerebellar cortex, affecting both Purkinje and granule cells and beginning prenatally.

Reduced number and altered morphology of microglial cells in colony stimulating factor-1-deficient osteopetrotic op/op mice.

The numerical density of microglial cells is reduced by 47% in the corpus callosum, by 37% in the parietal cortex and by 34% in the frontal cortex of mice mutant at the op locus which are totally devoid of colony stimulating factor-1 (CSF-1), the major growth factor for macrophages. Moreover, microglia in the frontal cortex of the op/op mice are smaller and have shorter cytoplasmic processes compared to control mice. Study suggests that CSF-1 plays a role in vivo in the formation and maturation of microglia and has little or no effect on perivascular cells.

The role of taurine in the survival and function of cerebellar cells in cultures of early postnatal cat.

The role of taurine and beta-alanine was analyzed in kitten cerebellar cultures. Since in contrast to mouse, cats (and primates including man) cannot synthesize sufficient taurine to maintain their body pools, we considered the cat an ideal species for the analysis of the role of taurine during early postnatal cerebellar development under controlled conditions. Unexpectedly, we found that the presence of taurine was toxic to neurons but that compounds, considered to be competitors for the beta-amino acid uptake system, support cell survival and cell function in vitro, the opposite of the results found in mice. This could be explained by the finding that only minute amounts of [3H]taurine were taken up by both cat neurons and glial cells under optimal culture conditions but that in the presence of the taurine analogues beta-alanine and guanidinoethane sulfonic acid (GES) significant amounts of taurine were found in all cell types. These differences between mouse cerebellar cells and cat cerebellar cells in vitro suggest that a re-evaluation of the mechanisms that control taurine function in cats and primates is warranted.

Balanced interaction of growth factors and taurine regulate energy metabolism, neuronal survival, and function of cultured mouse cerebellar cells under depolarizing conditions.

The development of neuronal cells in a given cellular environment requires mechanisms that dynamically regulate the balanced interactions of multiple factors which are known to control maintenance and plasticity in function of neurons throughout constantly changing extracellular conditions. Periodic release of excitatory amino acids from both developing glial and neuronal cells into the extracellular environment and their uptake has been shown to stimulate neuronal function in concert with growth factors that control the degree of depolarization and, therefore, neuronal function. This study attempts to characterize the critical concentrations of these factors either alone or together in relation to energy metabolism, cell survival and function. We demonstrate a close correlation between energy metabolism of neuronal cells, controlled by the combination of growth-factors (beta FGF, BDNF), and glutamate-taurine as well as K+ in depolarizing concentrations (10-25 mM), during the balancing act of neuronal survival or death, and neuronal function. These functions depend on medium conditions (energy sources, ion composition), the ratio of glial cells versus neurons and cell density. Granule cell migration as a measure of developmental neuronal function was analyzed in the presence of various combinations of growth factors and taurine under various depolarizing conditions (glutamate, K+). We found that K+ concentrations > 7 mM in BME and 10% horse serum blocked migration in less than 30 min. Taurine did not prevent this effect. However, in the presence of HEPES as well as in F12-medium with HEPES, taurine restored granule cell migration. On the other hand, glutamate-or NMDA-mediated depolarization stopped migrating granule cells while NMDA antagonists extended the period of migration. Taurine amplified the stop-signal in the presence of glutamate agonists but increased the number of migrating cells in the absence of glutamate. Thus, the mechanisms of glutamate receptor mediated excitotoxicity, possibly by reducing Ca2+ influx under depolarizing conditions, but amplifies the stop-signal, Ca2+ levels may not control granule cell migration.

Microbial carbohydrate specific antibodies distinguish between different stages of differentiating mouse cerebellum.

High titered anticarbohydrate antibodies were used to identify cell surface carbohydrates during different stages in histogenesis of mouse cerebellum in a micro tissue-culture system which mimics selected features of in vivo cerebellum development. Blockage of fiber formation within the first few days in vitro and inhibition of cell migrations by carbohydrate-specific antibodies served as an assay system for possible contributions of surface carbohydrates to the behavior of developing cerebellar cells. Microbial strains were selected on the basis of carbohydrate structures of their cell wall antigens, and anticarbohydrate antibodies were raised against treated whole bacteria and yeast in rabbits. We found that antibodies to mannan were active at all stages of development tested (embryonic day 13, E13; the day of birth, PO; and postnatal day 7, P7). Antibodies to sialic acids prepared against strains B and C of Neisseria meningitidis distinguish different subterminal structures: anti-B reacted with E13 and PO cerebellar cells, and anti-C mostly with cells older than P7. Antifetuin antibody recognized E13 and PO but not P7 cell populations. Pneumococcus C strain R36A-specific antibodies were effective only after coating cells to C type carbohydrate before application of the antibody. The results demonstrate that antimicrobiol carbohydrate antibodies cross-react with mammalian cell surface carbohydrate structures and therefore can be used as a powerful tool in tissue culture to analyse those structures which might control cell behaviors pertinent to cerebellar development.

Changes in particulate neuraminidase activity during normal and staggerer mutant mouse development.

The activity of particulate neuraminidase (sialidase, EC 3.2.1.18) in wild-type mice and the neurological mutant Staggerer was studied during development. Peak activity of this enzyme was observed at postnatal day 3 (P3) in three tissues of normal mice: cerebellum, cerebrum, and liver. In Staggerer, however, neuraminidase peak activity was observed at P27 in the cerebellum, whereas the activity was close to normal in Staggerer cerebrum and liver. Activities of the other glycosidases in Staggerer (alpha-glucosidase (pH 3.7), alpha-glucosidase (pH 6.0), N-acetyl-beta-hexosaminidase, beta-glucosidase, and beta-galactosidase) did not show significant variation compared with wild-type at P27 in any of the three tissues. This indicates that the late activity peak of particulate neuraminidase activity in the Staggerer cerebellum is neuraminidase-specific and not due to a general increase of lysosomal enzymes.