Immuno-electron microscopic study of the distribution of the S 100 protein in brain glial cells.

The distribution of the brain-specific S 100 protein was studied by an immunohistochemical method at the ultrastructural level. The S 100 protein was localized in the plasma membrane of astrocytes, oligodendrocytes and neurons, but not in that of endothelial cells and pericytes. Astrocytic filaments also possessed S 100 activity. The vascular basement membrane lacked traces of S 100.     

Regulation of specific functions of glial cells in somatic hybrids. I. Control of S100 protein

Hybrids were isolated between rat glial cells and mouse fibroblasts. Micro complement (C′) fixation was used to assay S100, a highly acidic protein specific for nervous tissues. The glial cells contain large amounts of S100. Extracts of the fibroblasts contain some C′ fixing material which is detected only at very high protein concentrations and which fixes C′ only weakly. The identity of this material is not known. The hybrids contain some C′ fixing material, but the concentrations of protein necessary to reach the point of antigen-antibody equivalence is ten times greater with an extract of hybrids than with an extract of glial cells. This increase in the concentration of protein is associated with a decrease in the amount of C′ fixed. The possible significance of the C′ fixing material in the hybrids is discussed.     

The effect of antiserum to S 100 protein on behavior and amount of S 100 in brain cells

Since antiserum raised against the S 100 protein has an impairing effect on acquisition in behavioral tests, when interacting with S 100 on hippocampal cells, the effect of S 100 antiserum was studied in rats on the S 100 content of the hippocampus and thalamus, as well as on behavior. The operant reversal of handedness test and a light discrimination test were used. S 100 antiserum, 2 × 30 μl, was injected intraventricularly before and during the sessions of two different learning tests. The S 100 protein was determined by quantitative immunoelectrophoresis. In the antiserum-injected animals, the levels of S 100 protein was increased by up to 30%, the incorporation values of ³H-valine increased in proteins of high molecular weight. Further acquisition was inhibited compared to controls, in which antiserum absorbed with pure S 100 protein was injected intraventricularly. The stimulation of S 100 synthesis, probably by the glia, may have occurred by a negative feedback effect, as has been observed in thymocytes.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

The hetero-oligomeric complex of the S100A8/S100A9 protein is extremely protease resistant

S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.     

Appearance and accumulation of the brain specific S 100 protein in the developing nervous systems of rat, rabbit and guinea pig.

The appearance and accumulation of S 100 protein during postnatal development of rats and rabbit, and during pre- and postnatal development of guinea pig, was studied quantitatively by immunoelectrophoresis. High amounts of S 100 were found in newborn guinea pig brain. In rabbit and rat central nervous systems the content of S 100 rose linearily between the 1st and the 3rd and 4th weeks of postnatal life, respectively. From this later period to the adult animal there was a small increase in the amount of S 100 in rabbit and rat brain. The results of this study are compared with other studies on S 100 accumulation during postnatal development of rat, rabbit and guinea pig, using immunofluorescence and immunoelectron microscopic techniques.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections

Summary Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study

Abstract— A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [¹⁴C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the ¹⁴C-labelled proteins in both cell types were greater, the younger the brain. The ¹⁴C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [¹⁴C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [¹⁴C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of ¹⁴C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [¹⁴C]phenylalanine, the specific radioactivities of all ¹⁴C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.     

Regulation of synthesis of a brain-specific protein in monolayer cultures of clonal rat glial cells

C6 cells were grown in monolayer culture under conditions permitting continued exponential cell division after attainment of a density at which extensive intercellular contacts were formed. An increase in the relative synthesis of S100 protein coincided with the time of formation of extensive intercellular contacts and preceded the onset of the stationary phase of growth by three generations. These observations suggested that the induction of S100 protein synthesis was mediated by cell contact and not by an arrest of cellular growth. The mechanism of this induction was first studied in a homologous non-initiating cell-free protein-synthesizing system from C6 cells, using fixed amounts of free amino acids or fully charged rat liver aminoacyl-tRNA as a source of precursors for protein synthesis. Real synthesis of total soluble proteins decreased as the cells progressed from logarithmic to stationary growth while synthesis of S100 protein increased during this period. The capacity of poly(A)+ RNA from logarithmic and stationary cultures to direct the synthesis of S100 protein was estimated in a cell-free protein-synthesizing system derived from wheat embryos. Increased synthesis of S100 protein in stationary cultures was directly correlated with an increase in translatable S100 protein mRNA.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.     

S100 beta stimulates calcium fluxes in glial and neuronal cells.

The glial-derived protein S100 beta can act as a mitogen or a neurotrophic factor, stimulating proliferation of glial cells or differentiation of immature neurons. We report here that dimeric S100 beta evokes increases in intracellular free calcium concentrations ([Ca2+]i) in both glial cells and neuronal cells. The [Ca2+]i increase exhibited a rapid transient component which was not affected by removal of extracellular calcium and a sustained component which appeared to require influx of extracellular calcium through Ni(2+)-sensitive channels. S100 beta also stimulated hydrolysis of phosphoinositides, suggesting a mobilization of calcium from intracellular stores. These data suggest that although the final biological responses of neuronal and glial cells to S100 beta are different, transduction of the S100 beta signal in both cell types involves changes in [Ca2+]i.     

Age-dependent changes in brain protein synthesis in the rat

Brain protein synthesis was studied in vivo, in brain slices, and in cell-free systems in rats aged 1, 16, and 24 months. We observed a highly significant reduction in amino acid incorporation with advancing age. This reduction was observed in vivo, in slices, in postmitochondrial supernatant, microsomes, and membrane-bound polysomes. Free heavy polysomes showed no age-dependent decline but formed a smaller proportion of total ribosomes in older animals. These studies suggest that in the rat brain protein synthesis declines before senescence, possibly due to an impairment in the initiation process.     

The protein of glial intermediate filaments in different areas of the rat brain at experimental neurosis

The content and polypeptide composition of glial fibrillary acidic protein (GFAP) in the rat cerebral cortex, cerebellum, hippocampus, and mesencephalon were studied under conditions of experimental neurosis. Significant changes of the total GFAP content were observed in the hippocampus, mesencephalon, and cerebellum. Both the content and polypeptide composition of soluble GFAP form were markedly modified. These changes of glial filament protein apparently reflect the peculiarities of the reorganization of the astrocyte intermediate filaments at the animal’s long-term neurotization.