Gabrielle Ulrich

Chromogranin A induces a neurotoxic phenotype in brain microglial cells.

Chromogranin A (CGA) belongs to a multifunctional protein family widely distributed in secretory vesicles in neurons and neuroendocrine cells. Within the brain, CGA is localized in neurodegenerative areas associated with reactive microglia. By using cultured rodent microglia, we recently described that CGA induces an activated phenotype and the generation of nitric oxide. These findings led us to examine whether CGA might affect neuronal survival, expression of neurofilaments, and high affinity gamma-aminobutyric acid uptake in neurons cultured in the presence or absence of microglial cells. We found that CGA was unable to exert a direct toxic effect on neurons but provoked neuronal injury and degeneration in the presence of microglial cells. These effects were observed with natural and recombinant CGA and with a recombinant N-terminal fragment corresponding to residues 1-78. CGA stimulated microglial cells to secrete heat-stable diffusible neurotoxic agents. CGA also induced a marked accumulation of nitric oxide and tumor necrosis factor-alpha by microglia, but we could not establish a direct correlation between the levels of nitric oxide and tumor necrosis factor-alpha and the neuronal damage. The possibility that CGA represents an endogenous factor that triggers the microglial responses responsible for the pathogenesis of neuronal degeneration is discussed.

The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture

SummaryThe organization of intermediate filaments (IF) and microtubules (MT) and the solubility of intermediate filament proteins and tubulin in astrocytes which develop from cerebral hemispheres of neonatal rats in culture were examined using immunocytochemical and immunochemical approaches. Results of immunocytochemical studies demonstrated that in flat astrocytes which develop after 3 weeks of culturing in serum-supplemented medium, the IF containing vimentin and glial fibrillary acidic protein (GFAP) are concentrated around the nucleus and dispersed in an irregular fashion throughout the cytoplasm. Astrocytes which develop in serum-free hormonally-defined medium irrespective of whether they are bipolar, multipolar or flattened, have IF organized as a fibrous network of filaments distributed from the nuclear regions to the cell periphery. Under both culture conditions, vimentin and GFAP are resistant to extraction with low salt buffer containing nonionic detergent, indicating that the different cytoplasmic distribution of IF is unrelated to the solubility properties of vimentin and GFAP. Double immunolabelling experiments with polyclonal antibody to GFAP and monoclonal antibody to each α-tubulin or β-tubulin reveal an extensive codistribution and parallel organization of IF and MT in all morphological types of astrocytes studied. Stabilization of MT with taxol, or depolymerization of MT with colchicine, cause dramatic changes in the distribution of IF and inhibit the extension of astrocyte processes in response to dibutyryl cyclic AMP (dBcAMP). In early stages of treatment with dBcAMP, renewal of culture medium without dBcAMP produces a rapid and permanent retraction of astrocyte processes, whereas in later stages the processes only retract partially and are then restored and maintained for several days in the absence of dBcAMP. The retraction of processes is accompanied by changes of immunocytochemical staining of IF with antibody to GFAP, which appears more intense and diffuse. However, electrophoretic and immunoblot analyses of detergent-extracted proteins from parallel cultures demonstrate that neither the amount nor the solubility of GFAP and vimentin are changed. Detergent extraction in MT stabilizing conditions shows that a substantial proportion of tubulin in astrocytes cultured in serum-containing and serum-free media is assembled into MT, most of which depolymerize on treatment with low temperature and Ca2+. Following long exposure to dBcAMP the proportion of cold/Ca2+-stable MT increases. The results suggest that the IF of astrocytes in culture are dependent on MT with respect to their cytoplasmic distribution. The MT play an important role in the organization and the maintenance of asymmetric shape of astrocytes.

Fibronectin and collagens modulate the proliferation and morphology of astroglial cells in culture

The proliferation and morphology of astroglia derived from neonatal rat cortex and cultured in serum‐free medium on either untreated, or fibronectin‐, or collagen I‐, or collagen IV‐treated substrates were investigated using tritiated thymidine autoradiography and immunocytochemical staining of glial fibrillary acidic protein (GFAP) and actin. Modification of culture substratum with fibronectin enhanced the rate of proliferation of astroglial cells and increased the proportion of process‐bearing astroglial cells. The distribution of actin and patterns of adhesion observed were typical for motile cells. Both types of collagen decreased the proportion of astroglial cells undergoing mitosis. Many of the astroglial cells exhibited a flat morphology and displayed prominent stress fibres in the cell body and processes. The data suggest that specific interactions with the substratum modulate the proliferation and morphological behaviour of astroglial cells.

Acquisition of vimentin in astrocytes cultured from postnatal rat brain

SummaryVimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin—GFAP transition which occurs during glial developmentin situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.

Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [(35)S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.

Differences in vimentin distribution in glial cells in culture revealed with an antibody against a phosphorylated epitope

We have previously described that spatial and temporal changes in the organization of vimentin that are correlated with protein kinase C (PKC)‐induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski‐Treska et al. (1991) J. Neurosci. Res.29, 362–378]. The antibodies were generated to native form of vimentin‐containing filaments and antibody B3 reveals a restricted immunostaining of vimentin in glial cells from human, rat and mouse origin. In the present study we show the distribution of epitope B3 analyzed by immunofluorescence within defined populations of rat glial cells. Relatively high immunoreactivity was found in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had low immunoreactivity. Although the results suggested that in Type 1 astrocytes the phosphorylated epitope is prominent, its phosphorylation was not found to be cell cycle‐dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the morphological conversion of polygonal to stellate astrocytes. As expected, the immunofluorescence was increased by exposition of astrocyte cultures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC‐mediated phosphorylation. In addition, the finding that the immunofluorescence of vimentin was more homogeneous in quiescent, serum‐deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycloheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescence studies on cultures enriched in Type 2 astrocytes and in oligodendrocytes indicate that the epitope B3 is hypophosphorylated in glial cells of this lineage and becomes dephosphorylated after terminal differentiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation of vimentin could be related to the down regulation in vimentin expression.

Monoamine regulation of adenosine 3′,5′-monophosphate in homogeneous neuronal cultures from chick brain hemispheres

The effect of isoproterenol, norepinephrine, dopamine, and serotonin on the accumulation of adenosine 3′,5′-monophosphate (cAMP) was studied in homogeneous neuronal cultures from 8-day chick embryo hemispheres. Among the catecholamines, isoproterenol had a more pronounced effect on the accumulation of cAMP. Norepinephrine and dopamine were considerably less potent and serotonin was ineffective. The response of neuronal cells to isoproterenol was inhibited by propranolol, suggesting that the cAMP increase was mediated by β-adrenergic receptors. Maximally effective concentration of isoproterenol (10 μM) produced a 2.5-fold increase in cAMP content which is in contrast to the much greater cAMP response elicited by isoproterenol in chick brain tissue. These results suggest that in chick embryo hemispheres the nonneuronal cells are the major sites of the effect of β-adrenergic agonists. The low responsiveness of the cAMP-generating system found in neuronal cultures in interpreted as reflecting either the number of β-adrenergic receptors or the regulation of β-adrenergic receptors and adenylate cyclase at the membrane level.