Mitochondrial impairment and recovery after heat shock treatment in a human microglial cell line.

The application of a heat shock on the human microglial cell line (CHME 5) has been shown to cause cytoskeleton modifications and alterations in phosphorylated metabolite content (Macouillard-Poulletier de Gannes et al., 1998a Metabolic and cellular characterization of immortalized human microglial cells under heat stress. Neurochem. Int. 33, 61-73). In this study, we focused on the possible involvement of mitochondria in this heat stress response. The cell respiratory properties were followed during the recovering period and the possible relationships between mitochondria and the cytoskeleton were studied. We observed that the heat shock induced changes in mitochondrial activity due to protein denaturation, rather than mitochondrial loss. Furthermore, these alterations were correlated with cytoskeleton disorganization since vimentine, tubuline and mitochondria shift, simultaneously, to a perinuclear location. The perturbations of the mitochondrial distribution persisted until cytoskeleton networks had recovered. Nevertheless, the respiratory properties recovered rapidly suggesting a renaturation of mitochondrial proteins in connection with mitochondrial cytoplasmic redistribution.

Metabolic and cellular characterization of immortalized human microglial cells under heat stress.

Microglia are able to shift from a resting to an activated state during which they acquire functions typical of primitive forms of mononuclear phagocytes, as well as highly differentiated forms of these cell types. Activation of the microglia after a trauma involves functional readjustments including both the resurrection of basic cell functions and the development of functions implicated in cell-cell interactions. Here, the behaviour of the human microglial cell line CHME-5 was observed after hyperthermia. The strong expression of the inducible form of the hsp70 proteins and their nuclear location, like the more nucleolar presence of the hsc70 proteins seemed to confer heat resistance to the cells. Following minor morphological changes, the observation of the cytoskeleton network during stress recovery revealed a differential sensitivity of actin and tubuline to heat shock. The energy metabolism of both the microglial cell line and the monocyte cell line U937 were compared, under basal conditions, using phosphorus-31 NMR, to discern those phosphorylated metabolites which could be specific of either the monocytic or the macrophagic phenotype. A very high content of phosphocreatine was observed in the microglial clone whereas this compound was absent in monocytes. The lower CHME-5 phosphomonoester content as compared to monocytes also suggested that the microglial cell line displayed a more macrophagic metabolic pattern. After heat shock, the CHME-5 phosphorylated compounds showed large, transient, perturbations. Cells recovered their basal metabolic content 24 h after heat stress. This behaviour, in addition to the expression of inducible hsp70 proteins and the cytoskeleton rearrangement, are indicative of the particular adaptation of the microglial cell line to stress situations.

Energetic and morphological plasticity of C6 glioma cells grown on 3-D support; effect of transient glutamine deprivation.

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum.

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4-5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Developmental characterization of thymosin beta 4 and beta 10 expression in enriched neuronal cultures from rat cerebella.

The beta 4 and beta 10 thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin beta 4 is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin beta 10 is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the beta-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both beta-thymosins were initially expressed in granule cells, although expression, especially of thymosin beta 4, was truncated compared with the in vivo time course. As in vivo, thymosin beta 4 was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin beta 10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the beta-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating beta-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.

Beta-adrenergic receptors of cerebellar astrocytes in culture: intact cells versus membrane preparation.

Experiments were carried out to assess: the influence of culture conditions on the expression of beta-adrenergic receptors in intact glial cells from the central nervous system; and the extent to which quantitation of receptor sites in membrane preparations reflects the receptor population of the whole cells they are derived from. Cerebellar astrocytes were chosen for this study since essentially one receptor subtype, the beta 2 one, is present in adult cerebellum. Intact, attached cerebellar astrocytes exhibit only one class of binding sites for the beta-adrenergic antagonist, [3H]CGP 12177. Replating of the astrocytes after a few days of culture in vitro induces an up-regulation of the receptors. This effect is particularly important when astrocytes are maintained for 6 days in the presence of horse serum, a condition that favors cellular differentiation. Only 30-50% of the beta-adrenergic receptors of the intact cells can be detected on membrane preparations. When membranes are prepared from astrocytes grown either in the presence of horse serum or under chemically controlled medium (i.e. under differentiation promoting conditions) two classes of binding sites for [125I](-)-iodocyanopindolol are revealed. Several hypotheses, mainly related to the morphology of the cells, may provide an explanation for such differences. Studies of the pharmacological specificity of receptors of membrane fractions show that cerebellar astrocytes cultured in vitro exhibit both beta 1 and beta 2 receptor subtypes. The beta 1 subtype receptors are slightly more abundant when astrocytes are grown in fetal calf serum (FCS), a condition under which they exhibit a polygonal, poorly differentiated morphology. When culture conditions favor cellular differentiation, more receptors of the beta 2 subtype are seen, which can be related to what is observed in the adult in vivo where the astrocytes exhibit a differentiated morphology.