Cornelis H. Langeveld

Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency

Astrocytes promote the survival of neurons. Conditions characterized by loss of neurons, such as aging and aging-related neurodegenerative disorders, are accompanied by both disturbances in astrocyte-neuron interactions and signs of oxidative damage. Neuronal glutathione, a major antioxidant in the brain, is maintained by astrocytes and brain levels of glutathione are reduced in named conditions. Therefore, we focused on a possible link between glutathione deficiency and loss of astrocyte-derived neuronal support. For this purpose, we used a coculture system consisting of rat striatal astrocytes and mesencephalic, dopaminergic (DAergic) neurons. Using tyrosine hydroxylase immunocytochemistry and radiolabeled dopamine uptake as parameters, an increase in the number and outgrowth of DAergic neurons was noted in cocultures as compared to cultures of mesencephalic neurons alone. This enhanced survival of DAergic neurons in cocultures was abolished following depletion of glutathione with buthionine sulfoximine. As demonstrated by glial fibrillary acidic protein immunocytochemistry and a microtiter tetrazolium assay, under these conditions no change in astrocyte survival occurred. However, glutathione depletion in cocultures was accompanied by loss of astrocyte-mediated neuroprotection against hydrogen peroxide toxicity. Thus, our results indicate that glutathione is important for the maintenance of the neuronal support function of astrocytes and that glutathione deficiency in the brain may lead to enhanced vulnerability of neurons to (oxidative) damage.

ASTROCYTE-ENHANCED NEURONAL SURVIVAL IS MEDIATED BY SCAVENGING OF EXTRACELLULAR REACTIVE OXYGEN SPECIES

The survival of cultured neurons is promoted by the presence of antioxidants or astrocytes. This indicates that extracellular reactive oxygen species (ROS) impair neuronal survival and suggests that astrocytes exert their survival-enhancing effect through inactivation of these toxicants. However, to our knowledge, data supporting this hypothesis are lacking. Previously, we showed that loss of the antioxidant glutathione abolishes the neuronal survival-stimulating action of astrocytes in cocultures, consisting of rat striatal astrocytes and mesencephalic, dopaminergic neurons. Using uptake of [3H]dopamine as marker of neuronal survival, we presently investigated whether this effect of glutathione depletion is mediated by extracellular ROS. For this purpose, we incubated glutathione-depleted cocultures with superoxide dismutase, catalase or both. Whereas superoxide dismutase had no effect and catalase only partially protected, addition of the enzymes together completely prevented the impairment of neuronal survival caused by glutathione loss. No change in neuronal survival occurred upon exposure of control cocultures to superoxide dismutase and/or catalase. These data strongly implicate scavenging of extracellular ROS in astrocyte-stimulated neuronal survival and moreover suggest a crucial role for glutathione in this process.

Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization

We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3–9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte‐specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP‐positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1–3%) were present in the human adult astrocyte cultures, using a panel of macrophage‐specific markers. However, the monoclonal antibodies (mAbs KP1, EBM11, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non‐lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay). J. Neurosci. Res. 49:342–354, 1997.

Cultured rat striatal and cortical astrocytes protect mesencephalic dopaminergic neurons against hydrogen peroxide toxicity independent of their effect on neuronal development

Reactive oxygen species (ROS), including hydrogen peroxide, are supposed to be involved in the degeneration of dopaminergic neurons in Parkinsons disease. The potential role of astrocytes against neurotoxic effects of ROS was studied in cocultures of rat mesencephalic neurons and rat striatal or cortical astrocytes. Neuronal [3H]dopamine uptake, a marker of dopaminergic neuron integrity, was enhanced by striatal astrocytes, but not by cortical astrocytes, compared to uptake in mesencephalic neurons cultured alone. Whereas hydrogen peroxide at concentrations up to 100 microM reduced the [3H]dopamine uptake in neuronal cultures, no reduction of the uptake was observed in cocultures, regardless of the origin of the supporting astrocytes. These results suggest that astrocyte mediated protection of neurons against hydrogen peroxide induced toxicity is not directly related to a region-specific neurotrophic effect.

The antiproliferative effect of 8-chloro-adenosine, an active metabolite of 8-chloro-cyclic adenosine monophosphate and disturbances in nucleic acid synthesis and cell cycle kinetics

8-Chloro-adenosine, the dephosphorylated metabolite of the antineoplastic agent 8-chloro-cyclic AMP, has been proposed to act on the regulatory subunits of cyclic AMP-dependent protein kinase. 8-Chloro-adenosine has a growth-inhibitory effect, the mechanism of which is unclear. We investigated the effects of 8-chloro-cyclic AMP and 8-chloro-adenosine on nucleic acid synthesis and cell cycle kinetics in two human glioma cell lines. These effects were compared to those of the cyclic AMP analogue 8-(4-chlorophenyl)-thio-cyclic AMP (8-CPTcAMP), which is less susceptible to dephosphorylation. Whereas 8-CPTcAMP almost completely inhibited RNA and DNA synthesis, both 8-chloro-adenosine and 8-chloro-cyclic AMP only partly inhibited synthesis of RNA and DNA at growth-inhibitory concentrations, as demonstrated by using [5-1H] uridine and [14C]thymidine incorporation. Therefore, the growth-inhibitory effect of 8-chloro-cyclic AMP is not (or not completely) due to activation of cyclic AMP-dependent protein kinase nor to the inhibition of nucleic acid synthesis. Flow cytometric analysis revealed that 8-chloro-cyclic AMP and 8-chloro-adenosine probably block cell cycle progression at the G2M phase. The effects of 8-chloro-cyclic AMP on nucleic acid synthesis and cell cycle progression were largely prevented by adenosine deaminase, which inactivates 8-chloro-adenosine. This indicates that the effects of 8-chloro-cyclic AMP were at least in part due to its metabolite 8-chloro-adenosine. Incorporation of 8-chloro-adenosine into RNA and DNA might contribute to the disturbance of the cell cycle kinetics and growth-inhibitory effect of 8-chloro-adenosine.

Anethole dithiolethione prevents oxidative damage in glutathione-depleted astrocytes.

Astrocytes protect neurons against reactive oxygen species such as hydrogen peroxide, a capacity which reportedly is abolished following loss of the antioxidant glutathione. Anethole dithiolethione, a sulfur-containing compound which is used in humans, is known to increase cellular glutathione levels and thought thereby to protect against oxidative damage. In the present study we found that anethole dithiolethione increased the glutathione content of cultured rat striatal astrocytes. This effect was abolished by coincubation with the glutathione synthesis inhibitor buthionine sulfoximine. Nevertheless, in the presence of buthionine sulfoximine, despite the lack of an increase in the lowered glutathione level, anethole dithiolethione fully protected the astrocytes against the enhanced toxicity of hydrogen peroxide. Thus, apparently other mechanisms than stimulation of glutathione synthesis are involved in the compounds protective action in astrocytes. Considering the occurrence of lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of anethole dithiolethione is warranted.

Differential sensitivity to hydrogen peroxide of dopaminergic and noradrenergic neurotransmission in rat brain slices.

Oxidative stress, induced by hydrogen peroxide, has been implicated in the pathogenesis of Parkinsons disease. Only scarce information is available if and how hydrogen peroxide, a side product of catecholamine (CA) breakdown, interferes with CAergic neurotransmission. Therefore, we investigated the effect of hydrogen peroxide on the release of [3H]dopamine (DA) and [3H]noradrenaline (NA) from rat striatal and cortical tissue slices, respectively. Hydrogen peroxide (0.01-1 mM) stimulated the spontaneous release of [3H]DA from striatal slices. Its effect on [3H]NA release from cortical slices, however, was much smaller than on DA release and occurred only in concentrations above 0.1 mM. Furthermore, only in concentrations of 1 mM or higher did a stimulation of spontaneous release of radioactivity from striatal slices incubated with [3H]choline occur. Omission of calcium significantly enhanced the effect on DA release, and an increase of calcium significantly reduced it. Blockade of vesicular storage with reserpine (0.3 microM) almost completely abolished [3H]DA release induced by hydrogen peroxide. Following incubation of striatal slices with [3H]NA in the presence of the NA (re)uptake blocker desmethylimipramine (0.3 microM), NA release was observed at a concentration (0.1 mM) at which no effect occurred in cortical slices. Moreover, under these conditions [3]NA and [3H]DA release from striatal slices reached comparable levels. Our results show that hydrogen peroxide induces a nonexocytotic release of DA and NA by interfering with the vesicular uptake and/or storage of these CAs. However, the striatal DA storage system, irrespective of the presence of either DA or NA, appeared to be substantially more sensitive to this effect than its cortical equivalent for storage of NA.

8-chloro-cyclic adenosine monophosphate, a novel cyclic AMP analog that inhibits human glioma cell growth in concentrations that do not induce differentiation

Cyclic AMP is supposed to play a role in cell growth and differentiation via activation of protein kinase A. The cAMP signal transduction pathway may therefore be used as a target for the development of anticancer drugs. We compared the effects of 8ClcAMP, a newly developed cAMP analog, to the effects of more commonly used cAMP analogs on the morphology and the proliferation of three human glioma cell lines. 8ClcAMP was the most potent growth inhibitor, exhibiting an IC50 of approximately 10 microM and inducing growth arrest in all three glioma cell lines at a concentration of 100 microM. The cAMP analogs 8CPTcAMP, dibutyryl cAMP, and 8BrcAMP were much less potent. If used in concentrations that induce growth arrest, both 8CPTcAMP and IBMX, but not 8ClcAMP, induced morphological differentiation of the glioma cells. Apparently, the growth-inhibiting effect of 8ClcAMP is not paralleled by its ability to induce morphological differentiation. The explanation for this phenomenon may be that 8ClcAMP does not exert its growth-inhibiting effect via activation of cAMP-dependent protein kinase. Two alternative mechanisms of action are discussed. Since 100 microM 8ClcAMP retarded the growth of normal rat astrocytes only to a marginal extent, without cytotoxic effects, it is concluded that 8ClcAMP may offer interesting perspectives in the treatment of malignant glioma.