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Max Planck Institute of Psychiatry, Clinical Institute, Munich, Germany
Address all correspondence and requests for reprints to: Christian Behl Ph.D., Clinical Institute, Department of Neuroendocrinology, Max Planck Institute of Psychiatry, Kraepelinstrasse 10, 80804 Munich, Germany. E-mail: chris{at}mpipsykl.mpg.de
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| Introduction |
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Amyloid ß-protein (Aß) is a 40- to 43-amino acid peptide that is associated with plaques in the brains of patients with AD and is cytotoxic to neurons (6, 7, 8, 9, 10). Aß-induced cytotoxicity has been shown to be caused by the intracellular accumulation of H2O2, ultimately leading to peroxidation of membrane lipids and cell death (6). In addition to its direct toxic effect, Aß increases the vulnerability of rodent and human neurons to excitotoxins (8, 9). Excitatory amino acids, such as glutamate, may be directly toxic to cultured neuronal cells via two different processes. The classical pathway is mediated by specific glutamate receptors that can be blocked by specific glutamate receptor antagonists (11). Excitatory amino acid neurotoxicity may also be caused by the induction of an imbalance in antioxidant enzyme systems and a reduction in intracellular glutathione levels that ultimately lead to the intracellular accumulation of peroxides and cell death. The drop in glutathione levels has been shown to arise from the competition by glutamate for a glutamate/cystine antiporter, leading to an imbalance in the homeostasis of cystine, the precursor of glutathione formation (12). This second pathway can be blocked by the addition of antioxidants, which demonstrates the involvement of free radicals and oxidative stress (12, 13). In addition, several reports suggest that independently of the pathway, glutamate can induce the generation of several reactive oxygen species in neurons, such as the superoxide anion (14) and hydrogen peroxide (15). Glutamate has been implicated in various neurodegenerative diseases, such as AD, Huntingtons disease, and Parkinsons disease (16). HT22 cells are sensitive to glutamate (13). Glutamate receptor antagonists do not protect HT22 cells from glutamate toxicity. This observation together with the finding that antioxidants, such as vitamin E, and high extracellular cystine levels protect these hippocampal cells from glutamate toxicity indicate that glutamate-induced cell death occurs via an oxidative pathway (13). Therefore, the toxicities of Aß and glutamate were used as paradigms of oxidative stress-induced cell death in hippocampal neurons.
Data are accumulating that the activities of different steroids can have an impact on neuronal function and the sensitivity of neurons to different toxic insults (4, 17, 18, 19, 20). Recently, we showed that the sex hormone 17ß-estradiol is an effective neuroprotector against oxidative stress-induced cell death (21). To investigate whether glucocorticoids can increase the sensitivity of hippocampal neurons to Aß and glutamate, we studied the influence of glucocorticoids on the cell death caused by these neurotoxins using the clonal mouse hippocampal cell line HT22 and rat primary hippocampal neurons.
| Materials and Methods |
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Transfections and assays for luciferase and ß-galactosidase
activities
HT22 cells were grown in DMEM supplemented with 10% FCS.
Transient transfections were performed using an electroporation system
(Biotechnologies and Experimental Research, San Diego, CA) after
determination of the optimal electric field strength. Five micrograms
of steroid-responsive luciferase reporter gene (MTV-LUC) were
cotransfected with 5 µg pCH110 (Pharmacia LKB, Freiburg, Germany), a
simian virus 40 promoter-driven ß-galactosidase expression vector.
Electroporated cells were replated in DMEM supplemented with 10%
steroid-free (charcoal-stripped) FCS and incubated immediately with
various concentrations of dexamethasone. Charcoal stripping of FCS was
performed using Dextran T-70 (Pharmacia, Uppsala, Sweden) as described
previously (23). After 24 h, cells were harvested, and extracts
were assayed for luciferase and ß-galactosidase activities as a
control for transfection efficiency, as previously described
(23, 24, 25, 26).
Cell survival assays
Neuronal cell death was estimated by three different assays: 1)
microscopical examination of the cells with phase contrast microscopy
to monitor for morphological changes in the absence of knowledge of the
condition, 2) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay (6, 27), and 3) trypan blue exclusion staining
followed by cell counting (6, 21). For all of these assays, culture
medium was switched 24 h before hormone addition to DMEM (1000
mg/liter glucose) supplemented with 10% charcoal-stripped steroid-free
FCS. Primary cultures were cultivated in FCS-free DMEM (1000 mg/liter
glucose) 24 h before cell survival assessment.
The MTT assay was performed as described previously (6, 21). Briefly, 10003000 HT22 cells or 10,000 primary neurons were plated in 96-well microtiter dishes with 100 µl medium/well. Hormones were added the next day. The toxins were added 24 h later. After 6 h, 10 µl MTT (5 mg/ml stock) were added to each well, and the incubation was continued for 4 h. Finally, 100 µl solubilization solution (50% dimethylformamide and 20% SDS, pH 4.8) were added. Adsorption readings were performed at 570 nm. To assess cell lysis, trypan blue staining was performed as previously described (6, 21). Briefly, cells were plated in 60-mm dishes and left untreated overnight. Then hormones were added for 24 h, followed by the addition of glutamate. After an additional 24 h, trypan blue at a concentration of 0.12% was added, and the number of viable cells (trypan blue-excluding) per low magnification field was determined. Because dexamethasone, corticosterone, and RU486 stock solutions (10-2 M) were prepared in ethanol, a possible ethanol effect was also assessed. Cell viability was not influenced by 0.1% ethanol in the incubation medium. Also, there was no interference of corticosterone, dexamethasone, or RU486 with the colorimetric MTT assay. All MTT and trypan blue exclusion assays were repeated three to five times in triplicate, with similar results. An ANOVA with post-hoc tests was performed, and P values are presented.
| Results |
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| Discussion |
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It has been shown repeatedly that under certain conditions glucocorticoids exacerbate different types of neurological insults (2, 3, 4). As the hippocampus is a major target for neuronal degeneration in the brains of patients with AD, and as it is richly endowed with GRs, it is also a principal target site for glucocorticoids. The goal of our study was to investigate the influence of glucocorticoids on the sensitivity of hippocampal neurons to the potential oxidative stressors Aß and glutamate. In addition to rat primary hippocampal neurons, we used cells of the clonal mouse hippocampal cell line HT22.
Here we report that the glucocorticoids corticosterone and dexamethasone enhance cell death induced by the oxidative stressors Aß and glutamate in HT22 cells and cell death induced by Aß in rat primary hippocampal neurons. The concentrations of glucocorticoids used are consistent with those used in a previous study, showing that 10-5-10-7 M corticosterone can increase the vulnerability of hippocampal neurons (19). This increased vulnerability of hippocampal neurons was quantified with the sensitive MTT assay to detect rather early mitochondrial disturbances and changes in cellular viability, and the trypan blue exclusion assay was used to describe cell lysis. The MTT assay measures the reduction of MTT to a colored formazan (27). As the major site of MTT reduction is thought to be at two stages of electron transport (31), the MTT assay is a very sensitive first assay of changes in mitochondrial activity and, therefore, of cellular viability. Moreover, this assay has been shown repeatedly to be a very sensitive indicator of the cell death induced by Aß and other amyloidogenic peptides as well as by other oxidative stressors (6, 32, 33, 34, 35). Cell lysis induced by Aß has been demonstrated previously (6, 10, 15). The excitotoxin glutamate can induce the generation of several reactive oxygen species in neurons independently of the pathway (receptor-mediated or nonreceptor-mediated) of toxicity (12, 13, 14, 15).
The increase in sensitivity after GR activation could be prevented by the GR antagonist RU486, indicating the receptor specificity of this effect. Hence, the results of the present study support and extend our initial observations (36). In addition, the present data are consistent with those of a very recently published report showing that corticosterone can exacerbate oxidative neuronal injury (37). Taken together, this evidence clearly suggests, as also proposed for other neurotoxic insults, an important role for glucocorticoid homeostasis in degenerative processes resulting from the oxidative challenges imposed by glutamate and Aß.
The hippocampus is almost always damaged in patients with AD (38, 39, 40). This area of the brain is important in regulation of the stress-adaptive and stress-responsive HPA system (41), and its activity is controlled mainly by corticosteroid receptors (1). An altered response of the HPA system may lead to increased glucocorticoid levels (5). Although additional mechanisms cannot be ruled out, the ability of glucocorticoids to increase the vulnerability of cells has been reported to be mediated via broad catabolic disruptions of cell biology, such as energy depletion, rather than through the activation of relatively discrete mechanisms of cell death (42, 43, 44, 45). We agree with the hypothesis that GR activation can induce an energy crisis that resembles glucocorticoid-induced cellular stress (19, 43, 44, 45), as the enhancement of glutamate and Aß toxicity through glucocorticoids in our cellular systems could be observed only after reducing glucose levels approximately 4-fold. The use of reduced glucose concentrations in the cell survival assays is consistent with previous studies (19). Hippocampal neurons may, therefore, be left glucose deprived after activation of the GRs, rendering them more vulnerable to subsequent insults.
In addition to the increased glucocorticoid levels that may occur in patients with AD (5), it is well known that the human hippocampus loses neurons with aging (38) and that there is an age-related increase in HPA system activity (46). Because the primary risk factor for AD is age, any model designed for the study of neuronal cell death in AD must account for age-related physiological changes. These changes also include a drop in antioxidant defense system expression and activity, a reduced plasticity of hippocampal GR regulation, and, ultimately, a hypersecretion of glucocorticoids (47, 48, 49). A decrease in some antioxidant enzyme activities can be directly caused by high levels of glucocorticoids in the adult rat brain, as there is a 50% decrease in glutathione peroxidase activity reported for the hippocampi of glucocorticoid-treated rats (50). Glutathione peroxidase is the major H2O2-detoxifying enzyme in the brain and a decrease in its activity has been shown to be involved in glutamate toxicity in neuronal cell lines (12, 13). Recently, we have been able to show that increased expression and activity of cellular antioxidant enzymes, such as glutathione peroxidase and catalase, are sufficient to afford resistance of neuronal cells to Aß and that neuronal cells stably double transfected with glutathione peroxidase and catalase are significantly less sensitive to H2O2 and Aß than are control transfectants (35). The important role of an antioxidant defense for overall cellular protection is further strengthened by several other in vitro investigations (6, 12, 13, 15). All of these in vivo and in vitro data are complemented by the well documented fact that one major change in the CNS that is associated with aging is free radical-induced oxidative damage (see Ref. 49 for review). Therefore, we hypothesize that oxidative stressors, such as Aß and glutamate, can add to accumulating oxidative stress and bring additional oxidative challenges into play, thus increasing the neurodegeneration observed in AD in age-compromised neurons that may be additionally compromised by a preexisting hypercortisolism.
In summary, our data show that the neurotoxic challenges induced by the oxidative stressors Aß and glutamate, two neurotoxins that have been implicated in AD (6, 7, 8, 9, 11, 15, 16), are enhanced by pretreatment of the hippocampal neurons with glucocorticoids. Our findings add to the growing body of evidence that supports the idea that physiological interactions between the brain and the HPA system play a crucial role in brain aging and possibly also in the etiology of AD and other neurodegenerative and age-related neurological diseases.
| Acknowledgments |
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plasmid, and Dr.
M. Renoir for providing RU486. | Footnotes |
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2 Present address: Institute for Anatomy, Charite Hospital, Humboldt
University, 10098 Berlin, Germany. ![]()
Received June 19, 1996.
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