Different effects of oxidative stress on activation of transcription factors in primary cultured rat neuronal and glial cells
Introduction
Oxidative stress is known to be involved in neuronal damage mediated through reactive oxygen species (ROS) such as free radicals. Free radicals, i.e. superoxide anion (O–2) and hydroxyl radical (•OH), react at great speed with DNA, membrane lipids, enzymes and other essential cell components, resulting in cell death.
Several recent studies have suggested that free radicals are deeply involved in the pathophysiology of several neurodegenerative diseases, such as Alzheimer's diseases [17], stroke [8,29], amyotrophic lateral sclerosis [41] and Parkinson's disease (PD). In the substantia nigra of PD patients, there is increased lipid peroxide and iron deposition, and marked decrease in the level of free radical scavenging enzymes such as glutathione peroxidase and catalase [1,5,9,10,14,18,26,39]. Because of these changes, free radicals are likely to be formed in and poorly eliminated from the brain of PD patients [36]. PD is characterized by a selective loss of dopaminergic neurons of the nigrostriatal pathway [45]. Neurotoxin 6-hydroxydopamine (6-OHDA)-induced lesions of the nigrostriatal pathway result in a neurochemical profile similar to that seen in patients with PD. It is well known that the toxicity is mediated through the oxygen radical species, O–2,H2O2 and •OH, formed from autoxidation of 6-OHDA [11,19,20].
In the brain, several antioxidant molecules, i.e. ascor-bate, α-tocopherol, superoxide dismutase, catalase, and glutathione peroxidase, protect against oxidative stress of free radicals. Furthermore, the concentration and activity of such free radical scavengers are different in neuronal and glial cells [31]. The response to oxidative stress is also different between neuronal and glial cells; neuronal cells are more susceptible to injury, ischemia and hypoxia than glial cells [43].
A number of reports have demonstrated that oxidative stress and antioxidant reagents induce the expression of early-response genes, such as c-fos, c-jun and egr-1, and may influence DNA-binding activity of AP-1 and NFkB [2,13,32,33,35]. Changes in DNA-binding activity of transcription factors may lead the changes in the expression level of damage-associated genes, which induce functional and morphological modification of cells.
Brain-derived neurotrophic factor (BDNF) promotes the survival and differentiation of neurons in many populations of the peripheral and the central nervous systems [3,30]. Most strikingly, BDNF promotes the survival of dopaminergic neurons in vivo and in vitro [4,21,27]. In addition, BDNF protects dopaminergic neurons and dopaminergic neuroblastoma cell line (SH-SY5Y) against the cytotoxic effects of 6-OHDA and N-methyl-4-phenyl-pyridinium ion (MPP+). These neuroprotective effects appear to be mediated by the glutathione system, suggesting that the effects may be associated with a decrease in ROS [44].
To examine the effects of oxidative stress on DNA-binding activity of transcription factors in the brain, we initially examined the effects of 6-OHDA or H2O2on transcription factors, activating protein-1 (AP-1) and cAMP-responsive element binding protein (CREB), in primary cultured rat mesencephalic neuronal and glial cells using electrophoretic mobility-shift assay (EMSA). We also compared the effect of BDNF on 6-OHDA and H2O2-induced changes on the level of transcriptional activation between cultured mesencephalic neuronal and glial cells.
Section snippets
Cell culture
The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of our institution. The mesencephalon area was dissected from Sprague-Dawley rat embryos (14 or 15 days of gestation). The tissue was incubated at 37°C for 15 min in 0.125% trypsin. Following centrifugation (1500 × g, 5 min), the cell pellet was treated with 0.004% DNase I at 37°C for 7 min. After centrifugation (1500× g, 5 min), the cell suspension was gently resuspended in a small volume of tissue
Effect of 6-OHDA and H2O2 on cell viability
Fig. 1A shows that 6-OHDA and H2O2 reduced the survival of neuronal cells in a dose-dependent manner. The effect was also time-dependent. Although both agents were toxic to glial cells, these cells were more resistant against 6-OHDA and H2O2compared with neuronal cells (Fig. 1B). In particular, exposure to 500 μM H2O2caused a marked reduction of cell viability of neuronal cells (2 h, 12.7 ± 3.4%; 6 h, 8.3 ± 1.4% of control), but caused a less severe form of inhibition of cell viability in glial cells
Discussion
The finding of the present study was that cultured glial cells were more resistant to oxidative stress than cultured neuronal cells (Fig. 1). In particular, glial cells were more resistant to H2O2 than to 6-OHDA. Makar et al. [31] indicated that the activity of the glutathione system was at a higher level in cultured chick astrocytes than in cultured chick neurons. These results indicate that the effect of either the catalase system or glutathione peroxidase/glutathione reductase system or
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research (C) from the Japanese Ministry of Education, Science, Sports and Culture, and grants from the Research Committee on CNS Degenerative Diseases and Research Projects on Aging and Health from the Japanese Ministry of Health and Welfare.
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