Different effects of oxidative stress on activation of transcription factors in primary cultured rat neuronal and glial cells

Molecular Brain Research 50 Ž1997. 213–220

Research report

Different effects of oxidative stress on activation of transcription factors in primary cultured rat neuronal and glial cells Emi Iwata ) , Masato Asanuma, Sakiko Nishibayashi, Yoichi Kondo, Norio Ogawa Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama UniÕersity Medical School, 2-5-1 Shikatacho, Okayama 700, Japan Accepted 13 May 1997

Abstract We compared the cytotoxic effects of oxidative stress on neuronal and glial cells in vitro by examining the cell viability and changes in DNA-binding activities of transcription factors, AP-1 and CREB, using Trypan blue exclusion and electrophoretic mobility shift assay ŽEMSA., respectively. Neurotoxin 6-hydroxydopamine Ž6-OHDA. and H 2 O 2 reduced the viability of both types of cells in time- and concentration-dependent manner. Both neurotoxins dose-dependently decreased DNA-binding activities in neuronal cells. The results of cell viability assay suggested that these changes may reflect the reduction in neuronal cell viability. In contrast, both reagents increased DNA-binding activities in glial cells, although they decreased cell numbers. These results suggest that the effects of oxidative stress on transcription factors is different in neuronal and glial cells. We also examined the effect of brain-derived neurotrophic factor ŽBDNF. on 6-OHDA- or H 2 O 2-induced changes in DNA-binding activities. In neuronal cells, pre-treatment with BDNF prevented the decrease in DNA-binding activities induced by 6-OHDA or H 2 O 2 . In glial cells, the effect of BDNF on oxidative stress-induced changes in DNA-binding activities in the 6-OHDA-treated group were opposite to those in H 2 O 2-treated group. Our results suggest that 6-OHDA and H 2 O 2 may exert their cytotoxic mechanisms through different signal transduction systems. q 1997 Elsevier Science B.V. Keywords: Oxidative stress; Brain derived neurotrophic factor; Cell viability; Transcription factor; DNA binding activity; Culture; Neuron; Glia

1. Introduction Oxidative stress is known to be involved in neuronal damage mediated through reactive oxygen species ŽROS. such as free radicals. Free radicals, i.e. superoxide anion ŽOy . and hydroxyl radical Ž v OH., react at great speed 2 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 w17x, stroke w8,29x, amyotrophic lateral sclerosis w41x and Parkinson’s disease ŽPD.. 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 w1,5,9,10,14,18,26,39 x. Because of these changes, free radicals are likely to be formed in and poorly elimi-

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Corresponding author. Fax: q81 Ž86. 234-2426.

nated from the brain of PD patients w36x. PD is characterized by a selective loss of dopaminergic neurons of the nigrostriatal pathway w45x. Neurotoxin 6-hydroxydopamine Ž6-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, Oy 2 , H 2 O2 and v OH, formed from autoxidation of 6-OHDA w11,19,20x. In the brain, several antioxidant molecules, i.e. ascorbate, a-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 w31x. 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 w43x. 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

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w2,13,32,33,35x. 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 ŽBDNF. promotes the survival and differentiation of neurons in many populations of the peripheral and the central nervous systems w3,30x. Most strikingly, BDNF promotes the survival of dopaminergic neurons in vivo and in vitro w4,21,27x. In addition, BDNF protects dopaminergic neurons and dopaminergic neuroblastoma cell line ŽSH-SY5Y. against the cytotoxic effects of 6-OHDA and N-methyl-4-phenylpyridinium ion ŽMPPq. . These neuroprotective effects appear to be mediated by the glutathione system, suggesting that the effects may be associated with a decrease in ROS w44x. 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 H 2 O 2 on transcription factors, activating protein-1 ŽAP-1. and cAMP-responsive element binding protein ŽCREB., in primary cultured rat mesencephalic neuronal and glial cells using electrophoretic mobility-shift assay ŽEMSA.. We also compared the effect of BDNF on 6-OHDA and H 2 O 2-induced changes on the level of transcriptional activation between cultured mesencephalic neuronal and glial cells.

2.2. OxidatiÕe stress and analysis of cell Õiability Cultured neuronal cells were grown for 5 or 6 days and then treated with different amounts of 6-OHDA Ž75, 150 or 300 m M. or H 2 O 2 Ž125, 250 or 500 m M.. On the other hand, cultured glial cells were exposed to the same drug using the same doses, after 1 week of subculture. Following a 2 or 6 h incubation with the agent, cell viability was determined by Trypan blue exclusion and counting in a hemocytometer. To investigate the effects of BDNF in oxidative stress-induced toxicity, neuronal or glial cells were exposed to BDNF for 24 h before either 6-OHDA or H 2 O 2 treatment. Six hours later, we assessed cell viability as described above. 2.3. Preparation of nuclear extracts After a 2 h exposure to 6-OHDA or H 2 O 2 , nuclear extracts were prepared from the cultured neuronal or glial cells according to the method of Schreiber et al. w42x with minor modifications. Briefly, the cultured cells were collected in 1 ml of Tris-buffered saline Ž25 mM Tris-HCl pH 7.4, 130 mM NaCl and 5 mM KCl. by centrifugation at 7000 = g for 30 s. The pellet was suspend in 400 m l of buffer A Ž10 mM HEPES pH 7.9., 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol ŽDTT. and 1 mM phenylmethylsulfonyl fluoride ŽPMSF... The suspen-

2. Materials and methods 2.1. 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 Ž14 or 15 days of gestation.. The tissue was incubated at 378C for 15 min in 0.125% trypsin. Following centrifugation Ž1500 = g, 5 min., the cell pellet was treated with 0.004% DNase I at 378C for 7 min. After centrifugation Ž1500 = g, 5 min., the cell suspension was gently resuspended in a small volume of tissue growth medium; Dulbecco’s modified Eagle’s medium ŽDMEM., containing 10% fetal bovine serum, followed by plating the cell suspension Ž1–2 = 10 6 cellrdish. in 35-mm Corning culture dishes pre-coated with 100 m grml poly-L-lysine. The cells were maintained in the growth medium at 378C in a 5%–95% CO 2 –air gas mixture. For cultures containing neuronal cells, DMEM was replaced with a fresh medium supplemented with 2 m M cytosine-b-D-arabinofuranoside to inhibit the replication of non-neuronal cells within 24 h and incubated for 5–6 days. For glial cell cultures, the incubation mixture was subcultured on day 7 and incubated for another 1 week in 10% FBS-DMEM. Under these conditions, most glial cells were astrocytes.

Fig. 1. Effects of 6-OHDA or H 2 O 2 on cell viability in cultured mesencephalic neuronal ŽA. and glial ŽB. cells. Cultured cells were treated with various amounts of 6-OHDA Ž75, 150 or 300 m M. or H 2 O 2 Ž125, 250 or 500 m M. for 2 or 6 h. Cell viability was assessed by cell counting using Trypan blue exclusion. Data are expressed as percentages of control values and represent the mean"S.E.M. of 4–6 experiments in each group. ) P - 0.05, ) ) P - 0.01 compared with untreated control Ž2 h treated groups.. q P - 0.01, qq P - 0.01 compared with untreated control Ž6 h treated groups.. Statistical analyses were performed by Mann–Whitney U-test.

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sion was placed on ice for 15 min. Then, 30 m l of 10% Nonidet P-40 was added and mixed vigorously for 10 s. The nuclear fraction was precipitated by centrifugation at 12 000 = g for 5 min and suspended in 50 m l of ice-cold buffer B Ž20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF.. The mixture was placed on ice for 15 min with frequent agitation. In the next step, the supernatants of the nuclear extracts were prepared by centrifugation at 12 000 = g for 5 min and stored in aliquots at y808C. The protein concentration was determined by using the Bio-Rad protein assay kit with bovine serum albumin as a standard. 2.4. Electrophoretic mobility-shift assay (EMSA) The procedures used for EMSA were as described previously reported w6,23x. Oligonucleotides encoding TRE and CRE were synthesized with the following sequences: TRE 5X-GATTCGTGACTCAGCACAGG-3X , 3X-CTAAGCACTGAGTCGTGTCC-5X ; CRE 5X-GATTCGTGACGTCAGCACAG-3X , 3X-CGAAGCACTGCAGTCGTGTC-5X . Consensus sequences are indicated in bold letters. Each double-stranded oligonucleotide was labeled with w a-

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PxdCTP using DNA polymerase I ŽKlenow fragment. and purified by Sephadex G-50 column chromatography. The DNA-protein binding reaction was performed for 15 min at 208C in a reaction mixture Ž20 m l. containing 20 mM HEPES-NaOH pH 7.9, 2 mM Tris-HCl, 1 mM DTT, 0.4 mM EDTA, 0.2 mM EGTA, 80 mM NaCl, 2 m g of polywdI-dCx, 10% glycerol, 0.2 mM PMSF, 200 pg of 32 P-labeled double-stranded oligonucleotide probes, and 2 m g of nuclear extract. The DNA-protein complexes were resolved on 4% polyacrylamide gel Ž29 : 1 cross-linking ratio. containing 6.7 mM Tris-HCl pH 7.6, 3.3 mM sodium acetate, 2.5% glycerol, 1 mM EDTA and 0.06% ammonium persulfate. Electrophoresis was carried out at 11 Vrcm for 2.5 h at 48C. After gel electrophoresis, the gel was dried and exposed to X-ray film ŽKodak X-OMAT. with intensifying screens for 1 day at y808C. The reactive density of detected bands on the autoradiograms was measured and analyzed with an image scanner ŽEpson GT6500. and a computerized image analysis software ŽImage 1.56, NIH.. 32

2.5. Statistical analysis Data were expressed as percentages of the control as mean " S.E.M. for each group. Statistical analysis of the data was performed using Mann-Whitney U-test. A P

Fig. 2. Changes in DNA-binding activities in nuclear extracts of cultured neuronal cells treated with 6-OHDA or H 2 O 2 . Nuclear extracts were prepared from cultured neuronal cells 2 h after incubation with 6-OHDA Ž75, 150 or 300 m M. or H 2 O 2 Ž125, 250 or 500 m M.. Two micrograms of protein from nuclear extracts was incubated with 32 P-labeled double-strand oligonucleotide probes ŽTRE and CRE. and subjected to electrophoresis. The gels were exposed to X-ray film Župper panels. and analyzed with an image scanner Žlower panels.. The results are expressed as percentages of untreated control values and represent the mean " S.E.M. of 3–4 experiments in each group. ) P - 0.05 compared with untreated control ŽMann–Whitney U-test..

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value of - 0.05 denoted the presence of a statistically significant difference between the groups.

3. Results 3.1. Effect of 6-OHDA and H2 O2 on cell Õiability Fig. 1A shows that 6-OHDA and H 2 O 2 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 H 2 O 2 compared with neuronal cells ŽFig. 1B.. In particular, exposure to 500 m M H 2 O 2 caused a marked reduction of cell viability of neuronal cells Ž2 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 Ž2 h, 106 " 7.8%; and 6 h, 60.5 " 4.2% of control.. 3.2. TRE- and CRE-binding actiÕities induced by 6-OHDA or H2 O2 We examined the changes in DNA-binding activities of AP-1 and CREB using EMSA. Cultured cells were incubated with 6-OHDA Ž75, 150 or 300 m M. or H 2 O 2 Ž125, 250 or 500 m M. and harvested 2 h later to prepare nuclear

extracts. Fig. 2 shows that both 6-OHDA and H 2 O 2 treatments decreased DNA-binding activities of AP-1 and CREB in cultured neuronal cells. In contrast, both agents increased DNA-binding activities in cultured glial cells in a dose-dependent manner ŽFig. 3.. When glial cells were exposed to 300 m M 6-OHDA for 2 h, DNA-binding activities of AP-1 and CREB increased significantly to 241.6 " 17.6 and 246.5 " 39.0% of control levels, respectively, whereas cell viability decreased to 16.2 " 3.5% of control ŽFig. 1B.. 3.3. Effect of BDNF on cell Õiability Primary cultured cells were treated with 100 ngrml BDNF for 24 h followed by treatment of neuronal and glial cells with 75 m M 6-OHDA or 125 m M H 2 O 2 , or with 150 m M 6-OHDA or 500 m M H 2 O 2 , respectively. Under these conditions, the proportion of surviving cells was f 40–60% of the control in each case. Thus, BDNF pre-treatment had no significant protective effect on the survival of both neuronal and glial cells after a 6 h exposure to either 6-OHDA or H 2 O 2 ŽFig. 4.. 3.4. Effect of BDNF on DNA-binding actiÕity BDNF alone decreased DNA-binding activities in neuronal cells but had no effect on DNA-binding activities in

Fig. 3. Changes in DNA-binding activities in nuclear extracts of cultured glial cells treated with 6-OHDA or H 2 O 2 . Nuclear extracts were prepared from cultured glial cells 2 h after incubation with 6-OHDA Ž75, 150 or 300 m M. or H 2 O 2 Ž125, 250 or 500 m M.. Two micrograms of proteins from nuclear extracts were incubated with 32 P-labeled double-strand oligonucleotide probes ŽTRE and CRE. and subjected to electrophoresis. The gels were exposed to X-ray film Župper panels. and analyzed with an image scanner Žlower panels.. The results are expressed as percentages of untreated control values and represent the mean " S.E.M. of 3–4 experiments in each group. ) P - 0.05 compared with untreated control ŽMann–Whitney U-test..

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Fig. 4. Effects of BDNF on 6-OHDA or H 2 O 2 toxicity in cultured neuronal Žleft panels. and glial Žright panels. cells. Cultured cells were pretreated with 100 ngrml BDNF for 24 h, followed by exposure of neuronal cells to 75 m M 6-OHDA or 125 m M H 2 O 2 for 6 h, glial cells were exposed to 150 m M 6-OHDA or 500 m M H 2 O 2 for 6 h. The cell viability was assessed by cell counting using Trypan blue exclusion. The results were expressed as percentages of untreated control values and represent the mean " S.E.M. of 6 experiments in each group. ) P - 0.05, ) ) P - 0.01 compared with untreated control ŽMann–Whitney U-test..

glial cells. The effect of BDNF on 6-OHDA- or H 2 O 2-induced change in TRE-binding activity was studied using EMSA ŽFig. 5.. In neuronal cells, pre-treatment with 100 ngrml BDNF for 24 h reversed 6-OHDA or H 2 O 2-induced reduction in TRE-binding activity to the control level. In contrast, there was a differential effect for BDNF on 6-OHDA- and H 2 O 2-induced changes in TRE-binding activity in glial cells. Pre-treatment of glial cells with BDNF suppressed the activation of TRE-binding activity induced by 6-OHDA, whereas BDNF pre-treatment enhanced activation of TRE-binding activity induced by H 2 O 2 . Similar results were observed when we examined CRE-binding activity Ždata not shown..

4. Discussion The finding of the present study was that cultured glial cells were more resistant to oxidative stress than cultured neuronal cells ŽFig. 1.. In particular, glial cells were more resistant to H 2 O 2 than to 6-OHDA. Makar et al. w31x 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 peroxidaser

glutathione reductase system or both, which catalyze the decomposition of H 2 O 2 into H 2 O and O 2 , may be operational in glial cells. Our results also showed that DNA-binding activities of AP-1 and CREB decreased in a dose-dependent manner in cultured mesencephalic neuronal cells treated with 6OHDA or H 2 O 2 ŽFig. 2., reflecting a decrease in the number of surviving neuronal cells ŽFig. 1.. This indicates that DNA-binding activity does not change per cell in neuronal cells. While cell viability was reduced, DNA-binding activities of both transcription factors increased in a dose-dependent manner in glial cells treated with 6-OHDA or H 2 O 2 ŽFigs. 1 and 3.. Several investigators have demonstrated that exposure of mammalian peripheral cells to adverse environmental conditions activated the onset of specific genetic responses which protect against permanent cell damage and death w12,22,24,40x. The addition of exogenous H 2 O 2 is known to increase the expression of early response genes, c-fos, c-jun and egr-1, in various types of peripheral cells, such as Balbr3T3; mouse embryo cells, HeLa; human epithelial-like cells, MC3T3; mouse osteoblastoma cells, and JB6; mouse epidermal cells w35x. Hydrogen peroxide also increases TRE-binding activity in HeLa nuclear extracts w13x. We speculate that the rise in DNA-binding activities of AP-1 and CREB in

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glial cells induced by 6-OHDA or H 2 O 2 noted in our experiment was also due to the induction of c-fos and c-jun. Thus, such activation may regulate the expression of oxidative stress-related genes acting as antioxidants andror exert cell protective effects. Our results also showed a preferential effect by 6-OHDA or H 2 O 2 on glial cells compared with neuronal cells. Such a difference may reflect differences in mechanisms regulating signal transduction or differences in the variety of genes induced after exposure to oxidative stress in glial cells compared with neuronal cells. While these differences were studied in vitro in the present study, they may also be present in vivo. Glial cells are resistant to oxidative stress w31,43x and this resistance might explain glial survival and proliferation in PD or ischemic injured brain w38x. BDNF pre-treatment did not prevent 6-OHDA- or H 2 O 2-induced reduction in cell viability ŽFig. 4., although several reports indicated that BDNF promotes the survival of dopaminergic neurons and protect these cells against neurotoxins w21,27,44x. The exact mechanisms for the discrepancies between our results and those of previous studies are not clear, but they may represent several methodological differences, for example, dose or exposure periods of oxidative agents used in the present experiments. Furthermore, because of the low population of dopaminergic

neurons in the cultured mesencephalic neuronal cells Ž0.5– 2%. w37x, most cells counted on Trypan blue exclusion represented viable non-dopaminergic neurons. Thus, the protective effects of BDNF against 6-OHDA or H 2 O 2 in dopaminergic neurons appear to be masked by a decline in non-dopaminergic neurons. In contrast to the failure of BDNF in protecting against cell death, in neuronal cells, the decrease in DNA-binding activity induced by both oxidative agents was totally inhibited by pre-treatment with BDNF ŽFigs. 4 and 5.. These results indicate that BDNF might up-regulate DNA-binding activity per cell in cultured neuronal cells under these conditions. In glial cells, BDNF pre-treatment inhibited 6-OHDA-induced increase in DNA-binding activity, while it enhanced similar activities induced by H 2 O 2 ŽFig. 5.. These effects indicate that the mechanisms of activation of transcription factors induced by 6-OHDA are different from those induced by H 2 O 2 . Hydrogen peroxide may generate v OH which is the most reactive and cytotoxic oxygen species w17x via metal-catalyzed Fenton reaction w7,16x: H 2 O 2 q Fe 2 q ™ v OH q OHyq Fe 3q. However, the toxicity of 6-OHDA also involves the production of oxygen species w11x. Super oxide anion and H 2 O 2 are formed during the autoxidation of 6-OHDA, and

Fig. 5. Effects of BDNF on 6 OHDA- or H 2 O 2 -induced change in TRE-binding activity in neuronal and glial cells. Cultured cells were pretreated with 100 ngrml BDNF for 24 h, and then neuronal cells were exposed to 75 m M 6-OHDA or 125 m M H 2 O 2 , and glial cells were exposed to 150 m M 6-OHDA or 500 m M H 2 O 2 . Nuclear extracts were prepared from the cells 2 h after incubation with 6-OHDA or H 2 O 2 . Two micrograms of proteins from nuclear extracts were incubated with 32 P-labeled double-strand oligonucleotide probes ŽTRE. and subjected to electrophoresis. The gels were exposed to X-ray film Župper panels. and analyzed with an image scanner Žlower panels.. Data are the mean" S.E.M. of 4–6 experiments in each group and expressed as percentages of untreated control values. ) P - 0.05, ) ) P - 0.01 ŽMann–Whitney U-test..

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then Oy 2 further catalyze the autoxidation. Hydroxyl radical is also arise from the reaction between Oy 2 and H 2 O 2 ŽHaber-Weiss reaction. w11,20,44x: q 6 y OHDAq O 2 ™ semiquinone Ž SQ . q Oy 2 qH

Hqq 6 y OHDAq Oy 2 ™ SQ q H 2 O 2 q SQ q O 2 ™ quinoneq Oy 2 qH q SQ q Oy 2 q H ™ quinoneq H 2 O 2 v y H 2 O 2 q Oy 2 ™ OH q OH q O 2 .

Superoxide dismutase ŽSOD. or catalase inhibit this generation of v OH by 6-OHDA w11x. This observation support Haber-Weiss reaction as the major source of v OH arising from 6-OHDA. Such differences in the oxygen species-generating systems of 6-OHDA and H 2 O 2 may explain the opposite effects of BDNF on DNA-binding activities in 6-OHDAand H 2 O 2-treated groups. It is well known that neurotrophic factors increase activations of antioxidant enzymes SOD, catalase, glutathione peroxidase and glutathione reductase w25,34x. In this study, BDNF pre-treatment may activate SOD to prevent the v OH generation in Haber-Weiss reaction and then inhibit the activation of the DNA-binding activities caused by 6-OHDA. On the other hand, SOD cannot block the Fenton reaction so that BDNF pre-treatment might not inhibit the v OH generation by H 2 O 2 . Moreover, various oxidants are known to increase cytosolic Ca2q and neurotrophic factors enhance the intracellular concentration of Ca2q w15,28x. These observations suggest that BDNF pre-treatment may augment the increase of intracellular level of Ca2q and then enhance the activation of DNA-binding activities in H 2 O 2-treated group. An alternative mechanism is that BDNF might trap 6-OHDA itself or oxygen species generated through 6OHDA. BDNF could not trap v OH as examined by ESR spectrometer Ždata not shown.. However, whether BDNF traps 6-OHDA and Oy 2 is still unclear at present. It is also possible that DNA-binding activity may be enhanced in BDNF-pretreated cells through other as yet unknown mechanisms. The relationship between the paradoxical mechanisms of BDNF on 6-OHDA and H 2 O 2 and oxygen species-generation systems remains to be investigated. It is not known at present whether BDNF reacts with drugs Ži.e. trapping the oxygen species. or influences cultured neuronal and glial cells Ži.e. activation of enzymes.; however, it is certain that BDNF itself changes DNA-binding activity of transcription factors and regulates the expression of functional products that play an important role against oxidative stress. Our results also showed that BDNF influenced the level of transcription factors not only in neuronal cells, but also glial cells. Thus, it is possible that certain in vivo effects of BDNF on neuronal cells may, at least in part, be exerted through its stimulatory effect on glial cells.

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In conclusion, our study showed that glial cells were more resistant to oxidative stress than neuronal cells, and distinct effects of oxidative stress on the activation of transcription factors were recognized in neuronal and glial cells. It is possible that 6-OHDA and H 2 O 2 produce glial cell injury via different mechanisms. The findings in vitro assay seem to reflect differences between neuronal and glial cells response to oxidative stress conditions. These in vitro studies may provide useful information for future therapeutic research related to neurodegenerative diseases, such as PD and Alzheimer’s diseases.

Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research ŽC. 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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