Kainate excitotoxicity in organotypic hippocampal slice cultures: evidence for multiple apoptotic pathways

Brain Research 916 (2001) 239–248 www.bres-interactive.com

Interactive report

Kainate excitotoxicity in organotypic hippocampal slice cultures: evidence for multiple apoptotic pathways q Wei Liu c , Ruolan Liu a , Jong Tai Chun b , Ruifen Bi a , Warren Hoe a , Steven S. Schreiber b , a, Michel Baudry * a Neuroscience Program, University of Southern California, Los Angeles, CA 90089 -2520, USA Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA c Department of Pharmacology, School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA b

Accepted 16 August 2001

Abstract The mechanisms underlying kainate (KA) neurotoxicity are still not well understood. We previously reported that KA-mediated neuronal damage in organotypic cultures of hippocampal slices was associated with p53 induction. Recently, both bax and caspase-3 have been demonstrated to be key components of the p53-dependent neuronal death pathway. Caspase activation has also been causally related to the release of mitochondrial cytochrome c (Cyto C) in the cytoplasm as a result of the collapse of the mitochondrial membrane potential (DcM ) and the opening of mitochondrial permeability transition pores (mPTP). In the present study, we observed a rapid induction of bax in hippocampal slice cultures after KA treatment. In addition, the levels of Cyto C and caspase-3 were increased in the cytosol while the level of the caspase-9 precursor was decreased. There was also a complete reduction of Rhodamine 123 fluorescence after KA treatment, an indication of DcM dissipation. Furthermore, inhibition of mPTP opening by cyclosporin A partially prevented Cyto C release, caspase activation and neuronal death. These data suggest the involvement of bax, several caspases, as well as Cyto C release in KA-elicited neuronal death. Finally, inhibition of caspase-3 activity by z-VAD-fmk only partially protected neurons from KA toxicity, implying that multiple mechanisms may be involved in KA excitotoxicity.  2001 Published by Elsevier Science B.V. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Excitatory amino acids, excitotoxicity Keywords: Excitotoxicity; Caspase; Bax; Cytochrome c; Mitochondrial membrane potential and kainate

1. Introduction Excitotoxicity is widely considered to be a contributing factor in neuronal death associated with a number of central nervous system (CNS) insults or disorders, including stroke, epileptic seizures, and Huntington’s disease [6].

Abbreviations: KA, kainic acid; OHC, organotypic hippocampal culture; mPTP, mitochondrial membrane transition pore; Cyto C, cytochrome c; R-123, Rhodamine 123 q Published on the World Wide Web on 10 September 2001. *Corresponding author. HNB124, University of Southern California, Los Angeles, CA 90089-2520, USA. Tel.: 11-213-7409-188; fax: 11213-740-5687. E-mail address: [email protected] (M. Baudry).

An accumulating body of evidence suggests that apoptosis is a key event in excitotoxin-induced neurotoxicity [31,33,40,49], although the distinction between apoptosis and necrosis remains a difficult task [24]. We have previously shown that kainate (KA)-induced pyramidal cell death observed in organotypic hippocampal slice cultures (OHCs) exhibits several apoptotic characteristics, including DNA fragmentation and induction of the tumor suppressor gene, p53 [38]. In vivo and in vitro studies support the notion that p53 plays an important role in mediating neuronal apoptosis induced by excitotoxins [15,17,42], ischemia [7,27] and hypoxia [2]. In particular, the transcriptional activity of p53 protein increased in KA-induced excitotoxicity [28], suggesting that p53 may promote neuronal death by activating p53-responsive genes. However, the exact molecular mechanisms underly-

0006-8993 / 01 / $ – see front matter  2001 Published by Elsevier Science B.V. PII: S0006-8993( 01 )03006-2

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ing KA-induced neuronal apoptosis still remain to be completely understood. In recent years, several pro-apoptotic genes, which were identified in a variety of mammalian non-neuronal cell types, have been implicated in several neuronal models of neurodegeneration including a p53-inducible gene, bax, as well as members of the caspase family [20,46]. Accordingly, activation of the bax gene and increased bax expression result in the release of cytochrome c (Cyto C) from mitochondria into the cytoplasm [14,25]. The binding of Cyto C with a complex of Apaf-1 and caspase-9 precursor produces the activation of caspase-9 and the subsequent activation of caspase-3 via a cascade of proteolytic cleavages [47,50]. Caspase-3 is a potent effector of apoptosis, and cleaves specific aspartate residues in a variety of structural, housekeeping, and regulatory proteins [10,41]. The mechanism by which Cyto C is released into the cytoplasm may be related to the opening of mitochondrial permeability transition pores (mPTP) and the collapse of mitochondrial membrane potential (DcM ) [23]. Interestingly, the opening of mPTP and the collapse of DcM have been observed in cultured hippocampal neurons following excitotoxin exposure [1,34,45]. However, studies with various knock-out mice have shown that the recruitment of different death effectors during apoptosis is highly celltype- and stimulus-specific. In the present study, we used OHCs to examine the molecular events involved in KAinduced neuronal death. The OHC is a useful system to study mechanisms of neurodegeneration as several features of hippocampal circuitries are preserved, and the preparation is well suited for prolonged pharmacological treatment and recovery, which are difficult to perform in intact animal experiments. Our results indicate that bax, caspase9, and caspase-3 genes, as well as the collapse of DcM and the release of mitochondrial Cyto C into the cytosol are involved in KA-induced neurodegeneration. In addition, blockade of mPTP by cyclosporin A (CsA) prevented DcM collapse, partially inhibited caspase activation, and provided a significant degree of neuronal protection. Likewise, partial neuronal protection was provided by an inhibitor specific for caspase-1, 3 and 4. Our data indicate that there are multiple mechanisms involved in KA-induced neuronal death in OHCs.

2. Materials and methods

2.1. Materials Anti-bax polyclonal antibodies were obtained from Upstate Biotechnology (Lake Placid, NY, USA). Anticaspase-3 polyclonal antibodies and anti-Cyto C monoclonal antibodies were purchased from PharMingen (San Diego, CA, USA), and anti-caspase-9 polyclonal antibodies from StressGene Biotechnologies (Victoria, Canada). Minimal essential medium (MEM) was obtained

from Gibco (Rockville, MD, USA). Alkaline phosphataseconjugated secondary antibodies, nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate toluidine (BCIP) were purchased from Bio-Rad (Hercules, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Amersham (Piscataway, NJ, USA). Benzyloxycarbonyl-Val–Ala–Asp (Ome)-fluoromethylketone (z-VAD-fmk) was purchased from Enzyme Systems Products (Livermore, CA, USA). Cyclosporin A was obtained from RBI (Natick, MA, USA). FK 506 was purchased from Calbiochem (La Jolla, CA, USA). Other reagents were purchased from Sigma (St. Louis, MO, USA).

2.2. Preparation of organotypic hippocampal slice cultures Hippocampal slice cultures were prepared as described [3]. Briefly, transverse slices (400 mm thick) were prepared from the hippocampi of 6–8 day-old rats, using a McIlwain tissue slicer, and were placed on a membrane insert (Millicell-CM, Millipore). The culture medium was GIBCO MEM containing (in mM) HEPES (30), D-glucose (30), glutamine (3), NaHCO 3 (5), MgCl 2 (2.5), L-ascorbate (0.5), CaCl 2 (2), 1 mg / ml insulin and 20% horse serum. Cultures were maintained at 358C in a humidified incubator with 5% CO 2 , and the medium was changed every 2 or 3 days. The effects of KA and other agents were tested in mature cultures, 20–25 days in vitro (DIV).

2.3. Kainic acid treatment and assessment of KA neurotoxicity KA (50 mM) was applied for 3 h after mature cultures were incubated in serum-free culture medium overnight. After the treatment, cultures were allowed to recover for 24 h in fresh, serum-free medium containing 4.6 mg / ml of the fluorescent dye propidium iodide (P.I.). The caspase inhibitor, z-VAD-fmk (100 mM), the blocker of the mitochondrial permeability transition pore, CsA (100 mM) or the anti-inflammatory agent, FK 506 (20 mM), were applied in cultures overnight before KA application, and re-applied for 24 h immediately after KA treatment. These concentrations were chosen based on maximal effects reported for these compounds in the literature. Previous studies have addressed the principle of staining by P.I., and its usefulness for evaluating neuronal damage in hippocampal cultures [3,44]. In the current study, toxicity was observed by microscopic evaluation of P.I. uptake 24 h after KA treatment. Results were scored semi-quantitatively with a scale of 0–4, with 05no toxicity and 45 maximum toxicity. Sections were also photographed. Another measure of cellular injury consisted in determining lactate dehydrogenase (LDH) release in the culture medium [19]. LDH activity was expressed as units per ml medium, where one unit of activity is the amount of LDH

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that causes a decrease of 0.001 AU per min in the presence of sodium pyruvate and NADH at 340 nm. z-VAD-fmk, CsA, or Fk 506 had no effect on measurement of P.I. uptake and LDH activity. Data are generally reported as means6S.E.M. from the indicated number of independent experiments, and each treatment consisted of 3–4 replicate slices. Analysis of variance (ANOVA) was used to verify statistically significant differences between treatments.

2.4. Western blots 14–16 cultured hippocampal slices were homogenized in a buffer containing (mM) HEPES (20), pH 7.5, sucrose (250), KCl (10), MgCl 2 (1.5), EDTA (1), EGTA (1), DTT (0.5), phenylmethylsulfonyl fluoride (PMSF) (0.5), 2 mg / ml leupeptin, and 2 mg / ml antipain. Homogenates were centrifuged at 1000 g for 10 min at 48C to remove nuclei. The supernatants were then centrifuged at 8000 g for 30 min at 48C to remove the crude mitochondrial fraction. The supernatants were further centrifuged at 100000 g for 60 min at 48C, and the resulting supernatant was used as the cytosolic fraction. Aliquots from cytosolic fractions were diluted with equal amounts of 23 sample buffer [4% sodium dodecyl sulfate (SDS), 100 mM Tris–HCl (pH 6.8), 20% 2-mercaptoethanol, 20% glycerol and 0.2% bromophenol blue] and boiled for 10 min. Aliquots from each sample (15 mg protein / lane) were subjected to SDS– polyacrylamide gel electrophoresis (PAGE) (8% polyacrylamide for Cyto C and 15% for caspases) and transferred onto nitrocellulose membranes. For bax and caspase-3 immunoblotting, the blots were blocked in Trisbuffered saline (TBS) containing 5% dry milk and 0.05% Tween at room temperature (RT) for 2 h. They were then probed overnight with primary antibodies (1:500) at RT in TBS containing 5% non-fat milk and 0.05% Tween 20. After washing three times with TBS containing 0.05% and Tween 20 for 10 min each, blots were incubated with anti-rabbit IgG (1:3000) conjugated to horseradish peroxidase for 2 h at RT and the antigen visualized using the Enhanced Chemiluminescence (ECL) Western Blot Detection Kit from Amersham (Piscataway, NJ). For caspase9 and Cyto C immunoblotting, the blots were blocked in TBS containing 3% gelatin at RT for 2 h. After three washes with TBS containing 0.05% Tween 20, the blots were probed with primary antibodies (1:250) at RT in TBS containing 1% gelatin and 0.05% Tween 20 overnight. Blots were then incubated with alkaline phosphataseconjugated appropriate IgG (1:2000) for 2 h at RT and visualized with NBT / BCIP. Antibodies recognizing the caspase-9 precursor (46 kDa) and the active form (17 kDa) of caspase-3 were used in Western blots of OHC cytosolic fractions. We were unable to detect the active form of caspase-9 even using ECL development to enhance the detection sensitivity. Immunoblots were scanned and the digitized images were analyzed quantitatively by densitometry with ImageQuant program providing peak areas.

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Statistical analysis of the immunoblotting data was performed with ANOVA followed by post-hoc analysis, and the level of statistical significance was defined as P,0.05.

2.5. RNA probe preparation Sense or antisense RNA probes complementary to the rat bax mRNA coding sequence were synthesized by in vitro transcription from a linearized plasmid template (generous gift from Dr. Jonathan L. Tilly). Transcription reaction mixtures (10 ml) contained 25 mM uridine 59-(a[35S]thio) triphosphate (1300 Ci / mmol, NEN; 1 Ci537 GBq), 50–100 ng of linearized template, 40 mM Tris–HCl (pH 8.0), 8 mM MgCl 2 , 2 mM spermidine, 50 mM NaCl, 500 mM nonradioactive GTP, CTP and ATP, 10 mM dithiothreitol (DTT), 20–40 units of RNAsin (Promega, Madison, WI, USA), and 1 ml of T3 or T7 RNA polymerase (Stragene, La Jolla, CA, USA) and were incubated for 1 h at 378C. Unincorporated nucleotides were removed by ethanol precipitation. The incorporation ratio of the probe was measured by acid-precipitable cpm. Specific activities averaged between 1 and 2?10 9 cpm / mg.

2.6. In situ hybridization After assessment of P.I. uptake and LDH release, hippocampal slice cultures were rinsed with cold PBS once, fixed in 2% (w / v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS) containing 20% sucrose, sectioned on a cryostat (14 mm), and the sections mounted onto microscope slides. Sections were then incubated with 4% PFA in PBS for 30 min at RT. After three washes in PBS (5 min each), sections were treated with freshly made 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min at RT and dehydrated in an ethanol series (30–100%) and air-dried. Probes were diluted to 0.2 mg / ml in hybridization buffer (50% formamide, 43 SSC, 53 Denhardt’s solution, 1% SDS, 10% dextran sulfate, 0.1 M DTT, 25 mg / ml poly A, 25 mg / ml poly C and 0.25 mg / ml tRNA) and heated at 708C for 5 min. Each slide was covered with 60 ml of radioactive hybridization solution and incubated at 508C for 3 h in a humidified chamber. After washes in 43 SSC, sections were incubated in a buffer containing 10 mg / ml RNase A, 0.5 M NaCl and 50 mM PBS at 378C for 30 min and washed in a high stringency solution (0.23 SSC, 20 mM b-mercaptoethanol) at RT overnight. Sections were then dehydrated through graded ethanol concentrations containing 0.3 M ammonium acetate and exposed to Hyperfilm bmax (Amersham, Arlington Heights, IL, USA). Autoradiograms were quantified using a computerized image system (Brain software running on DUMAS system from Drexel University). Data were analyzed by two-way ANOVA with STATISTICA software (Statsoft, Tulsa, OK, USA) with respect to animal groups followed by post-hoc analyses with the LSD test.

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2.7. Digital imaging of mitochondrial membrane potential DcM The mPTP regulates mitochondrial membrane potential DcM , which can be measured by a number of fluorochromes including Rhodamine 123 (R-123) [34]. To evaluate the involvement of mPTP in KA-induced neuronal death, slice cultures were loaded with the cationic and voltage-sensitive fluorescent dye, R-123 (3 mM), for 2 min and washed three times with fresh serum-free medium before KA application. Under normal conditions, R-123 is confined within the mitochondrial matrix. Collapse of DcM following depolarization of mitochondrial membranes results in the release of R-123. Slice cultures were mounted using VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA) 3 h after KA treatment. R-123 fluorescence was examined using a confocal microscope with appropriate filter blocks and irradiated with light from an argon–krypton laser. All slices were initially surveyed at low magnification to identify the specific region and overall distribution of fluorescent label within that region. CA3 was further examined with a 603 objective and zoom 1 setting on the computer. Images were routinely Kalman averaged to reduce background noise level. Release of R-123 was defined as reduction in R-123 fluorescence.

3. Results

3.1. Induction of bax in degenerating pyramidal neurons after KA treatment in OHC To evaluate the involvement of bax in KA-induced neuronal death in OHC, we examined the induction of bax in hippocampus after KA application. A significant increase in bax levels was detected as early as 3 h after KA treatment (Fig. 1). Bax levels continued to increase and reached a maximum about 9 h after KA treatment. We also examined the expression of bax mRNA in pyramidal cells 24 h after KA application (Fig. 2). A significant increase in bax mRNA expression in CA1 and CA3 regions was detected 24 h after KA treatment by in situ hybridization. In control experiments, sections hybridized with the sense cRNA probe showed a low-level background signal that was homogeneous throughout the whole section (data not shown). The pattern of bax mRNA expression matched the distribution of pyramidal cells in CA1 and CA3, suggesting that bax induction was taking place in neurons and not in other cell types.

3.2. Depolarization of mitochondrial membranes following KA treatment Previous studies in several apoptotic models have shown that overexpression of bax results in collapse of the

Fig. 1. Changes in bax levels in OHC following KA treatment OHC treatment and Western blots were performed as described in Materials and methods. (A) Representative Western blots of control (left lane) and KA-treated OHC (same time-points as below the graph shown in B). (B) Western blots were quantified by measuring the intensity of the 21 kDa band corresponding to bax. Data were expressed as relative optical densities and represent means6S.E.M. of five experiments. *Significantly different from control (P,0.05).

mitochondrial membrane potential, DcM , followed by Cyto C release and caspase activation [14,16]. Changes in mitochondrial DcM were determined with the fluorescent dye R-123. In untreated OHC, R-123 was retained inside mitochondria as indicated by a punctate and stable fluorescence pattern under the confocal microscope (603). By contrast, in KA-treated OHC, R-123 fluorescence decreased significantly, and the pattern of staining became more diffuse 3 h after treatment (Fig. 3). Similar changes were observed in CA3 and in CA1.

3.3. Partial neuronal protection of KA-induced neuronal toxicity by cyclosporin A It has been suggested that opening of mPTP initiates apoptosis via the release of Cyto C into the cytosol and, subsequently, activation of a caspase cascade [48]. To study the role of mPTP opening in KA-induced toxicity in OHC, we examined neuronal viability by measuring P.I. uptake and LDH activity release after blocking mPTP with CsA. In control, untreated cultures, no P.I. fluorescence was observed (Fig. 4). In contrast, in KA-treated cultures, increased P.I. fluorescence was clearly evident in pyramidal cells 24 h after treatment. The pattern of P.I. fluorescence corresponded well with the distribution of the morphological changes detected by hematoxylin–eosin staining (data not shown). CsA pretreatment produced a significant decrease in the level of P.I. fluorescence,

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although it remained significantly higher than in control (Fig. 4). LDH activity was increased in culture medium from KA-treated OHC, and CsA pretreatment resulted in a decrease in LDH activity by 32% (Fig. 5). There was a good agreement between changes in P.I. fluorescence uptake and LDH release in medium. Since CsA is also a potent anti-inflammatory agent, we compared its effects with those of another anti-inflammatory agent, FK 506 (Fig. 5). In contrast to the effects of CsA, FK 506 had no effect on KA-induced increase in P.I. uptake or LDH release, suggesting that the effects of CsA are due to its blockade of mPTP. In addition, no additional protection was observed when 200 mM CsA was used instead of 100 mM. These results suggest that the opening of mPTP is an important, but not exclusive, event in mediating KAinduced neuronal apoptosis.

3.4. Release of Cyto C into cytosol and activation of caspase-9 and caspase-3

Fig. 2. Changes in bax mRNA expression in OHC following KA treatment. OHC treatment and in situ hybridization were performed as described in Materials and methods. (A) Representative film autoradiographs of control (CONT) and KA-treated OHC. (B) Autoradiographs similar to those shown in (A) were analyzed as described. Data were expressed as relative optical densities and represent means6S.E.M. of 5–7 slices per group. *Significantly different from control (P,0.05). CA1: Pyramidal cell layer of the CA1 region of the hippocampus; CA3: pyramidal cell layer of the CA3 region of the hippocampus; DG: granule cell layer of the dentate gyrus (DG).

To characterize events downstream from the opening of mPTP in KA-induced neurotoxicity, we determined changes in levels of Cyto C and cleavage of caspase-9 and caspase-3 in cytosolic fractions. The levels of cytoplasmic Cyto C were assessed by Western blots stained with an antibody against the native form of Cyto C. They were significantly increased in cytosolic fractions prepared 24 h after KA treatment as compared to control. CsA pretreatment resulted in a significant decrease (64%) in the amount of Cyto C (Fig. 6). However, the levels of cytoplasmic Cyto C were still statistically higher in CsA

Fig. 3. Changes in mitochondrial membrane potential in CA3 region following KA treatment. OHCs were treated with KA and then loaded with the fluorescent probe, R-123, and examined by confocal microscopy as described in Materials and methods 3 h after KA treatment. (A) Representative image of control OHC (CONT) CA3 region. (B) Representative image of a KA-treated OHC CA3 region. Note the high fluorescent yield with a punctate pattern in control OHC (A). Treatment with KA (B) markedly extinguished R-123 fluorescence. (Magnification5603).

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Fig. 4. Effects of CsA on KA-induced P.I. uptake in OHC. Organotypic hippocampal slice cultures were loaded with P.I. (4.6 mg / ml) and examined under fluorescent microscopy (2.53) as described in Materials and methods 24 h after KA treatment. (A) Untreated cultures; (B) KA: 50 mM for 3 h; (C) KA1CSA (100 mM). Representative images (magnification52.53).

pre-treated OHC than in control OHC. Increasing CsA concentrations to 200 mM did not provide a larger reduction in Cyto C release (not shown). These results suggest that there may be other mechanisms than mPTP opening for KA-induced release of Cyto C into cytoplasm.

Proteins of the caspase family are synthesized as inactive precursors that are cleaved to form active proteases. For example, the caspase-3 precursor (32 kDa) is cleaved at two specific aspartic acid residues in the C-terminal domain to form two mature subunits, p17 (17 kDa) and p12 (12 kDa). The p17 subunit contains the catalytic site [13]. Untreated cultures exhibited high levels of caspase-9 precursor and very low levels of active caspase-3 protein. Twenty-four hours after KA treatment, the levels of caspase-9 precursor decreased and those of active caspase3 increased significantly (Fig. 6). In CsA pre-treated cultures, the levels of caspase-9 precursor were higher (19%) while the levels of active caspase-3 (p17) were lower (40%) when compared to the values found in the KA-treated group. However, there was still a significant difference in caspase-9 precursor and active caspase-3 levels when compared to control (Fig. 6).

3.5. Neuronal protection against KA-induced neuronal toxicity by a caspase inhibitor, z-VAD-fmk

Fig. 5. Effects of CsA and FK 506 on KA-induced P.I. uptake and LDH release in OHC. P.I. uptake (A) and LDH activity released in culture medium (B) were determined as described in Materials and methods. Data represent means6S.E.M. of 6–8 experiments. * and † significantly different from control (P,0.05).

To address the potential role of caspase-3 in KA-induced cell death, we applied a caspase inhibitor, z-VADfmk, relatively specific for caspase-1, 3 and 4, in OHC before and after KA treatment. We found that inhibition of caspase-3 activity significantly decreased the levels of P.I. uptake and LDH release (40%), as compared to KAtreated alone group, suggesting that caspase-3 is a critical mediator of KA-induced neuronal death (Fig. 5). Interestingly, the pattern of P.I. uptake in z-VAD-fmk pre-treated OHC was similar to that in CsA pre-treated cultures. In particular, both compounds produced more protection in CA3 than in CA1. Increasing the concentration of z-VADfmk to 200 mM did not provide more protection than 100 mM.

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Fig. 6. Effects of KA treatment on levels of cytosolic cytochrome c, caspase-3 (active) and caspase-9 (inactive) in OHC. Cytosolic fractions were prepared from OHCs after KA treatment as described in Materials and Methods. (A) Representative blots from untreated (CONT), KA-treated (KA) and CsA-pretreated (CsA / KA) cultures. Arrows indicate relative molecular weights. (B) Quantitative analysis of blots similar to those shown in (A). Blots were scanned and the intensities of bands were quantified and expressed as means6S.E.M. of six experiments. *Significantly different from KA-treated group (P,0.05).

4. Discussion The mechanisms underlying KA-induced neuronal damage are not yet understood. Several processes, including formation of free radicals, caspase activation, inhibition of mitochondrial function, activation of p53, have been proposed to play critical roles in KA-induced neuronal death. Furthermore, there is still some debate regarding the apoptotic versus necrotic nature of KA-induced neuronal death, although the distinction between these two forms of cell death is still a subject of intense discussion [24]. We previously showed that KA-induced neurotoxicity in OHC is associated with increased p53 expression and sequencespecific DNA binding activity [28,38], indicating that the p53 gene product may transcriptionally activate other genes that regulate cell death. The present results indicate that there was a rapid increase in the expression of bax in degenerating pyramidal cells following KA treatment. We also observed Cyto C release into the cytosol, and activation of caspase-9 and caspase-3. In addition, pretreatment with CsA partially blocked the release of Cyto C, caspase activation, and partially protected neurons from KA toxici-

ty, indicating that opening of mPTP contributed to the release of Cyto C and activation of caspase-3. Furthermore, inhibition of caspase-3 activity partially prevented neuronal death, suggesting that activation of caspase-3 is a critical but not exclusive, event in KA-mediated toxicity. The bax gene, a member of the pro-apoptotic BCL-2 gene family, contains a consensus sequence within its promoter that is recognized and activated by p53 protein in non-neuronal cell death models [29]. Although many studies have shown the importance of bax in neuronal apoptosis, whether bax is regulated transcriptionally, or post-transcriptionally, remains unclear [5,11,13,21,35]. In this study, we found that KA-induced toxicity in OHC is associated with the expression of bax mRNA in pyramidal neurons in CA1 and CA3, which are vulnerable to KA toxicity. In addition, bax gene induction was also associated with increased expression of bax protein. This induction was temporally and regionally correlated with the pattern of KA-induced expression of p53 [38], suggesting that the bax gene was transcriptionally activated in response to KA toxicity. A large body of evidence suggests that death signals

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triggered by bax activation include the release of Cyto C into the cytosol and the resulting activation of caspases [12,14]. It has been hypothesized that translocation of a BH-3 containing, pro-apoptotic BCL-2 family member, such as bax, to mitochondrial membranes might trigger the collapse of mitochondrial membrane, DcM , and the release of mitochondrial proteins including Cyto C via the opening of mPTP [30] and / or a channel formed by proapoptotic BCL-2 family members [9]. The opening of mPTP would cause the mitochondrial inner membrane to become permeable to protons, resulting in a persistent depolarization of the mitochondrial membrane. Here, we provide evidence that there was a nearly complete loss of R-123 fluorescence in the CA3 and CA1 region of OHC after KA treatment, indicating that mitochondrial membranes of pyramidal cells were depolarized. These results are in good agreement with other reports indicating loss of mitochondrial membrane potential following KA treatment [4,18,34]. On the other hand, no reduction of DcM was reported in neuronal apoptosis induced by KA toxicity [39]. This discrepancy may be due to the type of tissue studied. Recent reports have suggested a novel function for Cyto C in promoting apoptosis via binding with Apaf-1 and subsequent cleavage and activation of caspase-9 in neuronal cultures [22,25,37]. Activation of caspase-9 further cleaves and activates other caspases, especially caspase-3, to cause apoptosis [51]. In agreement with this idea we found increased levels of Cyto C and activated caspase-3, and a decreased level of the inactive form of the caspase-9 precursor in cytosolic fractions from KA-treated OHC. Although the caspase-9 antibody we used was reported to recognize both inactive and active forms of caspase-9, we were unable to detect any cleaved product of the caspase-9 precursor by Western blot. One possibility could be due to differences between the conformation of caspase-9 after cleavage in neurons and other cell types. We also cannot exclude the possibility that caspase-9 may be further cleaved by other proteases including other activated caspases. These observations provide further evidence to support the proposal that cytoplasmic Cyto C and activation of caspases are involved in excitotoxic neuronal death. Previous studies suggested that CsA prevents apoptosis by inhibiting mPTP opening [32]. However, other reports recently argued that cytoplasmic Cyto C release and activation of caspases occur independently from mitochondrial depolarization and mPTP opening [9]. Our results indicate that inhibition of mPTP opening by CsA partially reduced Cyto C release, caspase activation, and neuronal death following KA treatment. These observations suggest that mPTP opening is involved in regulating the release of Cyto C from mitochondria into the cytosol, and the subsequent activation of caspases following KA treatment. However, the fact that we only observed partial inhibition of Cyto C release by CsA suggests that it may also be regulated by other mechanisms such as mito-

chondrial swelling [43], or pore formation by bax or other proteins localized in mitochondrial membranes [36]. Moreover, we cannot rule out the possibility that other nonmitochondrial signals, including oxygen free radicals, may activate caspase-9 and caspase-3 [26]. In particular, we also observed that co-treatment of OHC with CsA and a scavenger of oxygen free radicals resulted in complete protection against KA-induced cell death (Liu et al., unpublished observations). Several lines of evidence suggest that caspase-3 is a predominant protease in p53-dependent apoptosis [8]. In our study, inhibition of caspase-3 activity by a potent blocker, z-VAD-fmk, produced only partial protection against KA-induced neurotoxicity. This result is consistent with observations that neurons from caspase-3 knockout mice displayed more, albeit not complete, resistance against p53-mediated apoptosis than those from wild-type mice [8]. Another interesting finding of the present study is that pretreatment with either CsA or Z-VAD-fmk provided more protection in CA3 than in CA1. However, there was no significant difference in bax mRNA induction in both regions, which is also consistent with the pattern of p53 mRNA expression [38]. These results may indicate that, although the opening of mPTP and caspase-3 activation are important events in p53-mediated neuronal death following KA treatment, there are additional downstream events in p53-mediated apoptosis that may vary with brain region [16]. In addition, there may exist p53-independent pathways promoting KA-induced apoptosis. Treatment of OHC with p53 antisense oligonucleotides and / or testing the effects of KA in OHC prepared from p53-knockout mouse will be useful to differentiate these possibilities. Our present studies provide evidence that induction of the bax gene, DcM collapse with opening of the mPTP, followed by cytoplasmic Cyto C release and caspase activation are involved in KA-induced excitotoxic neuronal death. However, the mechanisms of Cyto C release remain to be identified. Furthermore, it would be of great interest to investigate the signaling pathways in p53-mediated neuronal apoptosis in order to fully understand the mechanisms of excitotoxicity.

Acknowledgements This work was supported by grant NS18427 from NINDS and a grant from Sankyo Pharmaceuticals, Ltd.

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