Although the shape of PC12h cells before differentiation was comparatively round and thick, after the addition of nerve growth factor (NGF) to the culture medium and undergoing differentiation, the cells became flat and thin with processes, resembling neuronal cells (Figs. 1A–C). To confirm that PC12h cells differentiated into neuron-like cells, NGF-treated PC12h cells were immunofluorescently labeled with phosphorylated neurofilament (pNF) (Fig. 1D). The cell bodies and processes were neurofilament positive, indicating the ability of these neuronal cells to differentiate.#

The viability of the differentiated PC12h cells was analyzed using the MTS assay. The optical density (OD) of the cells significantly decreased after the addition of glutamate to the culture medium compared with unexposed cells. The addition of glutamate to differentiated PC12h cell cultures resulted in dose-dependent cell death (Figs. 2A–D). Glutamate 1 mM induced significant decreases in cell viability 2, 3, and 5 days after exposure. The TUNEL assay showed that glutamate induced apoptosis in differentiated PC12h cells in a dose-dependent manner (Figs. 3A, B). Significant increases were observed in the number of TUNEL-positive cells in the glutamate 1- and 5-mM-exposed cells compared with the unexposed cells 1 and 2 days after addition. DNA ladder study showed that glutamate 1 mM treatment leads to the generation of low-molecular weight DNA fragments (180–200 bp) (Fig. 3C).#

We used ELISA methods to investigate whether TNF-α is produced from differentiated PC12h cells after the addition of glutamate. TNF-α levels in the supernatant of glutamate (1 and 5 mM)-exposed cells were significantly increased compared with those of unexposed cells (Fig. 4). The levels of TNF-α in PC12h cell culture supernatants after the addition of glutamate 1 and 5 mM were 6.15 and 6.16 pg/ml, respectively. Similar to the results of the TUNEL assay, glutamate 0.1 mM did not alter the level of TNF-α (3.94 pg/ml) when compared with control cells (3.64 pg/ml). To determine whether TNF-α was produced via the NMDA receptor or AMPA/KA receptor in differentiated PC12h cells, d(−)-2-amino-5-phosphonopentanoic acid (APV, a competitive antagonist of the NMDA receptor) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPA/kainate receptor antagonist) was added to the cell cultures 2 h before the addition of glutamate. APV 1 mM significantly attenuated the glutamate-induced increase in TNF-α production. However, CNQX did not have any effect on the levels of TNF-α (Fig. 5).#

Since caspase-8 is a downstream effector of TNF-α, we examined the expression of caspase-8 in differentiated PC12h cells after glutamate exposure. Glutamate caused dose-dependent increases in the levels of caspase-8 protein (Fig. 6), although glutamate 0.1 mM did not alter the level of caspase-8 protein. Soluble TNF receptor 1 (sTNF-R1), which is a scavenger of TNF-α, was added to the cells 2 h before glutamate treatment. sTNF-R1 significantly ameliorated the increases in caspase-8 protein induced by glutamate in a dose-dependent manner (Fig. 7). Moreover, we performed the MTS assay 3 days after treatment to examine the effects of sTNF-R1 on cell viability. sTNF-R1 significantly ameliorated the cell death induced by glutamate (Fig. 8).#

Glutamate was purchased from Wako Pure Pharmaceuticals (Osaka, Japan), NGF from Invitrogen (Carlsbad, CA, USA), anti-caspase-8 rabbit polyclonal antibody from Santa Cruz Biotech (Santa Cruz, CA, USA), and anti-pNF mouse monoclonal antibody from Sternberger Monoclonals Incorporated (SMI31, Deisenhofen, Germany). TO-PRO-3 iodide was purchased from Molecular Probe (Eugene, OR, USA). Blockace was purchased from Dai-nippon Pharmaceutical Company (Osaka, Japan). APV, an NMDA receptor-specific antagonist, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, were purchased from Sigma Chemical (St. Louis, MO, USA). sTNF-R1 was purchased from R&D Systems (Minneapolis, MN, USA).#

PC12 cells are a well-established neuronal model originally isolated from a catecholamine-secreting tumor (pheochromocytoma) in rats (Greene and Tischler, 1976) and have been widely used to study the molecular mechanism of neuronal cell death (Ohmichi et al. 1993; Xia et al. 1995; Gollapudi and Neet 1997; Macdonald et al. 1999). Although cell lines generally have the ability to proliferate, neurons in vivo do not. For this reason, we used NGF-differentiated PC12h cells because the cells cease to proliferate, extend neurites, and express a battery of neuronal genes in response to NGF (Greene and Tischler 1976; Greene and Kaplan 1995). PC12h cells (Hatanaka, 1981), a subclone of PC12 cells, also undergo some NGF-responsive cellular events, including neurite outgrowth. PC12h cells were maintained in a mixed medium (Dulbecco's modified Eagle's medium and F12) containing 10% fetal calf serum (FCS), penicillin 100 U/ml, streptomycin 100 μl/ml, and amphotericin B 0.25 μl/ml at 37 °C with 5% CO2 in air. For differentiation, the cells were cultured in the presence of NGF 100 ng/ml for 3 days in mixed medium containing 1% FCS in a 100-mm collagen-coated dish for Western blot analysis or 24-well plate for ELISA analysis.#

The cells were seeded on a collagen-coated 35-mm glass-based dish (Iwaki Glass, Tokyo, Japan). After differentiation, the cells were washed three times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) fixative in PBS 0.1 M for 1 h. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 30 min and blocked with Blockace in PBS for 30 min. The cells were incubated with anti-pNF monoclonal antibody (1:1000) overnight at 4 °C. The cells were washed three times with PBS 0.1 M, incubated for 1 h with FITC-conjugated anti-rabbit IgG (Cappel Research Products, Durham, NC, USA, 1:100), and subsequently examined with confocal microscopy (LSM510, Carl Zeiss, Jena, Germany).#

The metabolism in a tetrazolium dye (MTS) assay evaluated the cell viability in neuronal differentiated PC12h cells. Cells were seeded and allowed to differentiate on collagen-coated 96-well plates for 3 days. To investigate the effects of glutamate on cell viability, several concentrations of glutamate were added to the medium to a total volume of 100 μl in each well. At different times (24-, 48-, 72- or 120-h exposure), the culture medium was removed and 20 μl of MTS reagent mixed with 100 μl of growth medium (mixed medium containing 1% FCS) was added to each well. Then the cells were incubated at 37 °C in a humidified, 5% CO2 atmosphere for 2–3 h. Absorbance was measured at 490 nm with a microplate reader (Multiskan, ThermoLabsystems, Ventaa, Finland).#

The TUNEL assay was performed with a fluorescein apoptosis detection system (Promega, Madison, WI, USA), according to the manufacturer's instructions. Briefly, PC12h cells were seeded on collagen-coated 35-mm glass-based dishes. After undergoing differentiation as previously described, the cells were washed with PBS, and medium containing 1% FCS with several concentrations of glutamate was added. After continuous 24-h exposure, the cells were washed three times with PBS, fixed in 4% PFA in PBS 0.2 M for 25 min, and again washed three times with PBS. Then the cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature and washed with PBS for 10 min. They were incubated with the terminal dUTP transferase enzyme together with a mixture of nucleotides in an equilibration buffer for 60 min at 37 °C in a humidified incubator. After terminating the reaction by immersing the cell mixture in 2× saline sodium citrate for 15 min, they were washed three times with PBS for 5 min each. For nuclear staining, they were counterstained with To-Pro-3 iodide diluted to 1:3000 for 15 min. The percentage of FITC-labeled (TUNEL-positive) cells was determined by averaging the values obtained from three random regions per 35-mm glass-based dish containing 50–100 cells/region using a confocal microscope (LSM510, Carl Zeiss).#

Total genomic DNA was isolated from differentiated PC12h cells in the presence or absence of glutamate using an Apoptotic DNA Ladder kit (Roche Diagnostics GmbH, Penzberg, Germany) according to manufacturer’s protocol. Briefly, the cells were collected by centrifugation and washed twice with ice-cold PBS. The cell pellets were suspended in 200 μl of PBS and 200 μl of lysis buffer (pH 4.4) at room temperature for 10 min. The lysates were centrifuged at 8000 rpm for 1 min to separate the fragmented DNA (supernatant) and intact DNA (pellet). Then, washing buffer was added to the fragmented DNA, and the mixture was centrifuged at 8000 rpm for 1 min and finally centrifuged at 13,000 rpm for 10 s to remove residual washing buffer. DNA bound to glass fibers in the presence of chaotropic salts was eluted in 200 μl of prewarmed (70 °C) elution buffer (pH 8.5). An equal amount of DNA sample was then electrophoresed on 2% agarose gel containing 0.1 μg/ml ethidium bromide. The agarose gel was run at 100 V for 75 min in a TBE buffer (Tris 90 mM/boric acid 64.6 mM/EDTA 2.5 mM, pH 8.3) and evaluated and photographed under ultraviolet illumination.#

Cells were seeded on 24-well plates and after differentiation were exposed to glutamate. Supernatants of control and glutamate-exposed differentiated PC12h cells were collected, harvested, centrifuged to remove any debris, filtered (pore size 0.22 μm), and stored at −20 °C until analysis. A commercial ELISA kit (Biosource, Camarillo, CA, USA) was used to detect TNF-α production in culture supernatants following the manufacturer's instructions. The sensitivity of TNF-α detection in this assay system is about 0.7–150 pg/ml and the minimum detectable level is <0.7 pg/ml. OD readings were measured at 450 nm with a microplate reader (Multiskan, ThermoLabsystems).#

Cells were grown to 60–70% confluence in 100-mm culture dishes (Iwaki Glass) and differentiated with NGF for 3 days. For experiments examining caspase-8 expression after glutamate exposure for 24 h, the cells were collected and washed twice with ice-cold PBS. The cells were then lysed with protein extraction buffer (Tris–HCl 50 mM, pH 7.4, EGTA 1 mM, 0.001% leupeptin) and centrifuged at 15,000×g for 30 min at 4 °C. Protein concentrations were determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA). Protein samples (35 μg each) were boiled with gel loading buffer for 5 min and subjected to SDS-PAGE on 10% polyacrylamide gels. The samples were transferred to enhanced chemiluminescence (ECL; Amersham-Pharmacia Biotech, Piscataway, NJ, USA) membranes, and the membranes were blocked overnight at 4 °C with Tris-buffered saline (TBS)–0.1% Tween 20 (TBS-T) containing 2% skim milk. The membranes were then incubated for 2 h with anti-caspase-8 antibody diluted to 1:200 in TBS. After washing five times with TBS-T, the membranes were incubated for 1 h with peroxidase-labeled anti-rabbit IgG antibody (1:5000, Cappel, Aurora, OH, USA) in TBS-T. After washing five times with TBS-T, the immune complex was visualized with an ECL Plus Western Blotting Detection Systems kit (Amersham-Pharmacia Biotech). The signals were detected using FujiFilm LAS-3000 (FujiFilm, Tokyo, Japan). The intensity of the detected bands was analyzed using Image Gauge (FujiFilm).#

Data are presented as mean±SEM. Differences among groups were analyzed using one-way ANOVA, followed by the Mann–Whitney U method or Scheffe's method. A probability value of less than 0.05 was considered to represent a statistically significant difference.#