Cell Culture

Sympathetic ganglia of thoracic regions were dissected from 10–15 chick embryos (White Leghorn) at 11 to 11.5 embryonic-day and collected in Ca²⁺, Mg²⁺-free Hanks' balanced salt solution (HBSS (−)). The obtained 100–150 ganglia in 100 ml of HBSS (−) were incubated with 0.05% trypsin (GIBCO) at 37°C for 10 min and then gently triturated by pipetting. After centrifugation for 10 sec at 1000 rpm, the supernatant containing dissociated cells was transferred to a tube containing 1 ml of culture medium supplemented with 30 ng/ml mouse 2.5S NGF (Chemicon), and this procedure was repeated until dissociation was completed. Collected supernatant was centrifuged at 10°C for 10 min at 1000 rpm and cell pellet was suspended in 4 ml of culture medium and plated on non-coated 100 mm plastic dish (Corning). The dish was kept at 37°C for 1.5 hr in a humidified 5% CO₂ incubator. During this incubation, most non-neuronal cells attached to the plastic surface, whereas neurons remained in suspension. The collected suspension was diluted with the same medium to 6.0–7.5 × 10⁵ cells/ml and aliquots were plated on a poly-lysine coated culture dish (Corning), followed by addition of NGF at the final concentration of 30 ng/ml. Culture medium was composed of 90% Eagle's minimum essential medium (Nissui Inc.) and 10% dialyzed and heat-inactivated FCS (JRH Biosciences) supplemented with 18 μM fluorodeoxyuridine and 10 μM uridine (Nakarai Tesque Inc.) to kill dividing cells such as Schwann cells and fibroblasts.

Neurons began to extend neuritic processes within 10 hr after plating. Cell bodies aggregated in clusters of 3–10 cells and were connected by neurite to each other in 2 days. The number of cells in culture was reduced to approximately 50% after 2 days, because cells that had been severely damaged before plating degenerated within the first 2 days (they adhered to living cells as debris). Although the culture initially contained non-neuronal cells comprising 15% of the total cells, most of them were killed by fluorodeoxyuridine during the 5-day incubation so that finally more than 98% of the cells in the culture were neurons. Every 2.5–3 days, two-thirds of culture medium was exchanged for NGF-free medium and NGF was immediately added at 25 ng/ml. Neurons required NGF for the first 2 weeks, and thereafter, they began to lose the NGF-dependency. Therefore, we used the 5 to 6-day-old culture for all of the following experiments.

Deprivation and Readdition of NGF

For the sake of rapid and complete removal of the activity, IgG fraction from the sheep immunized by mouse NGF (CIDtech Research Inc.) was added to the culture at the final concentration of 12.5 μg/ml, after exchanging two-thirds of culture medium for NGF-free medium. For readdition of NGF, two-thirds of culture medium was exchanged for NGF-free medium and then NGF was added at 40 ng/ml. Thereafter, every 2.5–3 days medium was exchanged in the same manner and NGF was added at 25 ng/ml.

Analysis of phosphorylated proteins

Each 48-well containing 1.2–1.5×10⁴ neurons received 7.4 MBq of [³²P]orthophosphate for 6–7 hr (ARC Radiochemicals) and cells were lysed with a buffer containing 1% Triton X-100 and 1% sodium deoxycholate. After centrifugation at 100,000 rpm for 1 hr in a Beckman model TLA 120.2 rotor at 4°C, the precipitates were electrophoresed through a 10–20% linear polyacrylamide gradient gel containing 0.1% SDS and 7 M urea. The gel was rinsed with 50% methanol-10% acetic acid, dried and exposed to an image plate of Fujix Bas 2000 to take an autoradiography.

Inhibitors

Inhibitors were purchased from the following corporations; actinomycin D (WAKO Pure Chemical), anisomycin and puromycin (Sigma Chem. Co.), a-amanitin (Boehringer Ingelheim), blasticidin S (Funakoshi Co. Ltd.) and emetine (Nakarai Tesque Inc.). For use of each translational inhibitor, a dose which inhibited approximately 80% of [³⁵S]Met/Cys incorporation in 3 hr was determined and defined as a minimal dose.

NGF-deprivation and puromycin induced apoptosis of developing chick sympathetic neurons

Apoptosis of developing neurons after NGF-deprivation has not been characterized in vitro, except for rat sympathetic neurons [4]. In order to investigate the mechanism of apoptosis caused by NGF-deprivation without limit to mammalian system, we established the culture system of sympathetic neurons from chick embryo. Observation of morphological alterations under a microscope revealed that most cell bodies fragmented into pieces between 45–65 hr after NGF-deprivation (Fig. 1). This and other morphological alterations (refer to the legend of Fig. 1) were quite similar to those during apoptosis of rat sympathetic neuron after NGF-deprivation [4]. Like the death of rat sympathetic neuron, this death was also apoptotic, because DNA degradation characteristic to apoptosis occurred. When total DNA extracted from NGF-deprived neurons was analyzed by agarose gel electrophoresis, a DNA ladder, each band of which corresponded to a multimer of nucleosome-sized DNA, was detected with DNA extracted from neurons deprived of NGF for longer than 12 hr (Fig. 2). In addition, DNA longer than 20 Kb was very scarce in the total DNA from neurons at 48 hr, indicating that extensive degradation of DNA had occurred.

Fig. 1

Phase-contrast micrographs of cultured sympathetic neurons after NGF-deprivation. NGF was deprived at time 0 (A). The first alterations were detected at 25 hr; the neurites became slightly thinner and locally disrupted in places, leaving behind bits of neuritic debris (B). Cell bodies became phase-dark, and the distortion of nuclear perimeter became noticeable in 45 hr (C). At 65 hr, most intact cell bodies disappeared and were replaced by scattered debris (D). The neurons comprising 10–15% of initial culture remained intact at 65 hr and for at least 6 more days in the absence of NGF (data not shown), indicating that the culture initially contained 10–15% neurons whose survival did not depend on NGF. Scale bar in photograph (A), 50 μm.

Fig. 2

Apoptotic DNA degradation after NGF-deprivation or puromycin-addition. Each 35 mm plastic dish containing 6.0–7.5 × 10⁵ neurons was deprived of NGF or received 3 μM puromycin at time 0. At various timepoints indicated above the figure, neurons were collected and lysed with 20 μl of buffer containing 0.2% SDS. After incubation at 37°C for 3.5 hr with 0.5 mg/ml proteinase K and further incubation for 1 hr with 0.2 mg/ml pancreatic RNaseA, all the lysate was electrophoresed through a 2% agarose gel in TAE buffer and DNAs were stained with ethidium bromide. DNA size markers were Lambda phage DNAs prepared by digestion with two restriction enzymes, EcoRI and HindIII.

During the course to examine effects of translational inhibitors on apoptosis caused by NGF-deprivation (see below), we found that puromycin itself caused fragmentation of cell bodies between 30 to 50 hr eventually after morphological alterations similar to those with apoptosis induced by NGF-deprivation (Fig. 5D). This death was also apoptotic, because DNA degradation was discernible by 12 hr (Fig. 2). Apoptosis was induced by puromycin in the dose-dependent manner: 1, 2 or 3 μM puromycin induced fragmentation of cell bodies of 30%, 80% or more than 95% neurons, respectively.

The decrease of protein synthesis rate preceded committment to Apoptosis

Preceding to DNA degradation, an activity of protein synthesis of neurons was found to decrease rapidly after NGF-deprivation. Figure 3 shows that the incorporation rate of [³⁵S]Met/Cys into the TCA-insoluble fraction began to decrease by 6 hr and reached approximately 30% level at 18 hr after NGF-deprivation. Taking into account the activity of NGF-independent cells that comprised 10–15% in this culture (refer to the legend for Fig. 1), NGF-dependent neurons appear to lose approximately 80% of their original protein synthesis activity within 18 hr.

Fig. 3

Characterization of apoptosis after NGF-deprivation. Each 96 half-area well containing 2.5–3.0 × 10³ neurons was deprived of NGF at time 0. To examine the activity of protein synthesis (•), each well received 185 KBq of [³⁵S]Met/Cys (41.7 MBq/mM, 70% of L-methionine and 30% of L-cystein, ARC Radiochemicals) for 20 min. After removing the medium, cells were washed twice with ice-cold HBSS (−) and lysed with a solution of 10 mM EDTA, 0.2% SDS. The radioactivity incorporated into the TCA-insoluble fraction was measured as described previously [16] and was corrected by subtracting the radioactivity obtained from cell-free wells. To examine the time course of commitment to apoptosis (○) NGF was readded at the timepoints indicated in abscissa, and to assess rescue effects of actinomycin D and anisomycin either 20 nM actinomycin D (▵) or 10 μM anisomycin (▴) was added at the timepoints indicated in abscissa. The number of intact neurons at day 3 was counted and expressed as the percentage of that for control cultures which received 25 ng/ml NGF instead of anti-NGF antibody. Results represent the mean ± SD of 2–3 separate experiments each done in duplicate.

Next, we examined the time course of commitment to apoptosis. The commitment is defined as entering an irreversible step to apoptosis, which is not suppressed by NGF [4]. At various timepoints after NGF-deprivation, NGF was readded to measure the percentage of neurons that could be rescued. The results shown in Figure 3 indicate that the survival began to decrease at 8 hr and reached to approximately 25% at 24 hr after NGF-deprivation. Taking into account NGF-independent neurons in this culture, NGF-dependent neurons become committed to apoptosis during 8–24 hr.

To examine the time course of commitment to apoptosis triggered by puromycin, neurons were exposed to puromycin for various periods of time and then puromycin was removed by exchanging culture medium. Figure 4 presents the survival at day 3 (closed circles) and the incorporation rate of [³⁵S]Met/Cys (open circles) after puromycin-addition, and indicates that the commitment to apoptosis started soon after the decrease in protein synthesis.

Fig. 4

Characterization of puromycin-induced apoptosis. Culture received 3 mM puromycin in the presence of NGF at time 0. The time course of commitment to apoptosis (○) was investigated as in Fig. 3 except that puromycin was removed by exchanging medium for puromycin-free medium at each time-points. The incorporation rate of [³⁵S]Met/Cys (•) and the rescue-effect of actinomycin D (▵) were measured as in Fig. 3. All the data were expressed as the percentage to that obtained for a control culture which did not receive puromycin. Results represent the mean ± SD of 2–3 separate experiments each done in duplicate.

Fig. 5

Phase-contrast micrographs of sympathetic neurons treated with actinomycin D, anisomycin or puromycin in the presence or absence of NGF for 3 days. (A) 20 nM actinomycin D, (B) 10 μM anisomycin or (C) 3 μM puromycin was added simultaneously with NGF-deprivation. In the presence of 25 ng/ml NGF, (D) puromycin or (E) both puromycin and anisomycin was added. The culture which was solely deprived of NGF is also presented in photograph (F). It should be noted that a fibroblast remains intact in photograph (D). Scale bar in photograph (A), 50 μm.

Transcription was required for apoptosis

Inhibitors of transcription and translation are reported to block or delay apoptosis induced by deprivation of neurotrophic factors, proposing the requirement of gene expression for such dying processes [7]. To assess the requirement of transcription for apoptosis of chick sympathetic neurons, transcriptional inhibitors were added simultaneously with either NGF-deprivation or puromycin-addition and then the survival at day 3 was measured. The results indicate that by addition of 20 nM actinomycin D, approximately 85% or 70% of neurons remained intact (Fig. 3 open triangle at time 0 and Fig. 5 A) or puromycin-addition (Fig. 4, open triangle at time 0). 20 nM actinomycin D suppressed approximately 80% of [³H]uridine incorporation within 3 hr, and inhibitory effect continued throughout the experimental period. We also found that a-amanitin (13.6 nM) rescued 70–80% of NGF-deprived or puromycin-exposed neurons from apoptosis.

The above results suggest that transcription is required for apoptosis of either NGF-deprived or puromycin-exposed neurons. In order to determine when transcription is essential for both apoptoses, actinomycin D was added at various timepoints after triggering of apoptosis. Open triangles in Figure 3 indicate that addition of actinomycin D within 6 hr effectively prevented apoptosis triggered by NGF-deprivation, but thereafter, the effect rapidly diminished by addition at 18 hr only 20% neurons remained intact. Considering 10–15% NGF-independent neurons in this culture, this result indicates that addition of actinomycin D at 18 hr is almost unable to prevent neuronal apoptosis. These results strongly suggest that the transcription during 6–18 hr after NGF-deprivation is indispensable for apoptosis. Open triangles in Figure 4 represents the results obtained from the same experiment except that apoptosis was triggered by puromycin. The results suggest that transcription between 6–18 hr after puromycin-addition is crucial for neurons to be committed to apoptosis.

Anisomycin and Blasticidin S, but not Emetine, Prevented Apoptosis

Following to transcriptional inhibitors, the rescue effect of translational inhibitors was tested. Since translational inhibitors have complicated effects on cellular physiology, inhibitors whose action was well characterized were selected. Puromycin at the minimal dose (3 μM, see Materials and Methods) and 20 μM, puromycin did not prevent apoptosis caused by NGF-deprivation (Fig. 5C). Anisomycin and blasticidin S were effective on prevention. At the minimal dose (10 μM for anisomycin and 300 μM for blasticidin S) and 10-fold higher dose, simultaneous addition of anisomycin or blasticidin S with NGF-deprivation rescued 70–80% of neurons from apoptosis (as for anisomycin, see Fig. 3 closed triangle at time 0 and Fig. 5B). However, at the one-fifth of each minimal dose, they could not rescue the neurons at all (data not shown). Figure 3 indicates the rescue effect of anisomycin which was added at various timepoints after NGF-deprivation: the rescue effect declined when added after 6 hr, and addition at 24 hr was no longer effective at all. Similarly to NGF-deprived neurons, anisomycin and blasticidin S had a rescue-effect on puromycin-exposed neurons: 3 days after simultaneous addition of anisomycin and puromycin, approximately 65% of neurons remained intact (Fig. 5E for anisomycin). Addition of blasticidin S rescued approximately 80% of neurons from apoptosis. These results indicate that anisomycin and blasticidin S apparently antagonize the effect of NGF-deprivation or puromycin on induction of apoptosis.

By contrast, emetine did not prevent apoptosis. When emetine was added at the minimal dose (0.1 μM) or 10-fold higher dose simultaneously with either NGF-deprivation or puromycin-addition, only less than 10% neurons remained intact (data not shown). These results are summarized in Table 1.

Table 1

Rescue effects of translational inhibitors on apoptosis

Change of phosphorylated proteins after NGF-deprivation and Puromycin-addition

NGF binds to two cell surface receptors and its action is mediated by tyrosine kinase activity associated with one of the receptors [15]. Since this signal transduction would be mediated by serial phosphorylation/dephosphorylation of proteins [15], we addressed phosphorylated proteins before and after NGF-deprivation (Fig. 6, lanes 1–4). Among many phosphorylated proteins detected in a soluble fraction, three phosphorylated proteins (marked with a closed arrow-head) disappeared after NGF-deprivation, indicating that they underwent dephosphorylation or were eliminated.

Fig. 6

Change of phorphorylated proteins. Proteins were labeled with [³²P]orthophosphate; lane 1, for 6 hr in the presence of NGF; lanes 2–4, during 0–6 hr, 6–12 hr and 12–18 hr after NGF-deprivation, respectively; lane 5, during 12–19 hr after NGF-deprivation in the presence of 20 μM anisomycin; lane 6–8, during 0–6 hr, 6–12 hr and 12–18 hr after addition of 3 μM puromycin, respectively; lane 9, during 12–19 hr after addition of 3 μM puromycin in the presence of 50 μM anisomycin; lane 10, during 12–19 hr after addition of 3 μM puromycin in the presence of 0.1 μM emetine; lane 11, during 12–19 hr after addition of 3 μM puromycin in the presence of 30 μM actinomycin D. Phosphorylated proteins were analysed as described in Materials and Methods. Numbers marked in the left margin are molecular weights (Kd) deduced from Kaleidoscope prestained proteins (Bio-Rad).

The effect of puromycin on phosphorylation was examined whether puromycin mimicked NGF-deprivation. Lanes 6–8 show that puromycin affected the same three proteins in the same manner as NGF-deprivation did. Thus, the similarity of NGF-deprivation and puromycin in their effects on neurons further extended. Besides, we found that actinomycin D and anisomycin suppressed the disapearance of these phosphorylated proteins (lanes 5, 10, 11), but emetine did not (lane 10). Thus, the effects of these inhibitors on the three phosphorylated proteins paralleled their effects on suppression of apoptosis, implying that these proteins are crucial for apoptosis.