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Keywords:

  • aging;
  • nerve growth factor;
  • neuronal death;
  • p75ntr;
  • trka;
  • tumor necrosis factor alpha

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Physiological and pathological aging of the central nervous system (CNS) is characterized by functional neuronal impairments which may lead to perturbed cell homeostasis and eventually to neuronal death. Many toxic events may underlie age-related neurodegeneration. These include the effects of beta amyloid, Tau and mutated presenilin proteins, free radicals and oxidative stress, pro-inflammatory cytokines and lack of growth factor support, which can be individually or collectively involved. Taken individually, these toxicants can induce very diverse cell responses, thus requiring individually targeted corrective interventions upstream of common cell death (apoptotic) pathways. Recent preliminary evidence suggests that the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) and growth factor withdrawal can both activate a common apoptotic pathway in nerve growth factor (NGF)-responsive PC12 cells involving caspase 3, albeit through very distinct upstream pathways: the former through active signalling and the latter through passive or lack of survival signalling. Here, we show that NGF can rescue PC12 cells from both growth factor withdrawal- and TNFα-promoted cell death. However, NGF rescue from growth factor withdrawal requires NGF signalling through the high-affinity tyrosine kinase receptor (TrkA), while NGF rescue from TNFα-promoted cell death requires NGF signalling through the low-affinity p75NTR receptor. These results strengthen the idea that prevention of age- or pathology-associated neurodegeneration may require varied molecular approaches reflecting the diversity of the toxicants involved, possibly acting simultaneously.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Neurotrophins (NT) are essential for the development and maintenance of certain neurones (Levi-Montalcini, 1987; Levi-Montalcini et al., 1996). Consequently, aberrant NT function has been suggested to play an important role in the onset of age-related neurodegenerative diseases such as Alzheimer's disease (Hefti, 1994; Cotman & Su, 1996; Tong et al., 1998). Age-associated neurodegenerative diseases are also characterized by high CNS levels of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNFα) (Akiyama et al., 2000; Fillit et al., 1991; Tanoiri et al., 1994; Hays, 1998; Malek-Ahmadi, 1998). Because TNFα has been reported to be either neuroprotective (Barger et al., 1995; Liu et al., 1996; Mattson et al., 1997; Sullivan et al., 1999) or neurotoxic (D’Souza et al., 1995; Wagner et al., 1995; Srisawasdi et al., 1996; Viviani et al., 1998; Barker et al., 2001), it is possible that TNFα may play a role in the onset of neuronal impairments during neurodegenerative diseases.

Members of the NT family – which includes brain-derived neurotrophic factor (BDNF) (Leibrock et al., 1989), NT-3 (Hohn et al., 1990; Maisonpierre et al., 1990), NT-4/5 (Hallbook et al., 1991), NT-6 (Gotz et al., 1994), NT-7 (Lai et al., 1998) and the more thoroughly characterized NGF (Levi-Montalcini, 1987; Levi-Montalcini et al., 1996) – act via binding to specific high-affinity membrane tyrosine kinase receptors (Trk) (Barbacid, 1994; Stephens & Kaplan, 1994; Chao & Hempstead, 1995), as well as a common low-affinity receptor p75NTR (Chao, 1994). While Trk receptors mediate most of the characterized effects of NT, the role of p75NTR is intimately associated with the modulation of apoptosis, although its exact function in mediating NT-promoted cell death or cell rescue is still inconclusive and deserving of further investigation. For example, it has been shown that NGF binding to p75NTR can either suppress (Rabizadeh et al., 1993; Cortazzo et al., 1996; Taglialatela et al., 1996; Khursigara et al., 2001) or promote neural cell apoptosis (Casaccia-Bonnefil et al., 1996; Frade et al., 1996; Bamji et al., 1998). This dual effect of p75NTR in modulating neural apoptosis appears to be developmentally regulated (Barrett & Bartlett, 1994; Ladiwala et al., 1998) and affected by the concomitant presence of Trk receptors (Taglialatela et al., 1996; Yoon et al., 1998; Eggert, 2000).

We have preliminary evidence that in the NGF-responsive PC12 cells both TNFα and growth factor withdrawal (two toxicant events associated with the effects of aging and/or disease in the CNS) induce a common downstream apoptosis pathway impinging upon caspase-3 activation, even though through very distinct upstream signalling events. TNFα induces signalling pathways proactively, while growth factor withdrawal depresses the same pathways (N. J. Macdonald et al., unpublished data). This observation is consistent with the hypothesis that common neuronal impairments may derive from different toxicants acting through distinct yet converging pathways. Also, it suggests that any successful treatment for age-associated neurodegeneration should reflect the different specific molecular events, often acting simultaneously, that multiple toxicants may influence. Thus, understanding such molecular events and signalling pathways is crucial to developing specific successful treatments.

Considering this, as well as other evidence suggesting that NGF can rescue cells from apoptosis induced by either TNFα or growth factor withdrawal, we reason that NGF should protect via different mechanisms, probably leading on the one hand to the suppression of an active death signal delivered by TNFα or on the other hand to re-establishment of survival signalling depressed by growth factor withdrawal. Our results confirm that while TrkA (the mediator of NGF trophic effects) is required for NGF-mediated rescue of cell death induced by growth factor withdrawal, the p75NTR (which modulates apoptosis) appears to be sufficient to mediate NGF rescue from TNFα-promoted cell death. These results suggest that different neurotoxic pathways may affect aged or diseased neurones simultaneously.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

TNFα mediates cell death in PC12 cells

We first determined whether PC12 cells express detectable levels of the type I TNF receptor (TNFR-I). We focused our attention on TNFR-I because, like p75NTR, it modulates cellular apoptosis by activating downstream signalling elements such as sphingomyelinase ceramide, c-jun N-terminal kinase (JNK), and nuclear factor kappa B (NFκB) that are involved in the modulation of cell apoptosis (Cosman, 1994; Wallach et al., 1996; Orlinick & Chao, 1998; Schulze-Osthoff et al., 1998; Warzocha & Salles, 1998). Using Western blot analysis we found that our PC12 cells express TNFR-I at levels that are comparable to those observed in dorsal root ganglion neurones, a positive control (data not shown).

The effects of increasing concentrations (10–1000 ng mL−1) of human recombinant TNFα on PC12 cell survival were tested on PC12 cells cultured in either serum-containing media or serum-free media in the presence of 25 ng mL−1 NGF (Fig. 1). PC12 cells cultured in serum-containing media derive trophic support from factors present in the serum, whereas serum-free PC12 cells supplemented with NGF are supported solely by the NGF added to the culture medium (Levi et al., 1988; Levi-Montalcini et al., 1996; Taglialatela et al., 1996), thus representing a useful tool to study NGF-mediated trophic support. As shown in Fig. 1(A), the presence of TNFα reduced PC12 cell survival in a dose-dependent fashion in both serum-cultured cells and in serum-free cells supplemented with NGF, as assessed by the reduction of the vital mitochondria dye MTT. This decrease in cell survival was statistically significant at both 100 and 1000 ng mL−1 TNFα, but it did not reach statistical significance when TNFα was added at a concentration of 10 ng mL−1.

image

Figure 1. A: PC12 cell survival as assessed by MTT reduction assay after 24 h treatment with 10, 100 or 1000 ng mL−1 human TNFα. PC12 cells were either cultured in serum containing medium (open bars) or in serum-free medium supplemented with 25 ng mL−1 NGF (closed bars). N= 6–8 per group. Statistical significance was assessed by anova followed by the Fischer's LSD test. B: PC12 cell death as assessed by the ratio between LDH release and MTT reduction assay 4, 8, 24 or 48 h after serum withdrawal from the culture medium. Upon serum starvation cells were additionally treated with 1000 ng mL−1 human TNFα. N= 6–8 per group. Statistical significance was assessed by anova followed by the Fischer's LSD test. C: Agarose gel electrophoresis of 32P-ATP-labelled low-molecular-weight DNA extracted from PC12 cells after 24 h of serum withdrawal in the presence of 25 ng mL−1 NGF, 1000 ng mL−1 human TNFα or both.

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TNFα also increased the extent of PC12 cell death induced by 48 h of serum deprivation (Fig. 1B). In this experiment, and in some of those shown below, the extent of cell death was assessed by calculating the ratio between LDH released into the medium and MTT reduction in the same cell culture dish. The release of LDH into the culture medium is proportional to the number of dead cells, while MTT reduction values are proportional to the number of live cells. Therefore, the ratio LDH/MTT is an index of cell death that is corrected for possible differences in cell number or proliferation rates due to the experimental manipulations. In addition, the ability of TNFα to induce cell death in both serum-containing and serum-free NGF-treated PC12 cells was confirmed by the appearance of fragmented DNA as revealed by agarose gel electrophoresis (Fig. 1C).

NGF prevents TNF α-mediated cell death in a TrkA-independent fashion

The experiments described above showed that TNFα induced cell death in serum-cultured PC12 cells and also affected the ability of NGF to rescue serum-free PC12 cells. We then wanted to determine whether higher concentrations of NGF (> 25 ng mL−1) might, in turn, protect serum-cultured PC12 cells from TNFα-induced cell death.

In the experiment shown in Fig. 2(A), PC12 cells were treated with 1000 ng mL−1 TNFα, a concentration of TNFα that significantly reduces cell viability in serum-containing culture conditions. Cells were additionally treated with increasing concentrations of NGF (from 10 to 2000 ng mL−1) in the presence or absence of 100 nm K252a, an inhibitor of TrkA phosphorylation and signal transduction (Ohmichi et al., 1992). As expected, TNFα induced a significant decrease of cell viability. Whereas NGF at concentrations of 10 or 100 ng mL−1 did not protect PC12 cells, NGF at 1000 or 2000 ng mL−1 completely abolished the cell death induced by 1000 ng mL−1 TNFα (Fig. 2A, grey bars). In the presence of 100 nm K252a, NGF was still able to rescue PC12 cell from TNFα-induced cell death at concentrations of 1000 or 2000 ng mL−1 (Fig. 2A, black bars), indicating that TrkA signalling was not essential to promote NGF rescue from TNFα-induced cell death.

image

Figure 2. A: PC12 cell death as assessed by the ratio between LDH release and MTT reduction assay 24 h after treatment with 1000 ng mL−1 human TNFα (grey and dark bars) and 0, 10, 100, 1000 or 2000 ng mL−1 NGF. The experiment was performed either in the absence (grey bars) or in the presence (dark bars) of the tyrosine kinase inhibitor K252a (100 nm). N= 7–8 per group. Statistical significance was assessed by anova followed by the Fischer's LSD test. B: PC12 cell death as assessed by the ratio between LDH release and MTT reduction assay 24 h after serum deprivation and replacement with 1000 ng mL−1 NGF in the presence or absence of 100 nm K252a. N= 4 per group. Statistical significance was assessed by anova followed by the Fischer's LSD test.

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Inhibition of TrkA signal transduction has been shown to abolish most of the effects elicited by NGF in PC12 cells, including rescue from cell death induced by serum withdrawal (Ohmichi et al., 1992). We tested the ability of K252a to abolish NGF-promoted rescue of serum-free PC12 cells (Fig. 2B) to confirm that this signal transduction pathway is active in our cells. In this experiment, serum-free PC12 cells were treated with 1000 ng mL−1 NGF in the presence or absence of 100 nm K252a, the same NGF and K252a concentrations included in the experiment depicted in Fig. 2(A). Under these serum-free conditions, however, K252a was able to abolish NGF-induced rescue of PC12 cells.

The induction of cell death by TNFα and the K252a-independent rescue by NGF in serum-cultured PC12 cells was confirmed by assaying the presence of fragmented DNA by ELISA. PC12 cells were treated for 24 h with 1000 ng mL−1 TNFα and 1000 ng mL−1 NGF in the presence or absence of 100 nm K252a. As Fig. 3 shows, there was a significant increase in fragmented DNA in PC12 cells treated with TNFα, and this was completely prevented by NGF. The presence of K252a did not affect the ability of NGF to abolish the appearance of fragmented DNA in PC12 cells treated with TNFα. These results were consistent with those observed in the experiment depicted in Fig. 2(A).

image

Figure 3. Cell death as assessed by ELISA detecting nucleosome-associated low-molecular-weight DNA in PC12 cells deprived of serum for 24 h and treated with 100 ng mL−1 rat TNFα (T) and 1000 ng mL−1 NGF (N) in the presence of absence of 100 nm K252a. N= 4 per group. Statistical significance was assessed by anova followed by the Fischer's LSD test.

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To confirm the role of TrkA and/or p75NTR in the rescue by NGF of PC12 cells from TNFα-promoted cell death, we used a mutant NGF (Δ9–13) that has been shown to bind p75NTR but not TrkA (Hughes et al., 2001). Figure 4 shows the survival of PC12 cells treated with TNFα and Δ9–13 or NGF, in the presence of K252a. Both Δ9–13 and NGF were able to rescue PC12 cells from TNFα-induced cell death, and in both instances K252a did not affect the ability of either factor to rescue cells.

image

Figure 4. PC12 cell death as assessed by the ratio between LDH release and MTT reduction assay 24 h after treatment with 100 ng mL−1 rat TNFα (hatched and black bars) and 500 ng mL−1 NGF or 500 ng mL−1 D9-13, a mutant NGF that binds to p75NTR but not to TrkA. The experiment was performed either in the absence (open and hatched bars) or the presence (black bars) of the tyrosine kinase inhibitor K252a (100 nm). N= 5–6 per group. Statistical significance was assessed by anova followed by the Fischer's LSD test.

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Furthermore, the presence of a p75NTR antibody (Ab 9651) that blocks the binding of NGF to p75NTR but not to TrkA (Brann et al., 1999) significantly reduced the ability of NGF to rescue PC12 cells from TNFα-promoted cell death (Fig. 5).

image

Figure 5. PC12 cell death as assessed by the release of LDH in the culture medium 24 h after treatment with 100 ng mL−1 of rat TNFα in the presence of 500 ng mL−1 NGF. Cells were additionally treated with a rabbit p75NTR antibody (Ab9651, dilution 1 : 10 000 v:v) that selectively blocks NGF binding to p75NTR. Control cells were treated with an equal volume of pre-immune serum. N= 8 per group. *P < 0.05 (anova followed by the Fischer's LSD test).

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Lastly, NGF rescue from TNFα-induced toxicity was also abolished by the simultaneous presence of either BDNF or NT-4 (Fig. 6), both of which compete with NGF for binding to p75NTR but fail to activate p75NTR-associated signal transduction pathways (Dobrowsky et al., 1994; Taglialatela et al., 1996). From the experiments shown in Figs 2 and 3 we conclude that while the rescue from serum-free cell death induced by 1000 ng mL−1 NGF is abolished by 100 ng mL−1 K252a, the rescue of cells by 1000 ng mL−1 NGF from TNFα-induced cell death is not. In addition, considering the experiments shown in Figs 4–6 we also conclude that NGF binding to p75NTR is sufficient to rescue cells from TNFα-promoted cell death.

image

Figure 6. PC12 cell death as assessed by the release of LDH in the culture medium 24 h after treatment with 100 ng mL−1 of rat TNFα in the presence of 500 ng mL−1 NGF. Cells were additionally treated with 500 ng mL−1 BDNF or 500 ng mL−1 NT-4, NTs that compete with NGF for binding to p75NTR but do not induce p75NTR-associated signalling events. N= 4–8 per group. *P < 0.05 (anova followed by the Fischer's LSD test).

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NGF-promoted neurite outgrowth is independent of NGF rescue from TNFα-induced cell death

Figure 7 shows phase-contrast photomicrographs of serum-cultured PC12 cells treated for 24 h with 1000 ng mL−1 TNFα in the presence or absence of 1000 ng mL−1 NGF and 100 nm K252a. Panel A shows untreated control cells. When the cells were treated with TNFα there was a significant cell death, and many cell bodies appeared shrunken and pyknotic (panel B). Both panels C and D show PC12 cells treated with TNFα in the presence of 1000 ng mL−1 NGF. NGF induced abundant neurite outgrowth and most of the cells showed morphological signs of differentiation. Panel E shows cells treated with both TNFα and NGF in the presence of 100 nm K252, while panel F shows PC12 cells treated with K252a alone. K252a completely abolished the neurite outgrowth and cell differentiation induced by 1000 ng mL−1 NGF but did not affect NGF rescue as assessed by cell morphology (panel E) and cell survival quantification (Fig. 2A).

image

Figure 7. Representative phase contrast photomicrographs of PC12 cells treated for 24 h with 1000 ng mL−1 human TNFα and 1000 ng mL−1 NGF in the presence or absence of 100 nm K252a. A: control cells; B: TNFα; C & D: TNFα plus NGF; E: TNFα plus NGF plus K252a; F: K252a alone. Magnification 200×.

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These results illustrate that the high NGF concentrations needed to rescue from TNFα-induced cell death also stimulate neurite outgrowth in PC12 cells. However, while NGF rescue from TNFα toxicity is not affected by K252a, K252a abolishes GF-promoted neurite outgrowth under similar experimental conditions. This suggests that the NGF rescue from TNFα-induced cell death and the NGF-promoted neurite outgrowth are regulated via distinct pathways that are independent of (in the case of rescue) or dependent on (in the case of neurite outgrowth) TrkA phosphorylation.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

TNFα induced cell death in PC12 cells in a dose-dependent fashion, confirming previously published evidence (Haviv & Stein, 1999). When PC12 cells are cultured in serum-free medium supplemented with NGF, cell survival is provided solely by NGF (Levi et al., 1988; Levi-Montalcini et al., 1996; Taglialatela et al., 1996). Thus, serum-free/NGF-treated PC12 cells represent a useful model to study NT-dependent cell survival. In our experiments, TNFα decreased survival of NGF-treated serum-free PC12 cells, suggesting that TNFα affected the signalling pathways associated with NGF-mediated trophic support to cells. TNFα also induced cell death in serum-cultured PC12 cells, and the presence of TNFα exacerbated the degree of cell death that PC12 normally undergo upon serum starvation. Thus, our observations suggest that TNFα is capable of inducing an independent cell death pathway in PC12 cells, as well as affecting NGF support of cells. The idea that TNFα may suppress survival signalling delivered by NGF in PC12 cells is consistent with a similar effect proposed for TNFα on IGF-promoted neuronal survival (Venters et al., 2000). In addition, that TNFα may selectively affect NGF-dependent neurones in vivo has been recently reported (Barker et al., 2001).

We found that high, but not low, concentrations of NGF were able to rescue PC12 cells from TNFα-induced cell death, suggesting an effect mediated by binding to the p75NTR low-affinity receptor. In addition, we found that NGF-mediated rescue of PC12 cells from TNFα-induced cell death could not be abolished by K252a. This suggests that TrkA phosphorylation and the subsequent activation of its associated signalling pathways may not play a crucial role in mediating NGF rescue of PC12 cells from TNFα. The TrkA-independent rescue of PC12 cells from TNFα-induced cell death can also be achieved using a mutant NGF that binds to p75NTR but not to TrkA (Hughes et al., 2001). Finally, we show that competing NGF off the p75NTR using a p75NTR blocking antibody (Brann et al., 1999) or neurotrophins such as BDNF or NT-4 that bind to p75NTR but do not induce p75NTR-mediated signalling events (Carter et al., 1996; Casaccia-Bonnefil et al., 1996) significantly reduces the ability of NGF to rescue PC12 cells from TNFα-promoted cell death. On the basis of the above results, it is therefore reasonable to speculate that p75NTR alone may be sufficient to mediate NGF-induced rescue of PC12 cells from TNFα-induced cell death. Thus, this would suggest that NGF acting through p75NTR is required to affect those signalling pathways proactively induced by TNFα in promoting PC12 cell death.

On the other hand, we were able to confirm that K252a completely abolished the NGF-mediated rescue of serum-free PC12 cells, consistent with other reports (Ohmichi et al., 1992; Taglialatela et al., 1996). Thus, NGF signalling through TrkA is neurotrophic in that it is necessary and sufficient to replace the trophic signalling lost after growth factor withdrawal. We also demonstrated extensive growth of neurites in PC12 cells treated with high doses of NGF and TNFα. The presence of K252a, while not affecting the ability of NGF to rescue PC12 cells from TNFα-induced toxicity, completely blocked the NGF-induced neurite outgrowth. These results indicate that the pathway promoting NGF rescue from TNFα toxicity is distinct from the pathway promoting NGF-induced neurite outgrowth and rescue from growth factor withdrawal. This is consistent with reports showing that inhibition of TrkA-activated signal pathways involving the MAP kinase signal cascade completely abolished NGF-induced neurite outgrowth and neuronal differentiation (Pang et al., 1995), further illustrating the idea that TrkA is essential to mediate the tropic and trophic effects of NGF.

The common NT receptor p75NTR and the TNFα receptor TNFR-I share sequence similarity in their respective intracellular domains, suggesting that both receptors may induce similar signal transduction pathways. In fact, it has been reported that both p75NTR and TNFR-I induce intracellular ceramide by stimulating sphingomyelin hydrolysis via activation of SMase, promote JNK activity, induce cPLA2, and stimulate activation and nuclear translocation of NFκB (Wallach et al., 1996; Orlinick & Chao, 1998; Warzocha & Salles, 1998). While induction of NFκB activity has been associated with the maintenance of cell survival induced by NGF and TNFα (Barger et al., 1995; Liu et al., 1996; Mattson et al., 1997; Taglialatela et al., 1997, 1998; Mattson et al., 2000; Mattson & Camandola, 2001), activation of cPLA2 and JNK seems to associate with NGF- and TNFα-induced cell death (Guo et al., 1998; MacEwan, 1996; Wissing et al., 1997; Yoon et al., 1998). Thus it appears that both p75NTR and TNFR-I can initiate multiple signal pathways in response to NGF or TNFα, respectively, likely in a cell type-dependent fashion.

Thus, we propose that in PC12 cells, p75NTR and TNFR-I can activate common signalling pathways leading to either cell death or cell survival. The idea that NGF acting via p75NTR can promote distinct pathways in PC12 cells leading either to cell death or cell survival is consistent with recent evidence suggesting that, in stressed but not unstressed PCNA cells, NGF acting through p75NTR promotes survival via activation of NFκB (Cosgaya & Shooter, 2001). In addition, NGF acting through p75NTR may promote survival via recruitment of RIP2 to the cytopasmic domain of p75NTR (Khursigara et al., 2001). Thus, it is reasonable to propose that under conditions of stress (e.g. TNFα exposure that results in PC12 cell death) p75NTR may convey a survival signal as reported here, possibly by recruiting selective cytoplasmic accessory signalling proteins.

In conclusion, our results suggest that there may be specific receptor requirements for NGF-promoted rescue of PC12 cells. Such requirements are dependent on the nature of the apoptotic stimulus applied: TrkA is required to replace the lack of trophic support resulting from growth factor withdrawal; in contrast, p75NTR is necessary when the apoptotic stimulus is pro-active, such as following stimulation of the TNFα-promoted signalling cascade, regardless of the presence of appropriate trophic stimuli such as serum or TrkA-binding concentrations of NGF. These results are consistent with the existence of distinct signalling pathways mediating apoptosis promoted by growth factor withdrawal (passive) or TNFα (active). Preliminary evidence (N. J. Macdonald et al., unpublished data) suggests that both pathways are upstream of, and converge on, caspase-3. The exact signalling events involved are currently being investigated. Understanding such mechanisms is important to determine the molecular events involved in age-associated neurodegeneration where pro-active toxicants such as TNFα and passive toxic events such as lack of trophic factor support have been reported, often coexisting to contribute to neuronal impairments and associated cognitive deficits.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Materials Human and rat tumour necrosis factor alpha (TNFα) were from R & D (Minneapolis, MN, USA); human TNFα was used at concentrations up to 1000 ng mL−1, whereas the same effects in PC12 cells may be observed using rat TNFα at concentrations up to 100 ng mL−1. The polyclonal antibody against type I TNFα receptor (TNFR-I) was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); Mouse α(2.5S)NGF purified from male mouse submaxillary gland was a kind gift from Dr J. R. Perez-Polo of the University of Texas Medical Branch at Galveston, TX, USA. The mouse D9-13 NGF mutant was prepared in the baculovirus sf21 insect cell system and purified to near homogeneity by ion exchange and immunoaffinity chromatography as described (Woo et al., 1995). All chemicals were either molecular biology or analytical grade.

Cell cultures Rat pheochromocytoma PC12 cells were a kind gift of Dr L. A. Greene of Columbia University, New York, NY, USA. Stock cultures of PC12 cells were maintained in 125-cm2 tissue culture flasks in 10 mL of RPMI-1640 culture medium supplemented with 5% (v:v) heat-inactivated horse serum, 5% heat-inactivated fetal calf serum and 1% antibiotic mixture PNS (Penicillin, Neomycin, Streptomycin) in a humidified cell incubator at 37 °C under a 5% CO2 atmosphere. For serum-free experiments, cells were plated in complete medium and allowed to attach to the plastic substrate overnight prior to changing the medium to fresh RPMI 1640 without serum.

DNA fragmentation assay by enzyme-linked immune-sorbant assay (ELISA) The presence of nucleosome-associated low-molecular-weight fragmented DNA in PC12 cells was assayed by a specific two-site ELISA employing antihistone as primary antibody and anti-DNA as secondary antibody according to the manufacturer's instructions (Boehringer Mannheim, Terre Haute, CA, USA).

DNA fragmentation assay by agarose gel electrophoresis Cells were lysed in 0.5 mL of lysis buffer (0.5% Triton X-100; 5 mm Tris buffer, pH 7.4; 20 mm EDTA) and, after centrifugation for 20 min at 1200 r.p.m., the low-molecular-weight DNA extracted by a standard buffered phenol/chloroform extraction protocol and resuspended in 1 mm Tris-EDTA buffer, pH 8.0. Equal amounts of nucleic acids from each sample (as assayed by spectrophotometric extinction at 260 nm wavelength) were treated with 20 µg mL−1 DNase-free RNase and the DNA was then labelled with 32P-dATP using DNA polymerase I. After labelling, samples were subjected to electrophoresis onto a 1.5% agarose submerged slab gel. After electrophoresis, the gel was vacuum-dried and exposed to autoradiographic film for 48–72 h to visualize the DNA.

MTT reduction cell viability assay and LDH release For cell survival/cell death assays, experiments were performed in poly d-lysine-coated 96-well plates seeded at a density of 104 cells well−1 100 µL−1. At the end of the experiment, 10 µL of the dye MTT (3,(4,5-dimethylthiazol-2-yl-)diphenyltetrazolium bromide, 5 mg mL−1) was added to each well and the plates incubated for 3 h at 37 °C. One hundred microlitres of lysis buffer (20% SDS in 50% N,N-dimethylformamide, containing 0.5% (v:v) 80% acetic acid and 0.4% (v:v) 1 n (HCl) was then added to each well and the colour intensity (proportional to the number of live cells) assessed with an ELISA plate reader at 570 nm wavelength. Lactate dehydrogenase (LDH) release was assayed in the cultured medium using a commercially available kit (Boehringer Mannheim) according to the manufacturer's instructions. MTT reduction assays and LDH release assays were performed, whenever possible, on the same cell culture and the final evaluation of the extent of cell death was assessed by the ratio LDH/MTT so as to normalize the values for possible differences in cell density.

Western blot analysis Nuclear and cytosolic extracts (20 µg of protein) or total cell protein extracts (20–40 µg of protein) were diluted in 1× SDS loading buffer (250 mm Tris-HCL pH 6.0, 8%[v:v] SDS; 162 mm DTT; 0.05%[wt:vol] Bromophenol blue; 40%[v:v] glycerol) and loaded onto a 10% SDS-polyacrylamide denaturing gel. After electrophoresis, gels were blotted onto nitrocellulose membranes by electrophoretic transfer. Filters were then incubated with the appropriate antibody and immunoreactive bands detected by a chemiluminescent Western blot detection kit (Amersham) according to the manufacturer's instruction.

Statistical analysis Statistical differences among groups were assessed by analysis of variance (anova) followed by the Fischer's LSD test for multiple comparisons. Student's t-test was applied where appropriate.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

This work was supported by NIH/NIA grant AG14165, by a Research Development Grant from the Sealy Endowed Fund for Biomedical Research and by an Advanced Research Program Grant of the Texas Higher Education Council Board to G.T.; the expression, purification, characterization and experiments reported here involving the NGF mutant Δ9–13 were supported by NIH grant NS24380 to K.N. We wish to thank Sang B. Woo and Debbie Messineo-Jones for their technical support and Ms. Bonnie Walters for clerical help.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
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