Multiple Pathways of Apoptosis in PC12 Cells
CrmA INHIBITS APOPTOSIS INDUCED BY beta -AMYLOID*

Kathryn J. IvinsDagger §, Jonathan K. Ivins, Jason P. SharpDagger , and Carl W. CotmanDagger

From the Dagger  Institute for Brain Aging and Dementia and the  Department of Developmental and Cell Biology, University of California at Irvine, Irvine, California 92697

    ABSTRACT
Top
Abstract
Introduction
References

Stable transfectants of PC12 cells expressing bcl-2 or crmA were generated and tested for their susceptibility to various apoptotic insults. Bcl-2 expression conferred resistance to apoptosis induced by staurosporine and by oxidative insults including hydrogen peroxide and peroxynitrite, but was less effective in inhibition of activation-induced programmed cell death induced by concanavalin A. Concanavalin A-induced apoptosis was abated, however, in cells expressing very high levels of bcl-2. In contrast, cells expressing crmA were protected from concanavalin A-induced apoptosis, but were as susceptible as control cells to apoptosis induced by staurosporine and oxidative insults. Therefore, at least two apoptotic pathways in PC12 cells can be discerned by their differential sensitivity to blockade by bcl-2 and crmA. The ability of beta -amyloid (Abeta ) to induce apoptosis in these cells was assessed. CrmA transfectants were protected from apoptosis induced by Abeta 1-42, but only cells expressing very high levels of bcl-2 were similarly protected. These results suggest that the apoptotic pathway activated by Abeta 1-42 in PC12 cells can be differentiated from the apoptotic pathway activated by oxidative insults. Gene transfer experiments also demonstrated that expression of crmA in primary cultures of hippocampal neurons is protective against cell death induced by Abeta 1-42. Together these results support the hypothesis that Abeta -induced apoptosis occurs through activation-induced programmed cell death.

    INTRODUCTION
Top
Abstract
Introduction
References

Alzheimer's disease (AD)1 is characterized by a pronounced loss of neurons in susceptible regions of the brain (1). Evidence suggests that this neuronal loss occurs through apoptosis (2, 3), a type of cell death with distinct morphological and biochemical characteristics. The principle component of senile plaques in AD brain is beta -amyloid (Abeta ), a 39-43-amino acid peptide derived from amyloid precursor protein. Abeta has been shown to be neurotoxic in vivo (4) and in vitro (5-8), and is generally believed to contribute to the etiology of AD. Understanding how Abeta induces neuronal apoptosis, therefore, may be important for clinical interventions in AD.

Abeta must aggregate into a beta -pleated sheet structure to induce the death of cultured hippocampal neurons (6), and Abeta is not toxic when immobilized as a neuronal substrate (9). These findings have led to the suggestion that aggregated Abeta might cross-link transmembrane plasma membrane receptors to initiate a death program, in a type of activation-induced programmed cell death (APCD) (10). Studies of Abeta effects on cells have documented changes in tyrosine phosphorylation of cellular substrates (11, 12), suggesting that activation of signal transduction pathways might also be involved. Because Abeta causes oxidative stress in neurons, Abeta has been proposed to cause death through an oxidative mechanism (13), and antioxidants have sometimes (13) but not always (14) been reported to block Abeta -induced cell death. Other studies have suggested that Abeta perturbs calcium homeostasis in neurons to cause cell death (15).

Recent studies have demonstrated that multiple pathways of apoptosis can be discerned by their differential sensitivity to blockade. In one model of APCD, Fas ligand binds to its receptor, causing receptor aggregation, recruitment of death domain- and death effector domain-containing proteins, and activation of a cascade of caspase proteases (16). APCD induced by Fas can be blocked by the viral serpin crmA (17), a caspase inhibitor whose physiological target is likely to be the apical caspase in the cascade (18), but it is insensitive to blockade by bcl-2 (Refs. 19-21, but also see Refs. 22-24). In contrast, apoptosis induced by the protein kinase inhibitor staurosporine (STS) is not inhibited by crmA (25), but is very efficiently blocked by bcl-2 (26). Other insults initiate apoptosis which is similarly sensitive to blockade by bcl-2 but insensitive to crmA, including etoposide, a DNA topoisomerase inhibitor (27).

In order to differentiate pathways used by apoptotic insults, stable transfectant PC12 cell lines expressing crmA or bcl-2 were generated. The results shown here demonstrate that APCD and non-APCD pathways of apoptosis differentially sensitive to inhibition by crmA and bcl-2 exist in PC12 cells. APCD induced by concanavalin A (ConA) is blocked by crmA, while apoptosis elicited by STS and oxidative insults is blocked by bcl-2. Importantly, Abeta -induced cell death is blocked by crmA, suggesting that Abeta may cross-link cell surface receptors to engage an APCD apoptotic pathway.

    EXPERIMENTAL PROCEDURES

Vectors-- The complete coding sequence of human bcl-2 (provided by D. Hockenbery; Fred Hutchinson Cancer Research Center, Seattle, WA) (28) was subcloned into the pCDNA3 expression vector (Invitrogen) which uses the cytomegalovirus promoter to direct high levels of transgene expression in many types of cells. Similarly, a cDNA encoding the viral serpin crmA (provided by D. J. Pickup, Duke University) (29) was subcloned into pCDNA3. An IRES (internal ribosome entry site)-lacZ reporter sequence (30) was subcloned into the pHSVpuc amplicon (generously provided by F. Lim, Universidad Autonoma de Madrid). CrmA or bcl-2 cDNAs were subcloned upstream of the IRES-lacZ sequence to generate amplicons directing expression of bicistronic mRNAs.

PC12 Cell Culture-- The PC6 subline of PC12 cells obtained from R. N. Pittman (University of Pennsylvania) (31) was grown in DMEM containing 10% horse serum, 5% fetal calf serum, and penicillin/streptomycin in 5% CO2. Cells were transfected with bcl-2/pcDNA3, crmA/pcDNA3, or pcDNA3 (the vector control) using LipofectAMINE (Life Technologies, Inc.) and stable transfectants were selected with 0.5 mg/ml G418. Several independent clones were isolated, recloned, and selected for use.

Drugs-- For assays using PC12 cells, a 1 mM stock solution of STS (Calbiochem) was made in dimethyl sulfoxide. ConA (Sigma) was dissolved in Opti-MEM. Hydrogen peroxide (37%, Sigma) was diluted in Opti-MEM immediately before use. Peroxynitrite (Upstate Biotechnology) was used at 100 µM. A 1.32 mM stock solution of FeSO4 was made in Opti-MEM. Abeta 1-42 and Abeta 25-35, synthesized and purified as described previously (6), were obtained from C. Glabe (University of California, Irvine). A stock solution of Abeta 1-42 was made in Opti-MEM and used after one freeze-thaw cycle. Abeta 25-35 was dissolved in ddH2O at 2.5 mM and used after several freeze-thaw cycles. zVAD-fmk (Bachem) was dissolved in dimethyl sulfoxide at 100 mM. Glutathione ethyl ester (GSH) and propyl gallate (Sigma) were dissolved in Opti-MEM. For assays using primary cultures of hippocampal neurons, drugs were prepared similarly, with the exceptions that Opti-MEM was replaced by DMEM-B27 medium, and Abeta 1-42 was dissolved in ddH2O and used after 1 week at 4 °C.

PC12 Cell Toxicity Assays-- Cells were passaged at 1e4 cells/cm2 into 48-well tissue culture dishes coated with 50 µg/ml poly-D-lysine and 20 µg/ml type 1 collagen (Sigma). The following day, cells were rinsed and the medium was replaced with Opti-MEM (Life Technologies, Inc.) supplemented with 2 mM CaCl2. After 4 h, drugs were added, and 24 to 28 h later cell viability was assessed by nuclear morphology. Nuclei in live cells or cells previously fixed with 4% paraformaldehyde in phosphate-buffered saline were stained by the addition of 0.5 µM SYTO11 (Molecular Probes), and cells were visualized with phase-contrast and epifluorescence microscopy. For each condition, live/dead cell counts were obtained from 2-3 fields of 3-4 wells. Cells with large nuclei containing uniformly stained chromatin were counted as live cells, while cells containing fragmented nuclei and/or condensed chromatin were counted as dead cells. None of the drugs used caused changes in the total cell number (live + dead) over the course of the assay. Therefore, the number of live treated cells, expressed as the percentage of the number of live vehicle-treated cells, was used as a measurement of cell viability. Data shown are from representative experiments. Each experiment was repeated several times with similar results. In some experiments the ability of cells to oxidize 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used as a measurement of cell viability (32). Similar results were obtained regardless of the method used to assess cell viability when STS and ConA were used to induce cell death.

Western Blots-- Cells were rinsed with Opti-MEM and total cell lysates were prepared by scraping cells into 1% SDS, 10 mM Tris, pH 7.5, 1 mM EDTA. Protein was estimated by the BCA assay (Pierce), and 1-15 µg of total cellular protein was separated by 12% SDS-polyacrylamide gel electrophoresis and blotted onto Immobilon-P polyvinylidene difluoride (Invitrogen). Blots were blocked with 3% goat serum, 2% bovine serum albumin in TBS containing 0.1% Tween 20 and incubated with rabbit anti-bcl-2 (Santa Cruz 492 at 1:1000) in block. Antibody was detected using horseradish peroxidase-conjugated secondary antibody and ECL reagent (Amersham). Films were scanned and analyzed using NIH Image.

Primary Cultures of Hippocampal Neurons-- Hippocampi of E18 rat embryos were dissected in CMF (calcium- and magnesium-free Hanks'-balanced salt solution, containing 20 mM HEPES, 1 mM pyruvate, 4.2 mM sodium bicarbonate, and 0.3% bovine serum albumin), rinsed with CMF, and resuspended in a trypsin solution (0.125% trypsin in CMF containing 0.5 mM EDTA) for 8 min at 37 °C. The trypsinization was stopped by the addition of DMEM containing 10% fetal calf serum, and the tissue was centrifuged at 200 × g for 5 min. The resulting cell pellet was resuspended in 2 ml of culture medium (DMEM, Life Technologies, Inc., 12100-046, containing 20 mM HEPES, 26.2 mM sodium bicarbonate, 1 mM sodium pyruvate, and B27 supplement, Life Technologies, Inc.). Following trituration through fire-polished Pasteur pipettes with the diameter maximally 50% constricted, the cell suspension was filtered through a 40-µm cell strainer (Falcon), and viable cells were counted using trypan blue. Cells were plated at 5-8e4 cells/cm2 in 48-well tissue culture dishes (Costar).

Transduction of Neurons with Herpes Simplex Virus-1 Amplicons and Toxicity Assays-- Amplicons were packaged using replication incompetent 5dl1.2 helper virus (obtained from P. A. Schaffer, University of Pennsylvania) and 2-2 cells (provided by R. Sandri-Goldin, University of California, Irvine) and purified through sucrose gradient centrifugation as described previously (33). Viral vectors were titered on PC12 cells as described (33). Hippocampal neurons were infected at a multiplicity of infection of 0.1-0.01 with crmA-IRES-lacZ, bcl2-IRES-lacZ, or IRES-lacZ vectors after 2 days in culture. Cells were treated with drugs the next day and fixed 24 h later by underlay with 4% paraformaldehyde in phosphate-buffered saline containing 5% sucrose for 30 min. Fixed cultures were blocked in TBS containing 0.3% Tween 30, 3% goat serum, and 2% bovine serum albumin and then incubated with 40-1a (1:4000 ascites) anti-beta -galactosidase (developed by Joshua Sanes and obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa Department of Biological Sciences under contract NO1-HD-7-3263 from the National Institute of Child Health and Human Development, National Institutes of Health). beta -Galactosidase immunoreactivity was detected by incubation with 7 µg/ml cy2-antimouse IgG (Jackson Immunoresearch Laboratories). Nuclear morphology was assayed by inclusion of 1.25 µg/ml Hoechst 33258 (Sigma) in the secondary antibody incubation. Cells were visualized by epifluorescence microscopy. Noninfected cells and cells infected with 5dl1.2 helper virus were uniformly negative for beta -galactosidase immunoreactivity. Cells transduced by amplicons, identified by the presence of beta -galactosidase immunoreactivity, were counted and their nuclear morphology (normal or apoptotic) recorded. Results are presented as the number of infected cells with normal nuclei after drug treatment as a percentage of the number of infected cells with normal nuclei after vehicle control treatment.

    RESULTS

In preliminary experiments to establish dose-response curves for cell death, PC12 cells were exposed to increasing concentrations of STS, ConA, Abeta 1-42, Abeta 25-35, or hydrogen peroxide in Opti-MEM, a low protein-containing chemically defined medium. Cell viability was assessed after 24 h by nuclear morphology visualized with the fluorescent dye SYTO11, since this method allows clear visualization of apoptotic nuclei (Fig. 1). Over 95% of control cells were viable after serum withdrawal into Opti-MEM, but exposure to these insults caused dose-dependent apoptotic death of the cells, evidenced by somal shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear fragmentation. Drug concentrations that caused 50-90% of the cells to die over the course of 24 h were chosen for use in subsequent assays. Preincubation with the cell permeable caspase inhibitor zVAD-fmk completely blocked cell death induced by these concentrations of ConA, STS, Abeta 1-42, and Abeta 25-35, and partially blocked cell death induced by this concentration of hydrogen peroxide, consistent with the involvement of apoptotic pathways in the observed cell death (Fig. 2).


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Fig. 1.   Changes in nuclear morphology produced by apoptotic insults in PC12 cells. Nuclear morphology in fixed cultures of PC12 cells was visualized by epifluorescence of the chromatin dye SYTO11. The nuclei of untreated PC12 cells are large and chromatin is diffusely stained. Cell death produced by exposure to ConA (100 nM), STS (250 nM), hydrogen peroxide (150 µM), Abeta 1-42 (50 µM), or Abeta 25-35 (25 µM) is associated with chromatin condensation and nuclear fragmentation. Arrowheads indicate healthy nuclei; arrows indicate condensed or fragmented nuclei.


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Fig. 2.   Inhibition of cell death by zVAD-fmk. Cell viability was assessed in PC12 cells pretreated for 2 h with 100 µM zVAD-fmk or vehicle then exposed to ConA (125 nM), STS (250 nM), hydrogen peroxide (150 µM), Abeta 1-42 (50 µM), or Abeta 25-35 (25 µM). Data shown are means ± S.E. *, p < 0.01 versus control; +, p < 0.01 versus vehicle; 2-way ANOVA with Tukey HSD post-hoc tests.

To differentiate pathways of apoptosis in PC12 cells, cells were transfected with an expression vector encoding bcl-2 or an empty vector control, and stable transfectants that expressed various levels of bcl-2 were selected (Fig. 3A). These cells lines were tested for their susceptibility to 2 prototypical apoptotic insults: STS and ConA. Cell lines that produced moderate levels of bcl-2, bcl-2#11 and bcl-2#12, were better protected from STS-induced apoptosis than from ConA-induced apoptosis (Fig. 3B). Cell lines expressing approximately 6-fold higher levels of bcl-2 (bcl-2#3 and bcl-2#10), however, were protected from apoptosis induced by both STS and ConA.


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Fig. 3.   Effect of bcl-2 expression on cell viability. A, Western analysis of bcl-2 levels in the transfected cell lines. B, cell viability was assessed in bcl-2 transfectants and vector control transfectants exposed to 125 nM ConA or 250 nM STS. Data shown are means ± S.E. *, p < 0.01 versus survival after STS exposure in this cell line; +, p < 0.01 versus values obtained with this drug in control cell lines; 2-way ANOVA with Tukey HSD posthoc tests.

An expression vector encoding crmA was used to transfect PC12 cells, and stable transfectants were selected and screened for their susceptibility to apoptotic insults. All of the crmA-transfected cell lines were protected from ConA-induced apoptosis (not shown). Two lines, crmA#3 and crmA#5, that showed the highest level of protection from ConA were used in subsequent assays. Neither crmA#3 nor crmA#5 cells were protected from STS-induced apoptosis (Fig. 4). Together, these results suggested that at least 2 pathways of apoptosis exist in PC12 cells. One pathway, activated by STS, is preferentially blocked by bcl-2 and is not blocked by crmA. The second pathway, activated by ConA, is blocked by crmA but is less efficiently blocked by bcl-2.


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Fig. 4.   Comparison of the ability of crmA and bcl-2 to prevent cell death. Cell viability was determined in crmA and bcl-2 transfected PC12 cell lines exposed to ConA (100 nM) or STS (250 nM). Data shown are mean ± S.E. *, p < 0.01 versus survival after ConA exposure in this cell line and versus survival after STS exposure in control cells; +, p < 0.01 versus survival after STS exposure in this cell line and versus survival after ConA exposure in vector controls; 2-way ANOVA with Tukey HSD posthoc tests.

To determine whether the apoptotic pathway activated by Abeta is sensitive to inhibition by bcl-2 or crmA, transfectants were exposed to 50 µM Abeta 1-42. Cell viability was assessed by nuclear morphology, because Abeta causes rapid decreases in the MTT assay that are not associated with cell death (Refs. 24 and 25, and data not shown). Approximately 50% of control cells were apoptotic after exposure to Abeta 1-42. Significantly, crmA#3 and crmA#5 cell lines were protected from this cell death, while bcl-2#11 and bcl-2#12 cell lines expressing moderate levels of bcl-2 were as susceptible to Abeta 1-42 as control cells (Fig. 5A). Bcl-2#3 and bcl-2#10 cell lines expressing high levels of bcl-2 were also protected from Abeta 1-42 induced apoptosis. Because the Abeta 25-35 fragment of Abeta 1-42 is toxic and has frequently been used in studies of the mechanisms of Abeta induced cell death (34), we wanted to determine whether crmA blocked cell death induced by Abeta 25-35. Surprisingly, although the bcl-2#3 and bcl-2#10 cell lines expressing high levels of bcl-2 were protected from cell death induced by Abeta 25-35, crmA transfectants were not protected from Abeta 25-35 toxicity (Fig. 5B).


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Fig. 5.   Susceptibility of crmA and bcl-2 transfectants to Abeta toxicity. Cell viability was assessed in PC12 cell lines exposed to 50 µM Abeta 1-42 (A) or 25 µM Abeta 25-35 (B). Data shown are mean ± S.E. *, p < 0.01 versus values obtained in control cell lines; +, p < 0.05 versus values obtained in control cell lines; 1-way ANOVA with Tukey HSD tests.

Cell death induced by Abeta has been associated with oxidative damage to cells (35-37), yet under at least some assay conditions antioxidants protect against hydrogen peroxide- and iron-induced cell death, but do not protect against Abeta -induced cell death (14). This suggests that the mechanism of Abeta toxicity is less dependent on oxidative damage. To determine whether bcl-2 and crmA transfectants were protected from cell death induced by oxidative stress, cell survival was assessed after exposure to hydrogen peroxide (Fig. 6A). As expected, cells expressing bcl-2 were protected from cell death induced by this oxidative insult, but crmA transfectants were not protected from cell death. Moreover, in other experiments cell lines expressing moderate levels of bcl-2 were observed to be protected from cell death induced by the oxidative insults peroxynitrite and FeSO4, while crmA transfectants were not protected from death. Cell viability after exposure to 100 µM peroxynitrite was 95.4 ± 2.8% in bcl-2 transfectants, 42.1 ± 1.5% in crmA transfectants, and 47.6 ± 0.8% in control transfectants (mean ± S.D., n = 2, p < 0.01 survival of bcl-2 transfectants versus control transfectants), while cell viability after exposure to 120 µM FeSO4 was 94.5 ± 3.9% in bcl-2 transfectants, 46.3 ± 3.3% in crmA transfectants, and 49.5 ± 4.8% in control transfectants (mean ± S.D., n = 2, p < 0.01 survival of bcl-2 transfectants versus control transfectants). These data show that bcl-2 levels sufficient to block apoptotic pathway(s) activated by oxidative insults do not block the apoptotic pathway activated by Abeta 1-42, while crmA does block the apoptotic pathway activated by Abeta 1-42, but does not block apoptotic pathway(s) activated by oxidative insults. Therefore, these results strongly suggest that oxidative stress does not mediate the initial activation of an apoptotic program by Abeta 1-42. Accordingly, antioxidants including GSH (Fig. 6B) and propyl gallate (data not shown) did not block cell death induced by Abeta 1-42 in these cells.


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Fig. 6.   Ability of bcl-2 and antioxidants to block cell death induced by oxidative insults. A, cell viability was assessed in crmA and bcl-2 transfected PC12 cell lines exposed to 150 µM hydrogen peroxide. Data shown are mean ± S.E. *, p < 0.01 versus survival after hydrogen peroxide in vector control lines and in crmA transfectants; 1-way ANOVA with Tukey HSD posthoc tests. B, cell viability was assessed in PC12 cells pretreated with GSH for 2 h then exposed to ConA (125 nM), STS (250 nM), hydrogen peroxide (100 or 150 µM), Abeta 1-42 (50 µM), or Abeta 25-35 (25 µM). Data shown are mean ± S.E. *, p < 0.05 versus control; **, p < 0.01 versus control; +, p < 0.05 versus vehicle; ++, p < 0.01 versus vehicle; 2-way ANOVA with Tukey HSD posthoc tests.

Taken together, these results indicate that multiple apoptotic pathways can be discerned in PC12 cells, and that the apoptotic pathway activated by Abeta 1-42 is susceptible to inhibition by crmA. However, although PC12 cells are often used to model aspects of neuronal physiology, there is always the possibility that cell death might be differentially regulated in immortalized "neuronal" cell lines and neurons. Therefore, in order to confirm that crmA blocks the apoptotic pathway activated by Abeta 1-42 in neurons, primary cultures of hippocampal neurons were transduced with crmA using a herpes simplex virus amplicon containing an IRES-lacZ reporter. Infected cells were identified by their expression of beta -galactosidase and their viability determined by nuclear morphology (Fig. 7). Cells infected with the control IRES-lacZ amplicon and uninfected cells were similarly susceptible to apoptosis induced by ConA, STS, Abeta 1-42, and Abeta 25-35 (Fig. 8). However, as predicted, neurons transduced with crmA were protected from cell death induced by ConA and Abeta 1-42, but were not protected from cell death induced by STS or Abeta 25-35 (Fig. 8). In other experiments, hippocampal neurons transduced with bcl-2 were protected from cell death induced by each of these insults (data not shown). Because the IE 4/5 promoter used in the amplicon directs a high level of gene expression in neurons (38), these results appear consistent with the results obtained in PC12 cells expressing high levels of bcl-2.


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Fig. 7.   Viral-mediated expression of crmA by cultured hippocampal neurons. Hippocampal neurons infected with IRES-lacZ (A-D) or crmA-IRES-lacZ (E-H) herpes simplex virus vectors were exposed to vehicle (A, B, E, and F) or 100 nM ConA (C, D, G, and H) for 24 h. A, C, E, and G, beta -galactosidase immunoreactivity was used to identify infected cells. B, D, F, and H, nuclear morphology of these cells was visualized using Hoechst 33258. Arrows indicate infected cells with normal nuclear morphology; arrowheads indicate infected cells with apoptotic nuclear morphology.


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Fig. 8.   Effect of crmA expression on the susceptibility of hippocampal neurons to apoptotic insults. Uninfected primary cultures of hippocampal neurons and cultures infected with crmA-IRES-lacZ or IRES-lacZ herpes simplex virus vectors were exposed to ConA (100 nM), STS (250 nM), Abeta 25-35 (50 µM), or Abeta 1-42 (25 µM) for 24 h. Cell viability was assessed by nuclear morphology. Data shown are mean ± S.E. (n = 4). +, p < 0.05 versus uninfected; ++, p < 0.01 versus uninfected; **, p < 0.01 versus lacZ.


    DISCUSSION

The accumulation of plaques containing Abeta is an invariant feature of AD pathology, and there is abundant evidence suggesting that Abeta contributes to the etiology of AD. Most significantly, chromosome 21-linked familial AD is caused by amyloid precursor protein alleles with mutations in or near the Abeta coding sequence (39), and transgenic mice expressing these amyloid precursor protein alleles exhibit pathological changes and memory deficits reminiscent of AD (40, 41). Furthermore, AD is characterized by a profound loss of neurons in susceptible regions of the brain (1), and Abeta causes the apoptotic death of cultured neurons (5, 7, 8). Therefore, understanding the mechanisms by which Abeta induces apoptosis may be relevant to discovery of clinical interventions to delay or alleviate AD.

We have previously suggested that cell death induced by Abeta may represent a type of APCD (10, 42). Neurons have been shown to be susceptible to APCD: transforming growth factor beta  causes the apoptotic death of cerebellar granule neurons (43), and ConA, a lectin which binds mannose and cross-links glycoproteins on the cell surface, causes the apoptotic death of cortical neurons (42). Other insults including uv radiation (44) and growth factor withdrawal (45, 46) may also cause apoptosis through APCD. However, apoptosis has also been demonstrated in neurons exposed to oxidative stress (47), etoposide (48), heat shock (49), and decreased extracellular K+ (50), indicating that non-APCD apoptotic pathways also exist in neurons. Alternate pathways of apoptosis can be discerned by their differential sensitivity to blockade by bcl-2, crmA, and various caspase inhibitors (20, 51). Anti-apoptotic members of the bcl-2 family of proteins are very effective in blocking apoptosis induced by oxidative stress (30, 52) and STS (26), while the viral serpin and caspase inhibitor crmA effectively inhibits Fas- and TNF-mediated apoptosis (17). Both bcl-2 and crmA, however, block apoptosis prior to activation of caspase-3 (25, 53). Therefore, although multiple pathways are involved in the induction of apoptosis, these pathways converge on the activation of caspases which are the effectors of apoptosis.

The results presented here demonstrate that in PC12 cells, ConA and STS activate separate apoptotic pathways that are differentially blocked by crmA and bcl-2. Because the physiological target of crmA is likely to be caspase-8, the apical caspase activated by Fas and TNFR (18), the ability of crmA to inhibit ConA-induced apoptosis in PC12 cells suggests the possibility that ConA might cross-link death domain containing receptors of the Fas/TNFR family to initiate a death program. In addition to inhibiting Fas- and TNF-initiated apoptosis, crmA inhibits anoikis (54), apoptosis induced by NGF withdrawal from sympathetic neurons (55), and apoptosis induced by serum withdrawal from PC12 cells.2 These insults may also cause death through inappropriate activation of cell surface receptors. For example, death produced by NGF withdrawal has been proposed to be mediated by the low affinity NGF receptor (p75 NGFR), a member of the fas/TNF superfamily of receptors that has been reported to cause apoptosis in the absence of NGF (45).

Significantly, expression of crmA inhibited apoptosis induced by Abeta 1-42. The ability of crmA to block Abeta 1-42-induced cell death supports the suggestion that Abeta 1-42 stimulates APCD by binding to and cross-linking transmembrane receptor(s) (10). Interestingly, expression of crmA did not inhibit apoptosis induced by Abeta 25-35. Because Abeta 25-35 often appears more potent than Abeta 1-42 and causes more rapid cell death (56), it is possible that levels of crmA were insufficient to block Abeta 25-35 toxicity. Alternatively, the mechanisms of Abeta 25-35 toxicity in PC12 cells may differ from the mechanisms of Abeta 1-42 toxicity. Because previous studies have also suggested the possibility that Abeta 25-35 and Abeta 1-42 may have distinct mechanisms of toxicity (56, 57), caution should be used in interpreting the results of investigations which use only the non-physiological Abeta 25-35 to study Abeta toxicity.

In a previous study, overexpression of bcl-2 did not inhibit Abeta 25-35-induced death of PC12 cells or of human neuroblastoma IMR-5 cells (58). Cell death was assessed by the MTT assay, however, which has been shown to be an inaccurate indicator of cell death induced by Abeta (59, 60). The results shown here demonstrate that the ability of bcl-2 to inhibit Abeta -induced death of PC12 cells is dependent on its level of expression: relatively high levels of bcl-2 expression are required to block apoptosis induced by Abeta 25-35 or Abeta 1-42. Interestingly, moderate levels of bcl-2 sufficient to inhibit apoptosis induced by oxidative insults in PC12 cells were not able to inhibit apoptosis induced by Abeta 1-42. Moreover, and in agreement with the results we have obtained in primary cultures of hippocampal neurons (14), antioxidants did not block apoptosis induced by Abeta 1-42 or Abeta 25-35. In contrast, a recent study demonstrated that Abeta 25-35 toxicity in PC12 cells was blocked by antioxidants (37). As previously discussed (14), discrepancies in the effectiveness of antioxidants in blocking Abeta toxicity may be related to methodological differences between experiments. In the experiments described here, cells were exposed to insults in Opti-MEM, a medium which supports the growth of the cells for at least several days. These cells might be healthier and more resistant to oxidative damage than cells assayed after serum and NGF withdrawal into RPMI (37), since PC12 cells deprived of trophic support in this manner undergo apoptosis within 2 to 3 days (61, 62).

There has been some ambiguity in the literature as to whether bcl-2 and related anti-apoptotic members of this family of proteins are able to block Fas- or TNF-induced apoptosis (19-24). The results here demonstrate that PC12 cells expressing moderate levels of bcl-2 were preferentially protected against apoptosis induced by oxidative insults and STS, but cells expressing higher levels of bcl-2 were also completely protected against apoptosis induced by ConA. These results suggest that the level of bcl-2 expression may be critical to its ability to suppress APCD, and that the ability of bcl-2 to block Fas- or TNF-induced apoptosis is likely to require high levels of expression.

It is not known whether apoptotic pathways in neurons are identical to those demonstrated here in PC12 cells. However, because apoptotic execution may involve inappropriate activity of proteins involved in the regulation of the cell cycle (63, 64), it has been suggested that differentiated and cycling cells use similar apoptotic programs, and what varies is whether new protein synthesis is required or whether the necessary proteins are available because they are constitutively made in cycling cells (63). In fact, our results demonstrate that crmA protects hippocampal neurons as well as PC12 cells from death induced by ConA and Abeta 1-42, but does not block cell death induced by STS or Abeta 25-35 in either type of cell culture. Therefore, our results suggest that central nervous system neurons and PC12 cells use similar apoptotic pathways, and that Abeta 1-42 causes APCD in neurons as well as in PC12 cells.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant RO1-AG13007.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 949-824-6071; Fax: 949-824-2071; E-mail: kjivins{at}uci.edu.

The abbreviations used are: AD, Alzheimer's disease; Abeta , beta -amyloid; APCD, activation-induced programmed cell death; STS, staurosporine; ConA, concanavalin A; IRES, internal ribosome entry site; GSH, glutathione ethyl ester; DMEM, Dulbecco's modified Eagle's medium; NGF, nerve growth factor; TNF, tumor necrosis factor; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

2 K. J. Ivins, unpublished observations.

    REFERENCES
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Abstract
Introduction
References

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