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Keywords:

  • Alzheimer's disease;
  • amyloid β-protein;
  • amyloid β-protein precursor;
  • apoptosis;
  • caspase;
  • C31

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

The amyloid β-protein precursor (APP) is proteolytically cleaved to generate the amyloid β-protein (Aβ), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Aβ in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Aβ induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Aβ. Interestingly, both Aβ toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Aβ toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Aβ-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Aβ cytotoxicity, the leading mechanism proposed for neuronal death in AD.

Abbreviations used

amyloid β-protein

AD

Alzheimer's disease

APP

amyloid β-protein precursor

DMEM

Dulbecco's modified Eagle medium

DMSO

dimethyl sulfoxide

FBS

fetal bovine serum

GFP

green fluorescent protein

Prominent histopathological hallmarks of Alzheimer's disease (AD) are the extracellular deposition of amyloid plaques, accumulation of intracellular neurofibrillary tangles, and loss of synapses. Although the temporal order and relationship of these events to each other are not clear, evidence suggests that amyloid β-peptide (Aβ), the principal protein component of amyloid plaques, plays a role in neuronal toxicity.

In the cell culture setting, the toxicity of Aβ has been characterized as either apoptotic (Loo et al. 1993; Gschwind and Huber 1995; Estus et al. 1997; reviewed in Mattson and Duan 1999; Ivins et al. 1999) or necrotic (Behl et al. 1994; Gschwind and Huber 1995; Suzuki 1997). Thus, Aβ is likely to trigger toxicity via multiple mechanisms, including but not limited to oxidative stress, tau phosphorylation, changes in signal transduction, and by activation of caspases, a family of cysteine proteases which execute the apoptotic pathway (Ii et al. 1996; Ferreira et al. 1997; reviewed in Ivins et al. 1999; Nakagawa et al. 2000; Troy et al. 2000; Allen et al. 2001).

Recently, we and others have shown that APP is a substrate for caspase cleavage (Barnes et al. 1998; Gervais et al. 1999; LeBlanc et al. 1999; Pellegrini et al. 1999; Weidemann et al. 1999; Lu et al. 2000). Our results also demonstrated that following cleavage, a cytotoxic C-terminal APP peptide of 31 amino acids residues in length unrelated to Aβ, designed as C31, is generated (Lu et al. 2000). Furthermore, there was evidence that this cleavage event and caspase activation occur in brains of AD but not in control individuals (Lu et al. 2000). It is unclear, however, when APP cleavage with the release of cytotoxic APP C-terminal fragments might be physiologically activated.

Because the progressive accumulation of Aβ in brain is currently the leading theory of AD pathogenesis, we asked in this study whether there is any relationship between C31-mediated cell death and Aβ-mediated toxicity. This is because C31 should be generated in response to Aβ insult under conditions that induce apoptosis, which, in the process, activate caspases that can cleave the cytoplasmic domain of APP. Furthermore, we sought to elucidate further the mechanism by which C31 increases the susceptibility to cell death in culture cells, using caspase activation assays and taking advantage of certain specific antiapoptosis reagents. Our results confirmed that Aβ-induced caspase-mediated cleavage of APP in the course of causing cell death. Moreover, we demonstrated that this cleavage event of APP (herein designated as C31 pathway) may contribute significantly to Aβ toxicity under certain conditions. Consistent with this idea, the C31 pathway and Aβ toxicity share similar caspase requirements for cell death. Therefore, our results suggest that Aβ toxicity, caspase cleavage of APP, and the C31 pathway may be closely linked together.

Plasmid construction and mutagenesis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

APP695, APP-D664A, a point mutation of APP695 in which the P1 caspase consensus site at the 664 amino acid is mutated from D to A, APP-ΔC31, a deletion of the last 31 amino acids of APP695, C100, C31, caspases, and catalytic-mutant caspases were generated as previously described (Lu et al. 2000). APP C100 construct consists of the signal peptide sequence of APP fused to the C-terminal 99 amino acid residues beginning at the aspartate residue of Aβ. The mutation of the aspartate residue at codon 664 to alanine (by APP695 numbering) or the deletion of the last 31 amino acids of APP were made by using QuikChange method (Stratagene, La Jolla, CA, USA) to generate C100-D664A and C100-ΔC31 constructs, respectively, similar to the constructs above for APP695. CrmA and bcl-2 were subcloned into pcDNA3.

Antibodies

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

APP antibodies included the following: CT15, polyclonal rabbit antibody recognizing the C-terminal 15 amino acids of APP (Sisodia et al. 1993); a monoclonal antibody 26D6 recognizing the Aβ peptide sequence of amino acids 1–12 (Lu et al. 2000); and a polyclonal antibody α664 directed against amino acids 657–664 of APP (Soriano et al. 2001). The latter antibody is end-specific and only recognizes APP when it has been cleaved after residue 664. Additional monoclonal antibodies include β-tubulin (Sigma-Aldrich, St Louis, MO, USA), HA tag (Roche, Indianapolis, IN, USA), and FLAG M2 (Sigma-Aldrich).

Cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

Rat neuroblastoma B103 cells (kind gift from Dr David Schubert) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), while B103 cells stably expressing APP695 and APP695-D664A were maintained in the same medium and supplemented with 700 µg/mL of G418 (Calbiochem, San Diego, CA, USA). Mouse N2a neuroblastoma cells were cultured in 45% DMEM, 45% OptiMEM I (Life Technologies, Rockville, MD, USA), and 10% fetal bovine serum (FBS). Plasmid constructs were transiently transfected into the various cell lines by LipofectAMINE 2000 transfection reagent for B103 cells or Fugene-6 for N2a cells (Roche Diagnostics). Generation of B103 cells stably transfected with APP, APP-D664A, or APP-ΔC31 have been described previously (Soriano et al. 2001). These cell lines represent pooled stably transfected cells collected without clonal selection and were selected for comparable APP expression by western blotting. Quantitation of western blot was performed by CCD coupled imaging system (Genegnome, Syngene Bioimaging, Frederick, MD, USA). Signals from quantitation were in the linear range, as determined from standards within each experiment.

1−42 peptide (kind gift from Dr Charles Glabe) was dissolved in dimethyl sulfoxide (DMSO) and added immediately without prior aggregation to culture medium of N2a or B103 cells at the desired concentration for 18–24 h for caspase cleavage and cell death studies. A concentration of 50 µm of Aβ in B103 cells and 10 µm in N2a cells was found to be optimal in inducing cell death (> 40%) in the above time frame and was used throughout the study. Control cells not treated with Aβ1−42 peptide were subjected to the same concentration of DMSO.

Assessment of cell death and caspase activity measurement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

After co-transfection with the desired construct and 0.25 µg of green fluorescent protein (GFP) tracer (pEGFP-N1, Clontech, Palo Alto, CA, USA), cell death was assessed at 48 h by Hoechst staining or trypan blue staining as previously described with 25 µm tamoxifen treatment (Lu et al. 2000) for B103 or without tamoxifen for N2a cells. Trypan blue staining is an assessment of membrane permeability, a reflection of cell death. To elucidate the type of cell death, Hoechst staining was used. Cells that were determined to be Hoechst-positive (5 µg/mL) had bright condensed chromatin and deemed early apoptotic as they excluded propidium iodide (0.5 µg/mL), reflective of intact cell membrane (Deshmukh and Johnson 1998). Necrotic cells, on the other hand, excluded Hoechst staining but were propidium iodide-positive. Hoechst-postive and GFP-positive cells were counted as apoptotic against the total number of GFP-positive cells to arrive at the percentage of cell death. There was no difference in transfection efficiencies among the different constructs (e.g. vector, APP, APP-D664A) and cell death among these constructs was not different during the course of transfection. Experiments were performed four to six times in duplicate or triplicate wells. Average of all experiments (± SEM) are presented below. Statistical analysis was performed by one-way anova and post-hoc analysis by Tukey–Kramer.

Caspase-8 and caspase-3 activity were measured by using ApoAlert caspase-8 and caspase-3 from Clontech according to manufacturer's directions. These assays use AC-IETC-AFC (caspase-8) and AC-DEVD (caspase-3) substrates. Caspase activation is presented as ratio between the caspase activity of the sample and that measured in N2a or B103 cells transfected with pcDNA3 empty vector. No sensitizing agent to apoptosis (tamoxifen or staurosporine) was used when assaying for caspase activation in C31-transfected cells.

1−42 peptide induced cell death is associated with caspase cleavage of APP

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

Aβ peptide has been shown to induce apoptosis when given to a variety of cell under cultured conditions (reviewed in Cotman 1998). To demonstrate whether APP can be cleaved at the consensus caspase site in the cytoplasmic domain, we examined N2a neuroblastoma cells transiently transfected with either full-length APP or the C-terminal C100 fragment and treated with Aβ (10 µm). In these cells, Aβ was able to induce caspase cleavage of full-length APP and APP C100 fragment when assayed after 48 h. In both cases, Aβ or co-transfection with caspase-8 resulted in the generation of a C-terminally truncated APP species that was recognized by a N-terminal Aβ antibody (26D6) and an end-specific antibody (α664) that specifically recognized the C-terminus of the APP fragment after caspase cleavage at position 664, but not to the APP C-terminal antibody (CT15; Figs 1a and b). This cleavage event was abolished with the treatment of a pan-caspase inhibitor, zVAD.fmk, or by mutating the caspase cleavage site at codon 664 from D to A (APP D664A).

image

Figure 1. Aβ-induced APP intracytoplasmic caspase cleavage in cultured cells at DEVD/A. (a)  In N2a cells transiently expressing C100, Aβ at 10 µ m induced intracytoplasmic cleavage of this CTF (top panel, lane 4) and is represented by a doublet to 26D6 antibody. The lower band co-migrated with C100ΔC31 and a c-terminal truncated band in cells co-transfected with caspase-8, which is known to induce APP intracytoplasmic cleavage. This lower band is not reactive to CT15 antibody (middle gel, lane 4), but reactive to the cleavage specific α664 antibody (bottom panel, lane 4). This cleavage is absent in the C100 D664A mutant or when the pan-caspase inhibitor, zVAD.fmk, is added. (b)  In N2a cells transiently expressing APP, Aβ at 10 µ m induced intracytoplasmic cleavage of full length APP, as demonstrated by positive reactivity to cleavage specific α664 antibody (lane 4, top panel) In cells expressing APP D664A caspase mutant construct, such cleavage event is not observed as demonstrated by the lack of reactivity to α664 antibody (lane 5, top panel). Additionally, this cleavage event is also inhibited by a pan-caspase inhibitor zVAD.fmk. Comparable level expression of the APP constructs is seen by immunoblotting with 26D6 antibody (bottom panel).

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The above results indicated that Aβ resulted in caspase activation and subsequent cleavage of APP at the consensus caspase cleavage site in the C-terminus. To demonstrate that the Aβ treatment was also associated with cytotoxicity, N2a cells were transfected with APP, APP-D664A, or control vector and treated with Aβ (10 µm) for 48 h (Fig. 2a). In APP-transfected cells, cell death was approximately 40%, almost threefold higher than control cells (14%). In APP-D664A- and APPΔC31-transfected cells, cell death was essentially the same as control cells (17% and 18%, respectively). In cells not exposed to Aβ, there was essentially no cell death for all conditions (approximately 3% for all constructs: APP, APP-D664A, APP-ΔC31, and control). The level of expression of the three APP constructs were comparable after transient transfection (Fig. 2b). Thus, augmenting APP levels by transfection increased cell death substantially after Aβ treatment.

image

Figure 2. Aβ-induced apoptosis in N2a cells. (a)  Transient expression of APP in N2a cells followed by Aβ treatment increased cell death significantly above control (* p  < 0.001; one-way anova , p  < 0.0001; F = 126.44). In contrast, expression of APP-D664A or APPΔC31 showed no increase in cell death as compared to control ( p  > 0.5). (b)  Representative western blotting for caspase cleavage of APP following Aβ treatment shown in (a). As seen before, APP caspase fragment detected by α664 antibody is observed in cells treated with Aβ (lane 2, top panel) and in the control APPΔC31 cells but not in cells expressing the caspase mutant APP-D664A (lane 3). Expression of APP constructs is comparable in these cells (compare lanes 2–4, bottom panel).

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Dose–response of Aβ in B103 cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

The above results indicate that Aβ-induced apoptosis is associated with caspase cleavage of APP. The apparent contribution of APP, specifically the caspase cleavage of APP, to Aβ toxicity was unexpected. To verify that this effect was not cell line-specific, the studies were repeated with B103 rat neuroblastoma cells that do not express APP or APLPs (Ninomiya et al. 1994). Four different stably transfected B103 cell lines were examined: cells expressing APP, APP-D664A mutant, APP-ΔC31, or empty control vector (Fig. 3a). The expression level of the three different transfected APP constructs in B103 cells was comparable to each other (Figs 3b and c). Consistent with the results from N2a cells, there was a significant increase in cell death (45%) in B103-APP cells exposed to Aβ (50 µm) as compared to B103 cells transfected with either empty control vector, the APP caspase-site mutant APP-D664A, or the truncated APP molecule without the last 31 amino acids, APPΔC31 (Fig. 3a). This cell death in B103 cells was apoptotic in nature, as the cells with DNA condensation brightly stained by bis-benzamide excluded propidium iodide (Fig. 3d). As was seen in N2a cells, cell death in B103 cells after Aβ treatment was similarly associated with caspase cleavage of APP, but not with the APP caspase-site mutant APP-D664A (Fig. 3b). Therefore, these findings from B103 cells are consistent with the results obtained from N2a cells and demonstrate that the APP-dependent component of Aβ toxicity is not cell type-specific.

image

Figure 3. Response of B103 cells to Aβ treatment. (a)  Cell death is seen only in Aβ-treated B103 cells stably expressing APP but not in B103 cells expressing control pcDNA3 vector, APP-D664A, or APP-ΔC31 constructs. Cell death in B103-APP-transfected cells in response to Aβ treatment is significantly different from B103 control cells, B103 cells expressing APP-D664A or APP-ΔC31 (* p  < 0.001; one-way anovap  < 0.0001, F = 58.458, post-hoc Tukey–Kramer). (b)  In same above conditions, APP caspase cleavage fragment is present in B103 cells expressing APP as detected by α664 antibody (bottom panel, lane 2). No cleavage is seen in the caspase mutant APP-D664A-expressing cells. Control B103 cells expressing APP C31 is immunoreactive to α664 antibody as this construct is truncated at position 664. As expected, B103 cells lack APP and there is no reactivity to 26D6 APP antibody in control B103 cells (top panel, lane 1). (c)  APP expression in B103 stably transfected cells express approximately fourfold more APP than mixed primary cortical cells as detected by APP antibody (compare lane 1 with lanes 3 and 6). Control B103 cells has no detectable APP expression (lane 2). To confirm equal protein loading, the same lysates are immunoblotted with a tubulin antibody (lower panel). (d)  Photomicrographs of B103 APP-expressing cells exposed to 50 µ m of Aβ. Following Aβ treatment, B103 APP cells are shrunken with neurite retraction as observed by brightfield microscopy (top row, second column) when compared to control (top row, first column). Aβ-induced apoptosis is demonstrated by the lack of staining to propidium iodide (PI) but positive to Hoechst staining (second column, lower two panels). As control, B103 APP-expressing cells not exposed to Aβ are negative to both PI and Hoechst staining (first column, lower two panels). Scale bar represents 100 µm.

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C31 increased susceptibility to cell death in B103 cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

The above findings suggested that Aβ-induced apoptosis is in part attributable to both APP and surprisingly, to C31 generation as an intact caspase cleavage site in the cytoplasmic domain is required. Therefore, we next asked whether susceptibility to C31 toxicity itself also shows this dependence on APP. This would occur if continued generation of C31 by cleavage from APP is necessary for cell death as, for example, observed in huntingtin-associated toxicity (Wellington et al. 1997). Indeed, in native B103 cells, which do not express APP or APLPs, transfection of C31 alone did not induce any measurable cell death as compared to control following low-dose tamoxifen (25 µm) treatment (Fig. 4a). However, susceptibility to tamoxifen toxicity was markedly increased when C31 was transfected with APP (∼80%), much higher than the transfection with APP alone (∼55%). This amplifying effect was abolished when the caspase-mutant APP-D664A was substituted for APP, suggesting that the effect requires an intact caspase cleavage site and by inference, caspase cleavage.

image

Figure 4. Increased susceptibility to C31 toxicity in B103 cells. (a)  Transient expression of APP or co-expression of APP and C31 in B103 cells causes significant cell death as compared to control, C31, D664A or D664A + C31 cells ( p  < 0.001; one way anova , p  < 0.0001, F = 87.25, post-hoc Tukey–Kramer). Co-expression of APP and C31 increase cell death approximately 20% (* p  < 0.001) more than APP expression alone. (b)  APP potentiates susceptibility to C31-induced cell death in B103 cells. Increasing concentrations (1, 5, 10, 20 µg) of full-length APP constructs or vector control were co-transfected with a constant amount of C31 cDNA (2 µg) in tamoxifen-treated (25 µ m ) B103 cells. Increasing amount of APP increase cell death to 68% at 20 µg of APP as compared to 22% at 1 µg of APP ( p  < 0.001), while increasing concentrations of APP-D664A, APP-ΔC31, or control vector does not change C31 toxicity.

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To further test the hypothesis that the susceptibility to cell death induced by C31 is dependent on APP, a dose–response study was performed. In B103 cells transiently transfected with a constant level of C31-expressing plasmid (2 µg), increasing amounts of co-transfected APP (1, 5, 10, 20 µg) were associated with increasing cell death after low-dose tamoxifen treatment (Fig. 4b). This effect was absent when the cells were co-transfected with either APP-D664A or APP-ΔC31. Thus, there is an element of APP dependency in both the susceptibility to cell death seen by C31 expression and Aβ-induced apoptosis that was unexpected.

Caspase activation by Aβ and C31

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

The above results suggested that C31 and Aβ toxicity share certain common features or may induce cytotoxicity via a common pathway. To obtain more evidence to support this concept of APP-dependent toxicity induced by both C31 and Aβ via cleavage of APP by caspases, we first examined activation of specific caspases after C31 expression or Aβ treatment.

In our previous study, we demonstrated that APP cleavage appeared to require caspase-8 and caspase-9. This suggests that C31 and Aβ toxicity may similarly require activity of the upstream caspases-8 and -9. Aβ-induced caspase activation has been extensively studied. In these studies, caspase-3 has been found to be dispensable in Aβ-mediated cell death (LeBlanc et al. 1999; Troy et al. 2000), while caspase-8 (Ivins et al. 1999; Su et al. 2003) and caspase-9 (Kim et al. 2002; Zhang et al. 2002) are required. Thus, if C31- and Aβ-induced apoptosis were to share a common cell death pathway, then we predict C31 should have a similar caspase activation or requirement profile as Aβ. For these reasons, we focused the remaining studies on caspase-3, -8, and -9.

We initially assessed the caspase activation profile of Aβ- and C31-induced apoptosis by using caspase fluorescent substrate assays. In N2a cells or B103 cells stably transfected with APP, expression of C31 showed minimal activation of caspase-3 despite substantial cell death (Fig. 5a). In Aβ-treated N2a or B103 cells, caspase-3 activation was approximately threefold over control. In contrast, tamoxifen given as a positive control (250 µm) induced nine- to 10-fold increase in caspase-3 activity over untreated N2a and B103 cells. Using a similar fluorescence-based assay, both C31 transfection and Aβ treatment in N2a cells and B103 cells stably expressing APP-induced substantial caspase-8 activation (Fig. 5b), comparable to staurosporine (100 µm) treatment given as a positive control.

image

Figure 5. Caspase activation of C31 in N2a and B103 cells. (a)  Capase-3 activation in N2a and B103 cells after C31 transfection or Aβ treatment assayed by ApoAlert detection. As positive control, cells expressing control vector were induced with tamoxifen to undergo cell death and assayed for caspase-3 activity. A significant increase in caspase-3 activity is seen in tamoxifen-treated N2a cells (□) and B103 cells (▪) as compared to C31-transfected or Aβ-treated cells ( p  < 0.05 for both conditions and cell lines). Data represent caspase activation after C31 transfection or Aβ treatment that corresponded to 70% cell death by bis-benzamide staining. Data are expressed as relative caspase activities to N2a or B103 cells expressing pcDNA3 vector and are representative of one of four experiments. (b)  Caspase-8 activation N2a and B103 cells after C31 transfection or Aβ treatment assayed by ApoAlert detection. Caspase-8 activity is approximately four- and fivefold increased over basal untreated condition after Aβ treatment and C31 transfection in N2a (□) and B103 cells (▪), respectively. Cells treated with staurosporine as positive control show about fivefold increase in caspase-8 activity. Data represent caspase activation after C31 transfection of Aβ treatment that corresponded to 40% cell death by bis-benzamide staining. Data are expressed as relative caspase activities to N2a or B103 cells expressing pcDNA3 vector and are representative of one of four experiments.

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Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

In the preceding experiment, Aβ treatment and C31 expression both led to little to modest activation of caspase-3 but robust activation of caspase-8. While a useful tool, assays of caspase activation do not necessarily implicate a requirement of the particular caspase in apoptosis. To assess which caspases are required for Aβ- and C31-associated cell death, we took two different approaches. First, if C31 acts to specifically activate certain caspases to affect cell death, we predict that dominant negative mutants of these caspases, enzymatically inactivated by mutating the catalytic cysteine residue to an alanine, would suppress C31-mediated toxicity (Pan et al. 1998; Lu et al. 2000). When transiently expressed in N2a cells, these dominant negative catalytic mutants potently inhibited staurosporine-induced cell death in control experiments (Fig. 6a). When N2a cells were transiently co-transfected with C31 and caspase-3 catalytic mutant, there was no reduction in cell death as compared to vector control (75% vs. 72%; Fig. 6b). Neither was there an effect of caspase-3 catalytic mutant on Aβ toxicity (Fig. 6c). In contrast, catalytic mutants of caspase-8 and -9 both reduced C31 cell death significantly, although the effect of caspase-8 mutant was only partial (∼45%; Fig. 6b). The effect of caspase-8 and -9 catalytic mutants on Aβ toxicity was more dramatic, reducing cell death to almost control levels (15.6% and 10.7% vs. 9.6%, respectively; Fig. 6c).

image

Figure 6. Expression of caspase catalytic mutants and inhibitors of apoptosis constructs attenuate C31 and Aβ toxicity in N2a cells. (a)  Expression of catalytic mutant caspases inhibited staurosporine (stauro.)-induced cell death in N2a cells by bis-benzamide staining. Catalytic mutant caspase-3, -8, and -9 all inhibited staurosporine-induced cell death and is significantly different from control staurosporine treated cells ( p  < 0.001 for all three comparisons; by one-way anovap  < 0.0001, F = 83.57). (b)  Co-expression of C31 with either bcl-2 or CrmA in N2a cells reduced C31 toxicity to control levels. However, co-expression of C31 with catalytic mutant caspase-3 has no effect in inhibiting C31 toxicity (control vs. C31 + CM3, p  < 0.001). Co-expression of C31 with catalytic mutant caspase-8 and -9 partially inhibited C31 toxicity (C31 vs. C31 + CM8 and C31 + CM9, p  < 0.001 for both comparisons). Control represents N2a cells transfected with control vector pcDNA3. (c)  Expression of catalytic mutant caspase-8 and -9 but not catalytic mutant caspase-3 inhibited Aβ toxicity to control levels (control vs. Aβ + CM8 or Aβ + CM9, p  < 0.001; by one-way anovap  < 0.0001, F = 95.35). Expression of bcl-2 or CrmA partially inhibited Aβ toxicity as compared to control cells (Aβ vs. Aβ + CrmA and Aβ vs. Aβ + bcl-2, p  < 0.001). Control cells represent native N2a cells not exposed to Aβ but treated with the equivalent amount of DMSO.

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Second, expression of bcl-2 and CrmA, which are well documented inhibitors of caspase-9 and caspase-8 pathway, respectively (Zhou et al. 1997; Adams and Cory 1998) should similarly impair C31 toxicity. Indeed, the results were even more striking as co-expression of bcl-2 or CrmA in N2a cells completely inhibited C31 toxicity to control levels (14.5% or 13.4% vs. 16% cell death, respectively; Fig. 6b). Similarly, expression of bcl-2 or CrmA also reduced Aβ induced toxicity to near control levels (Fig. 6c). Taken together, these results demonstrated that caspases-8 and -9 are required to activate the cell death pathway initiated by either Aβ or C31 in N2a cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

An invariant feature of Alzheimer's disease pathology is the accumulation of Aβ in senile plaques in cerebral cortex. However, whether Aβ is neurotoxic in a fibrillar form as present in senile plaques or in an unaggregated oligomeric state remains controversial. In brains of AD patients, we and others have detected evidence of a cleavage event in APP within the C-terminus, unrelated to Aβ, that appeared to be mediated by caspases. Following cleavage, a C-terminal APP polypeptide with cytotoxic properties, termed C31, is generated (Lu et al. 2000; McPhie et al. 2001; Galvan et al. 2002; Nishimura et al. 2002). The relationship between Aβ toxicity and C31 generation is unclear and may be pathologically relevant. In this study, we were able to link C31 with Aβ toxicity by demonstrating that Aβ-induced caspase cleavage of APP at position 664 in the C-terminus with release of C31. We found a surprising dependency of both C31- and Aβ-induced apoptosis on the presence of APP. In particular, the APP-dependent event requires an intact caspase site in the APP C-terminus. Loss of caspase cleavage at this site decreased both Aβ and C31 toxicity. Finally, we found that both Aβ and C31 toxicity was in part mediated by caspase-8 and -9 but not caspase-3, suggesting that the initiation of caspase cleavage of APP and subsequent generation of C31 contribute to Aβ toxicity.

Based on the activation assays, a number of studies have implicated caspases-2, -3, -6, -8, -9, and -12 in Aβ toxicity (Barnes et al. 1998; Gervais et al. 1999; Harada and Sugimoto 1999; Ivins et al. 1999; LeBlanc et al. 1999; Nakagawa et al. 2000; Selznick et al. 2000; Troy et al. 2000; Allen et al. 2001). It is possible that some of the caspases are activated as part of the overall cell death program following Aβ treatment but were not an absolute requirement for cytotoxicity. In this study, we utilized specific antagonists to ascertain which caspases were required for Aβ toxicity as well as assaying which caspases were activated upon exposure to Aβ. By caspase activation assay, both Aβ- and C31-induced caspase-8 activity, while caspase-3 was not strongly activated. Consistent with this finding, by expressing various dominant negative caspase mutants and either bcl-2 or CrmA, which have been reported to attenuate Aβ toxicity (Bruce-Keller et al. 1998; Ivins et al. 1998), we found that caspases-8 and -9 were required for both Aβ and C31 toxicity, while caspase-3 was not. This requirement for caspase-8 and caspase-9 in C31-mediated toxicity is consistent with our previous data that APP cleavage at position 664 could be induced by these apical caspases (Lu et al. 2000). In this study, we did not ascertain the precise downstream caspases that are required for C31 toxicity. Our results suggested that caspase-3 is not required for either C31- or Aβ-induced cytotoxicity, a finding that is consistent with a recent report that examined C31 toxicity and Aβ formation (Dumanchin-Njock et al. 2001). It is likely that other caspases such as caspase-2 (Troy et al. 2000) as well as caspases-6 and -7 participate in this pathway as dominant negative catalytic mutants of these two caspases were able to attenuate C31-mediated toxicity (data not shown). Taken together, we hypothesize that in certain culture conditions, both Aβ- and C31-induced cytotoxicity may share a common pathway which requires caspase cleavage of APP, thereby indicating a strong link between Aβ toxicity and the caspase cleavage of APP and C31 pathway.

Interestingly, the caspase requirements for C31 and Aβ toxicity we observed in cultured cells have parallels in brain. For example, in several studies, caspase-3 activation was relatively minor in the brains of AD patients and did not appear to account for the widespread neuronal loss that occurs in AD (Chan et al. 1999; Stadelmann et al. 1999; Selznick et al. 2000). However, both caspases-6 and -9 have been shown to be activated in AD but not in control brains (LeBlanc et al. 1999, 2000; Lu et al. 2000). Thus, there are in vivo observations that are consistent with our cell culture findings.

In primary neurons derived from transgenic mice deficient in APP, it was recently proposed that APP contributes to Aβ toxicity (Lorenzo et al. 2000) and in another study, Aβ possessing the E22Q Dutch type mutation was found to bind to APP to effect cell death (Melchor and Van Nostrand 2000). Our results are entirely consistent with these observations. At a dose of Aβ (5–10 µm), an increase in toxicity of almost threefold was observed in APP-transfected as compared to untransfected N2a cells. This effect was considerably more than that described in the APP-deficient neurons (Lorenzo 2000) and may be due to the higher level of APP expression in our transfected system as compared to endogenous APP. More interesting, however, was the finding that expression of a caspase-resistant form of APP (APP-D664A) demonstrated reduced Aβ toxicity. One possibility suggested by these findings is that upon further cleavage of APP, more C31 is generated, hence greater cell death. However, additional APP C-terminal peptides can also be generated. Specifically, after γ-secretase cleavage of APP in the transmembrane domain, the APP intracellular domain (AID or AICD) is generated. It has been reported that these AID/AICD peptides or fragments are also cytotoxic (Passer et al. 2000; Bertrand et al. 2001). Lastly, AID/AICD have also been found to translocate into the nucleus and modulate gene expression by interacting with Fe65 (Cao and Sudhof 2001; Baek et al. 2002). Therefore, it is conceivable that C31, following cleavage from APP, induces cell death through a nuclear signaling mechanism (Kinoshita et al. 2002). Testing these interesting and varied hypotheses of C31 toxicity will have to await further investigations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References

We thank Dr Charles Glabe for generous gift of Aβ peptide, Dr David Teplow and Dr Guy Salvesen for helpful discussions, Dr David Schubert for kind gift of B103 cells, Kathy Shin and Rana Shayya for technical assistance. This work was supported in part by NIH grants AG05131 (EHK and DEB) and NS28121 (EHK). DCL is an MD/PhD candidate in the Medical Scientist Training Program at the University of California, San Diego (NIH GM07198) and is the recipient of a grant from the Paul and Daisy Soros Fellowships for New Americans; the program is not responsible for the views expressed.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid construction and mutagenesis
  5. Antibodies
  6. Cell culture
  7. Assessment of cell death and caspase activity measurement
  8. Results
  9. 1−42 peptide induced cell death is associated with caspase cleavage of APP
  10. Dose–response of Aβ in B103 cells
  11. C31 increased susceptibility to cell death in B103 cells
  12. Caspase activation by Aβ and C31
  13. Inhibition of Aβ and C31-mediated toxicity by catalytic-mutant caspases
  14. Discussion
  15. Acknowledgements
  16. References
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