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J. Biol. Chem., Vol. 278, Issue 46, 46074-46080, November 14, 2003
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-Amyloid Generation Independently of Caspase Cleavage of the -Amyloid Precursor Protein (APP)*
From the Genetics and Aging Research Unit, Center for Aging, Genetics and Neurodegeneration, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachussetts 02129
Received for publication, July 18, 2003 , and in revised form, September 2, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased A production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted A and A1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to A generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase A generation but, in contrast, dramatically decreased A production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced A production following etoposide-induced apoptosis. These findings indicate that the enhanced A generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amyloid deposition, intracellular neurofibrillary tangles, and extensive loss of neurons. Amyloid plaques are formed by aggregates of amyloid--peptide, a 37-43 amino acid fragment (primarily A40 and A42) generated by proteolytic processing of the amyloid precursor protein (APP). APP is cleaved sequentially by -secretase and 
-secretase to release A constitutively from neuronal and non-neuronal cells. APP proteolysis by - and 
-secretases results in the production of - and -APP secreted fragments (APPs) as well as the C99 and C83 APP-C-terminal fragments (APP-CTFs), respectively. The C99 and C83 APP-CTFs then serve as substrates for -secretase, resulting in the production of A or p3, respectively (1). In addition to the normal APP processing, "alternative" processing of APP by caspases has also been reported. Apoptosis is a form of cell death triggered by the activation of an intrinsic cellular program deliberately invoked by the cell in response to environmental and or developmentally associated signals (2). The death machinery can be activated by diverse stimuli and has as its central component a group of proteolytic enzymes called caspases. Caspases exhibit primary specificity for aspartic acid residues (3). To date, more then 280 proteins have been shown to be substrates of one or more caspases in mammalian cells. Among them are proteins involved in cell death, cell cycle regulation, cytoskeleton organization, DNA and RNA metabolism, signal transduction, cytokine maturation, and gene transcription (4).
More recently, it has been shown that APP is also a substrate for caspase cleavage. Each of the four caspases, 3, 6, 7, and 8, have been shown to cleave APP in in vitro assays, and a major caspase site has been identified at Asp-720 (VEVD), resulting in the release of a fragment containing the last 31 amino-acids of APP (C31) and the production of APP
C31 (lacking Ala721 to Asn751) (5-8). Two other major caspase sites have been recently identified at the N-terminal domain of APP Asp197 and Asp219, leading to the production of a 
90 kDa fragment (APPN). APP caspase-dependent cleavage at Asp720 has been shown to occur in cultured cells stimulated to undergo apoptosis, in vivo in brains of human AD subjects, and following the induction of brain ischemic injury in animal models (5). Gervais et al. (5) generated a synthetic mutant of APP with a stop codon at the Asp720 site to mimic the product of apoptotic cleavage. Expression of this mutant in cells yielded 5-fold more A peptide compared with wild-type APP, providing the molecular basis for a positive feedback loop in the AD pathogenic process and raising the possibility that up-regulation of certain caspases precede A peptides secretion (5, 9-11).
In light of these findings, we set out to study the effects of apoptosis on A production and the potential role of caspase-dependent cleavage of APP in Chinese hamster ovary (CHO) cells. Here, we report that staurosporine- and etoposide-induced apoptosis increases levels of both Atotal and A1-42 independently of caspase cleavage of APP at Asp720, Asp219, and Asp197.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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region, was from Signet (Dedham, MA). The anti-CcspAPP antibody was raised against a synthetic peptide corresponding to the noveau C terminus generated by caspase-dependent cleavage at Asp720 of APP (for details, see Ref. 5). The C8 antibody, raised against the last 10 amino acids of APP, and R1155 (amino acids 705-720 of APP) were generous gifts of Dr. Selkoe (12). The 22C11 antibody was raised against amino acids 66-81 of APP (generous gift of Dr. Beyreuther) (13). Mammalian expression clones include the following: WT APP751-(Met1-Asn751):pCEP4; APP751-(D197A, D219A, D720A):pCEP4; and APP751-C-(Met1-Asn720):pCEP4 (for details, see Ref. 5).
Cell Culture, Transient Transfection, Western Blot Analysis, and Induction of Apoptosis—CHO cell lines stably transfected with APP751 and wild-type PS1 were cultured as described previously (14). We used CHO cell lines overexpressing APP to measure secreted Atotal and A42 during apoptosis. Because A42 normally represents only 10% of the Atotal, we expressed APP751 to generate amounts of A42 that could be detected by the ELISA. To induce apoptosis, we used staurosporine (STS) and etoposide (Eto) (Calbiochem), an inhibitor of topoisomerase II. CHO cells were seeded at the density of 0.5 x 106 cells per well in 6-well plates. After 48 h, cells were treated with STS (1 µM) or etoposide (100 µg/ml) for the indicated time (Figs. 1 and 3). To inhibit caspase activation, a sister plate of cells was pre-treated with zVAD (200 µM, Enzyme Systems Products) for 1 h before induction of apoptosis. For transient transfection, CHO naive cells were seeded at the density of 0.5 x 106 cells per well in 6-well plates, and after 24 h the APP cDNA clones were transfected using Superfect reagent (Qiagen) following the manufacturer's instructions. Cells were treated with etoposide 24 h after transfection. Western blot analysis and immunoprecipitation were performed as described previously (15).
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ELISA—Secreted A was measured in the conditioned media of untreated cells or cells treated with STS or etoposide, with or without zVAD, using a sensitive ELISA (courtesy of the Selkoe Laboratory; see Ref. 14 for details). At least three different experiments were carried out, and each experiment was performed at least in triplicate.
Immunofluorescence and Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL) Staining—For immunofluorescence microscopy, cells were grown on glass coverslips for 48 h and then treated with STS or Eto, with or without zVAD. Cells were fixed for 15 min in 4% paraformaldehyde (Tousimis Research Corp.) and then washed three times with PBS. Nonspecific immunostaining was blocked by incubation for 1 h at room temperature in PBS with 4% normal goat serum and 0.2% Triton X-100. The primary antibody anti-CcspAPP or anti-caspase 3 active fragment was incubated for 2 h at room temperature 1:2000 in PBS containing 4% normal goat serum. After three washes with PBS, the cells were incubated with a donkey anti-rabbit-CY3-conjugated antibody (Jackson Laboratories) for 1 h at room temperature 1:400 in PBS containing 4% normal goat serum. After three washes in PBS, nuclear fragmentation was detected by in situ labeling with a TUNEL assay kit (Roche Applied Science) following the manufacturer's instructions. After three washes with PBS, coverslips were mounted with Fluoromount-G (Southern Biotechnology Associates) and examined by laser scanning confocal microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production observed during apoptosis. Thus, we set out to study caspase cleavage of APP in the CHO cell lines stably transfected with both wild-type APP751 and wild-type PS1. We first determined the temporal pattern of caspase-dependent cleavage of APP. Western blot (WB) analysis of lysates from various time points following STS treatment was performed using a battery of anti-APP antibodies. Full-length APP was cleaved to produce a 90 kDa fragment that was immunoreactive for the 6E10 antibody (amino acids 1-17 A region) at time points 12 and 24 h (indicated as APPNcas in Fig. 1A). Treatment with zVAD prevented the formation of this fragment, indicating that its generation required caspase activation (Fig. 1A). When the same blot was reprobed with a C8 antibody raised against the last 10 amino acids of APP, the 90 kDa fragment was only detected at the 12-h time point (Fig. 1A). The lack of detection of the 90 kDa fragment at the 24-h time point indicated that caspase cleavage at Asp720 had occurred, removing the last 31 amino acids of APP.
To further characterize the origin of the 90 kDa caspase-derived fragment, cell lysates from a separate time course experiment were immunoprecipitated with an R1155 antibody, raised against an epitope (amino acids 705-720) immediately N-terminal to the Asp720 caspase cleavage site. As expected, 6E10 detected the 90 kDa fragment at the 9- and 24-h time points (Fig. 1B). When the same blot was reprobed with 22C11, raised against amino acids 66-81 at the N-terminal fragment of APP, the caspase-dependent 90 kDa fragment was not detected (Fig. 1B). Thus, during STS-induced apoptosis, full-length APP undergoes caspase-dependent cleavage at the N terminus, resulting in the production of a 90kDa APPNcas caspase fragment. APP-CTFs (C99 and C83) are normally generated in cells by the cleavage of full-length APP by - and -secretases, respectively and they both serve as substrates for -secretase. Because APP-CTFs contain the caspase site at Asp720, we also studied caspase-dependent cleavage of APP-CTFs. C83 is the predominant APP-CTF detected in CHO cells. Using C8, we detected a slight decrease in the amount of APP-CTF at the 6-h time point; zVAD treatment prevented this decrease (Fig. 1C). More interestingly, when WB was performed using R1155, a caspase-derived fragment of 6.5 kDa was detected in the STS-treated cells at the 12-h time point, consistent with cleavage at Asp720 (APP-CTFC31) (Fig. 1C). At the same time point, the 90kDa fragment derived by caspase-dependent cleavage of full-length APP could still be detected by C8 (Fig. 1A). A nonspecific band just below APP was also present at several time points, including time point 0, but was not detected in either 6E10 or C8 immunoprecipitates at the 6-h time point (data not shown). Cleavage at Asp720 was detected initially (6 h) as a reduction in APP-CTF and by the generation of the 6.5 kDa fragment at the 12-h time point. We could not rule out the possibility that cleavage at Asp720 of full-length APP was occurring at the 6-h time point. A similar decrease in full-length APP, due to cleavage at Asp720, was not detected because the band corresponding to full-length APP was saturated, and APPC31 is likely to co-migrate with full-length APP under these SDS-PAGE conditions. Like others (6), we were not able to detect the C-terminal product of APP caspase cleavage at Asp720 (C31). In the same experiment, the time course for caspase 3 activation was determined using an antibody that specifically recognizes the 17-kDa caspase 3-active fragment. We found that caspase 3 activation coincided with cleavage of APP-CTF at Asp720 at the 6-h time point (Fig. 1D). These data indicate that cleavage of the APP-CTF at Asp720 occurs prior to cleavage of the full-length APP at Asp197 and Asp219, which generates the N terminus of the 90 kDa APP fragment (APPNcas).
Caspase 3 Activation and APP Asp720 Cleavage Are Temporally Coordinated—To show that caspase-dependent cleavage of APP occurs in intact cells, we performed immunofluorescence microscopy analysis using specific antibodies raised against APPC31 (Gervais et al.; Ref. 5) or the active fragment of caspase 3. Caspase 3 activation and APPC31 cleavage was detected in TUNEL positive (apoptotic) cells (Fig. 2A). Time course experiments revealed that caspase 3 activation and APPC31 cleavage (Fig. 2B) are temporally coordinated during staurosporine treatment in TUNEL-positive cells. Furthermore, this method was more sensitive than WB analysis, as we were able to detect caspase 3 activation and APPC31 cleavage as early as the 3-h time point. These results confirmed the WB data showing that the cleavage at Asp720 temporally coincides with caspase 3 activation.
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total and A1-42 Production—We next addressed the question of whether A generation was increased during apoptosis concurrently with APP Asp720 cleavage. Secreted A was measured in the conditioned media of cells treated with STS (1 µM) for 6 h using a sensitive ELISA (14). Both Atotal and A1-42 were significantly increased in STS-treated cells (p < 0.05; paired t test). When cells were pretreated with zVAD for 1 h before STS, Atotal and A1-42 were reduced compared with STS-treated cells; but the decrease was significant (p < 0.05; paired t test) only for Atotal (Fig. 3A). It has been suggested that A1-40 and A1-42 are generated in different cell compartments and that A1-42 is mainly generated in the ER (16, 17). Furthermore, peptide aldehyde -secretase inhibitors differentially block production of secreted A1-40 relative to secreted A1-42 and, at some doses, can stimulate production of A1-42 while inhibiting A1-40 generation (18-21). More recently, Wilson et al. (22) reported that presenilins are nor required for A1-42 production in the endoplasmic reticulum/intermediate compartment, raising the possibility that A1-42 might be generated by the activity of a different -secretase. Similarly, one could hypothesize that the increase in A1-42 may involve the activation of different caspases, which may have different affinities for the same inhibitor and/or be localized in distinct organelles.
Whereas 6 h of STS treatment clearly induced apoptosis (based on caspase 3 activation and TUNEL-positive cells) (Fig. 2), we next confirmed that the effect on A generation was directly due to apoptosis and was independent of the drug treatment. For this purpose, apoptosis was induced with etoposide, a topoisomerase II inhibitor. Cells were treated for 12 h with 100 µg/ml etoposide. Treatment with either STS for 6 h or etoposide for 12 h reduced cell viability by 10-20% as assayed by MTT reduction (data not shown). Both secreted Atotal and A1-42 were significantly increased (p < 0.05; paired t test) (Fig. 3B). Caspase cleavage of APP at Asp720 was detected by immunofluorescence using the anti-CcspAPP antibody (Fig. 3C). These data indicate that the increase in A and APP cleavage at Asp720 occurs at the same time point and concurrently with caspase 3 activation.
Deletion of the Last 31 Amino Acids of APP Decreases A Production in CHO Cells—We next asked whether APP cleavage at Asp720 was involved in the increased A generation observed during apoptosis. Gervais et al. (5) first proposed that caspase-dependent cleavage at APP Asp720 was responsible for the increased A generation associated with apoptosis, reporting that the expression of APP cDNA deleted at Asp720 (APPC31) results in a 5-fold increase in total A production in rat B103 cells. Thus, we set out to determine whether the expression of APPC31 in CHO cells would increase A generation. Naïve CHO cells were transiently transfected with APPWT, APPC31, or vector alone. Secreted A was measured by ELISA in conditioned media after 24 and 48 h of transfection. CHO cells transfected with APPWT underwent a large increase in total A generation. However, cells overexpressing APPC31 exhibited a dramatic decrease (Fig. 4A) of 90 and 75% at 24 and 48 h, respectively, in A total production as compared with APPWT (Fig. 4B). Western blot analysis of cell lysates from transfected cells showed that the different cDNAs overexpressed comparable amounts of APP (Fig. 4C). We found that removal of the last 31 amino acids of APP did not increase but, instead, decreased A production in CHO cells, indicating that cleavage at Asp720 does not underlie enhanced A production following caspase activation.
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Production during Apoptosis—We and others have shown that caspase inhibitors can reduce the ability of apoptosis to increase A generation. The latter effect could be due simply to the prevention of the cleavage of all caspase substrates, including APP. To determine whether caspase-dependent cleavage of APP is necessary for the increased A generation associated with apoptosis, we expressed APP that was mutagenized at all three major caspase sites (APP Asp720/Asp219/Asp197). CHO cells were transiently transfected with APPWT, APP mutagenized at Asp720, Asp219, and Asp197, or vector alone. 24 h after transfection, apoptosis was induced with etoposide treatment as described above. Secreted total A was measured by ELISA. The amount of total A was comparable in untreated cells indicating, that the APP D720A/D219A/D197A mutations did not affect the production of A under normal conditions (Fig. 5A). As expected, total A was increased in APPWT-treated cells. Cells overexpressing APP D720A/D219A/D197A also showed an increase in total A production following etoposide treatment that was similar to the increase observed with APPWT, i.e. 38 and 34% of untreated cells, respectively (Fig. 5A). The levels of APP expression were comparable for all the different cDNA clones (20 µg of total protein each lane; Fig. 5B). Etoposide treatment induced caspase dependent cleavage of APP at the N-terminal sites in WT but not in the triple mutant as indicated by the detection of APPNcas (WB with 6E10). Caspase cleavage at Asp720 was detected as a decrease in APP-CTF, which was absent in the triple mutant (WB C8). APP-CTFs in the triple mutant migrated faster than WT, most likely because the D720A mutation invokes a change in protein conformation (100 µg total protein each lane; Fig. 5C). The triple caspase site mutations did not affect - and -secretase-mediated APP processing (data not shown). Thus, the lack of caspase cleavage at all three major sites did not alter A generation during apoptosis. These findings argue against the premise that caspase-dependent cleavage of APP at Asp720, Asp197, and Asp219 of APP underlies the potentiation of A production observed during apoptosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production is increased during apoptosis. We further showed that apoptosis increases levels of A1-42 in CHO cells. These latter data are particularly interesting in view of the fact that A42 is more prone to aggregate and has been suggested as being the starting point of plaque formation (23). A42 has been reported as being increased in the brain and cerebrospinal fluid after stroke or head trauma (24). Interestingly, caspase activation has been shown to occur during cerebral ischemia (25) and head injury (26), both of which increase the risk for AD by possibly representing either precipitating or triggering events. However, it remains unclear how cerebral ischemia predisposes to AD. We hypothesize that caspase activation/apoptosis increases production of A42, consequently triggering and/or exacerbating AD pathology. Thus, identification of the mechanism underlying increased A generation associated with caspase activation/apoptosis could facilitate the treatment and prevention of AD.
Gervais et al. (5) first proposed that caspase-dependent cleavage at APP Asp720 was responsible for the increased A generation associated with apoptosis, reporting that the overexpression of APP cDNA deleted at Asp720 (APPC31) results in a 5-fold increase in total A production in rat B103 cells. In contrast, we found that the transfection of APPC31 in CHO cells led to a dramatic decrease in A. A previous study (27) showed that, in CHO cells, deletion of the cytoplasmic domain of APP or mutagenesis of the internalization motif (NPXY) significantly impaired APP endocytosis and A generation. Because caspase cleavage at Asp720 results in the elimination of the APP internalization motif, this could also explain our findings. Our data are also in agreement with a report by Soriano et al. (28) showing that overexpression of APPC31 leads to a decrease in A production in CHO cells. However, these findings contradict results previously reported for rat B103 cells in which A production was increased upon APPC31 overexpression (5). These contradictory results could be explained by possible differential APP processing in CHO and B103 cells. In fact, Soriano et al. (28) reported that constitutive APP internalization in B103 cells is less efficient than in CHO cells. In addition, we have shown that the ablation of caspase-dependent cleavage of APP at all three major caspase sites does not alter A generation during apoptosis. These findings argue against the premise that caspase cleavage at Asp720, Asp197, and Asp219 are implicated in the potentiation of A production observed during apoptosis in CHO cells.
Other caspase sites in APP have been recently identified and require further investigation. For example, a caspase 6-like site (VKMD) has been identified at Asp653 in the -secretase region of APP, and it has been shown that the APP Swedish mutation Lys-Met 
Asn-Leu improves the likelihood that caspase 6 can cleave at this site. LeBlanc et al. (8) provided evidence that the generation of a 6.5-kDa fragment, generated by cleavage at the -secretase region and Asp720, precedes the increased production of both secreted and intracellular A in apoptotic neurons and that pharmacological inhibition of caspase 6 activity prevents the increase in A. However, cleavage at Asp653 would generate A starting at position 2, and we did not observe such an A peptide during apoptosis (Koh et al.).2 Nevertheless, it is still possible that caspase-dependent cleavage at other sites may generate caspase-derived fragments that can serve as alternative substrates for the -secretase, leading to increased A generation during apoptosis. It is also possible that apoptosis/caspase activation may increase A generation independently of caspase-dependent cleavage of APP. For example, caspase activation can increase A production by potentiating - and -secretase activities. The ultimate elucidation of the biological pathways underlying enhanced A production during apoptosis should aid in the development of novel therapies for the treatment and prevention of AD.
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FOOTNOTES |
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To whom correspondence should be addressed: Genetics and Aging Research Unit, Massachusetts General Hospital, Bldg. 114, 16th St. C3009, Charlestown, MA 02129-4404. Tel.: 617-726-6845; Fax: 617-724-1949; Email: tanzi{at}helix.mgh.harvard.edu.
1 The abbreviations used are: AD, Alzheimer's disease; APP, -amyloid precursor protein; APPNcas, APP after caspase-dependent cleavage at the N terminus; A, -amyloid; CHO, Chinese hamster ovary; CTF, C-terminal fragment; ELISA, enzyme-linked immunosorbent assay; Eto, etoposide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PS1, presenilin 1; STS, staurosporine; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; WB, Western blot; WT, wild type; zVAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.
2 Y. H. Koh, R. E. Tanzi, and G. Tesco, manuscript in preparation.
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ACKNOWLEDGMENTS |
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CcspAPP antibody and Drs. Dennis Selkoe and Weiming Xia for providing us with cell lines and anti-APP antibodies and for carrying out the A ELISA. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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