Benjamin Wolozin, with Luciano D'Adamio and Eddie Koo, led this live discussion on 20 September 2000. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Journal of Alzheimer's Disease. 2000; 2(3-4): 289-301.

Brent Passer1*, Luca Pellegrini1*, Claudio Russo2, Richard M. Siegel3, Michael J. Lenardo3, Gennaro Schettini2, Martin Bachmann4, Massimo Tabaton5 & Luciano D'Adamio1,6

View Transcript of Live Discussion — Posted 31 August 2006

1T-cell apoptosis Unit, Laboratory of Cellular and Molecular Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892; 2Section of Pharmacology and Neuroscience, IST, CBA and Dept. of Oncology Univ. of Genova, Genova, Italy; 3Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, 4Cytos Biotechnology AG / ETH Zurich, Wagistrasse 21, CH-8952, Zurich-Schlieren, Switzerland; 5Istituto di Anatomia Umana and Dipartimento di Neuroscienze, Universita' di Genova, via De Toni 10, 16132 Genova, Italy; 6Present address, Albert Einstein College of Medicine, Dept. of Microbiology & Immunology, 1300 Morris Park Avenue, Bronx, N.Y. 10461

*B.P. and L.P. have equally contributed to this work.

Address correspondence to Luciano D'Adamio, Albert Einstein College of Medicine, Dept. of Microbiology & Immunology, 1300 Morris Park Avenue, Bronx, N.Y. 10461

E-mail: ldadamio@aecom.yu.edu

Abstract

The b-amyloid precursor protein (APP) is sequentially processed by β- and γ- secretases to generate the Aβ peptide. The biochemical path leading to Aβ formation has been extensively studied since extracellular aggregates of amyloidogenic forms of Aβ peptide (Aβ42) are considered the culprit of Alzheimer's Disease. Aside from its pathological relevance, the biological role of APP proteolysis is unknown. Although never previously described, cleavage of APP by γ-secretase should release, together with Ab, a COOH-terminal APP intracellular domain, herein termed AID. We have now identified AID-like peptides in brain tissue of normal control and patients with sporadic Alzheimer's disease and demonstrate that AID acts as a positive regulator of apoptosis. Thus, overproduction of AID, may add to the toxic effect of Aβ42 aggregates and further accelerate neurodegeneration.

Alzheimer's disease (AD) is believed to be caused by extracellular deposition of amyloidogenic forms of Aβ peptide (Aβ42) (1, 2). Aβ derives from cleavage of APP by β- and γ-secretase (3) (Fig. 1A, upper panels). This hypothesis of AD pathogenesis, known as "amyloid hypothesis", has found further support by the identification of the three genes linked to familial forms of AD (FAD). The first discovered was APP, the protein from which Aβ is derived (4). The others are presenilin-1 (PS1) and -2 (PS2), two highly homologous proteins that are required for γ-secretase activity and might indeed be the γ-secretase (5, 6, 7, 8, 9). Of more importance, presenilins and APP point mutations found in FAD patients augment APP processing and the formation of amyloidogenic Aβ (1, 2, 10, 11).

Extensive evidence has also supported a role for presenilins and APP in programmed cell death (PCD). A dominant negative PS2 fragment, named ALG-3, was shown to inhibit apoptosis (12). This COOH-terminal PS2 fragment contains the second aspartate residue that is essential for γ-secretase activity (9) and codes for a dominant negative repressor of γ-secretase activity (13). Depletion of PS2 protein levels by antisense RNA has been shown to protect cells against death (14). Conversely, overexpression of presenilins increased apoptosis (14). Moreover, FAD-associated mutations in presenilins and APP enhanced the pro-apoptotic activity of these molecules (14, 15, 16). Lastly, apoptosis induced by APP requires Presenilins (14). Together, these data suggest an alternative model for the pathogenesis of AD. According to this hypothesis, neurodegeneration in AD is facilitated by enhanced susceptibility of neurons to apoptotic stimuli.

The "amyloid" and "apoptotic" theories need not be mutually exclusive. An attractive possibility is that APP processing may generate peptides that regulate PCD. This hypothesis provides a unifying model of these two apparently conflicting theories of Alzheimer's pathogenesis and is supported by the following findings. Conditions that increase the generation of the amyloidogenic form of Aβ, such as those with Alzheimer's mutation in presenilins and APP, also promote cell death. Conversely, circumstances that inhibit apoptosis, such as overexpression of ALG-3, also repress γ-secretase activity (13).

In this paper we show that the COOH-terminal APP intracellular domain, herein termed AID, liberated after cleavage of APP by γ-secretase acts as a positive regulator of apoptosis. Thus, overproduction of AID, as in AD, might cause the neurodegeneration process observed in Alzheimer's patients.

Experimental procedures.

Antibodies, constructs and mutagenesi.

The C7 rabbit polyclonal antiserum, raised to a synthetic polypeptide of APP corresponding to amino acids 751-770, was kindly donated by Dr. Dennis Selkoe. The 6E10 (anti-APP) and C10 (anti-PARP) mouse monoclonal antibodies were purchased from Senetek and Enzyme System (Dublin, CA), respectively. The monoclonal antibody 718-770/Jonas was purchased from Roche Molecular Biochemicals. The mouse monoclonal antibody anti-caspase-8 (Pharmingen, San Diego, CA) and the rabbit polyclonal antiserum against caspase-6 (Upstate Biotecnology, Lake Placid, NY) are both specific for the pro-domains of caspase-8 and 6 and do not recognize the active form. cDNA coding for the various human APP fragments were obtained by PCR and cloned into either pcDNA3 (Invitrogen, San Diego, CA) or pEGF-N1 (Clonethec, Palo Alto, CA). The APP-Sw APPD664N, AID57mut and AID59mut constructs were obtained by site-directed mutagenesis as suggested by the manufacturer's (CLONTECH, Palo Alto, CA). The identity of each construct was confirmed by automatic sequencing using an ABI Prism 377 (Perkin Elmer, Foster City, CA).

Cell lines and transfection procedures

Jurkat T-cells were transfected with a BTX electroporator with 30 mg of DNA and a setting of 1050 mF, 250 V and 72 W. Transfection efficiency was assessed using a GFP vector and ranged between 65 and 80%. One hr after transfection, cells dead because of the electroporation were removed by Ficoll-Paque (Pharmacia, Piscataway, NJ) centrifugation. In some experiments, the compound 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (5(6) (CFDA, SE) (Molecular Probes Inc, Eugene, OR), a green fluorescent dye, was added prior to transfections to mark a population of cells. Sub confluent HeLa or MCF7 cells were transfected with 1 mg of the indicated plasmids with FuGene 6 (Roche Molecular Biochemicals).

Apoptosis Studies

Cell death in Jurkat cells was assessed by either measuring the DNA content of isolated nuclei or by staining cells with annexin V-PE (R&D System, Minneapolis, MN) as indicated by the manufacturer. Samples were analyzed with a FACScan (Becton Dickinson, San Jose, CA). For HeLa cells, 16 hours later Hoechst 33342 was added to stain nuclei and cells were fixed with 3% paraformaldehyde and examined by fluorescence microscopy. Viable and apoptotic cells were enumerated by cell morphology and confirmed by examining nuclear morphology. At least 100 cells in each experimental group were counted in duplicate. Photomicrographs were taken at 630x magnification. When indicated, 50 mM of the irreversible caspase inhibitor ZVAD-fmk (Enzyme System, Livermore, CA) was used to block caspase activity.

Immunoprecipitation, Western blot analysis and caspase activity assays

For immunoprecipitation of metabolically labeled proteins, cells were incubated with 300mCi/ml of 35S-labeled methionine (Amersham, Arlington Heights, IL) for 4 hr. Cells were analyzed by immunoprecipitation as described previously. Western blots of total cell lysates were developed using the SuperSignal system (Pierce, Rockford, IL). For caspase activity assay, cells were lysed in 25 mM HEPES pH 7.5, 50 mM ß-mercaptoethanol, and 0.1% CHAPS and 30 µg of proteins were used in the enzymatic assay in 500 µl of AFC buffer (50 mM HEPES pH 7.5, 1% sucrose, 0.1% CHAPS), 1 mM DTT, and 50 µM ZDEVD-AFC (Enzyme System). All enzymatic reactions were carried out at 25°C for 2 h. Release of the fluorescent group was measured with a Perkin-Elmer LS-5B luminescence spectrometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

MALDI (Matrix-Assisted Laser Desorption Ionization) Mass Spectroscopy (MS)

Human brain samples were homogenized in a TBS buffer containing 1% of Triton X100. After a preclearing step with 40ml/ml of protein G-agarose, the extracted proteins were immunoprecipitated with the monoclonal antibody 643-695 /Jonas (10mg/ml) for 3 hours at 4C. Then 10ml of magnetic beads (Dynal, A.S. Oslo, Norway) covalently coupled with anti-mouse IgG were added to the sample and rocked for 1 hour in the same conditions. The magnetic beads were collected, washed twice with RIPA 1X, twice with ddH2O and suspended in 10 ml of ddH2O. Two ml of this slurry were then incubated briefly with 2 ml of the matrix: a-cyano-4-hydroxycinnamic acid (10mg/ml), (Aldrich Chem.Co. MI) dissolved in HCl 0.05M acetonitrile: isopropanol 5:1.5. One ml of the incubation mixture was placed on the sample plate with 1 ml of the matrix solution, evaporated at room temperature and then analyzed. Similar results were achieved using the 3,5-dimethoxy-4-hydroxycinnamic acid as matrix with or without formic acid in the matrix dilution. The analysis was performed in linear positive mode and a minimum of 100 scans was averaged.

Results and Discussion

To investigate the role of γ-secretase activity and APP processing in PCD, we initially studied cell death induced by Fas-associated death domain protein (FADD) (12). Transfection of FADD induced PCD in a dose- and temporal-dependent manner (Fig. 2A). While APP alone had negligible consequences, it augmented apoptosis triggered by FADD (3 mg) (Fig. 2A) and induced significant cell death when cotransfected with non-toxic doses of FADD (0.3 and 1 mg) (Fig. 2A). Assessing the cleavage of poly [ADR-ribose] polymerase (PARP) by cell death protease known as caspases (18) also corroborated these results. By 8 hrs, PARP was completely processed in cells transfected with the combination of APP and FADD (3 mg) as compared to approximately 60% cleavage in cells expressing FADD alone (Fig. 2B).

APP is first cleaved by β-secretase, giving rise to C99 (Fig. 1A, upper left panel). Alternatively, APP can be cleaved by a-secretase within the Aβ domain, generating a COOH-terminal membrane bound molecule of 83 amino acids (C83) (Fig. 1A, lower left panel). Processing of C99 and C83 fragments by the γ-secretase results in the release and secretion of Aβ and P3, respectively (19, 20). Concomitantly, a putative intracellular product that we referred to as APP Intracellular Domain (AID) should be generated (Fig. 1A, upper and lower right panels). Such a peptide has so far never been described. We asked whether these processed intermediates of APP were responsible for the apoptotic phenotype observed above. Constructs encoding for C99 and C83 were transfected either alone or with FADD. Neither C83 nor C99 induced PCD when expressed alone and only C99 synergized with FADD in inducing apoptosis (Fig. 2C and D). Interestingly, we observed the appearance of a shorter COOH-terminal APP fragment in C99 transfected cells, whose pattern of immunoreactivity and molecular weight was consistent with that of AID (Fig. 2E and F, left panel). This fragment was absent in C83 transfected cells suggesting that C99 is a better γ-secretase substrate than C83. To further address this question, cells were transfected with either wild type APP or the Swedish APP (APP-Sw) mutant (1, 2.). This FAD-associated mutant is more efficiently processed by b-secretase giving rise to more C99 than wild type APP (Fig. 2F, right panel). Consistent with our hypothesis, APP-Sw is more effectively degraded to AID polypeptides (Fig. 2F, right panel) and possesses stronger pro-apoptotic activity (not shown) than the wild type protein. Together, these data suggest a correlation between the strength of the apoptotic signal and the processivity of APP by γ-secretase.

The above results are compatible with the hypothesis that processing of C99 by γ-secretase can produce APP fragment(s) with pro-apoptotic functions. We therefore investigated whether one or both of the C99-derived fragments, Aβ and AID, mediate the observed effect on PCD. As a large fraction of Aβ is secreted upon production, we first tested whether FADD-induced cell death was increased by the secretion of Aβ. To address this, Jurkat cells were either labeled with the green fluorescent dye, CFSE, and cotransfected with FADD and C99 (CFSE+) or remained unlabeled and transfected with FADD only (CFSE-). The two populations were mixed immediately following transfection and assessed for PCD. If the synergistic effect on apoptosis was a consequence of Aβ secretion, then equivalent levels of cell death should be observed in both CFSE+ and CFSE- populations. Regardless of cell ratio, apoptosis was consistently observed in ~55% and ~35% of the CFSE+ and the CFSE- cells, respectively (Fig. 3A). These results indicate that secreted Aβ does not facilitate FADD-induced apoptosis. As an alternative approach, synthetic Aβ40 or Ab42 was directly added to Jurkat cells transfected with either vector control or FADD. Our results show that the addition of Aβ in the range of 5-10 mM did not reproduce the observed synergistic effects (Fig. 3B). Finally, as a further attempt to investigate whether Aβ synergizes with FADD, we transfected Jurkat cells with a construct that encodes for APPNcas. APPNcas represents the NH2-terminal fragment of APP generated by caspase-6 cleavage (Fig. 1B) (21, 22, 23, 24) and has been shown to generate higher levels of Aβ than full-length APP (24). In agreement with the above studies, FADD-induced apoptosis was not enhanced by APPNcas (Fig. 2C). Subsequently, we proceeded to test whether the pro-apoptotic function of C99 was mediated by its cytoplasmic tail, the putative AID peptide. To this end, we transfected a construct encoding for AID into Jurkat cells either alone or with FADD and cell death was measured both by DNA fragmentation (Fig. 2C) and PARP cleavage (Fig. 2D, right panel). Consistently, we observed that AID acted as a stronger inducer of FADD-meditated apoptosis as compared to both APP and C99. Thus, the synergistic effect of APP does not correlate with Aβ production, but is rather mediated by the APP COOH-terminal tail.

Although enhanced cell death by AID required FADD in Jurkat cells, we sought to determine whether overexpression of AID alone could trigger PCD in other cell lines. HeLa and MCF7 cells were transfected with plasmids encoding various APP-derived fragments fused to green fluorescent protein (GFP) to directly visualize transfected cells. While overexpression of either APP (Fig. 4 and 5B) or APPNcas (not shown) did not affect cell viability, transfection of AID in either cell line consistently generated elevated levels of cell death (25-35%) as defined by cell shrinkage and nuclear condensation (Fig. 4 and 5B). From these studies, three lines of evidence demonstrate that AID induces an apoptotic form of cell death. First, overexpression of AID induced activation of caspases (Fig. 4C and D), which are cysteine proteases that implement PCD (18). Second, activation of caspases is required for the execution of cell death since the caspase inhibitors zVAD-fmk, Crma, p35 and MC159 blocked AID-induced apoptosis (Fig. 4A and B). Lastly, the anti-apoptotic protein Bcl-XL (Fig. 5B), a Bcl-2 family member, also inhibited AID-induced cell death.

C99 can be cleaved by the γ-secretase at two distinct positions to generate either Aβ40 or Ab42. The corresponding AID fragments would comprise either the 58 (AID59) or 56 (AID57) COOH-terminal amino acid of APP, respectively (Fig. 5A). FAD mutations in APP and presenilins all result in a shift in metabolism of APP such that more Aβ42 is produced. Consequently, increased amounts of AID57 will be released in the cytosol. If the shorter AID57 peptides were more toxic than the longer form, this could explain why APP and presenilins FAD mutants have enhanced pro-apoptotic activity than the corresponding wild type. To address this question, HeLa and MCF-7 cells were transfected with vectors expressing either AID59 or AID57 and analyzed for cell death. Strikingly, our data revealed that AID57 was significantly more potent than AID59 in inducing PCD (Fig. 5B). Moreover, in a mouse motor neuronal cell line (MN-1) (25), similar results were observed. That is, AID57 was more effective than AID59 in promoting apoptosis.

Interestingly, in all three cells lines, overexpression of APPCcas, a 31 amino acid COOH-terminal fragment of APP released by caspase-6 cleavage (Fig. 1B), was non-toxic. These results are contrary to those recently published (26), which demonstrated that C31, a COOH-terminal polypeptide corresponding to APPCcas, acts as an amplifier of PCD. We further addressed this discrepancy by asking whether disruption of the caspase cleavage site within the cytoplasmic tail of APP abrogates its inducing affect. A substitution of an aspartic acid residue for an asparagine was introduced at position 664 in APP (APPD664N), AID59 (AID59mut) and AID57 (AID57mut), and subsequently tested for cell death in Jurkat, HeLa and MCF-7 cells. In Jurkat cells, overexpression of either APP full-length or AID57-containing mutants were not compromised in their ability to augment FADD-induced apoptosis (Fig 5C). By contrast and in accordance with the above data, APPCcas was ineffective in amplifying the effects of FADD on cell death. Also, comparable levels of PCD were observed in HeLa cells bearing either AID57 or AID57mut (Fig. 5C). Lastly, both AID59 and AID59mut activated cell death in either HeLa or MCF-7 cells (Fig. 5C). Together, these results support a prerequisite for γ-secretase-mediated release of AID for induction of apoptosis, and, moreover, argue against the requirement of further processing.

Although the knowledge available on APP processing argues that one AID molecule must be produced for every Aβ peptide released (Fig. 1A), AID peptides have never been described previously. To substantiate the physiological and pathological significance of our findings, we investigated whether AID-like peptides are present in post-mortem sporadic AD and normal brain tissues (25-maldi) (27, 28, 29). As shown in Fig. 5D, four AID peptides were isolated from these tissues. These peptides were identified by MALDI-MS sequence analysis as AID fragments that undergo further proteolysis in vivo at both the NH2- and COOH-terminus.

Here we show that a natural product of γ-secretase cleavage, the cytoplasmic tail of APP, is a positive regulator of PCD. While in Jurkat cells it facilitates FADD-dependent apoptosis, AID directly triggers PCD in HeLa, MCF7 and MN-1 cells. Whether this difference is cell-type dependent it remains to be investigated. These data suggest that proteolysis of APP by secretases tunes the susceptibility of cells to apoptosis. In this scenario, presenilins might facilitate PCD by promoting cleavage of APP by the γ-secretase, thus governing the amount of AID generated. The biological and pathological relevance of this model is endorsed by the discovery that AID peptides are detected in normal and sporadic AD brain. The functional consequences of APP processing described above resembles that of Notch and Ire1, two other proteins whose processing is controlled by presenilins (30); release of the intracellular domain of Notch and Ire1 by cleavage within the transmembrane region results in downstream effector function.

Could these findings be applied to the pathogenesis of Alzheimer's disease? Our studies suggest that overproduction of AID, and especially the shorter AID57 peptide, makes cells more sensitive to apoptotic stimuli. This may add to the toxic burden caused by the amyloidogenic plaques and by Aβ released in the endoplasmic reticulum (31), further accelerating the neurodegenerative process observed in the brain of Alzheimer's patients.

Figure legend

Figure 1
Endoproteolysis of APP by secretases and caspase-6. (A) APP processing occurs by a series of cleavage events that lead to the formation of Aβ and AID. To initiate the generation of Aβ, APP is cleaved by β-secretase at the NH2-terminus of Aβ to release APPsb and C99, a membrane-bound fragment (upper left panel). Alternatively, cleavage by the α-secretase results in the generation of a large NH2-terminal fragment, APPsa, and a membrane-bound COOH-terminal polypeptide, C83 (lower left panel). C99 and C83 can be further cleaved by γ-secretase to release and secrete Aβ or P3 peptide, respectively (upper and lower right panel, respectively). In both cases, a ~6 kDa cytosolic AID fragment should be generated. (B) APP cleavage by caspase-6 occurs at a caspase consensus sequence (VEVD) within the cytoplasmic tail of APP. This cleavage event results in the generation of APPNcas, an NH2-terminal membrane-associated fragment, and APPCcas, a cytosolic fragment of 31 amino acids.

Figure 2
APP and its naturally processed derivatives augment FADD-induced PCD. (A) APP facilitates FADD-induced cell death. Jurkat cells were transfected with either APP (10 mg), FADD (0.1-3 mg) or in combination. At the indicated time points, apoptosis was measured by determining the percentage of nuclei undergoing DNA fragmentation. (B) FADD-induced PARP cleavage is increased by APP. 8 hrs post-transfection, Jurkat lysates were prepared from cells transfected with APP and/or FADD. Protein lysates (~7 mg/lane) were separated on a 4-12% PAGE, blotted and probed with an anti-PARP antibody. Note the complete cleavage of PARP (89 kDa) in cells cotransfected with APP and FADD. (D) Synergistic effects on FADD-induced PCD by APP-derived polypeptides. Jurkat cells were transfected with empty pcDNA3 vector (pc) or constructs encoding for various APP fragments (10 mg) either in the absence (-) or presence (+) of FADD (3 mg). The results represent the mean +/- S.D. of 5 independent experiments. Apoptosis was assessed 6 hrs post-transfection. A student's t-test was used to calculate the results and * indicates that P values were < 0.05. (D) C99 and AID augment FADD-induced PARP cleavage. Cell lysates were prepared from Jurkat cells transfected with pcDNA3 (pc) or C99 alone (-) or in combination with FADD (+) (left panel). In other experiments (right panel), lysates were prepared from control cells (pc) or cells transfected with FADD together with pcDNA3 (pc), C83 or AID. Samples were analyzed for PARP cleavage 8 hours post-transfection. C99 and AID increased the percentage of cellular PARP processed during FADD-induced apoptosis. Neither the vector control nor the various APP fragments alone induced PARP cleavage. (E) Cells were transfected with pcDNA3 (pc), C99, C83 or AID. Labeled proteins were immunoprecipitated with either 6E10 or C7 anti-APP antibodies. Since the epitope recognized by the anti-6E10 antibody is located between the β- and α- secretase sites of APP, only C99 was precipitated by this antiserum. Conversely, the C7 antiserum, specific for the COOH-terminal 20 amino acids of APP, precipitated all three APP-derived fragments. Interestingly, a polypeptide of molecular weight similar to AID was immunoprecipitated by C7 but not 6E10 antibodies in C99-transfected cells,

(F) Cells were transfected with C83, AID, C99 (left panel), APP wild type (W.t.), APP Swedish mutant (Sw.) or pcDNA3 (Vector) (right panel). Labeled proteins were immunoprecipitated with the C7 antiserum. While detectable levels of AID peptide were identified in C99 transfected cells, this fragment was not apparent in cells overexpressing C83 (Right panel). The APP-Sw mutant is processed at the b-secretase site more efficiently than W.t., APP, and consequently, generates higher levels of AID-like peptide.

Figure 3
The synergistic effect of APP and its processed forms on FADD-induced PCD is not dependent on secreted Aβ. (A) Secreted Aβ derived from CFSE+ cells does not enhance FADD-induced PCD in CFSE- cells. Jurkat cells labeled with the green fluorescent dye ,CFSE, were transfected with C99 and FADD, while unlabeled cells were transfected with FADD only. Immediately following transfection, unlabeled and labeled cells were mixed according to the ratios indicated (lower left and right quadrants). Apoptosis in CFSE+ (left quadrants) and CFSE- (right quadrants) populations was measured 6 hrs post-transfection by determining the percentage of AnnexinV+ cells. This protein binds phosphatidylserines that are "flipped" and exposed on the cell surface during PCD. CFSE+ cells (~55%) consistently induced higher levels of cell death than CFSE- cells (~35%), indicating a cis and not a trans-effect. (B) Apoptosis induced by FADD is not enhanced by addition of exogenous Aβ40 or Aβ42. Aβ40 or Aβ42 peptides (5-10 mM) were added immediately following transfection of Jurkat cells with either pcDNA3 vector control (pc) or FADD (3 mg). Of note, higher concentrations of Aβ42 (25 mM) induced significant apoptosis in Jurkat cells, indicating that the peptide preparation was cytotoxic (data not shown).

Figure 4
AID induces apoptosis in HeLa cells. HeLa cells were transfected with 2 mg of mammalian expression vectors coding for APP-GFP, AID-GFP, FADD-GFP or GFP. Cells were stained with Hoechst H33258 24 hrs after transfection, and apoptotic nuclei were scored with an inverted fluorescence microscope. At least 100 cells/sample were counted for each data point. Percentages are the average of three independently transfected wells. Similar results were obtained in two other experiments. (A) The caspase inhibitor, Z-VAD-fmk, blocks cell death triggered by AID. Note that Z-VAD-fmk blocks AID-induced apoptosis as efficiently as FADD. (B) Viral-derived caspase inhibitors protect HeLa cells from AID-induced PCD. Cotransfection of AID with plasmids encoding either CrmA (blocks caspase1 and 8), p35 (blocks multiple caspases) or MC159 (also called v-flip; blocks caspase 8 and FADD) completely blocked the effects of AID. (C) Protein levels of caspase-6 and caspase-8 precursors (pro-Cas.8 and pro-Cas.6) are diminished in AID transfected cells. Equivalent amounts of protein were determined by blotting with anti-BNIP2. Note that similar affects were observed with FADD. (D) AID activates caspases as determined by in vitro cleavage of the fluorescent substrate DEVD-AFC. Note that the same cell lysates from (C) were used to monitor for DEVD-AFCase activity.

Figure 5
(A) γ-secretase cleavage of APP can occur at two different positions. A cut occurring between residues 637-638(indicated as β-40) gives rise to the short Aβ (Aβ40) and long AID (AID59). Conversely, cleavage after residue 639 (indicated as g-42) yields the longer Aβ (Aβ42) isoform and shorter AID (AID57) fragment (numbering is according to the 695 amino acid long APP isoform). FAD mutations preferentially increase cleavage after residue 639, which result in the production of the highly amyloidogenic Ab42 peptide. In this model, we postulate that the resulting AID57, is more damaging to cells than its longer AID59 counterpart. Thus, FAD mutations will result in overproduction of two APP-derived peptides that exert their neurotoxic action both intra- (AID57) and extracellularly (Aβ42). Aβ peptides could also exert a pro-apoptotic activity that requires caspase-12 in the endoplasmic reticulum (E.R.) compartment (37). (B) (left panel) AID-induced apoptosis in MCF-7 cells is inhibited by the anti-apoptotic protein Bcl-XL (data not shown for HeLa and MN-1). Expression of an unrelated control protein (AIP1) did not influence cell death. (Middle panel) AID57 induces significantly more apoptosis than AID59 in HeLa cells (data not shown for MCF-7) (*P<0.05). Interestingly, APPCcas, a caspase-6 derived fragment, representing the last 31 COOH-terminal amino acids of APP (see fig. 1b), lacked pro-apoptotic activity. (Right panel) AID57 is more effective than AID59 in promoting PCD in the mouse motor neuronal cell line, MN-1. Note again, that APPCcas was not effective in promoting apoptosis. (C) Disruption of the caspase cleavage site within the cytoplasmic tail of APP and AID does not impair the execution of cell death. APPD664N and AID57mut were transfected into Jurkat cells (left panel) either alone (data not shown) or with FADD and analyzed at the indicated time points for apoptosis. As compared to their non-mutant counterparts, APPD664N and AID57mut were no different in their ability to implement apoptosis. Conversely, overexpression of APPCcas exhibited negligible effects on FADD-induced cell death. Note that the vector control background was subtracted from each time point. In HeLa cells, overexpression of either AID57mut (middle left panel) or AID59mut (middle right panel) induced cell death to the same extent of their non-mutant counterparts, whereas APPCcas was incapable in producing such effects. Similar results were also obtained in MCF-7 cells (right panel) where overexpression of AID59mut displayed comparable levels of cell death to AID59. (D) AID-like peptides are present in normal and sporadic AD brain. Sequence analysis of four peptides (peak 1-4) immunoprecipitated by the Jonas monoclonal antibody, which are recognized on western blot by an anti-APP antiserum (not shown) are compared to the sequences of AID59 and APPCcas. In the experiment shown, a post-mortem brain tissue from a 72 years old AD patient was analyzed. These AID-like peptides were also found in the three other post-mortem brains that were examined. Two were from normal controls (45 and 51 years of age) and one other AD (65 years of age).

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