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
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,
*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
Alzheimer's disease (AD) is believed to be caused by extracellular deposition
of amyloidogenic forms of Aβ peptide (Aβ42)
Aβ derives from cleavage of APP by β- and γ-secretase
(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
Of more importance, presenilins and APP point mutations found in FAD patients
augment APP processing and the formation of amyloidogenic Aβ (1,
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,
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
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.
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
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
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,
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
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.
(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).
Price, D. L., and Sisodia, S. S. (1998) Annu. Rev. Neurosci. 21,
Hardy, J. (1997) Trends Neurosci. 20, 154-159
Haass, C. and Selkoe, D.J. (1993) Cell 75,1039-1042
Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Crawford, F., Fidani,
L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P.,
Rooke, K., Roques, P., Talbot, C., Pericakvance, M., Roses, A., Williamson,
R., Rossor, M., Owen, M., and Hardy, J. (1991) Nature 349,
Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G.,
Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T., Mar, L., Foncin,
J.-F., Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L.,
Nee, L., Chumakov, I., Pollen, D., Brookes, A., Sanseau, P., Polinsky, R.
J., Wasco, W., Da Silva, H. A. R., Haines, J. L., Pericak-Vance, M. A.,
Tanzi, R. E., Roses, A. D., Fraser, P. E., Rommens, J. M., and St. George-Hyslop,
P. H. (1995) Nature 375, 754-760
Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell,
W. H., Yu, C., Jondro, P. D., Schmidt, S. D., Wang, K., Crowley, A. C.,
Fu, Y.-H., Guenette, S. Y., Galas, D., Nemens, E., Wijsman, E. M., Bird,
T. D., Schellenberg, G. D., and Tanzi, R. E. (1995) Science 269,
Rogaev, E. I., Sherrington, R., Rogaeva, E. A., Levesque, G., Ikeda, M.,
Liang, Y., Chi, H., Lin, C., Holman, K., Tsuda, T., Mar, L., Sorbl, S.,
Nacmias, B., Piacentini, S., Amaducci, L., Chumakov, I., Cohen, D., Lannfelt,
L., Fraser, P. E., Rommens, J. M., and St. George-Hyslop, P. H. (1995) Nature
De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde,
G., Annaert, W., Von Figura, K., and Van Leuven F. (1998) Nature
Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T.,
and Selkoe, D. J. (1999) Nature 398, 513-517.
Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C.-M., Perez-tur, J., Hutton,
M., Buee, L., Harigaya, Y., Yager, D., Morgan, D., Gordon, M. N., Holcomb,
L., Refolo, L., Zenk, B., Hardy, J., and Younkin, S. (1996) Nature
Scheuner ,D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N.,
Bird, T.D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E.,
Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco,
W., Lannfelt, L., Selkoe, D., and Younkin, S. (1996) Nat. Med. 2,
Vito, P., Lacaná, E., and D'Adamio, L. (1996) Science 271,
Palacino, J. J., Berechid, B. E., Alexander, P., Eckman, C., Younkin, S.,
Nye, J. S., and Wolozin, B. (2000) J. Biol. Chem. 275, 215-222.
Wolozin, B., Iwasaki, K., Vito, P., Ganjei, J. K., Lacaná, E., Sunderland,
T., Zhao, B., Kusiak, J. W., Wasco, W., and D'Adamio, L. (1996) Science
Guo, Q., Sopher, B. L., Furukawa, K., Pham, D. G., Robinson, N., Martin,
G. M., and Mattson, M. P. (1997) J. Neurosci. 17, 4212-4222
Yamatsuji, T., Okamoto, T., Takeda, S., Murayama, Y., Tanaka, N., and Nishimoto,
I. (1996) EMBO J. 15, 498-509.
Chinnaiyan, A. M., O'Rourke K., Tewari, M. V., and Dixit, M. (1995) Cell
Thornberry, N. A., Lazebnik, Y. (1998) Science 281, 1312-1313
Sinha, S., and Lieberburg, I. (1999) Proc. Natl. Acad. Sci. U. S. A.
Haass, C., and De Strooper, B. (1999) Science 286, 916-917
Weidemann, A., Paliga, K., Durrwang, U., Reinhard, F.B.M., Schuckert, O.,
Evin, E., and Masters, C.L. (1999) J. Biol. Chem. 274, 5823-5829
Pellegrini, L., Passer, B. J., Tabaton, M., Ganjei, J. K., and D'Adamio,
L. (1999) J. Biol. Chem. 274, 21011-21016
LeBlanc, A., Liu, H. , Goodyer, C., Bergeron, C., and Hammond, J. (1999)
J. Biol. Chem. 274, 23426-23431
Gervais, F. G., Xu, D., Robertson, G. S., Vaillancourt, J. P., Zhu, Y.,
Huang, J., LeBlanc, A., Smith, D., Rigby, M., Shearman, M. S., Clarke, E.E.,
Zheng, H., Van Der Ploeg, L. H., Ruffolo, S. C., Thornberry, N. A., Xanthoudakis,
S., Zamboni, R. J., Roy, S., and Nicholson, D. W. (1999) Cell 97,
Salazar-Grueso, E. F, Kim, S., and Kim, H. (1991) Neuroreport 2,
Lu, D. C., Rabizadeh, S., Chandra, S., Shayya, R. F., Ellerby, L.M., Ye,
X., Salvesen, G. S., Koo, E.H., and Bredesen, D.E. (2000) Nature Med.
De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm,
J.S., Schroeter, E.H., Schrijvers, V., Wolfe, M.S., Ray W.J., Goate, A.,
and Kopan, R. (1998) Nature 398, 518-522
Struhl, G., and Greenwald, I. (1999) Nature 398, 522-525
Ye, Y., Lukinova, N., and Fortini, M. E. (1999) Nature 398,
Niwa, M., Sidrauski, C., Kaufman, R. J., and Walter, P. A. (1999) Cell
Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, .J, Yankner, B. A., and
Yuan, J. (2000) Nature 403, 98-103