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INTRODUCTION |
Neurodegeneration in selected brain regions is one of the
pathological features of Alzheimer's disease, together with amyloid deposition in extracellular plaques, congophilic angiopathy, and intracellular tangles. Amyloid deposited in senile plaques is mainly
composed of
A4, a peptide consisting of 40-42 residues, which
derives by proteolytic processing from its cognate precursor protein
termed APP1 (1). Mutations in
the APP gene cause a rare form of early-onset familial Alzheimer's
disease (FAD) by raising the levels of total
A4 or by increasing the
ratio of the longer form of
A4 that ends at residue 42 to the form
ending at residue 40 (2-5). Additionally, the expression of APP
carrying those mutations has been reported to induce apoptosis in
cells, suggesting a probable link between apoptotic pathways and
neurodegeneration in Alzheimer's disease (6-8). Mutations causative
for the majority of FAD cases have been identified recently in genes
encoding presenilin (PS) 1 and PS2 (9-11). Missense mutations in these
genes were also found to alter APP processing in a pathological manner
by increasing the relative concentration of
A4 ending at residue 42 (12-16). Both proteins are endoproteolytically converted by two
alternative pathways: (i) in the first pathway, cleavage occurs within
a highly hydrophobic region of the presenilins (16-18), and (ii) in
the second pathway, presenilins become converted by caspase-3 family proteases at about 30 residues distal to the normal cleavage site (19-20). The corresponding caspase-derived fragments have been detected in all tissues and stages of animal development, suggesting a
role of PS processing in the cellular response to apoptotic signals
(19, 21). Furthermore, overexpression of PS2 in transfected cells has
been shown to increase susceptibility to apoptotic cell death. The
latter is even more pronounced in cells expressing mutant PS2-I that
encodes one of the known PS2 missense mutations changing
Asn141 into Ile (22, 23).
When we analyzed carboxyl-terminal fragments of APP in cells
coexpressing PS2 or PS2-I, we observed proteolytic processing of APP
within the cytoplasmic domain that was augmented for mutated PS2-I (see
below). To determine whether this result could be attributed to
apoptotic pathways and whether APP proteolysis was mediated by
caspases, cells expressing full-length APP and amino-terminal-truncated fragments were analyzed after the induction of apoptosis with different
compounds including staurosporine and doxorubicin. We report here that
proteolytic conversion of APP within its cytoplasmic domain is
increased in apoptotic cells and is inhibited by the addition of
specific caspase inhibitors. These results suggest that APP is a target
for caspase-like proteases and is involved in apoptotic pathways.
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EXPERIMENTAL PROCEDURES |
The cloning of PS2 and construction of mutant PS2-I have been
described previously (24). The point mutations in the cytoplasmic domain of SPA4CT (25) (SPA4CTD/A and
SPA4CTdel664) were introduced by polymerase chain reaction
and cloned into pBluescript SK+ (Stratagene). The open reading frames
of the resulting constructs were verified by sequencing, followed by
cloning into the expression vector pCEP4 (Invitrogen). Because of the
aberrant electrophoretic mobility of SPA4CTD/A protein on
SDS-PAGE, the corresponding pCEP4 derivative was again sequenced to
exclude any additional mutations during the cloning procedure. The APP
derivatives APPD/A and APPdel664 were derived
from the corresponding SPA4CT clones by using a unique EcoRI
site within the
A4 coding region.
Jurkat J16 T cells were cultivated in RPMI 1640 medium containing 10%
fetal calf serum. COS-7 cells were maintained and transfected as
described previously (24). Stably expressing cell lines were generated
by CaPO4 transfection of cells with the corresponding pCEP4
derivatives followed by selection with hygromycin (250 µg/ml). In
transient transfection experiments, cells were used the following day.
Apoptosis was induced for the time periods indicated by adding either
staurosporine (2 µM in dimethyl sulfoxide) or doxorubicin (100 µM in distilled water) in serum-free medium. Caspase
inhibitors DEVD-CHO, YVAD-CHO, IETD-CHO, and zVAD-fmk were purchased
from Bachem or Biomol and dissolved in dimethyl sulfoxide as 100 mM stock solutions. Cells were pretreated for 45 min with
the corresponding inhibitor before the addition of staurosporine or doxorubicin.
For in vitro experiments, COS-7 cells transfected with
SPA4CT were metabolically labeled with [35S]methionine,
and the lysates were immunoprecipitated with monoclonal antibody W02.
The immunoprecipitated SPA4CT was incubated with recombinant, purified
caspase-3 and caspase-8 (final concentration, 2 units/µl) for 30 min
at 30 °C under mild agitation in 25 µl of assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM dithiothreitol).
Apoptosis analysis was performed by using an Annexin V-FLUOS detection
kit (Boehringer). After stimulating apoptosis, about 106
cells were pelleted, washed once in phosphate-buffered saline, and
resuspended in 200 µl of binding buffer containing 4 µl of propidium iodide and 4 µl of Annexin V-FLUOS. Data were collected from 10,000 cells with a FACScan (Becton Dickinson) fluorescence accelerated cell scanner using the data acquisition program CELLQuest.
For immunoblotting, cells were harvested, resuspended in 8 M urea, briefly sonified, and boiled with SDS sample
buffer. SPA4CT-containing samples were separated on either 10-16.5%
SDS Tris-Tricine gradient gels or 12% Bis-Tris gels (Novex) followed
by immunodetection with monoclonal anti-
A4 antibody W02, which
recognizes the amino-terminal region of
A4 (26). For Western
blotting of APP, samples were separated on 7.5% Tris-glycine gels and
immunodetected with mAb 22C11. Radioactive labeling of cells,
immunoprecipitation with polyclonal rabbit anti-A4CT antiserum and
anti-CT and anti-APP antiserum, and immunodetection of PS2 were
performed as described previously (24, 25, 27).
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RESULTS |
Induction of Apoptosis Is Accompanied by Proteolytic Processing of
SPA4CT and APP within the Cytoplasmic Domain--
Structurally, APP
represents a type I integral membrane protein with a large ectodomain,
a transmembrane domain, and a short cytoplasmic tail (Fig.
1; numbered according to Ref. 1). In COS-7 cells stably transfected with an expression vector encoding APP695, most of the cellular APP695 was
detected as a band of about 95 kDa that corresponds to the immature,
N-glycosylated species (Fig. 2A,
lane 2). Additionally, signals at 100-120 kDa were observed that
represent posttranslationally modified APP species derived by
successive transport of APP through the Golgi apparatus and including
O-glycosylation, sulfation, and phosphorylation (27).

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Fig. 1.
Schematic representation of APP and its
carboxyl-terminal domain. The large extracellular domain of APP is
followed by the A4 sequence ( A4), the transmembrane
domain (TM), and the cytoplasmic domain (CT).
SP, signal peptide. The positions of the proteolytic
cleavage sites for -, - and -secretases are indicated. In its
cytoplasmic domain, APP encodes an internalization signal
(NPXY), a binding site for G0 proteins
(His657-Lys676), and a consensus sequence for
group III caspases (c) ((IVL)ExD).
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Fig. 2.
Apoptotic stimulation of cells induces
proteolytic conversion within the APP carboxyl-terminal domain.
A, proteolytic conversion of APP695 in cells
undergoing programmed cell death. COS-7 cells stably transfected with
APP695 expression vector (lanes 2-5) were
treated for 8 h with 2 µM staurosporine (lane
4) or 100 ng/ml doxorubicin (lane 5) in serum-free
medium (lanes 3-5). For comparison, cells were cultivated
in serum-free media (lane 3) or in fetal calf
serum-containing media (lanes 1 and 2). In
lane 1, vector-transfected COS-7 cells were analyzed as a
control. The band of about 100 kDa represents APP751 that
is endogenously expressed by COS-7 cells (lane 1).
APP695 was detected in the cell homogenates after
immunoprecipitation with anti-APP (lanes 2-5) or anti-CT
antiserum (lanes 6 and 7) followed by gel
electrophoresis and immunoblotting with mAb 22C11. Epitope mapping of
the APP isoforms reveals that the lower molecular mass species at about
90 kDa is not immunoreactive with anti-CT antiserum (lanes 6 and 7). The endogenously encoded APP751
(lane 1) is also detected in lane 8 after
prolonged ECL exposition (data not shown). B, conversion of
SPA4CT in apoptotic cells. Cells were transiently transfected with
SPA4CT cDNA (lanes 2 and 3), followed by
metabolic labeling with [35S]methionine for 7 h and
immunoprecipitation with anti- A4 mAb W02. Increased conversion of
SPA4CT into the 8-kDa species is observed in staurosporine-treated
cells (lane 2). C, proteolytic conversion of
SPA4CT in cells overexpressing PS2 and PS2-I. COS-7 cells were
transfected with cDNAs encoding SPA4CT alone (lane 1) or
in combination with PS2 (lanes 2 and 4) and
mutated PS2-I (lanes 3 and 5). In lanes
2 and 3, a 1:1 stoichiometry of expression vectors was
used, whereas in lanes 4 and 5, the cells were
transfected with only one-tenth of PS2- and PS2-I-encoding plasmids.
After radiolabeling the cells with [35S]methionine for
6 h, SPA4CT was immunoprecipitated with mAb W02 followed by
SDS-PAGE and phosphorimaging.
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To induce apoptotic cell death, cells were treated with either
staurosporine or doxorubicin. APP analysis revealed the apparition of
an additional band at about 90 kDa (Fig. 2, lanes 4 and
5). To test whether this band represents a
carboxyl-terminal-truncated species of APP, immunoprecipitations were
performed with an antiserum directed against the carboxyl terminus of
APP. The 90-kDa band was not recognized, suggesting that the APP
carboxyl terminus was cleaved in apoptotic cells (Fig. 2, lanes
6-8).
For further analysis, the last 103 carboxyl-terminal residues of APP,
which extend from the amino terminus of
A4 to the very end of the
APP cytoplasmic domain and mimic the
-secretase carboxyl-terminal product (Fig. 1, A4CT), were expressed in COS-7 cells. This
amino-terminal-truncated derivative of APP allowed a more accurate
investigation of the site of cleavage because of the lower molecular
weight and the higher resolution of the corresponding peptide on SDS
gels. To ensure correct membrane insertion, a signal peptide was fused to the amino terminus (SPA4CT (25)). After metabolic labeling of the
cells, the expressed peptide was immunoprecipitated with mAb W02, which
is directed to
A4 residues 1-16 (26). The antibody detected SPA4CT
as a band of 12 kDa (Fig. 2B, lane 3), a result consistent
with earlier studies (25). Additionally, a second band of about 8 kDa
was detected in nonapoptotic cells after immunoprecipitation with this
antibody, suggesting that this band represents a
carboxyl-terminal-truncated species lacking the last 20-30 residues of
the APP sequence as deduced from the differences in the relative
molecular masses. Treatment of cells with staurosporine resulted in a
strong increase in the relative intensity of the 8-kDa species (Fig.
2B, lane 2), suggesting elevated processing of SPA4CT within
its cytoplasmic domain in apoptotic cells.
Coexpression of PS2 and PS2-I with the SPA4CT Fragment Stimulates
Cleavage within the APP Cytoplasmic Domain--
Because overexpression
of PS2 has been reported to stimulate apoptotic pathways in cells, we
analyzed the conversion of SPA4CT in cells that were cotransfected with
a PS2-encoding expression plasmid. Additionally, PS2-I was coexpressed,
which encodes one of the two known missense mutations
(Asn141 to Ile) within the PS2 gene causative for
early-onset Alzheimer's disease (10, 11). Expression of PS2 and PS2-I
was monitored by immunoprecipitating lysates from radiolabeled cells
with anti-PS2 antiserum, as described previously (Ref. 24; data not shown).
In the first experiment, both PS2 and SPA4CT cDNA-encoding plasmids
were transiently cotransfected in an equal ratio. Here, the relative
intensity of the 8-kDa band compared with the 12-kDa band was increased
(Fig. 2C, lane 2). A similar result was obtained with the
cells cotransfected with PS2-I (lane 3). In a second experiment, PS2 cDNA and SPA4CT cDNA at a ratio of 0.1:1 were expressed in the cells. Under these conditions, mutant PS2-I was still
able to induce an increased conversion of SPA4CT into the 8-kDa species
(lane 5). In contrast, cells cotransfected with low amounts
of wild-type PS2 showed a pattern indistinguishable from that of the
cells expressing solely SPA4CT (lane 4). This finding is
consistent with reports that cells that overexpress mutant PS2-I are
more prone to undergo apoptosis than cells expressing wild-type PS2
(22, 23).
Proteolytic Conversion of SPA4CT upon Treatment by Apoptosis
Inducers Is Time Dependent and Is Inhibited by the Caspase Inhibitors
IETD-CHO and zVAD-fmk--
To further characterize the proteolytic
processing of the APP cytoplasmic domain, pulse-chase experiments were
performed in the presence of staurosporine and doxorubicin (Fig.
3A). Before the induction of
cell death, almost all SPA4CT was detected as a 12-kDa species
(lanes 1 and 4). After induction with
staurosporine for 9 h, about 50% of SPA4CT was converted into
the 8-kDa fragment (lane 2). Incubation for 30 h caused
a decrease in the intensities of both SPA4CT and the 8-kDa fragment, a
result attributed to the lower synthesis of SPA4CT in dying cells
(lane 3). A similar precursor-product relationship between
SPA4CT and its 8-kDa fragment was observed in doxorubicin-treated cells
(lanes 4-6).

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Fig. 3.
Proteolytic conversion of SPA4CT in apoptotic
stimulated cells: time course and inhibition by caspase inhibitors.
A, SPA4CT-transfected COS-7 cells were treated with either
staurosporine (2 µM; lanes 1-3) or
doxorubicin (100 ng/ml; lanes 4-6) for the time periods
indicated. Total cellular proteins were fractionated by SDS-PAGE and
analyzed by immunoblotting with mAb W02. B, inhibition of
apoptotic conversion by different caspase inhibitors. Transfected cells
were incubated for 8 h in the presence of staurosporine (2 µM) together with various caspase inhibitors (100 µM). Only IETD-CHO and zVAD-fmk displayed an inhibitory
effect on the conversion of SPA4CT. C, inhibition of
proteolytic conversion by zVAD-fmk is concentration dependent. Cells
were treated with either 100 ng/ml doxorubicin (lanes 1-5)
or 2 µM staurosporine (lanes 6-10) for 8 h in the presence of different zVAD-fmk concentrations, as indicated.
The relative conversion of SPA4CT was calculated after densitometric
analysis and is indicated for each lane. D, in
vitro cleavage of SPA4CT by recombinant caspases. SPA4CT was
immunoprecipitated from transfected, 35S-labeled COS-7
cells and incubated for 30 min either without caspases (lane
1) or with recombinant caspase-3 (lane 2) or caspase-8
(lane 3). The reaction products were separated by
Tris-Bis-Tris PAGE and detected by phosphorimaging. The rate of the
conversion was calculated after correction for the
[35S]methionine residues encoded by SPA4CT (4 residues)
and the proteolytic fragments (respective 2 residues).
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To determine whether a caspase was involved in the cleavage of the APP
cytoplasmic domain under apoptotic conditions, a subset of caspase
inhibitors was used such as a caspase-1-type inhibitor (YVAD-CHO), a
caspase-3-type inhibitor (DEVD-CHO), a caspase-8-type inhibitor
(IETD-CHO), and a broad spectrum inhibitor (zVAD-fmk). Of the three
tetrapeptide inhibitors, only IETD-CHO showed a clear inhibition of the
conversion of SPA4CT into the 8-kDa species (Fig. 3B, lanes
3-5). zVAD-fmk (lane 6) was the most potent inhibitor, probably because it can block all caspase activity by also inhibiting the upstream caspases in the proteolytic activation cascade, whereas IETD-CHO, due to its relative specificity, blocks the total activity of
the group III caspases less effectively. Therefore, zVAD-fmk was then
tested at lower concentrations. Reduced proteolytic conversion of
SPA4CT into the 8-kDa fragment was observed at concentrations ranging
between 3.3 and 100 µM (Fig. 3C). This result
again demonstrates the participation of activated caspases in the
apoptotic processing of APP.
In Vitro Conversion of the APP Carboxyl Terminus by Recombinant,
Activated Caspase-8--
To directly demonstrate the ability of
activated caspases to cleave the APP carboxyl terminus, SPA4CT
immunoprecipitated from radioactively labeled COS-7 cells was incubated
with purified, recombinant caspase-3 and caspase-8 (Fig.
3D). Incubation with buffer alone was used as a control and
showed no cleavage (lane 1). Incubation with caspase-8
resulted in the formation of two fragments of about 8 and 6 kDa
(lane 3). After a 30-min incubation, a 75% conversion of
SPA4CT was observed in the presence of caspase-8 (lane 3),
whereas a conversion of about only 20% occurred in the presence of
caspase-3 (lane 2). The lower molecular mass band apparently
represents the APP 31-residue carboxyl-terminal fragment resulting from
caspase cleavage. The rate of the caspase-8-mediated conversion is in
the same order of magnitude as that described for the in
vitro cleavage of Bid, a proximal substrate of caspase-8 in the
Fas and TNF apoptotic signaling pathway (28, 29). This result proves
formally that activated caspases, especially caspase-8, are capable of
cleaving the APP cytoplasmic domain.
Characterization of the APP Caspase Cleavage Site by Site-directed
Mutagenesis--
Examination of the cytoplasmic domain of APP revealed
the presence of a consensus sequence for caspases: the sequence
VEVD664 (numbered according to APP695; Ref. 1)
matches the sequence known for group III caspase-6, -8, and -9 ((IVL)ExD) (30-32). To test whether the observed proteolytic
processing of the APP cytoplasmic domain occurs at residue
Asp664, two constructs were generated by polymerase chain
reaction: in one clone, Asp664 was mutated into Ala
(SPA4CTD/A), whereas in the other clone, a stop codon was
introduced after Asp664, thereby terminating translation
after this residue (SPA4CTdel664). Both constructs were
expressed in cells, followed by analysis with anti-
A4 antibody (Fig.
4A). In contrast to nonmutated
SPA4CT (lanes 1 and 2), a single band was
observed for the D664A mutant (lane 3) even after the
induction of apoptosis with staurosporine (lane 4),
indicating that the single point mutation completely inhibited
proteolytic conversion. The faster migration of SPA4CTD/A on SDS gels compared with SPA4CT is most likely due to the removal of
the negatively charged Asp664 residue, resulting in an
altered mobility on SDS-PAGE. A similar increase in mobility has
been also observed for other proteins in which the critical Asp residue
was mutated into Ala (33). The electrophoretic mobility of the deletion
mutant SPA4CTdel664 was identical to the 8-kDa fragment
generated from wild-type SPA4CT (lane 5).

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Fig. 4.
APP and SPA4CT derivatives mutated at
Asp664 are resistant to proteolytic conversion.
A, cells were transfected with SPA4CT (lanes 1 and 2), SPA4CTD/A (lanes 3 and
4), and SPA4CTdel664 (lane 5). The
deletion construct SPA4CTdel664 (lane 5)
displays the same molecular mass on SDS-PAGE as the 8-kDa proteolytic
fragment generated from SPA4CT (lane 2). No additional
fragment was observed for SPA4CTD/A, even in the presence
of staurosporine (lane 4). The faster electrophoretic
mobility of SPA4CTD/A (lanes 3 and 4)
is likely to be due to the removal of the negatively charged Asp from
SPA4CT. B, analysis of the corresponding APP derivatives.
Cells were stably transfected with APP695 (lanes
1-4), APP695D/A (lanes 5-8), and
APP695del664 (lanes 9-12) and treated with
either staurosporine (lanes 3, 7, and 11) or
doxorubicin (lanes 4, 8, and 12). Total cellular
homogenates were subjected to SDS-PAGE, followed by immunoblotting with
mAb 22C11. The N-glycosylated moiety of APPdel664
(lanes 9-12) exhibits about the same electrophoretic
mobility as the caspase-derived fragments at about 90 kDa, as detected
in lanes 3 and 4. The additional signals of about
95 kDa in APPdel664-expressing cells represent
posttranslationally higher glycosylated APP species.
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The APP695 derivatives encoding both mutations
(APPD/A and APPdel664) were also expressed and
studied (Fig. 4B). In contrast to wild-type
APP695 (lanes 1-4), no additional lower
molecular mass species at about 90 kDa was detectable for
APPD/A (lanes 5-8), even in the presence of
staurosporine or doxorubicin, indicating a lack of proteolysis within
the cytoplasmic domain (lanes 7 and 8,
respectively). APPdel664 displayed the same relative
mobility (about 90 kDa) as the carboxyl-terminal-truncated
APP695 fragment that is observed in apoptotic cells
(lanes 9-12; compare with lanes 3 and
4). Neither staurosporine nor doxorubicin altered APPdel664 mobility (lanes 11 and
12).
Time Course of APP Cleavage versus Apoptosis--
To address the
question of whether the cleavage events described here occur early or
late in the apoptotic pathways, Annexin V assays were performed.
Annexin V staining of phosphatidylserine translocation on the outer
cell membrane represents a very early event in the apoptotic process,
before gross morphological features such as blebbing or nuclear
condensation become evident. Because we found that adherent growing
cells, including COS-7 cells, displayed different populations in flow
cytometric analysis, making a clear assertion difficult, we used Jurkat
T cells that are well established for studying apoptotic mechanisms.
Jurkat cells endogenously express several alternatively spliced
isoforms of APP (including APP695 and KPI-encoding APP,
Fig. 5A, lane 1). The identity
of the detected bands as APP isoforms was confirmed not only by
immunoprecipitation or immunoblotting with different anti-APP antisera,
but also by electroporation of APP-encoding plasmids, which resulted in
an increase of the band intensities (data not shown). To investigate the time course of APP cleavage in relation to the cell apoptotic status, Jurkat cells were treated with staurosporine for 3, 6, and
9 h, followed by APP immunoblotting and parallel Annexin V-based flow cytometry (Fig. 5, A and B, respectively).
After 3 h of induction, 30% of total APP695 and
APP-KPI were detected as carboxyl-terminal-truncated derivatives as
deduced from the small shift in molecular mass observed, which is
consistent with a loss of a 6-kDa carboxyl-terminal fragment (Fig.
5A, lane 2). After 6 and 9 h, most of the APP was detected as caspase-cleaved species (lanes 3 and
4). The corresponding flow cytometric analysis revealed that
3 h after the induction of apoptosis, about 66% of the cells were
stained with Annexin V (Fig. 5B). Because cleavage of APP
parallels the flipping of phosphatidylserine, we conclude that
proteolysis of APP occurs before gross changes in cell morphology
become detectable.

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Fig. 5.
Cleavage of APP versus apoptosis.
A, cleavage of APP. Jurkat cells were labeled with
[35S]methionine and treated for 0, 3, 6, or 9 h with
staurosporine, followed by immunoprecipitation with anti-APP polyclonal
antiserum. Endogenously expressed APP is detected in the cells as
alternatively spliced isoforms (APP695 and
APP-KPI, lane 1). 3 h after the induction of
apoptosis, about 30% of total APP was found in the cleaved form
(lane 2), whereas after 6 and 9 h, APP was completely
converted (lanes 3 and 4). The relative broad
appearance of the cleavage products is most likely due to a different
extent of posttranslational modifications. c-APP695 and
c-APP/KPI, caspase-cleaved derivatives. B,
fluorescence dot plots of Annexin V- and propidium iodide-stained
cells. The cells were treated with staurosporine for 0, 3, 6, or 9 h, as described in A, and the appearance of
phosphatidylserine on the outer cell membrane was measured by the
binding of fluorescein-labeled Annexin V. Additionally, the
fluorescence of DNA-binding propidium iodide was measured to determine
the membrane integrity of the cells. Data from 2,500 cells are depicted
in each graph. The relative percentage of cells is given within the
quadrants.
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DISCUSSION |
To date, evidence is accumulating that links apoptotic pathways to
neurodegeneration: (i) apoptotic cell death has been reported to be a
pathological feature of Alzheimer's disease, as determined by
histochemical studies (34-37). (ii) Similarly, Guo et al.
(38) have recently shown that the levels of apoptotic mediator proteins like Par-4 are increased in the neurons of Alzheimer's disease brains,
suggesting the involvement of apoptotic processes in neurodegeneration in vivo. (iii) Expression of mutant APP carrying FAD
mutations has been reported to induce apoptosis, indicating that
apoptosis may contribute to the neuronal loss in FAD (6-8). (iv)
Overexpression of PS2 in neuronal and non-neuronal cells was found to
enhance apoptosis, whereas transfection with a PS2 antisense construct rescued cells from apoptosis (22, 23, 39). Inhibitory effects on
apoptosis were also observed when a carboxyl-terminal portion of PS2
was expressed in cells (39, 40). Additionally, the PS2 N141I FAD
mutation was reported to confer enhanced basal activity for apoptosis
(22, 41) similarly to the L286V mutation in PS1 (42). (v) Both PS1 and
PS2 have been shown to be targets for caspase-mediated proteolytic
conversion (19-21). The mutant PS2-I might be even converted to a
higher extent as compared with the wild-type PS2 (20). The
corresponding PS fragments derived by caspase-mediated proteolysis were
detected in all tissues and stages of animal development (19, 21),
suggesting a role of PS processing in the cellular response to
apoptotic signals.
In this study, we present evidence that APP can be proteolytically
processed within its cytoplasmic domain by a caspase-like protease. The
involvement of caspase activity in APP processing was supported by the
following findings: (i) the induction of cellular apoptosis by chemical
agents such as staurosporine and doxorubicin, which are known to
activate the caspase cascade, or by the expression of PS2 and PS2-I
resulted in an increased proteolytic conversion (Fig. 2). (ii) APP
matches the consensus sequence for group III caspases (Fig. 1). (iii)
Mutation at the cleavage site of this canonical sequence completely
abolished proteolytic conversion (Fig. 4). (iv) Proteolysis is
inhibited by a caspase-specific inhibitor in a
concentration-dependent manner (Fig. 3, B and
C). (v) Activated caspase-8 recognizes APP in
vitro as a target molecule (Fig. 3D). Taken together,
the data strongly suggest a participation of caspase-like proteases in
the conversion of the APP cytoplasmic domain.
The caspase inhibitor profile shown here differs from that observed for
presenilins; the peptide inhibitor DEVD-CHO blocked the proteolytic
conversion of PS1 and PS2, suggesting the participation of a
caspase-3-like protease (19, 20). As shown in Fig. 3B, neither this inhibitor nor YVAD-CHO, which is specific for
caspase-1-like proteases, was effective at blocking the proteolytic
conversion of APP, making the involvement of group I and group II
caspases unlikely. However, zVAD-fmk was able to completely inhibit the conversion of APP. zVAD-fmk is a broad range inhibitor that blocks the
activation of all caspases including caspase-6, caspase-8, and
caspase-9 belonging to group III that recognize the consensus sequence
((IVL)ExD) (30-32). Such a motif is encoded within the APP carboxyl
terminus at residues 661-664 (VEVD; Fig. 1), making APP a likely
substrate for group III caspases. The latter conclusion is also
supported by two findings: (i) IETD-CHO, a more specific inhibitor for
group III caspases, was effective in blocking the conversion of APP
(Fig. 3B, lane 5); and (ii) the APP carboxyl-terminal domain
was more efficiently cleaved by a group III caspase (caspase-8) than a
group II caspase (caspase-3) in vitro (Fig. 3D).
However, it seems likely that other group III caspases, for example,
effector caspases such as caspase-6, contribute to the in
vivo conversion of the APP carboxyl terminus in cells.
Recent findings suggest that presenilins are targets for caspases and
that PS2 is capable of inducing programmed cell death. To study the
PS2-induced activation of caspases, both SPA4CT and PS2 were
coexpressed (Fig. 2C). The observed difference between PS2-
and PS2-I-transfected cells on the proteolytic conversion of SPA4CT is
consistent with the observation that cells overexpressing mutant PS2-I
are more prone to undergo apoptosis than wild-type PS2-expressing cells
(20, 22, 23, 41). Because APP and PS2 are known to interact and form
stable complexes (24, 43, 44), an alternative interpretation could be
that the carboxyl-terminal fragment of APP displays a different binding
capacity to PS2 versus PS2-I, causing the observed
dissimilarities. Such a difference was not observed; both PS2 and PS2-I
were equally able to form stable complexes with SPA4CT (data not
shown). However, it remains to be tested whether PS2 exerts its effects
on the apoptotic processing of APP as a APP/PS2 complex and whether the
PS mutations alter cellular routing or processing of APP in a
pathological manner. This could be accomplished by an analysis of cells
that do not express endogeneously encoded PS.
Two functional motifs have been described within the cytoplasmic domain
of APP: (i) a consensus sequence for internalization, and (ii) a site
for binding G0 proteins (Fig. 1). Removal of the internalization signal from the APP carboxyl terminus results in an
elevated secretion of APP into the extracellular space (45). In
agreement with those reports, increased levels of secretory APP are
observed in the conditioned media of APPdel664-transfected cells (data not shown).
As mentioned above, FAD mutations in the APP gene were found to induce
cellular apoptosis. Recently, this was shown to be dependent on a short
stretch within the cytosolic domain of APP: deletion of residues
His657-Lys676 of APP695 abolished
the apoptotic cell death provoked by FAD mutations. The same site has
been shown to be required for the induction of apoptosis by G-proteins
(7, 46). Correspondingly, a synthetic peptide comprising
His657-Lys676 activates G0 in
vivo (47). The ability of APP to activate G0 is
dependent on the continuous primary structure: synthetic peptides lacking the carboxyl-terminal part
Arg672-Lys676 were several times less potent in
activating G0 than peptides spanning the whole region from
His657-Lys676 (46). Because the proteolytic
conversion described here takes place at position Asp664,
the carboxyl-terminal part of the activating domain becomes cleaved in
apoptotic cells, which then results in an inability of APP to further
activate G0 proteins. Such a regulatory mechanism would
permit interruption of apoptotic stimuli and control of apoptosis. The
observation that cleavage of APP occurs very early in the course of
apoptosis (Fig. 5) may support such a hypothesis. Indeed, several
mechanisms have been discovered in which an initial apoptotic stimulus
does not cause cellular death but is controlled by antiapoptotic
proteins including members of the Bcl family, activation of nuclear
factor
B, or XIAP (reviewed recently in Ref. 48). The same mechanism
has also been proposed for PS2, which may either induce or increase
apoptosis but simultaneously serves as a target for caspases too (19,
20, 39, 40). Thus, apoptosis might be closely linked with
neurodegeneration not only in neuronal diseases such as amyotrophic
lateral sclerosis (49) or Huntington's disease (50) but also in
Alzheimer's disease.