From the Center for Molecular Biology Heidelberg
(ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, D-69120
Heidelberg, Germany, ¶ Departments of Biochemistry and Molecular
Biology, Merck Sharp and Dohme Research Laboratories, The
Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR,
United Kingdom, and
Adolf-Butenandt-Institut,
Ludwig-Maximilians-Universität München, Schillerstrasse 44, 80336 Munich, Germany
Received for publication, October 10, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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The final step in A Alzheimer's disease
(AD)1 is characterized
pathologically by the extracellular deposition of amyloid- In the present study we examined the cleavage site specificity of
intracellular Plasmid Construction--
Plasmids pBS/SPLEC99-WT, -I45F, and
-V50F were generated as described (38, 39). The
EcoRI/ClaI fragments of pBS/SPLEC99-WT, -I45F,
and -V50F were cloned into the vector pBS/SPDAC99-WT (36) containing
the two additional amino acids Asp and Ala instead of Leu and Glu, thus
resulting in the plasmids pBS/SPDAC99-WT, -I45F, and -V50F,
respectively. The KpnI/HindIII fragments of pBS/SPDAC99-WT, -I45F, and -V50F were cloned into the pCEP4 vector (Invitrogen) digested with KpnI and HindIII. The
dilysine motif at the C terminus of C99 (third and fourth amino
acid from the C terminus) was introduced via recombinant PCR, suitable
oligonucleotides, and pBS/SPDAC99-WT, pBS/SPDAC99-I45F, and
pBS/SPDAC99-V50F as PCR templates. The
KpnI/HindIII fragments of pBS/SPDAC99-WT KK, -I45F KK, and -V50F KK were cloned into the pCEP4 vector digested with
KpnI and HindIII to generate the expression
plasmids pCEP4/SPDAC99-WT KK, -I45F KK, and -V50F KK. The eukaryotic
expression vector pCEP4/APP695 SDYQRL was generated as described (40).
The APP695 SDYQRL insert was cloned in pBS/APP695 using the enzymes
BglII/ClaI. The EcoRI/ClaI fragment of pBS/APP695 SDYQRL was cloned in pBS/SPDAC99-WT digested with EcoRI/ClaI, resulting in pBS/SPDAC99-SDYQRL.
pCEP4/SPDAC99-SDYQRL was generated as described for pCEP4/SPDAC99-WT.
The identity of the constructs obtained by PCR was confirmed by DNA sequencing.
Cell Culture and Transfections--
Human SH-SY5Y neuroblastoma
cell line was maintained in Dulbecco's modified Eagle's medium (high
glucose) (Sigma) containing 10% fetal calf serum (PAA
Laboratories) and 1% nonessential amino acid solution (Sigma).
80% confluent cells were transfected with the expression vector pCEP4
(Invitrogen) alone or the pCEP4 vector carrying the SPDAC99 inserts
using Lipofectin (Invitrogen) as described by the producer. Stable
transfectants were selected using 300 µg/ml hygromycin (PAA
Laboratories). For each construct, at least two independent cell lines
were established.
Antibodies--
The following antibodies were used and diluted
for Western blot analysis as indicated: anti-calnexin (StressGen,
1:2500), anti- Preparation of Cell Lysates and Collection of Conditioned
Media--
Fresh culture medium (5 ml) was added to a confluent
monolayer of cells in a 10-cm culture dish. Conditioned media were
collected after 14-16 h. The conditioned media were centrifuged at
4 °C for 1 min at 13,000 rpm, and the supernatants were used for
immunoprecipitation of soluble secreted A Immunoprecipitation and Densitometric Quantification--
Equal
volumes of conditioned media and cell lysates were immunoprecipitated
with 20 µl of protein G-Sepharose (Sigma) and the antibodies W02,
G2-10, and G2-11, respectively (concentrations see above). The
immunoprecipitated proteins were separated on 12% Tris-Tricine gels
(45) or commercial 10-20% Tricine gels (Invitrogen). Western blot
analysis was performed with the antibody W02 (1 µg/ml) (42).
Densitometric quantification was performed using MACBAS 2 software.
Treatments with Organelle-specific Toxins--
Stably
transfected SH-SY5Y cells were preincubated with brefeldin A (BFA) (10 µg/ml, Sigma) or monensin (5 µM, Sigma) for 1.5 h
followed by a 10-h incubation period in the presence of the drugs in
fresh medium.
Metabolic Labeling and Preparation of Cellular
Membranes--
After 1.5 h of preincubation in methionine-free
minimum essential Eagle's medium (Sigma) in the presence or absence of
BFA (10 µg/ml), C99 WT-transfected SH-SY5Y cells were incubated for 6 h in methionine-free nonessential amino acid solution containing 5% fetal calf serum and 133 µCi/ml [35S]methionine
(Amersham Biosciences) in the presence or absence of BFA (10 µg/ml).
Cellular membranes were prepared as described by Mercken et
al. (46) and subjected to immunoprecipitation as described above.
Subcellular Fractionation and Sucrose Density
Centrifugation--
Subcellular fractionation and sucrose density
equilibrium centrifugation of SH-SY5Y cells stably expressing SPC99-WT
were performed essentially as described (43) with minor modifications. Postnuclear membranes were applied onto a continuous sucrose gradient (0.2-2 M) and 17 1-ml fractions were collected from the
bottom of the gradient after centrifugation overnight at 100,000 × gav (27,000 rpm, Beckman SW 28.1 rotor). Individual fractions were diluted into 5 mM HEPES,
pH 7.3, 0,15 M NaCl, and membranes were sedimented by
centrifugation for 1 h at 80,000 rpm in a MLA-80 rotor (Beckman).
The final membrane pellets were re-suspended in 200 µl of
phosphate-buffered saline, 5% glycerol and stored at Electrochemiluminescence Assay--
Equal aliquots of each
fraction were adjusted 2% CHAPSO and 0.5% CHAPSO for detection of
A Intracellular and Secreted A
Consistent with previously published data for non-neuronal cells (38),
both mutants (C99 I45F and C99 V50F) had an opposite effect on the
generation of the A
In contrast to the dramatic effect of both point mutations on the 42/40
ratio of secreted A Generation of A Brefeldin A Treatment Abolishes Intracellular A
The presence of BFA also dramatically affected the generation of A Monensin Increases Intracellular A
Using the A Generation of A
Furthermore, we could show that the A Subcellular Distribution of A
Using these organelle marker proteins we found that the The In the present study we show that point mutations close to the
Our finding that point mutations increasing The analysis of the intracellular sites of The identification of differences in generation is the cleavage
of the C-terminal 99 amino acid residues of the amyloid precursor
protein by
-secretase.
-Secretase activity is closely linked to
the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of
-secretase leading to the formation of secreted and intracellular
A
are identical, we made use of point mutations close to the
-cleavage site, known to have a dramatic effect on the 42/40 ratio
of secreted A
. We found that the selected point mutations only
marginally influenced the 42/40 ratio of intracellular A
, suggesting
differences in the
-secretase cleavage site specificity for the
generation of secreted and intracellular A
. The analysis of the
subcellular compartments involved in the generation of intracellular
A
revealed that A
is not generated in the early secretory pathway
in the human SH-SY5Y neuroblastoma cell line. In this study we
identified late Golgi compartments to be involved in the generation of
intracellular A
. Moreover, we demonstrate that the presence of
processed PS1 is not sufficient to obtain
-secretase processing of
the truncated amyloid precursor protein construct C99, proposing the
existence of an additional factor downstream of the endoplasmic
reticulum and early Golgi required for the formation of an active
-secretase complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide
(A
) in the brain (1, 2), derived by proteolytic processing from the amyloid precursor protein (APP) (3). APP processing by
-secretase BACE1 (
-site APP cleaving enzyme) generates the N terminus of A
,
releasing the ectodomain of APP (for review, see Ref. 4). The remaining
C-terminal membrane-bound fragment C99 is further cleaved by
-secretase, yielding two major species of A
peptides, A
40 and
A
42 (5-7). Alternatively, APP can be cleaved within its ectodomain
by
-secretases, identified as members of the ADAM family of
disintegrin metalloproteases, leading to
-secreted APP and a
truncated non-amyloidogenic peptide (p3) (for review, see Ref. 8). The
identity of
-secretase has been the subject of intensive
investigation; however, the exact nature of
-secretase has not been
definitively established. Recent studies implied that
-secretase
processing requires the presence of presenilin 1 (PS1) and PS2 (for
review, see Ref. 4). The PS holoproteins undergo highly regulated
endoproteolytic processing to yield N- and C-terminal fragments and are
thought to mediate
-secretase enzyme activity as part of a
multimeric high molecular weight complex (9, 10). It is still not
proven, however, whether PS itself is
-secretase or an essential
co-factor required for
-secretase enzyme activity, since other
proteins of the high molecular weight complex have been shown to
modulate
-secretase enzyme activity (11-13). Furthermore, a
discrepancy exists between the predominant subcellular localization of
PS1 in the endoplasmic reticulum (ER) and cis-Golgi
compartment (14-16) and PS1-dependent
-secretase
processing at or close to the plasma membrane where little PS1 seems to
be present (17). A
generated at or close to the plasma membrane is
rapidly secreted by cultured cells as well as in biological fluids and
aggregates into the characteristic extracellular protein deposits,
which are thought to be the cause of AD (for review, see Ref. 18).
Additionally, intracellular A
is generated and is discussed as
important in the pathogenesis of AD. Recent studies demonstrate an
intraneuronal accumulation of A
42, the predominant species of A
in senile plaques (19), in AD-vulnerable brain regions (20-22) as well
as in transgenic mice expressing mutant proteins that lead to a
familial form of Alzheimer's disease (23). Moreover, transgenic mice
show accelerated neurodegeneration without extracellular amyloid
deposition (24), indicating that intracellular amyloid-
peptides may
play a crucial role in the development of AD. Intracellular sites with
-secretase activity were identified in primary neurons, neuronal
cell lines, and peripheral cells (25-30). Evidence has been obtained
for the generation of A
42 within the ER (27-29, 31), where PS is
predominantly localized (14-16). However, it has also been reported
that A
42 is produced in different organelles later in the secretory
pathway and that the ER is not the major intracellular site of A
42
generation (26, 30, 32-34, 74). Additionally, recent work has
suggested that the A
42 generation in the ER may be independent of PS
(35). Therefore, the exact site of intracellular A
generation and
the subcellular compartments in which PS promotes A
generation
remain so far unclear. Furthermore, little is known about the cleavage site specificity of the
-secretase responsible for the generation of
intracellular A
, which may play a central part in the pathogenesis of AD.
-secretase as well as the subcellular compartments in
which the direct A
precursor C99 (36) is processed by
-secretase to intracellular A
. To address the cleavage site specificity of
-secretase we made use of point mutations close to the
-cleavage site known to have a dramatic effect on the 42/40 ratio of secreted A
(37, 38) and determined their effect on the 42/40 ratio of
intracellular A
. We found that these mutations only marginally influenced the 42/40 ratio of intracellular A
, indicating that the
cleavage site specificity of intracellular
-secretase is less
affected by point mutations close to the
-cleavage site. To
investigate the compartments involved in
-secretase processing, we
used different experimental strategies including treatments with
organelle-specific toxins and expression of C99 proteins directed to
different cellular compartments as well as subcellular fractionation.
Our results show that generation of intracellular A
was eliminated
when C99 was retained in the ER and early Golgi despite the presence of
processed endogenous PS1, indicating that PS1 is not sufficient to
mediate
-secretase processing in the early secretory pathway.
Instead, we obtained several lines of evidence that an active
-secretase complex is located in late Golgi compartments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP (Sigma, 1:500), anti-syntaxin 6 (Transduction
Laboratories, 1:500). PS1-FL and PS1-NTF were detected using antibody
98/1 raised against residues 1-20 of PS1 (1:2500) (41). The following
antibodies were used to detect APP, C-terminal fragments, and A
:
monoclonal W02, raised against residues 1-10 of A
(1 µg/ml) (42);
polyclonal R7334, raised against residues 659-694 of APP695 (1:750)
(43); monoclonal antibody 22C11, raised against residues 66-81 of APP (0.5 µg/ml) (44). Monoclonal antibodies W02 (5 µg/ml), G2-10, specific for A
ending residue 40 (12,5 µg/ml), and G2-11, specific for A
ending residue 42 (17,3 µg/ml) (42), were used for
immunoprecipitation, polyclonal antibody 98/1 was used for the
precipitation of PS1-NTF (5 µg/ml) (41), and polyclonal antibody
29414 was used for the precipitation of PS1-CTF (20 µl/ml) (provided
by C. Elle, Center for Molecular Biology, University of Heidelberg,
Heidelberg, Germany).
. In parallel to the
conditioned media, cell lysates were prepared. Cells were harvested and
lysed in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, and 2 mM EDTA) supplemented with protease inhibitor mixture
(Roche Molecular Biochemicals). The 13,000-rpm supernatants were used
for immunoprecipitation of detergent-soluble intracellular A
.
80 °C until
further use.
40 and A
42 peptides, respectively. Membrane-associated A
peptides were released by incubation for 20 min at 37 °C and
measured by an electrochemiluminescence assay as described (10, 47) in
a 96-well plate format. A
peptides were captured with biotinylated
monoclonal antibody 4G8 (Senetek) followed by detection of A
40 with
ruthenylated G2-10, whereas ruthenylated G2-11 was used for detection
of A
42. Nonspecific background signal was determined by using
non-biotinylated capture antibody 4G8 in the presence of
ruthenylated detection antibodies G2-10/G2-11 and subtracted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
42/40 Ratios Are
Differently Influenced by Point Mutations Close to the
-Cleavage
Site--
Point mutations close to the
-cleavage site of APP as
well as pathogenic mutations in the genes encoding PS1 and PS2 have been shown to alter the product ratio of A
42 and A
40 (A
42/40) (37-39, 48-50). However, in these studies, the authors analyzed the
42/40 ratios of secreted A
, which is generated via a
pH-sensitive and endocytosis-dependent pathway (17,
51-53). Far less is known about the cleavage site specificity of the
-secretase responsible for the generation of intracellular A
(A
i). To investigate the cleavage site specificity of intracellular
-secretase, we selected two point mutations close to the
-cleavage site (Fig. 1), known to have
a strong effect on the 42/40 ratio of secreted A
(A
sec42/40). These are the point mutations I45F and V50F, which have been identified by phenylalanine-scanning mutagenesis of the transmembrane domain of
APP (38).
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Fig. 1.
Schematic representation of wild-type and
mutant SPC99 proteins (I45F, V46F, and V50F) used for the study of
A i generation. SPC99 contains the signal
peptide (SP) of APP, two additional amino acids (Asp and
Ala), the A
domain (framed by black lines), and the
complete C terminus of APP. SPC99 only requires
-secretase activity
for A
generation; the N terminus of A
is produced by the activity
of the signal peptidase. Amino acids are shown in the single-letter
code. Protease cleavage sites are marked by black arrows.
Asterisks indicate the position of amino acid exchanges in
the SPC99 mutant proteins.
species secreted into the media of stably
transfected human SH-SY5Y neuroblastoma cells. C99 I45F is mainly
processed to secreted A
ending residue 42 (A
sec42) (Fig.
2A), resulting in a dramatic increase of
the A
sec42/40 ratio compared with C99 WT (relative ratio, 20.4 ± 3.6 (I45F), p < 0.001, n = 6) (Fig.
2C) (Table I). In
contrast, the V50F mutation is mainly cleaved after amino acid
residue 40 (Fig. 2A), causing a decrease of the A
sec42/40
ratio compared with C99 WT (relative ratio, 0.3 ± 0.1 (V50F),
p < 0.001, n = 6) (Fig. 2C) (Table I). As a negative control we used SH-SY5Y cells stably transfected with the expression vector alone measuring the amount of
endogenous secreted and intracellular A
(data not shown).
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Fig. 2.
Analysis of A 40 and
A
42 production in SH-SY5Y cells stably
transfected with the SPC99 WT and SPC99 mutant constructs (I45F and
V50F). A
40 and A
42 were immunoprecipitated from the
conditioned media (A) and cell lysates (B) using
the antibodies indicated above the gels (40, antibody G2-10, specific
for A
40; 42, antibody G2-11, specific for A
42). Both A
species
were detected by Western blot using antibody W02. C and
D, determination of A
sec42/40 and A
i42/40 ratios
relative to the A
42/40 ratios produced by the C99 WT protein.
A
sec42/40 and A
i42/40, respectively, were calculated by
densitometric quantification for each stably transfected cell line. The
determined A
42/40 values were divided by the corresponding A
42/40
ratios obtained for C99 WT in the same experiment. Thus, the values for
C99 WT were 1.0. Gray columns represent the mean values of 6 (A
sec) and 9 (A
i) independent experiments; black vertical
bars indicate the S.D. The asterisks show the
statistical significance (two-sided Student's t test)
relative to C99 WT (**, p < 0.01; ***,
p < 0.001). C, ratios of A
sec42/40
relative to the C99 WT construct. D, ratios of A
i42/40
relative to the C99 WT construct.
A42/40 ratios of intracellular and secreted A
, both mutations only marginally influenced the
42/40 ratio of intracellular A
(A
i42/40) (Fig. 2B).
The C99 WT protein produced roughly 1.5-fold more A
i40 than A
i42
(A
i42/40-WT, 0.65 ± 0.27, n = 9) (Table I),
indicating an elevated A
i42/40 ratio relative to the ratio of
A
sec42/40 (Table I), consistent with previously published data (27,
52, 54, 55). However, the point mutation at position 45 of A
that
dramatically influenced the 42/40 ratio of secreted A
by a factor of
20.4 compared with the WT-protein showed only a minor effect on the
42/40 ratio of intracellular A
. The ratio of A
i42/40 was
increased by a factor of 1.9 compared with C99 WT (relative ratio,
1.9 ± 0.5 (I45F), p < 0.01, n = 9) (Table I). A similar phenomenon was obtained for C99 V50F. Compared
with C99 WT, the A
sec42/40 ratio was reduced to 30%, whereas the
A
i42/40 ratio of C99 V50F was not significantly decreased compared
with the A
i42/40 ratio of the WT construct (relative ratio, 0.8 ± 0.5 (V50F), not significant, n = 9) (Table I),
indicating that both point mutations have a minor effect on the
cleavage site specificity of the
-secretase responsible for
intracellular A
generation. To verify our results that intracellular
-secretase is less affected by point mutations close to the
-cleavage site, we used C99 V46F, a familial Alzheimer's
disease-linked mutation at position 717 of APP770 (Fig. 1) (56). The
A
sec42/40 ratio of C99 V46F (A
sec42/40-V46F: 0.67 ± 0.17, p < 0.01, n = 7) was increased by a
factor of 3.1 relative to the WT protein (Table I), consistent with
previous findings (37, 38), whereas the A
i42/40 ratio was not
affected compared with the A
i42/40 ratio obtained for the WT protein
(A
i42/40-V46F: 0.66 ± 0.19, not significant, n = 3) (Table I), further confirming our results obtained for C99 I45F
and C99 V50F.
Is Inhibited by C99 Proteins Bearing an
ER/Intermediate Compartment (IC) Retrieval Signal--
To
analyze the subcellular compartments involved in A
generation, we
used different experimental strategies. To determine whether
-secretase processing can occur within the ER, we introduced a
dilysine motif at the C terminus of C99 (C99 KK) that leads to the
retention of proteins in the ER and IC (30, 57, 58). As shown in Fig.
3A the expression levels of
C99 KK proteins (C99 WT KK, C99 I45F KK, C99 V50F KK) were comparable
with the expression levels of C99 proteins without the ER/IC retrieval
signal (C99 WT, C99 I45F, C99 V50F) and did not affect the levels of
endogenous APP (Fig. 3A). However, significant amounts of
A
i could only be recovered from cell lysates of cells expressing C99
constructs without an ER/IC retrieval signal (C99 WT, C99 I45F, C99
V50F) (Fig. 3A). Cell lines expressing C99 proteins bearing
the dilysine motif did not produce A
i levels above the background
level of A
i produced from the endogenous APP, as verified by the
comparison with cells stably transfected with the expression vector
alone (Fig. 3A). These results clearly show that C99 has to
be transported out of the ER to get processed by
-secretase,
indicating that the ER is not a major intracellular site of
-secretase activity. This finding is supported by the observation
that A
secretion was blocked to endogenous background levels when
C99 was retained in the ER and IC (Fig. 3B), providing
evidence that A
was not generated in the ER and rapidly secreted
into the conditioned media.
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Fig. 3.
Addition of an ER/IC retrieval signal
abolishes the generation of A i and
A
sec. Western blot analysis of cell
lysates (A) and conditioned media (B) from
SH-SY5Y cells that were transfected with the expression vector alone
(control) or the indicated SPC99 constructs. Cell lysates
and conditioned media were immunoprecipitated with antibody W02. A
,
C99, and endogenous APP were detected in the Western blot using
antibody W02. A and B, respectively, represent
different exposure times of the same Western blot analysis.
Generation--
To verify our data that A
cannot be generated early
within the secretory pathway, we blocked protein transport using BFA. BFA blocks anterograde protein transport out of the ER, resulting in a
fusion of the proximal Golgi (cis- and
medial-Golgi) with the ER (59, 60). The efficacy of BFA
treatment was verified by different observations as follows (i) BFA
treatment affected the maturation of endogenous APP as seen by a
complete change of the observed band pattern of APP immunoreactivity
(Fig. 4A). (ii) The generation
of A
sec and
-secreted endogenous APP (sAPP
) was blocked in the
presence of BFA, whereas A
secretion could be detected in the
conditioned media of untreated cells (Fig. 4B). (iii) An
increase of endogenous APP was observed in cell lysates of treated
cells (Fig. 4C), indicating that the secretory pathway was
efficiently blocked by BFA treatment.
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Fig. 4.
Effects of brefeldin A treatment on
A generation, maturation of endogenous APP,
and endoproteolysis of PS1. SH-SY5Y cells stably transfected with
the indicated SPC99 constructs or the expression vector alone
(control) were preincubated with 10 µg/ml BFA for 1.5 h. After preincubation the cells were incubated for additional 10 h in fresh medium containing 10 µg/ml BFA. Control cells were
incubated in the absence of BFA. A, cell lysates of treated
and untreated cells, expressing C99 WT, were prepared and directly
loaded on a 8% Tris-Tricine gel. The immature (APP immat)
and mature (APP mat) isoforms of endogenous APP were
visualized by immunoblotting with antibody W02. B,
endogenous
-secreted APP (sAPP
) and A
secreted into
the conditioned media (A
sec) were
immunoprecipitated and detected by Western blot using antibody W02.
C, cell lysates were prepared after BFA treatment and
immunoprecipitated with the antibody W02. A
i, C99, and endogenous
APP were detected by immunoblotting with antibody W02. B and
C, respectively, represent different exposure times of the
same Western blot analysis. D, SH-SY5Y cells stably
transfected with C99 WT were metabolically labeled in the presence or
absence of BFA (10 µg/ml). Cellular membranes were prepared and
immunoprecipitated with the indicated antibodies. Precipitated proteins
were separated on a 12.5% SDS page. PS1-NTF and PS1-CTF were
visualized by autoradiography.
i.
A
i could be detected in untreated cells expressing C99 WT, I45F, and
V50F (Fig. 4C). However, A
i formation was strongly impaired in BFA-treated cells (Fig. 4C), indicating that
protein trafficking beyond the early secretory pathway is required for A
i generation and, thus, confirming our results obtained with C99 KK
mutant proteins. To verify that the lack of
-secretase processing in
BFA-treated cells is not simply caused by diminished endoproteolysis of
endogenous PS1, which seems to be important for PS function (61), we
analyzed the amount of N- and C-terminal PS1 fragments (PS1-NTF and
PS1-CTF, respectively) in cells treated or not treated with BFA.
Therefore, C99 WT-expressing cells were metabolically labeled in the
presence and absence of BFA, and cellular membranes were
immunoprecipitated with antibodies directed to the N or C terminus of
PS1. Processing of PS1 to its N- and C-terminal fragments was not
inhibited in the presence of BFA as shown in Fig. 4D,
indicating that the presence of processed PS1 is not sufficient to
obtain
-secretase activity.
Levels--
To investigate
further the intracellular compartments that are involved in A
i
generation, we incubated SH-SY5Y cells expressing the C99 WT protein or
the mutant proteins (C99 I45F, C99 V50F) in the presence or absence of
monensin. Monensin inhibits the maturation of newly synthesized
proteins in the trans-Golgi and blocks their transport out
of the trans-Golgi network (TGN) (62, 63). The analysis of
the conditioned media of treated and untreated cells showed that
secretion of A
as well as secretion of sAPP
(
-secreted
endogenous APP) was completely blocked in the presence of monensin
(data not shown), similar to treatment of cells with BFA. In contrast
to BFA, monensin treatment strongly increased A
i levels (Fig.
5A), indicating that late
Golgi compartments are involved in
-secretase processing of C99.
A
levels were increased by a factor of 3.0 in monensin-treated cells
stably expressing the C99 WT protein compared with untreated cells
(A
i + monensin/A
i
monensin, 3.0 ± 0.9 (WT),
p < 0.05; n = 4). Similar results were
obtained for C99 I45F and C99 V50F (A
i + monensin/A
i
monensin, 2.5 ± 0.4 (I45F), p < 0.05, n = 3; 2.9 ± 0.3 (V50F), p < 0.01; n = 3) (Fig. 5B).
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Fig. 5.
Effect of monensin treatment on
A i generation. SH-SY5Y cells stably
expressing the indicated SPC99 constructs were preincubated for
1.5 h with 5 µM monensin, and monensin treatment was
performed for an additional 10 h in fresh medium. A,
cell lysates of treated (+) and untreated (
) cells were
immunoprecipitated with the antibody W02 followed by Western blot
analysis using antibody W02. The figure represents different exposure
times of the same Western blot analysis as indicated by the black
line within the picture. B, quantification of at least
three independent experiments. A
i detected in the Western blot of
treated and untreated cells was quantified densitometrically. The
amount of A
i in the presence of monensin was divided by the amount
of A
i in the absence of monensin for each construct and each
experiment (A
i + monensin/A
i
monensin). Thus, the value
for untreated cells was 1.0 and was set to 100%. Gray
columns represent the mean values; black vertical bars
give the S.D. The asterisks indicate the statistical
significance (two-sided Student's t test) relative to
untreated cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001). C, SH-SY5Y cells
stably expressing C99 WT were incubated with monensin as described.
Equal volumes of cell lysates were immunoprecipitated (IP)
with the antibody G2-10, specific for A
40, and the antibody G2-11,
specific for A
42. The immunoprecipitated A
i40 and A
i42
peptides were detected with antibody W02 in the Western blot.
D, ratios of A
i42/40 after monensin treatment relative to
untreated cells of six independent experiments. For this, the same
graphical presentation was used as described under B.
40- and A
42-specific antibodies G2-10 and G2-11, we
showed that A
i40 and A
i42 were increased after monensin treatment
in C99 WT-expressing SH-SY5Y cells (Fig. 5C), indicating that late Golgi compartments (trans-Golgi, TGN) are involved
in cleavage at position 40 and position 42 of A
. However, A
i40 and A
i42 increased to a different extent, resulting in decreased A
i42/40 ratios in monensin-treated cells (A
i42/40 + monensin/A
i42/40
monensin = 0.49 ± 0.2;
p < 0.001; n = 6) (Fig.
5D).
by C99 Proteins Bearing a TGN-sorting
Signal--
The results obtained so far provide evidence that protein
transport to late Golgi compartments (trans-Golgi, TGN) is
essential for
-secretase processing of C99. To confirm these
results, we established SH-SY5Y cells, stably expressing a C99 protein,
bearing the sorting signal of TGN38 for recycling between the cell
surface and the TGN (C99 SDYQRL) (64, 65). It was previously shown that
the addition of the amino acid motif SDYQRL to the C terminus of APP
leads to an accumulation of APP in the TGN (34, 40). We found that
A
i levels were increased by a factor of 1.5 in cells stably
expressing the C99 SDYQRL construct compared with C99 WT (relative
ratio A
i/C99, 1.5 ± 0.3 (SDYQRL), p < 0.05, n = 7) (Fig.
6A), confirming the role of
the TGN in
-secretase processing of C99.
View larger version (16K):
[in a new window]
Fig. 6.
Generation of A from
a mutant C99 protein bearing a TGN-sorting signal. Cell lysates of
SH-SY5Y cells stably expressing the C99 WT protein or the mutant C99
protein combined with the TGN38 sorting signal (C99 SDYQRL) were
immunoprecipitated with antibody W02, G2-10, and G2-11, respectively.
The immunoprecipitated proteins were detected by Western blotting with
the antibody W02. The corresponding A
i and C99 bands were
densitometrically quantified. A, ratio of A
i/C99 relative
to the WT construct. Columns represent the mean values of
seven independent experiments; black vertical bars give the
S.D. The asterisks indicate the statistical significance
(two-sided Student's t test) relative to C99 WT (*,
p < 0.05). B, ratio of A
i42/40 relative
to the C99 WT construct. Columns represent the mean values
of four independent experiments. Black vertical bars
indicate the S.D., and asterisks give the statistical
significance (two-sided Student's t test) relative to C99
WT (*, p < 0.05).
i42/40 ratio was decreased in
C99 SDYQRL-expressing cells compared with the A
i42/40 ratio of the
WT construct (relative ratio, 0.7 ± 0.1 (SDYQRL), p < 0.05, n = 5) (Fig. 6B),
indicating that
-secretase cleaves preferentially at position 40 in
the TGN, consistent with our findings in monensin-treated cells.
i40, A
i42, and Endogenous
PS1--
To analyze the subcellular distribution of A
i40 and
A
i42 independent of sorting signals or drug treatments, membranous
organelles of SH-SY5Y cells transfected with C99 WT were separated by
sucrose density centrifugation. A
i40 and A
i42 were measured by a
highly sensitive electrochemiluminescence (ECL) assay (10, 47), whereas the corresponding fractions were probed by Western blotting for endogenous PS1, C99, C83 (
-cleaved C-terminal fragment), endogenous APP, and several well characterized organelle marker proteins. ER-rich
fractions were detected as expected at the bottom of the gradient using
an antibody against the ER marker protein calnexin (66).
Calnexin-reactive ER vesicles were most enriched in fractions 4-6
(Fig. 7A). Golgi-containing
fractions were identified by blotting for
-COP, a Golgi marker
protein (67, 68) most abundant in the early Golgi compartments, found
in fractions 12 and 13 (Fig. 7B). TGN-rich fractions were
defined by the TGN marker protein syntaxin 6 (69), enriched in
fractions 7-10 (Fig. 7C).
View larger version (23K):
[in a new window]
Fig. 7.
Subcellular fractionation of
A , PS1, and C99. Membranous organelles of
SH-SY5Y cells stably expressing C99 WT were separated by sucrose
density centrifugation. A, equal aliquots of individual
fractions were immunoblotted for calnexin, C99, and C83 as indicated.
The vertical bar indicates that the samples were loaded onto
two separate 10-20% Tris-Tricine gels, blotted onto the same
nitrocellulose membrane. As a standard (std) 1.0 ng of
recombinant C100FLAG (10) was loaded on each gel. Aliquots of
individual fractions were directly loaded on a 12% SDS-gel and
immunoblotted for
-COP and PS1 (PS1-FL and PS1-NTF) (B)
and on a 10% SDS-gel for the detection of endogenous immature and
mature APP (APP immat, APP mat) and syntaxin 6 (C). A, B, and C, the data
shown are representative of at least three independent experiments.
D, the individual fractions were analyzed by an ECL assay to
determine the subcellular distribution of A
i40 and A
i42. A
i
x-40 and A
i x-42 were detected by the use of the antibody
combination 4G8/G2-10 and 4G8/G2-11, respectively. The graph shows the
average of duplicate ECL measurements and is representative of at least
three independent experiments.
-secretase
substrates C99 and C83 accumulated in TGN-fractions, as defined by the
enrichment of the TGN marker protein syntaxin 6 (fractions 7-10) (Fig.
7, A and C). Exogenous expressed C99 was detected
to a smaller extent in ER fractions (fractions 4-6), defined by the
marker protein calnexin and the presence of immature endogenous APP
(APP immat) (Fig. 7, A and C). As
expected C83, the
-cleaved C-terminal fragment could not be detected
in the ER-rich vesicles (Fig. 7A), supporting previously
published data that
-secretase cleaves later within the secretory
pathway (70-72). PS1-NTF was detected across the whole sucrose
gradient; however, an accumulation of PS1-NTF as well as PS-FL was
observed in fractions 7-12 (Fig. 7B), overlapping with the
TGN marker protein syntaxin 6 (fractions 7-10) (Fig. 7C).
To detect all A
peptides ending at position 40 and 42 including
N-terminal-truncated A
species, for example Glu11-A
(73), we used
the antibody combinations G2-10/4G8 and G2-11/4G8, respectively.
A
i40 and A
i42 as measured by ECL are almost identically
distributed across the gradient (Fig. 7D). A peak for A
i
x-40 and A
i x-42 (intracellular A
peptides with variable N
terminus ending at residue 40 and 42, respectively) was observed in
fractions 7-9 (Fig. 7D), suggesting a TGN localization as
defined by the accumulation of the TGN marker protein syntaxin 6 in
fractions 7-10 (Fig. 7C). Neither A
i40 nor A
i42 could
be detected in fraction 5, which showed the strongest staining for the
ER marker protein calnexin (Fig. 7A). This excludes the
presence of significant amounts of A
i40 and A
i42 in the ER,
consistent with our findings that A
cannot be generated in the ER by
analyzing C99 KK mutant proteins. Additionally, A
i40 and A
i42
could not be detected in
-COP-rich fractions (fractions 12 and 13),
supporting our data that A
cannot be generated in early Golgi compartments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase is defined as proteolytic activity resulting in
cleavage of APP CTFs releasing the C terminus of A
and A
-like peptides (5-8). PS1 and PS2 have been found to be essential components of the high molecular weight complex mediating
-secretase activity (9, 10). The cleavage site specificity of the
-secretase enzyme
determines the ratio of A
40 and A
42. Pathogenic point mutations
in APP and presenilins increase the A
42/40 ratio of secreted A
(37, 48-50).
-cleavage site of APP have a minor effect on the cleavage site
specificity of intracellular
-secretase compared with their dramatic
effect on the cleavage site specificity of the
-secretase responsible for the generation of A
sec. The point mutations I45F and
V50F that revealed a dramatic effect on the A
sec42/40 ratio only
marginally affected the A
i42/40 ratio. The relevance of this
observation was confirmed by the use of the familial Alzheimer's disease mutant V717F of APP770 (56). Although secreted A
42 was
increased in the presence of the mutant, we observed no increase in
intracellular A
42 production, consistent with recent data (35).
Altered
-secretase cleavage site specificity may be caused by
different proteolytic activities for intracellular and secreted A
.
Wilson et al. (35) report that intracellular A
generation in the ER is independent of PS. However, PS-independent A
i42 production in the early secretory pathway is unlikely to be involved in
intracellular A
generation in the human SH-SY5Y neuroblastoma cells,
because we found late Golgi compartments responsible for intracellular
A
generation. Thus, it is not unlikely that presenilins are involved
in intracellular A
generation, and cleavage site specificity may be
affected by different co-factors of the high molecular weight
PS-containing complexes (9, 10). Alternatively, minor changes present
in subcellular compartments like lipid composition (75, 76), protein
glycosylation, or other modifications of
-secretase complex proteins
(11, 13, 77, 78) may be sufficient to modify cleavage-site specificity.
-cleavage at position 42 have a more pronounced effect on secretory A
compared with
intracellular A
may indicate that familial Alzheimer's
disease-linked mutant proteins initiate accumulation of A
42 in the
extracellular A
pool and that intracellular A
production has a
minor role in the pathogenesis of AD. However, we cannot exclude that
slightly elevated intracellular A
42 levels or additional mechanisms
that we did not observe in our in vitro cell culture system
may be sufficient to start A
aggregation. Indeed, intracellular
A
42 induces neurotoxicity in primary rat and human neurons (79, 80).
Moreover, studies on AD-vulnerable brain regions as well as animal
models expressing familial Alzheimer's disease-related mutant proteins
implicated intraneuronal accumulation of A
42 (20, 22, 23).
-cleavage shows that
intracellular A
generation was inhibited when cells were treated
with BFA or were stably transfected with constructs bearing an ER/IC
retrieval signal despite the presence of processed PS1. These results
are consistent with previously published data that A
i generation was
inhibited after BFA treatment in the mouse neuroblastoma cell line N2a
and COS1 cells as well as in different cell lines transiently
transfected with truncated APP constructs bearing an ER/IC retrieval
signal, including kidney 293 cells and N2a cells (26, 30, 32, 34).
However, A
42 generation was also found within the ER using very
similar approaches (27, 29, 31). A
42 generation within the ER seems
to be more abundant in differentiated neurons as shown for rat primary
neurons (28) and differentiated NT2N cells (27, 31, 81) derived from the human embryonal carcinoma cell line NT2 (82). Interestingly, the
less differentiated NT2 cells fail to produce intracellular A
(54).
However, a neuronal phenotype cannot explain the different results
obtained for NT2N cells infected with SFV/APP695 KK (27) versus primary cultures of mixed cortical neurons infected
with SFV/APP-C99-KK (17). When we take into account that even very closely related cell culture lines or primary cells show significant differences in
-secretase 42 activity in different subcellular compartments, it appears that intracellular A
42 generation can be
variable between different cell types. However, our data clearly show
that the ER/early Golgi is not the major intracellular site for
-secretase processing in the human SH-SY5Y neuroblastoma cells. Our
findings are further confirmed by our subcellular fractionation studies. Neither A
i40 nor A
i42 accumulation was detected in the
ER- and early Golgi-containing fractions. Moreover, we obtained several
lines of evidence that late Golgi compartments are involved in
-secretase processing. First, A
i levels were dramatically increased when we blocked protein transport late in the secretory pathway using monensin, consistent with studies proposing the involvement of late Golgi compartments in
-secretase processing (26,
32, 34, 84). Second, A
i generation was increased in cells expressing
a C99 mutant directed to the TGN. Third, subcellular fractionation of
organelles from C99 WT-transfected SH-SY5Y cells revealed that A
i40
and A
i42 accumulated in TGN-rich fractions. Interestingly, we found
that PS1 and the direct
-secretase substrate accumulated in TGN
fractions as well, indicating the possibility of an interaction between
PS1 and C99, resulting in
-secretase processing in these
compartments. These results are supported by findings that PS1 forms
complexes with APP C-terminal fragments in Golgi- and TGN-rich
fractions and that de novo A
i generation was found in the
same Golgi-/TGN-rich vesicles (85). In accordance to our ER-related
data we conclude that the presence of processed PS1 is not sufficient
to obtain
-secretase processing and that at least one additional
factor is required for the formation of an active
-secretase complex
in late Golgi compartments. Such additional factors could be the
transmembrane proteins aph-1, pen-2, and nicastrin that have been
recently shown to be essential
-secretase complex components (11,
13, 77, 78). Interestingly, the fully mature glycosylated form of
nicastrin preferentially interacts with PS1 (86). In contrast, only a
small proportion of PS1 is bound to the immature species of nicastrin,
indicating that mature nicastrin may be essential for the formation of
a functional
-secretase complex (86) and, thus, confirming our results that an active
-secretase complex is formed in late Golgi compartments where fully glycosylated mature nicastrin is present.
-secretase cleavage site
specificity for intracellular and secretory A
generation shows that
the
-secretase system is even more complex than previously assumed.
This may have important implications for AD. It shows that at least
partly different
-secretases exist. Because
-secretase cleaves a
number of different substrates, like Notch (for review, see Ref. 8) and
low density lipoprotein receptor-related protein (LRP) (83),
this finding may help to target A
processing in a manner avoiding
cross-inhibition with other
-secretase substrates.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Culvenor and C. Elle for providing the antibodies 98/1 and 29414.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and European Community Grant QLK 3-CT-2002-00172.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed. Tel.: 49-6221-546848; Fax: 49-6221-545891; E-mail: h.grimm@mail.zmbh.uni-heidelberg.de.
** To whom correspondence may be addressed. Tel.: 49-6221-546844; Fax: 49-6221-545891; E-mail: tobias.hartmann@zmbh.uni-heidelberg.de.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M210380200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid-
peptide;
A
i, intracellular A
;
APP, amyloid precursor protein;
C99, C-terminal 99 amino acid residues of
APP;
PS 1/2, presenilin 1/2;
PS1-FL, full-length presenilin;
NTF, N-terminal fragment;
CTF, C-terminal fragment;
ER, endoplasmic
reticulum;
IC, intermediate compartment;
BFA, brefeldin A;
TGN, trans-Golgi network: CHAPSO,
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic
acid;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
WT, wild type;
KK, ER/IC retrieval signal.
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