From the Department of Neuropathology and
Neuroscience, Graduate School of Pharmaceutical Sciences, University of
Tokyo, 7-3-1 Hongo Bunkyoku, Tokyo 113-0033, Japan and the
¶ Department of Pharmacology, Saitama Medical School, Moroyama,
Saitama 350-0495, Japan
Received for publication, August 31, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Increased production of amyloid Alzheimer's disease
(AD)1 is a progressive
dementing disorder characterized pathologically by a massive loss of
cortical neurons and an accumulation of two types of fibrillar lesions:
i.e. amyloid deposits composed of amyloid Although there is ample evidence that mutations in PS genes increase
the production of A Construction of Expression Plasmids--
A cDNA
encoding the C- terminal 99 amino acids of
cDNAs encoding C-terminally truncated C100 (i.e.
C100/stop68, C100/stop56, and C100/stop52) were generated by polymerase
chain reaction, using the following oligonucleotides as polymerase
chain reaction primers: 5'-TTTAAGCTTCCACCATGGCGCAGTTCCTG-3' as a
sense primer, 5'-TTTTCTAGACTAGTCAACCTCCAC-3' as an antisense primer for C100/stop68, 5'-CCCTCTAGACTACTGTTTCTTCTT-3' as an antisense primer
for C100/stop56, and 5'-CCCTCTAGACTACAGCATCACCAA-3' as an
antisense primer for C100/stop52. C-terminally truncated C100 peptides
were amplified by polymerase chain reaction and ligated into
pcDNA3.1-Hygro vector similarly as with C-terminally tagged C100.
C-terminally truncated Cell Culture, Transfection, and Caspase Inhibitor
Treatment--
Mouse neuro2a (N2a) neuroblastoma cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin at 37 °C in a 5%
CO2 atmosphere as described (32, 43, 44). Transient
transfection of C100 cDNAs into N2a cells and co-expression of C100
cDNAs in N2a cells stably expressing human PS2 cDNAs (43) were
performed using LipofectAMINE (Life Technologies, Inc.) according to
the manufacturer's instructions. Expression of transfected proteins
was enhanced by treatment with 10 mM butyric acid for
24 h prior to harvesting cells or culture supernatants. For the
inhibition of caspase activities, cells were treated with a 100 µM concentration of a pancaspase inhibitor, zVAD-fmk, for
24 h prior to analysis by Western blotting.
Immunoblot Analysis of Immunofluoresence Microscopy--
Transiently transfected N2a
cells were cultured on glass coverslips for 48 h. Cells were fixed
by incubation with phosphate-buffered saline (10 mM
phosphate buffer, pH 7.4) containing 4% paraformaldehyde for 30 min at
room temperature and then permeabilized and blocked with PBS-TB
(phosphate-buffered saline containing 150 mM NaCl, 0.1%
Triton X-100 and 3% bovine serum albumin) for 30 min at room temperature. Coverslips were then incubated with primary antibodies (a
rabbit polyclonal antibody, C4, against the C terminus of Quantitation of A Subcellular Fractionation and Differential
Extraction--
Subcellular fractionation was performed using
iodixanol as medium according to the previously described method (39)
with some modifications. N2a cells stably expressing either WT PS2 or
N141I mt PS2 and transiently transfected with C100 cDNAs were grown
on three 10-cm dishes. Cells were scraped in TS and resuspended in 4 ml
of homogenization buffer (10 mM HEPES (pH 7.4), 1 mM EDTA, 0.25 M sucrose, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 µg/ml pepstatin A, and 0.25 mM
phenylmethylsulfonyl fluoride). Cells were disrupted by a Polytron
homogenizer (Hitachi) at power level 2 for 10 s, and nuclei and
large cell debris were pelleted by centrifugation at 1500 × g for 10 min. The postnuclear supernatants were centrifuged
for 1 h at 65,000 × g. The resultant vesicle pellets (i.e. microsomal fractions) were resuspended in 0.8 ml of homogenization buffer. Gradients were set up in 13-ml Beckman SW41 centrifuge tubes by diluting iodixanol with homogenization buffer:
2.5%, 1 ml; 5%, 2 ml; 7.5%, 2 ml; 10%, 2 ml; 12.5%, 0.5 ml; 15%,
2 ml; 17.5%, 0.5 ml; 20%, 0.5 ml; 30%, 0.3 ml (iodixanol concentration/volume). The resuspended vesicle fractions were loaded on
the top of the gradients and centrifuged in a SW41 rotor at 40,000 × rpm for 2.5 h. The resulting gradients were collected in 1-ml
fractions. For the differential extraction of C-terminally truncated
C100, microsomal fractions of N2a cells transiently expressing
C100/WT, C100/stop68, C100/stop56, and C100/stop52 prepared as above
were extracted by 0.5 M Na2CO3 (pH
11.0) or 1% Triton X-100, and solubilized proteins in supernatants
after centrifugation at 100,000 × g for 15 min and
insoluble pellets were analyzed by Western blotting with BAN50.
Effects of FAD Mutant PS2 on A
We then expressed C100/ER, C100/TGN, or C100/WT in N2a cells that
stably express WT or N141I or M239V FAD mt PS2 and examined the
production of secreted or intracellular A
To verify the intracellular production of A
We next analyzed the relationship between the subcellular localization
of PS2 and the intracellular production site of A
To examine if the conclusions drawn from experiments using tagged C100
above are applicable to full-length Expression of C-terminally Truncated C100 and Effects of
Co-expression of mt PS2 on A
When transfected transiently in N2a cells, each C100 derivative was
expressed as ~6-13-kDa polypeptides of corresponding sizes on
Western blots. Notably, C100/stop68 co-migrated with the ~8-kDa band
derived from C100 (Fig. 4B), and the ~8-kDa band
diminished upon treatment of cells expressing C100 with a caspase
inhibitor zVAD-fmk (Fig. 4C), suggesting that the latter
polypeptide is cleaved by caspase from C100. Every C100 derivative was
solubilized by 1% Triton X-100, but not by
Na2CO3 (pH 11.0), suggesting that all types of
C-terminally truncated C100 were inserted into membranes (Fig.
4D). By immunocytochemistry with BAN50 that reacts with the
extracellular portion of C100, C100/wt, or C100/stop68, -56, or
-52 showed similar meshwork-like staining patterns accentuated in perinuclear areas, suggesting the ER/Golgi localization of these
C100 derivatives (Fig. 4E). We then quantitated A
We then transiently transfected cDNAs coding for C-terminally
deleted C100 in N2a cells stably expressing WT or N141I or M239V mt PS2
and quantitated A In this study, we showed that (i) TGN is one of the major
intracellular sites in which a secretable pool of A The intracellular site of A Our data indicating that TGN harbored elevated levels of
"secretable" A We and others have shown that co-expression of C100 that lacks the
majority of the extracellular domain of Finally, we have shown that the cytoplasmic domain of C100 is
dispensable for the abnormal effects of mt PS2 to increase production of A peptides
ending at position 42 (A
42) is one of the pathogenic phenotypes
caused by mutant forms of presenilins (PS) linked to familial
Alzheimer's disease. To identify the subcellular compartment(s)
in which familial Alzheimer's disease mutant PS2 (mt PS2) affects the
-cleavage of
APP to increase A
42, we co-expressed the
C-terminal 99-amino acid fragment of
APP (C100) tagged with sorting
signals to the endoplasmic reticulum (C100/ER) or to the
trans-Golgi network (C100/TGN) together with mt PS2 in N2a
cells. C100/TGN co-transfected with mt PS2 increased levels or ratios
of intracellular as well as secreted A
42 at similar levels to those
with C100 without signals (C100/WT), whereas C100/ER yielded a
negligible level of A
, which was not affected by co-transfection of
mt PS2. To identify the molecular subdomain of
APP required for the
effects of mt PS2, we next co-expressed C100 variously truncated at the
C-terminal cytoplasmic domain together with mt PS2. All types of
C-terminally truncated C100 variants including that lacking the entire
cytoplasmic domain yielded the secreted form of A
at levels
comparable with those from C100/WT, and co-transfection of mt PS2
increased the secretion of A
42. These results suggest that (i) late
intracellular compartments including TGN are the major sites in which
A
42 is produced and up-regulated by mt PS2 and that (ii) the
anterior half of C100 lacking the entire cytoplasmic domain is
sufficient for the overproduction of A
42 caused by mt PS2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptides
(A
) and tau-rich paired helical filaments (1). A
is produced from
-amyloid precursor proteins (
APP) through sequential cleavages by
proteases originally termed
- and
-secretases (1, 2);
-secretase has recently been identified as a novel aspartyl
protease, BACE (3-5). Deposition of
-amyloid is considered to be
closely related to the pathogenesis of AD because (i) deposition of
A
is a neuropathological change relatively specific to AD; (ii) the
diffuse type of senile plaque composed of highly aggregable A
42
species (6, 7), as opposed to A
40 that comprises the major portion
of the secreted form of A
(8, 9), is the initial lesion of AD
pathology; and (iii) mutations in genes coding for
APP (10-14) or
presenilin 1 (PS1) (15) or 2 (PS2) (16) are linked to some pedigrees of
autosomal dominantly inherited familial AD (FAD), and these mutations
increase the production of A
42 species (12-14, 17-20). Mutations
in PS genes that code for multipass integral membrane proteins account
for the majority of early onset FAD. Studies in knockout mice or
invertebrates demonstrated that PS is involved in
-cleavage of
APP (2, 21, 22) as well as in site 3 cleavage of the Notch receptor
(2, 23-25), both of which occur within the membrane or at the junction
with cytoplasm, although it has not been clear if PS is a co-factor for
-cleavage or if PS is identical to
-secretase. However, recent
data showing that transition state analogue
-secretase inhibitors
directly and exclusively bound fragment forms of PS strongly support
the hypothesis that PS represents the catalytic subunits of
-secretase (26-28).
42 (29-32), the intracellular compartment(s) in
which mutant forms of PS interact with
APP and promote
-cleavage at the A
42 position has not been clearly identified. Generation of
intracellular A
42 has been shown to occur in endoplasmic reticulum (ER) of cultured neurons (33, 34) or in human embryonic kidney 293 cells (35), whereas the trans-Golgi network (TGN) (36) or
endocytic pathway (37, 38) also are implicated in the generation of
secretable A
42. Although the ER localization of PS dovetails with
the former data, others have suggested that Golgi may be related to the
abnormal effect of mt PS1 to increase A
42 (39, 40). Furthermore,
subdomains in
APP proteins that are required for this interaction
with mt PS to increase production of A
42 have not been definitively
identified. In this study, we studied the intracellular compartment and
intramolecular subdomain of
APP that are relevant to the abnormal
effects of mutant PS2 to affect
-cleavage and increase production of
A
42. For these purposes, we expressed modified forms of a C-terminal
fragment of
APP tagged with targeting signals to specific
compartments or harboring deletion of defined cytoplasmic subdomains.
We show here that TGN and other late intracellular compartments are the
major sites where mt PS2 up-regulates A
42 production and that the
cytoplasmic domain of
APP is dispensable for the overproduction of
A
42 caused by mt PS2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
APP fused to a signal
peptide for rat preproenkephalin cDNA (C100) was previously
described (32, 41). C100 peptides tagged with sorting signals to ER or
TGN were generated using a C100 cDNA constructed in mammalian
vector p91023 as a template. Briefly, oligonucleotides encoding the rat
preproenkephalin signal peptide were used as a sense polymerase chain
reaction primer: 5'-TTTAAGCTTCCACCATGGCGCAGTTCCTG-3'. The following
oligonucleotides encoding the last four C-terminal amino acid residues
of C100 (i.e. QMQN) followed by the signal sequences KKLN
(for ER) or SDYQRL (for TGN) (42) were used as antisense polymerase
chain reaction primers: 5'-CCCGGATCCCTAATTCAGATTATTGTTCTGCATCTG-3' for C100/ER, 5'-CCCGGATCCCTAGAGCCGCTGATAATCGAAGTTCTGCATCTG-3' for
C100/TGN, and 5'-AAAGGATCCCTAGTTCTGCATCTG-3' for C100 (without signal
motif). Amplification of cDNAs was performed using PfuTurbo DNA
polymerase (Strategene). Amplified DNA fragments were digested with
HindIII and BamHI and ligated into a
pcDNA3.1-Hygro vector (Invitrogen).
APP695 tagged with KKLN or
SDYQRL motifs were constructed by ligating the
EcoRI/XbaI fragments of tagged C100 with those of
APP695 cDNA in pcDNA3.
APP695 cDNAs were constructed by ligating
the EcoRI/XbaI fragments of tagged C100 with
those of
APP695 cDNA in pcDNA3.
APP or PS2 Derivatives and
Cell-associated A
--
Cells were lysed in 2% SDS sample buffer
and briefly sonicated. Samples were separated by SDS-polyacrylamide gel
electrophoresis using a Tris-Tricine gel system, transferred to
polyvinylidene difluoride membrane (Millipore Corp.), and probed with
monoclonal antibodies BAN50 (specific for human A
1-16) for the
detection of C100 derivatives. A rabbit polyclonal antibody anti-G2N4
raised against a recombinant protein corresponding to the N-terminal residues 2-59 of human PS2 was used to probe PS2 and its derivatives. For the detection of A
in RIPA-soluble fractions of cell lysates, samples were initially lysed in RIPA (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate), and the supernatants after centrifugation at 15,000 × g for 5 min were immunoprecipitated by BAN50 using
protein G-agarose and then analyzed by immunoblotting with BA27, BC05,
or BAN50, using previously described procedures (44, 45, 47).
Extraction of cell-associated A
by formic acid was performed as
described (46). Briefly, cell pellets confluently grown in two 10-cm
dishes were solubilized by ultrasonication followed by incubation in
100 µl of 70% formic acid at room temperature for 30 min.
Supernatants after centrifugation at 100,000 × g for
20 min were desiccated and then solubilized in 100 µl of SDS sample
buffer. Samples containing A
were separated by SDS-polyacrylamide
gel electrophoresis, transferred to nitrocellulose membranes, and
reacted with antibodies after boiling (44, 45, 47). The immunoblots
were developed using an ECL system (Amersham Pharmacia Biotech) or
Immunostar (Wako Pure Chemicals).
APP (48)
and monoclonal antibodies specific for BiP or adaptin-
) for 2 h
followed by an incubation with a mixture of fluorescein isothiocyanate-conjugated anti-rabbit IgG and Texas Red-conjugated anti-mouse IgG antibodies in PBS-TB for 1 h and then mounted in PermaFlour aqueous mounting medium (Immunon) and viewed with a confocal
microscope (Fluoroview, Olympus, Tokyo) as described (43). BAN50 and
fluorescein isothiocyanate-conjugated secondary antibody were used for
single immunofluorescence detection of C100 derivatives.
by Two-site ELISAs--
Two-site ELISAs
that specifically detect the C terminus of A
were used as described.
BAN50, which was used as a capture antibody, binds only human
APP or
A
and does not cross-react with rodent A
or with an N-terminally
truncated fragment (e.g. p3). BA27 and BC05 that
specifically recognize the C terminus of A
40 and A
42,
respectively, were conjugated with horseradish peroxidase and used as
detector antibodies. Culture medium was collected after an appropriate
incubation period (48 h) and subjected to BAN50/BA27 or BAN50/BC05
ELISAs as described (32, 43, 44). Cell-associated A
was quantitated
after solubilization in 1% Nonidet P-40. ELISA data were statistically
analyzed by analysis of variance using StatView-J.4.11.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Production from
APP C100
Targeted to ER or TGN--
To identify the intracellular compartments
where A
, especially A
42, is generated and destined to be
secreted, we transiently expressed cDNAs coding for the C-terminal
99 amino acids of human
APP harboring a signal peptide at the N
terminus (C100) or C100 tagged with sorting signals for retention to ER
(C100/ER) or for recycling to TGN (C100/TGN) tagged at the C terminus
in mouse N2a cells (Fig. 1,
A-C). C100, C100/ER, or C100/TGN was expressed as a major
~13-kDa and a minor ~8-kDa polypeptide on immunoblots, the latter
corresponding to fragments cleaved by caspases (see below), and the
banding patterns were similar between C100 with and without sorting
signals (Fig. 1B). Immunocytochemistry by C4 (against the
cytoplasmic tail of
APP (48)) combined with anti-BiP antibody that
specifically reacts with a KDEL sequence (ER marker) or
anti-adaptin-
antibody (TGN marker) showed retention of C100/ER in a
meshwork like pattern overlapping with an immunolabeling with the ER
marker (Fig. 1C, top left), whereas
immunoreactive pattern for C100/TGN completely overlapped with that for
adaptin-
(Fig. 1C, middle right),
suggesting proper localization of C100 variants at the intended sites.
C100/WT showed a combined ER and TGN localization (Fig. 1C,
lower panels). We then quantitated A
-(1-40) and A
-(1-42) secreted from N2a cells expressing
C100 variants by two-site ELISAs using BAN50 as a capture antibody, which specifically detects human A
but not endogenous murine A
(Fig. 1D). Cells transfected with C100/TGN secreted ~2000
pM A
-(1-40) and ~200 pM A
-(1-42),
which were at comparable levels with those secreted from cells
expressing C100/WT. In contrast, C100/ER did not secrete detectable
levels of A
-(1-40) or A
-(1-42) as in N2a cells transfected with
an empty vector. These results suggested that TGN would be the major
intracellular site in which
-cleavage to yield A
-(1-40) as well
as A
-(1-42) that are destined for secretion takes place.
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Fig. 1.
Expression of APP
C100 harboring sorting signals to ER or TGN and A
secretion in N2a cells. A, schematic depiction of
C100 tagged with sorting signals to ER (C100/ER) or
trans-Golgi network (C100/TGN). C100/WT is C100 without a
sorting signal. The shaded area represents A
flanked by cleavage sites for
- and
-secretases
(arrows). B, Western blot analysis of C100
harboring sorting signals expressed in N2a cells with BAN50. An
arrow indicates the holoprotein of C100.
mock, cells expressing an empty vector alone. Molecular
markers are shown in kilodaltons. C, double fluorescence
immunocytochemistry of N2a cells expressing C100/ER, C100/TGN, or
C100/WT labeled by C4 (probe for C100; green) and anti-BiP
(ER marker, left lane; red) or
anti-adaptin-
(Ad
, TGN marker, right lane;
red) and viewed with a confocal microscope. Areas visualized
in yellow represent co-localization of C100 and ER or TGN
markers. Transfected cDNAs and primary antibodies are shown to the
left of and above the panels,
respectively. Scale bar, 10 µm. D,
levels of secreted A
-(1-40) (open column) and
A
-(1-42) (closed column) quantitated by
two-site ELISAs. Mean values ± S.E. in four independent
experiments are shown. Transfected C100 cDNAs are shown
below the columns.
. N2a cells expressing WT
PS2 transiently transfected with C100/WT or C100/TGN secreted similar
levels of A
-(1-40) and A
-(1-42), whereas those with C100/ER did
not secrete detectable levels of A
, in almost identical patterns to those observed in cells without exogenous PS2. In contrast,
cells expressing mt PS2 transiently transfected with C100/WT or
C100/TGN secreted larger amounts of A
-(1-42) compared with
A
-(1-40), whereas those with C100/ER did not secrete detectable A
(Fig. 2A). These data
suggest that TGN or later intracellular compartments, but not ER, are
the intracellular site where mt PS2 affects
-cleavage of
APP to
promote secretion of A
42 in N2a cells.
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Fig. 2.
Effects of FAD mutant PS2 on
A production from
APP
C100 targeted to ER or TGN. A, levels of A
-(1-40)
(open column) and A
-(1-42) (closed
column) secreted from N2a cells stably expressing WT PS2
(left), N141I (middle), or M239V
(right) FAD mt PS2 and transiently transfected with C100
with targeting signals (ER, C100/ER; TGN,
C100/TGN; wt, C100/WT) quantitated by two-site ELISAs. Mean
values ± S.E. in four independent experiments are shown.
Transfected C100 cDNAs are shown with lines
below the columns. B,
immunoprecipitation/Western blot analysis of RIPA-extractable
cell-associated A
(A
-(1-40), middle panel;
A
-(1-42), lower panel) and C100
(upper panel) in cells stably expressing WT
(left), N141I (middle), or M239V
(right) mt PS2 and transiently transfected with C100 with
targeting signals. Transfected C100 cDNAs are shown in each
lane. Proteins were first immunoprecipitated by BAN50 and
then probed by Western blotting with BA27 (for A
-(1-40)), BC05 (for
A
-(1-42)), or BAN50 (for C100), respectively. Molecular mass
markers are shown in kilodaltons. C, Western blot analysis
of formic acid-extracted A
40 by BA27 (middle
panel) and A
42 by BC05 (lower
panel) in cells overexpressing C100/ER (middle
lane; ER), C100/WT (right
lane; wt), or mock-transfected cells
(left lane; mock). Expression of C100
in cell lysates probed by BAN50 is shown in upper
panel. D, iodixanol density gradient
fractionation of N2a cells stably expressing WT (upper
two panels) or N141I FAD mt (middle
two panels) PS2 transiently transfected with
C100/WT. Each fraction was analyzed by Western blotting with anti-G2N4
(for full-length and N-terminal fragments of PS2), BAN50 (for
C100), anti-adaptin-
(marker for TGN), or anti-BiP (marker for ER).
The numbers of the fractions are shown above. Molecular mass
markers are shown in kilodaltons. E, levels of
cell-associated A
-(1-42) in N2a cell fractions stably expressing WT
(open column) or N141I FAD mt (closed
column) PS2 and transiently transfected with C100/WT
quantitated by two-site ELISAs. Mean values ± S.E. of four
independent experiments are shown. The numbers of the fractions are
indicated below the columns in the order shown in
D.
-(1-40) and A
-(1-42)
from C100 with or without sorting signal motifs, we analyzed RIPA-extracted lysates of N2a cells expressing C100/WT, C100/ER, or
C100/TGN together with WT or N141I mt PS2 by Western blotting with
antibodies to
APP (i.e. BAN50 against the A
N
terminus) or with those against C termini of A
after
immunoprecipitation with BAN50 (Fig. 2B). C100/WT, C100/ER,
or C100/TGN were expressed as ~13-kDa as well as ~8-kDa
polypeptides as observed without co-expression of PS2. In N2a cells
co-expressing WT PS2, C100/TGN yielded a ~4-kDa polypeptide positive
for A
40 as well as an equally intense A
42-positive ~4-kDa
polypeptide, in a similar pattern to those observed with C100/WT. In
N2a cells expressing N141I or M239V mt PS2, however, C100/WT and
C100/TGN yielded comparable levels of A
42-positive 4-kDa bands,
whereas only trace amounts of A
40-positive bands were detected in
cell lysates, despite robust expression of C100 and its derivatives. In
contrast, detectable levels of A
40 or A
42-positive polypeptides
were not observed in Western blots of cell lysates expressing C100/ER
together with WT or N141I or M239V mt PS2. To confirm the lack of
detectable cell-associated A
in cells expressing C100/ER, we
solubilized N2a cells expressing C100/ER or C100/WT in 70% formic acid
and analyzed the extracted proteins by Western blotting with BA27 and
BC05. Comparable levels of A
40-positive and A
42-positive ~4-kDa
proteins were detected in cells expressing C100/WT, whereas no
A
-positive bands were detectable in cells expressing C100/ER despite
robust expression of C100/ER holoprotein (Fig. 2C),
suggesting that the levels of formic acid-extractable ER-associated
A
are very low, if any, in our N2a cells overexpressing C100 derivatives.
42. To this end, we
expressed C100/WT together with WT or N141I mt PS2, separated the cells
by iodixanol density fractionation, and analyzed the fractions by
Western blotting (Fig. 2D). N-terminal fragments of PS2 were
chiefly distributed in fractions 4-10, which overlapped with those
positive for a TGN marker (adaptin-
; fractions 3-10). In contrast,
the distribution of full-length PS2 (fractions 11 and 12) was limited
to those positive for an ER marker (i.e. BiP; fractions
10-12). The distribution patterns of PS2 and its derivatives were
similar between WT and N141I mt PS2. ELISA quantitation of cell
fraction-associated A
42 showed that cells expressing N141I mt PS2
harbored elevated levels of A
-(1-42) in fractions 8 and 9, which
corresponded to those positive for PS2 N-terminal fragment as well as
for a TGN marker, supporting the notion that TGN is the site in which
mt PS2 affects
-cleavage of C100 to promote A
42 production (Fig.
2E).
APP, we transiently co-expressed
human
APP tagged with KKLN (
APP/ER) or SDYQRL (
APP/TGN) or
without the tags (
APP/WT) in N2a cells that stably express WT or
N141I or M239V FAD mt PS2 and examined the production of secreted A
.
N2a cells expressing WT PS2 transiently transfected with
APP/WT or
APP/TGN secreted similar levels of A
-(1-40) and A
-(1-42),
whereas those with
APP/ER did not secrete detectable levels of A
(Fig. 3A, left),
and the results were similar to those in cells expressing C100 and its
derivatives. In contrast, cells expressing N141I or M239V mt PS2
transiently transfected with
APP/WT or
APP/TGN secreted increased
levels of A
-(1-42), whereas those with
APP/ER again did not
secrete detectable A
(Fig. 3A, middle and
right). To examine whether the lack of ER-associated A
observed in experiments based on C100 is reproducible with full-length
APP in our N2a system, we solubilized N2a cells expressing
APP/ER
or
APP/WT in 70% formic acid and analyzed the extracted proteins by
Western blotting with BA27 and BC05. Comparable levels of
A
40-positive and A
42-positive ~4-kDa polypeptides were detected in cells expressing
APP/WT, whereas no A
-positive bands were detectable in cells expressing
APP/ER despite comparable levels of
expression of
APP/ER and
APP/WT (Fig. 3B). Taken
together, it was strongly suggested that late intracellular
compartments including TGN, but not ER, are the major intracellular
site of A
production and of mt PS2 effects on
-cleavage of
APP
to promote secretion of A
42 in N2a cells.
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Fig. 3.
A production in N2a
cells expressing full-length
APP tagged with
sorting signals. A, levels of A
-(1-40)
(open column) and A
-(1-42) (closed
column) secreted from N2a cells stably expressing WT PS2
(left), N141I (middle), or M239V
(right) FAD mt PS2 and transiently transfected with
full-length
APP with targeting signals (ER,
APP/ER;
TGN,
APP/TGN; wt,
APP/WT) quantitated by
two-site ELISAs. Mean values ± S.E. in four independent
experiments are shown. Transfected C100 cDNAs are shown by
lines below the columns. B,
Western blot analysis of formic acid-extracted A
40
(middle panel) by BA27 and A
42
(lower panel) by BC05 in cells overexpressing
APP/ER (middle lane; ER),
APP/WT
(right lane; wt), or mock-transfected
cells (left lane; mock). Expression of
full-length
APP in cell lysates probed by BAN50 is shown in the
upper panel. Molecular mass markers are shown in
kilodaltons.
Secretion--
Since C100 was fully
susceptible to
-cleavage as well as to the A
42-promoting effect
of mt PS2, we next examined if the C-terminal cytoplasmic domain of
APP, which has been implicated in a number of functions including
intermolecular association, caspase cleavage, and endocytosis, is
required for the abnormal function of mt PS2. For this purpose, we
constructed following C100 derivatives truncated at various positions
within the cytoplasmic domain: C100/stop68 truncated at
Asp68 (numbering starting from residue Asp1 of
A
) documented as a caspase-3 cleavage site (49), C100/stop56 retaining the KKKQ sequence flanking the membrane that is attributed to
membrane anchoring (50), and C100/stop52 that lacks the entire cytoplasmic domain (Fig.
4A).
View larger version (33K):
[in a new window]
Fig. 4.
Expression of APP
C100 truncated at the cytoplasmic domain and A
secretion in N2a cells. A, schematic depiction of
C100 truncated at the cytoplasmic domain. C100/stop68 is truncated at
Asp68 (starting from Asp1 of the A
sequence), which is inferred as the caspase-3 cleavage site;
C100/stop56 retains the membrane-flanking four amino acid residues
KKKQ; and C100/stop52 lacks the entire cytoplasmic domain.
B, Western blot analysis of C-terminally truncated C100
transiently expressed in N2a cells with BAN50. An asterisk
shows the co-migration of an ~8-kDa polypeptide derived from C100/WT
with C100/stop68. Molecular mass markers are shown in kilodaltons, and
the names of transfected cDNAs are indicated above each
lane. C, inhibition of the generation of ~8-kDa
band (*) from C100/WT by a caspase inhibitor zVAD-fmk.
, N2a cells
transfected with C100/WT without zVAD-fmk treatment. zVAD,
N2a cells transfected with C100/WT treated with 100 mM
zVAD-fmk for 24 h. D, differential extraction of
C-terminally truncated C100 by Na2CO3 or Triton
X-100. Microsomal fractions of N2a cells transiently expressed C100/WT
(wt), C100/stop68 (stop68), C100/stop56
(stop56), C100/stop52 (stop52) were extracted by
0.5 M Na2CO3 (pH 11.0) or 1%
Triton X-100, and solubilized proteins (S) and insoluble
pellets (P) were analyzed by Western blotting with BAN50.
E, immunofluorescence localization of C-terminally truncated
C100 in N2a cells revealed by BAN50. Scale bar,
10 µm. F, levels of A
-(1-40) (open
column) and A
-(1-42) (closed
column) secreted from transiently transfected N2a cells
quantitated by two-site ELISAs. Note that C100/stop52 yielded
significantly reduced levels of A
-(1-42) (*, p < 0.01 by analysis of variance). Mean values ± S.E. in four
independent experiments are shown. Transfected C100 cDNAs are shown
below each column.
secreted from N2a cells transiently transfected with these C100
derivatives (Fig. 4F). All C-terminally deleted C100
derivatives showed robust secretion of A
-(1-40) at similar levels
ranging from 1500 to 1800 pM. C100/stop68 and C100/stop56
produced similar levels of A
-(1-42), comprising ~11-13% that of
total A
(A
-(1-40) + A
-(1-42)). However, cells expressing
C100/stop52 secreted significantly lower levels of A
-(1-42), which
comprised only ~3.8% that of total A
(*, p < 0.01 by analysis of variance).
-(1-40) and A
-(1-42) secreted into culture media. In cells expressing WT PS2, levels of secreted
A
-(1-40) were similar among all C100 derivatives, and C100/stop52
yielded significantly reduced levels or ratios (~3%) of A
-(1-42)
compared with those from other C100 derivatives, as observed in cells
without transfection of PS2 (Fig.
5A; *, p = 0.011 in ratios by analysis of variance). In contrast, co-transfection
of FAD mt PS2 increased the percentage of secreted A
-(1-42) as a
fraction of total A
to ~60% (N141I) or ~50% (M239V) with all
types of C-terminally deleted C100, except that co-expression of
C100/stop52 and M239V mt PS2 yielded ~20% of A
42, although this
was still significantly higher than that with WT PS2 (Fig.
5A). To confirm that the cytoplasmic domain of
APP is
dispensable for
-cleavage and mt PS2 effect on A
42 generation on
a full-length
APP basis, we next co-expressed C-terminally deleted
full-length
APP in N2a cells stably expressing WT or N141I or M239V
mt PS2 and quantitated A
-(1-40) and A
-(1-42) in culture media
(Fig. 5B). Upon cotransfection with WT PS2, the levels of total secreted A
were significantly lower in cells expressing C-terminally truncated
APP compared with that with full-length
APP presumably due to lack of endocytosis as previously reported (51), whereas there was a uniform increase in the secretion of
A
42 in cells stably expressing N141I or M239V mt PS2. These data
suggest that the anterior half of the C100 (i.e. A
sequence plus the following intramembranous portion of
APP) is
sufficient for the full effect of mt PS2 to increase secretion of
A
-(1-42).
View larger version (30K):
[in a new window]
Fig. 5.
Secretion of
A -(1-40) and
A
-(1-42) from N2a cells stably expressing WT
or FAD mt PS2 and transiently transfected with C-terminally truncated
C100 or
APP. Levels of secreted
A
-(1-40) (open columns) and A
-(1-42)
(closed columns) from N2a cells stably expressing
WT (left columns), N141I (middle
columns), or M239V (right columns) FAD
mt PS2 and transiently transfected with C-terminally truncated
100 (A) or
APP (B) quantitated by
two-site ELISAs. Mean ± S.E. in four independent experiments are
shown in A and B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
42 is produced and up-regulated by the abnormal function of FAD mt PS2, (ii) lack of
the entire cytoplasmic domain of C100 selectively decreases the
production of A
42, and (iii) the anterior half of C100 lacking the
entire cytoplasmic domain is sufficient for the abnormal function of mt
PS2 to increase production of A
42.
42 generation in relation to PS function
has been a matter of controversy (38). Here we showed that C100
targeted to TGN (C100/TGN) yielded similar levels of intracellular as
well as secreted forms of A
-(1-42) and A
-(1-40), compared with
those derived from C100 without sorting signals, whereas C100 targeted
to ER (C100/ER) did not produce detectable levels of intracellular as
well as secreted A
. Moreover, FAD-linked N141I and M239V mt PS2
fully increased the production of A
-(1-42) from C100/TGN at an
extent comparable with that for C100 without signals, whereas
production of intracellular as well as secreted A
-(1-42) was not
up-regulated by co-expression of C100/ER. Use of C100 that does not
require
-cleavage, which is presumed to occur in the post-Golgi
compartments (52), to trigger
-cleavage enabled us to directly
address the intracellular site where
-cleavage takes place and is
affected by the abnormal effects of mt PS2. It has been reported that
cultured neurons (33, 34) as well as human embryonic kidney 293 cells
(35) produce A
-(1-42) in ER upon overexpression of
APP. These
findings were in good agreement with the predominant ER localization of
PS, which has been implicated in
-cleavage. However, it was
subsequently shown that the ER-associated A
42 was not directly
secreted and was considered to comprise a distinct pool from the
secreted A
(53). The reason for our failure to detect ER-associated
A
in our cell system is not clear at present; however, the following
possibilities could be considered to explain these discrepancies: (i)
full-length A
may be detected in ER only by extremely high
level expression of APP (e.g. by Semliki Forest virus
infection) (33), and modest levels of overexpression of
APP or C100
fail to produce detectable levels of A
; (ii) in N2a cells, small
amounts of A
42 truncated at the N terminus, but not full-length
species, have been detected in ER (36, 47), which escaped our detection
system specific for human full-length A
; and (iii) ER-derived A
is present at a relatively small amount compared with those in late
compartments, the former being lower than the detection limit of our
highly sensitive immunoblot assay.
42 upon co-expression of mt PS2 strongly support
the view that the active form of PS (i.e. endoproteolytic
fragments that are stabilized (44) and form a high molecular weight
complex (54)) resides in Golgi/TGN as well as in additional late
intracellular compartments and that mt PS1 up-regulates production and
secretion of A
42 in these compartments (39, 40). In this case, a
relatively small amount of "active" presenilin complex may be
sufficient for the generation of A
in the late compartments.
Notwithstanding the present data, the problem of the "spatial
paradox" (38) between the localization of PS and
-secretase
activities has not been completely clarified. Further careful studies
on the intracellular distribution of presenilin complex and
-cleavage activities for the processing of
APP as well as Notch
in different types of cells will be needed.
APP with FAD-associated mt
PS1 (40) or PS2 (32) is sufficient to induce overproduction of A
42.
To examine whether the cytoplasmic domain of
APP is required for the
abnormal effect of mt PS, we co-expressed C-terminally truncated forms
of C100 and evaluated the secretion of A
. Unexpectedly, we found
that the expression of C100 lacking the entire cytoplasmic domain
(C100/stop52), with or without co-expression of WT PS2, dramatically
reduced the secretion of A
42, although the total levels of A
secretion were not significantly altered. Similar results were obtained
also in COS cells (data not shown). The mechanism whereby
-secretase
differentially cleaves A
40 and A
42 within the transmembrane
segment of
APP is not well understood. However, accumulating data
suggest that
-cleavage occurs in a position-dependent
manner within the membranous portion, irrespective of the amino acid
sequences (54, 55). The lack of the cytoplasmic domain including the
KKKQ motif at the membrane-flanking portion, which is presumed to work
as a membrane anchor (50), may destabilize the positioning of the
transmembrane domain of
APP, thereby leading to predominant cleavage
at the A
40 position. Moreover, C100 ending at the putative caspase-3
cleavage site (C100/stop68) did not change the level or proportion of
secreted A
. It has been shown that
APP truncated at the caspase-3
cleavage site increased the secretion of A
40 (49). The reason for
this discrepancy is unknown, but it is possible that the
caspase-cleaved
APP may promote
-cleavage (49), thereby
increasing A
40 secretion.
42. This domain is implicated in a number of
APP functions including interaction with a number of binding proteins
(i.e. FE65 (57) or X11 (58, 59)) as well as endocytosis
(51), all of which are known to alter A
production (60). However, co-transfection of mt PS2 fully increased the secretion of A
42 from
all types of C100 truncated at the cytoplasmic domain. It has recently
been suggested that PS serves as a
-secretase harboring two
intramembranous aspartates in TM6 and TM7 domains as a catalytic center
(61). Taken together with our present data, shift of
-cleavage from
the predominant A
40 position to a more pathogenic A
42 position
caused by the abnormal gain-of-function of FAD mt PS may require solely
the intramembranous interaction between the TM domains of
APP and
PS. Further analysis on the molecular mechanism whereby mt PS leads to
increased production of A
42 should facilitate the understanding of
the pathogenesis of AD as well as of the unusual but important
proteolytic mechanism recently referred to as regulated intramembrane
endoproteolysis (62).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank N. Takasugi for anti-G2N4 antibody and Takeda Chemical Industries for continuous support.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants-in-Aid from the Ministry of Health and Welfare, the Ministry of Education, Science, Culture and Sports, CREST of Japan Science and Technology Corporation, and RIKEN, Japan.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.
§ A Research Fellow of the Japan Society for the Promotion of Science.
To whom correspondence should be addressed. Tel.:
81-3-5841-4877; Fax: 81-3-5841-4708; E-mail:
iwatsubo@mol.f.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M007989200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's disease;
A, amyloid
peptide;
APP,
-amyloid precursor protein(s);
C100, C-terminal 99-amino acid fragment of
APP;
ELISA, enzyme-linked
immunosorbent assay;
ER, endoplasmic reticulum;
FAD, familial
Alzheimer's disease;
N2a, mouse Neuro2a neuroblastoma;
PS, presenilin(s);
TGN, trans-Golgi network;
mt, mutant;
RIPA, radioimmune precipitation assay buffer;
WT, wild type;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp- (OMe)-fluoromethylketone.
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