Mutations in presenilin (PS) 1 or PS2 genes
account for the majority of early-onset familial Alzheimer's disease,
and these mutations have been shown to increase production of species
of amyloid
peptide (A
) ending at residue 42, i.e.
the most amyloidogenic form of A
. To gain insight into the molecular
mechanisms whereby mutant PS induces overproduction of A
42, we
constructed cDNAs encoding mutant and/or truncated forms of PS2 and
examined the secretion of A
42 from COS or neuro2a cells transfected
with these genes. Cells expressing full-length PS2 harboring both N141I
and M239V mutations in the same polypeptide induced overproduction of
A
42, although the levels of A
42 were comparable with those in
cells engineered to express PS2 with one or the other of these PS2
mutations. In contrast, cells engineered to express partially truncated
PS2 (eliminating the COOH-terminal third
of PS2 while retaining the endoproteolytic NH2-terminal
fragment) and harboring a N141I mutation, as well as cells expressing
COOH-terminal fragments of PS2, did not overproduce A
42, and the
levels of A
42 were comparable with those in cells that expressed
full-length, wild-type PS2 or fragments thereof. These data indicate
that: (i) the A
42-promoting effects of mutant PS2 proteins reach the
maximum level with a given single amino acid substitution
(i.e. N141I or M239V); and (ii) the expression of
full-length mutant PS2 is required for the overproduction of A
42.
Hence, cooperative interactions of NH2- and COOH-terminal
fragments generated from full-length mutant PS2 may be important for
the overproduction of A
42 that may underlie familial Alzheimer's
disease.
 |
INTRODUCTION |
Alzheimer's disease
(AD)1 is a progressive,
dementing, neurological disorder characterized pathologically by an
extensive neuronal loss in the cerebral cortex as well as a massive
deposition of amyloid
peptides (A
) as senile plaques and in the
walls of blood vessels (1). A subset of early-onset AD is inherited as
an autosomal dominant trait, and presenilin (PS) genes were identified
as the major causative genes for these early-onset familial AD (FAD).
PS1 gene, which is linked to the majority of early-onset FAD located on
chromosome 14 (2), and PS2 gene (3), which is responsible for a subtype
of FAD linked to chromosome 1, encode homologous polytopic membrane
proteins that predominantly localize to endoplasmic reticulum (4-6)
and span the membrane 8 times (7). More than 40 missense mutations (8),
as well as an exon 10 deletion (9) in PS1 and two missense mutations of
PS2 (3, 10), thus far have been identified in pedigrees of FAD.
The physiological function of PS proteins is unknown, although recent
data from studies in Caenorhabditis elegans (11, 12) and PS1
gene knock-out mice (13, 14) indicate that PS1 may play some role in
Notch signaling. The mechanisms whereby mutations in PS1 or
PS2 genes cause AD also remain elusive, but several lines of evidence
suggest that they may lead to AD by promoting
-amyloid deposition.
Amino acid substitutions, as well as an exon 10 deletion, of PS1
(15-17) and PS2 (17, 18) have been shown to increase the secretion of
a species of A
ending at residue 42 (A
42), i.e. the
most amyloidogenic form of A
(19-21). Recent findings that the
secretion of A
from primary neurons cultured from brains of mice
that lack PS1 is decreased, despite the normal level of full-length
-amyloid precursor protein (
APP) or the amyloidogenic
COOH-terminal fragment thereof, argue for the notion that PS is an
important co-factor for the proteolytic processing of
APP at the
COOH terminus of A
termed
-cleavage (22). However, the mechanisms
whereby mutant PS proteins affect
-cleavage and lead to the
increased production of A
42 is unknown. Some investigators have
shown the direct association of PS and
APP in cultured cells (23,
24), whereas others have not (25). Thus, one may speculate a direct or
indirect "chaperone"-like effect of mutant PS or effects on
intracellular vesicular trafficking to increase the susceptibility of
APP to be cleaved at position 42.
Several questions arise from these observations. The mechanistic
effects of a given single amino acid substitution in mutant PS protein
leading to AD are not yet understood. Previous data showing that either
of the two known PS2 mutations, i.e. the Volga German
mutation that substitutes Asn-141 for Ile (N141I) or the Italian
mutation causing a Met-239 to Val (M239V) mutation, considerably increase the percentage of secreted A
42 (17, 18, 26), suggesting that a single amino acid substitution on PS2 may lead to a significant change in the protein folding and/or interaction with other proteins of
PS2 compared with those with wild-type PS2. To gain insights into the
nature of pathogenic effects caused by PS2 mutations, we first examined
whether a mutant PS2 molecule harboring both the N141I and M239V
mutations would increase the overproduction of A
42, compared with
singly mutated PS2 with one or the other of these mutations.
PS1 and PS2 have been shown to undergo endoproteolytic cleavage that
yields a long NH2-terminal fragment (NTF) and a short COOH-terminal fragment (CTF) spanning the membrane 6 and 2 times, respectively (18, 27). These fragments are the predominant forms of PS1
or PS2 in cultured cells or brain tissues that do not overexpress PS
(27). However, the relationship between cleavage and function of PS is
not well understood. Next we sought to examine if the NTF or CTF forms
of mutant PS2 alone are capable of promoting the secretion of A
42.
To this end, we expressed partially truncated forms of PS2 (eliminating
the COOH-terminal third of PS2 while retaining the endoproteolytic NTF)
harboring a N141I mutation, as well as CTFs of PS2 in cultured cells
and examined the COOH-terminal properties of A
secreted from these
cells.
 |
EXPERIMENTAL PROCEDURES |
Construction of Expression Plasmids--
A full-length cDNA
encoding wild-type (WT) human PS2 was obtained by PCR from a normal
human cDNA library, and the N141I or M239V PS2 mutations were
introduced by the dU-template method as described previously (18). WT
as well as mutant (mt) PS2 cDNAs were subcloned into pBluescript,
and the coding region was then subcloned into a mammalian expression
vector pcDNA3. The N141I/M239V double mutation was introduced by
digesting the N141I mt PS2 cDNA in pBluescript with
BamHI and BstXI and then inserting the resultant
~0.5-kilobase pair product between the
BamHI-BstXI sites of M239V mt PS2 in
pcDNA3.
cDNAs encoding COOH-terminally truncated WT or N141I mt PS2
(i.e. PS2/270stop, PS2/303stop, and PS2/388stop) were
generated by PCR using Pfu polymerase (Stratagene), and
the following oligonucleotides were used as PCR primers:
5'-CCGGGATCCAGACCTCTCTGCGGCCCCAAGT-3' as a sense primer,
5'-CATTCTCTCGAGCTATTTGGGACACAG-3' for PS2/270stop, 5'-AGCTCGAGCTAGCCAACCGTCCACAC-3' for PS2/303stop, and
5'-GGCTCGAGCTACGTGGTATTCCAGTC-3' for PS2/388stop as antisense primers,
respectively. These primers were incubated with WT or N141I mt PS2
cDNAs in pcDNA3, and the PCR products were digested with
BamHI and XhoI. Purified fragments were ligated
into pcDNA3. cDNAs encoding varying lengths of PS2 COOH-terminal fragments (i.e. PS2/270ctf, PS2/304ctf, and
PS2/344ctf) were similarly generated by PCR using the following
primers: 5'-GGCACTCGAGTGTAAAACTATACAACTGC-3' as an antisense
primer and 5'-CCGGATCCACCATGGGGCCTCTGAGA-3' for PS2/270ctf,
5'-CCGGATCCACCATGGCGAAGCTGGAC-3' for PS2/304ctf, and 5'-ACGGATCCATGAGTTTTGGGGAGCCT-3' for PS2/344ctf as sense primers. These primers were incubated with WT PS2 cDNA in pcDNA3,
digested, and ligated into pcDNA3. Schematic depictions of
truncated and/or mutated PS2 derivatives are shown in Fig. 1.
To express derivative polypeptides of PS2 fused to glutathione
S-transferase (GST), cDNAs encoding the
NH2-terminal (2-84) or the loop (301-361) portions of PS2
were amplified by PCR and subcloned into an Escherichia coli
expression vector (pGEX-6P-1, Amersham Pharmacia Biotech). These
ligations resulted in the fusion of PS2 sequences COOH-terminal and
in-frame with GST.
All constructs were sequenced using Thermosequenase (Amersham Pharmacia
Biotech) on an automated sequencer (Li-Cor, Lincoln, NE) as described
(18).
Cell Culture and Transfection--
Monkey COS-1 cells or mouse
neuro2a (N2a) cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum and
penicillin/streptomycin at 37 °C in 5% CO2 atmosphere
as described (18). Transient expression in COS-1 cells was performed by
the DEAE-dextran method, and stable N2a cell lines were generated by
transfecting the cDNAs in pcDNA3 vector using a mammalian
transfection kit (Stratagene) or LipofectAMINE (Life Technologies,
Inc.) and selection in Dulbecco's modified Eagle's medium containing
G418 (Life Technologies, Inc.) at 400 or 500 µg/ml. Double
transfection of COS-1 cells with cDNAs encoding PS2 or its
derivatives in pcDNA3 and those encoding the COOH-terminal 100 amino acids (C100) of
APP in p91023(E) was performed as described previously (18). Co-expression of NTF and CTF of PS2 was performed by
transiently transfecting a cDNA encoding PS2/304ctf into N2a cells
stably expressing WT or N141I mt PS2/303stop using LipofectAMINE according to the manufacturer's instructions.
Antibodies and Immunoblot Analysis--
Polyclonal antibodies
were raised in rabbits using synthetic peptides conjugated to keyhole
limpet hemocyanin or GST fusion proteins corresponding to the following
predicted amino acid sequences of human PS2 as immunogens: anti-G2N2
against GST fused to amino acids 2-84 of PS2, anti-G2L against GST
fused to amino acids 301-361 of PS2, and anti-PS2C2 against peptides
corresponding to amino acids 443-448 of PS2. Anti-G2N2 and anti-PS2C2
antisera were further affinity-purified to their immunogens as
described (28). Anti-PS2loop antiserum raised against GST fused to the
loop domain of PS2 (29) was kindly provided by Dr. G. Thinakaran. The
locations of immunogen peptides/protein fragments within PS2 are shown
in Fig. 1.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic depictions of PS2 and truncated
and/or mutated derivatives thereof. A, the location of
the epitopes of antibodies used in this study (anti-G2N2, anti-G2L,
anti-PS2loop, and anti-PS2C2) is shown by broken underlines.
Arrowheads indicate the sites of standard (open
arrowhead) and alternative (caspase-type (32); closed
arrowhead) proteolytic processing. B, schematic
representations of mutant or truncated forms of PS2 encoded by the
cDNAs used in this study are shown. The names of cDNAs are
indicated at the left of each bar, and
squares with numbers represent putative TM domains.
Small arrows on each bar show the location of
amino acid substitutions linked to FAD mutations, and
arrowheads between the TM 6/7 domains represent the sites of
proteolytic processing shown in A.
|
|
Cells were lysed in 2% SDS sample buffer and briefly sonicated. The
samples were separated by SDS-PAGE without prior heating, transferred
to polyvinylidene difluoride membrane (Millipore), and probed with each
of the anti-PS antibodies as described (18). The immunoblots were
developed using an ECL system (Amersham Pharmacia Biotech).
Immunofluorescence Microscopy--
Transiently transfected COS-1
cells were cultured on glass coverslips. Cells were fixed by incubation
in phosphate-buffered saline (PBS) containing 4% paraformaldehyde at
room temperature for 30 min, permeabilized, and blocked with PBS-TB (10 mM phosphate buffer, pH 7.4, 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 against PS2 for 2 h and fluorescein isothiocyanate- or
Texas Red-conjugated secondary antibodies in PBS-TB for 1 h,
mounted in PermaFlour Aqueous Mounting Medium (IMMUNON), and viewed
with a confocal laser scanning microscope (Fluoview, Olympus, Tokyo) as
described (30).
Quantitation of A
by Two-site ELISAs--
Two-site ELISAs
that specifically detect the COOH terminus of A
were used. BNT77
raised against human A
11-28, which recognizes full-length as well
as NH2-terminally truncated A
, was used as a capture
antibody; BNT77 binds human as well as rodent-type A
but does not
react with the 3-kDa fragment (p3) beginning at the Leu-17 residue of
A
. BA27 and BC05, monoclonal antibodies that specifically recognize
the COOH termini of A
40 and A
42, respectively, were conjugated
with horseradish peroxidase and used as detector antibodies. The
specificity and sensitivity of these ELISAs have been characterized
previously (18, 31). Culture media were collected after appropriate
incubation periods (60 h in COS-1 and 12 or 24 h in N2a cells) and
subjected to BNT77/BA27 or BC05 ELISAs as described (18).
 |
RESULTS |
Expression of Full-length PS2 Harboring N141I, M239V, or Both of
the Mutations in Cultured Cells and Effects on A
Secretion--
We
transfected cDNAs encoding WT or N141I, M239V single mt as well as
N141I/M239V double mt PS2 cDNAs transiently into COS-1 cells or
stably into N2a cells and analyzed the cell lysates by Western blots
using anti-PS2 antibodies. As we have previously observed (18), when we
transfected cells with cDNAs encoding WT or N141I mt PS2,
50-55-kDa polypeptides corresponding to full-length (fl) PS2,
35-40-kDa NTFs migrating as a doublet as well as a ~19-kDa CTF were
detected in COS-1 cells (data not shown, but see Fig. 4 showing
identical patterns of processing of PS2 in COS-1 cells); this ~19-kDa
CTF was presumed to be produced by caspase-3-like proteolytic
activities (32) because its generation was inhibited by a caspase-3
inhibitor, DEVD-CHO.2 In
addition, a small amount of a ~23-kDa CTF was detected. In N2a cells,
50-55-kDa fl PS2 (Fig. 2,
arrow), 35-kDa NTF (Fig. 2,
closed arrowhead), as well as a 23-kDa CTF (Fig. 2,
open arrowhead) were detected. However, the amounts or
ratios of NH2- and COOH-terminal fragments were similar in
M239V or N141I/M239V double mt PS2 compared with N141I mt or WT PS2
both in COS-1 and N2a cells (Fig. 2).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Expression and metabolism of PS2 in N2a cells
stably transfected with single or double mutant PS2 genes. Western
blot analysis of stable N2a cells transfected with WT or mt PS2
cDNAs. Cell lysates (20 µg of protein) from N2a cells transfected
with an empty pcDNA3 vector or with wild-type, N141I mt, M239V mt,
or N141I/M239V double mt PS2 cDNAs were fractionated by SDS-PAGE
and analyzed by immunoblotting with anti-G2N2 or anti-PS2loop
antibodies. Full-length PS2 proteins are marked by arrows,
NTFs by closed arrowheads, and CTFs by
open arrowheads. The names of the transfected cDNA
constructs are indicated at the top of each lane.
Molecular mass standards are shown in kilodaltons.
|
|
We then quantitated the levels of A
40 and A
42 secreted from cells
transfected with these mt PS2 cDNAs. The percentages of A
42
secreted from COS-1 cells doubly transfected with
APP C100 and N141I
or M239V mt PS2 were elevated to almost similar levels (~30%) by
2.2-fold compared with those from cells with WT PS2 and
APP C100
(average of A
42, 13.8%), and the absolute levels of secreted A
42
were increased by 2.3 (N141I) and 1.7 (M239V) times, respectively,
compared with WT PS2. When COS-1 cells were transfected with
N141I/M239V double mutated PS2 and
APP C100, the A
42 was 31.4%,
and the A
42 level also was similar to those in cells with singly
mutated PS2 (Fig. 3A). Similar
results were obtained in COS-1 cells doubly transfected with
full-length WT or Swedish-type mutant (i.e. 595/596 KM-NL)
APP together with mt PS2 (data not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Secreted A 40 and A 42 from cells
expressing WT or single or double mutant PS2 genes. Levels of
A x-40 and A x-42 secreted from COS-1 cells doubly transfected with
APP C100 and PS2 genes (A) or N2a cells stably
transfected with PS2 genes or an empty vector (B)
quantitated by two-site ELISAs are shown. Mean values ± S.E. in
four independent experiments in A and two independent
experiments in B are shown. Transfected PS2 cDNAs are
indicated below the columns; PS2WT, wild-type
PS2; PS2N141I, N141I mutant PS2;
PS2M239V, M239V mutant PS2; and
PS2N141I/M239V, N141I and M239V double mutant
PS2.
|
|
We then examined the secretion of A
from stably transfected N2a cell
lines expressing WT, N141I, M239V, or N141I/M239V mt PS2. As described
previously (18), N2a cells expressing N141I mt PS2 secreted
considerably increased amounts or percentages of A
42 (71.6% of
total A
and 4.2 times compared with those with WT PS2: 17.0%). N2a
cells expressing M239V mt PS2 also secreted significantly increased
levels (3.4 times compared with those with WT PS2) or percentage (mean,
58.3%) of A
42. However, the secretion of A
42 from cells
expressing N141I/M239V double mutant PS2 were again similar to those
with either of the single PS2 mutations in terms of the absolute levels
(4.1 times compared with those with WT PS2) or percentages (mean,
59.3%) (Fig. 3B). The expression of endogenous
APP was
at similar levels between different N2a cell lines (data not
shown).
Characterization of Truncated Forms of WT or N141I mt PS2 Proteins
Expressed in Cultured Cells and Their Subcellular Localization--
To
gain insights into the biological significance of endoproteolytic
processing of PS2 and especially to examine whether the NTF or CTF of
PS2 is biologically active, we then expressed truncated forms of WT or
N141I mt PS2 in cultured cells and characterized their metabolism and
subcellular localization. Two categories of cDNAs encoding
truncated PS2 were used (see Fig. 1): (i) NTF constructs (WT or N141I
mt) ending at residues 270, 303, or 388 (designated PS2/270stop,
PS2/303stop, and PS2/388stop), retaining the NH2-terminal 6 (or 7) transmembrane (TM) domains and shorter than, close to, or longer
than the predicted size of PS2 NTF, respectively (33, 34); accordingly,
PS2/388stop retains the entire length of the loop region as well as the
7th TM domain of PS2; (ii) CTF constructs starting at residues 271, 304, or 344 (designated PS2/271ctf, PS2/304ctf, and PS2/344ctf), longer than, close to, or shorter than the predicted size of native CTF, respectively. PS2/344ctf is close to the size of the "ALG-3"
fragment, which was found to inhibit cellular apoptosis (35).
In COS-1 cells, PS2/270stop, PS2/303stop, and PS2/388stop (WT or mt)
were expressed as doublets migrating at 30-33, 32-35, and 45-50 kDa,
respectively (Fig. 4A).
PS2/271ctf, PS2/304ctf, and PS2/344ctf were expressed as ~27-, ~23-
and ~16-kDa fragments, respectively (Fig. 4B). These
polypeptides migrated at slightly slower positions than those estimated
from their predicted sizes (PS2/270stop, 30.6 kDa; PS2/303stop, 37.2 kDa; PS2/388stop, 43.5 kDa; PS2/271ctf, 19.6 kDa; PS2/304ctf, 15.9 kDa;
and PS2/344ctf, 11.3 kDa). In addition to the full-length transfected
proteins, PS2/388stop yielded 35-40-kDa doublet proteins that were
equivalent in size to the NTFs cleaved from fl PS2 (Fig. 4A,
arrowhead); PS2/271ctf (Fig. 4B) also yielded
proteolytic fragments of 23 (arrowhead) and 19 kDa
(asterisk) in size, and the 19-kDa CTF was also observed
with PS2/304ctf (Fig. 4B, asterisk).
Immunopositive bands at higher molecular weight ranges relative to
these polypeptides (Fig. 4, A and B) would
presumably represent dimeric forms and/or aggregates of these
proteins.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Expression and metabolism of PS2 in
transiently transfected COS-1 cells with cDNAs encoding truncated
PS2. A, Western blot analysis of expression of WT or mt
PS2 NTFs in transiently transfected COS-1 cells. Cell lysates (10 µg
of protein) from COS-1 cells transfected with an empty pcDNA3
vector or with WT or N141I mt fl, WT or N141I mt 270stop, WT or N141I
mt 303stop, and WT or N141I mt 388stop PS2 cDNAs were fractionated
by SDS-PAGE and analyzed by immunoblotting with anti-G2N2 antibody. The
positions of fl PS2 and NTFs are marked by arrows and
arrowheads, respectively. B, Western blot
analysis of expression of PS2 CTFs in transiently transfected COS-1
cells. Cell lysates (10 µg of protein) from COS-1 cells transfected
with an empty pcDNA3 vector or with WT fl, 271ctf, 304ctf, or
344ctf PS2 cDNAs were fractionated by SDS-PAGE and analyzed by
immunoblotting with anti-G2L or anti-PS2C2 antibodies. The positions of
fl PS2, 23-kDa standard CTF, and 19-kDa alternative CTF are marked by
arrows, arrowheads, and asterisks,
respectively. The names of the transfected cDNA constructs are
indicated at the top of each lane. Molecular mass
standards are shown in kilodaltons.
|
|
In stable N2a cells, the expression patterns of PS2 derivatives were
essentially similar to those in COS-1 cells with some differences (Fig.
5). Notably, 45-50-kDa polypeptides
corresponding to PS2/388stop were barely processed to form 35-kDa NTF
(Fig. 5A) that was present in cells with fl PS2 (Fig.
5A, arrowhead). PS2/271ctf (Fig. 5B)
and PS2/304ctf (Fig. 5B) also did not produce proteolytic
fragments of smaller sizes. The patterns of expression of endogenous
APP were almost similar between these cell lines (data not
shown).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
Expression and metabolism of PS2 in stably
transfected N2a cells with cDNAs encoding truncated PS2.
Western blot analysis of expression of WT or mt PS2 derivatives in
stably transfected N2a cells is shown. Cell lysates (20 µg of
protein) from N2a cells transfected with an empty pcDNA3 vector or
with WT or N141I mt fl, WT or N141I mt 270stop, WT or N141 mt 303stop,
WT or N141I mt 388stop, and 271ctf or 304ctf PS2 cDNAs were
fractionated by SDS-PAGE and analyzed by immunoblotting with anti-G2N2
(A) or anti-G2L (B) antibodies. The positions of
fl PS2 and NTFs (in A) or CTFs (in B) are marked
by arrows and arrowheads, respectively. Note that
PS2/303stop comigrates with the 35-kDa NTF in A and that
PS2/304ctf comigrates with the 23-kDa standard CTF in B,
respectively. The names of the transfected cDNA constructs are
indicated at the top of each lane. Molecular mass
standards are shown in kilodaltons.
|
|
Next we examined the subcellular localization of the PS2 NTFs or CTFs
in COS-1 cells by immunofluorescence microscopy. Remarkably, all
constructs encoding NTFs and CTFs of WT or N141I mt types of PS2 showed
similar distribution in a fine meshlike pattern throughout the
cytoplasm as well as dense immunostaining in the perikaryal areas,
which corresponded to those with BiP, a marker for endoplasmic
reticulum (Fig. 6). N2a stable cells also
showed similar patterns of ER localization of PS2 derivatives (data not shown).

View larger version (139K):
[in this window]
[in a new window]
|
Fig. 6.
Immunofluorescence localization of truncated
PS2 derivatives expressed in COS-1 cells. COS-1 cells transfected
with WT fl (A and B), N141I mt fl PS2
(C), WT 270stop (D), WT 303stop (E),
WT 388stop (F), N141I mt 270stop (G), N141I mt
303stop (H), N141I mt 388stop (I), 271ctf
(J), 304ctf (K), or 344ctf (L)
cDNAs were immunostained with appropriate anti-PS2 antibodies
(A and C-L; the primary antibodies
used are indicated in each panel) or doubly with an anti-BiP
monoclonal antibody (B) and observed with a confocal
microscope after labeling with fluorescein-conjugated anti-rabbit IgG
secondary antibody (in A and B, together with
Texas Red-conjugated anti-mouse IgG antibody). Scale bar, 10 µm.
|
|
Characterization of A
Secreted from Cells Expressing Truncated
Forms of WT or N141I mt PS2--
We then quantitated the levels and
percentages of A
40 and A
42 secreted from cells expressing
truncated forms of N141I mt or WT PS2. In COS-1 cells doubly
transfected with
APP C100 and each of the three types of truncated
mt PS2, the A
42 in total A
was ~10% in all, and they were
similar to those in cells with corresponding forms of truncated WT PS2,
whereas A
42 comprised 22.3% of total A
in cells expressing fl mt
PS2, which was 1.7 times relative to that in cells expressing fl WT PS2
(13.5%). However, the total levels of A
were increased by ~2-fold
in cells with truncated mt PS2 compared with those with truncated WT
PS2. When CTFs of PS2 were transfected together with
APP C100, the A
42 ranged between 11.9 and 14.6%, which also was similar to cells
with fl WT PS2 (Fig. 7A).
Similar results were obtained in COS-1 cells doubly transfected with WT
or Swedish-type mutant (i.e. 595/596 KM-NL)
APP together
with these PS2 derivatives (data not shown).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Secreted A 40 and A 42 from cells
expressing cDNAs encoding WT or mt truncated PS2 derivatives.
Levels of A x-40 and A x-42 secreted from COS-1 cells doubly
transfected with APP C100 and truncated PS2 genes (A) or
N2a cells stably transfected with truncated PS2 genes (B) or
co-transfected with NTF as well as CTF of PS2 (C)
quantitated by two-site ELISAs. Mean values ± S.E. in four
(A, B) or three (C) independent
experiments are shown. Transfected cDNAs encoding PS2 derivatives
are indicated below the columns; in C, stably expressed
cDNAs for NTF and transiently transfected cDNAs for CTF (with
"+") are shown in the lower and upper lanes,
respectively. Vector, empty pcDNA3
vector.
|
|
In N2a cells stably expressing three types of truncated forms of
N141I mt PS2, the percentage of A
42 that comprised the total A
ranged between 14.2 and 18.0%, which was similar to those in cells
with truncated (12.3-16.5%) or fl WT PS2 (20.5%), and the absolute
amounts of secreted A
were at similar levels between cells
expressing truncated WT or mt PS2 (Fig. 7B). This was in sharp contrast to the marked increase in the percentage or level of
A
42 from cells with fl mt PS2 (52.1%). The levels as well as
percentages of A
42 secreted from N2a cells expressing PS2 CTFs
(10.1-14.2%) also were similar to those with fl WT PS2 or mock-transfected cells (Fig. 7B).
To examine if coexpression of mt PS2 NTF together with CTF
reconstitutes overproduction of A
42, we transiently transfected PS2/304ctf in N2a cells stably expressing WT or mt PS2/303stop. Upon
co-transfection of PS2/304ctf, the total levels of secreted A
were
decreased by ~50% both in WT and mt PS2/303stop stable cells
relative to those in mock-transfected cells, whereas the A
42
remained unchanged both in cells expressing WT (9.7% in double transfection versus 11.3% in mock transfection) and mt
(11.1% in double transfection versus 10.3% in mock
transfection) PS2/303stop (Fig. 7C).
 |
DISCUSSION |
In this study, we have clearly shown that (i) full-length PS2
harboring both N141I and M239V mutations in the same polypeptide induced overproduction of A
42 at similar levels to those in cells expressing PS2 with one or the other of these PS2 mutations
(i.e. N141I or M239V); (ii) NTFs or CTFs of PS2 expressed in
cells predominantly localize in ER; and (iii) cells expressing
partially truncated PS2 (eliminating the COOH-terminal third of PS2
while retaining the endoproteolytic NH2-terminal fragment)
and harboring a N141I mutation, as well as cells expressing
COOH-terminal fragments of PS2, did not overproduce A
42, and the
levels or percentages of A
42 were comparable with those in cells
that expressed full-length and wild-type PS2 as well as fragments
thereof.
The nature of the structural or functional changes of the polytopic
membrane protein PS2 caused by the two known mutations is not fully
understood at present. Regarding the N141I Volga German PS2 mutation, a
PS1 mutation at the homologous site (N135D) was reported (36), and
these homologous residues in PS1 and PS2 are located at the
NH2-terminal flank (designated N-cap position) of the
second transmembrane (TM2) domain, which is believed to be important in
the accurate positioning of the transmembrane
-helix structure (37).
Another PS2 mutation of the Italian type (M239V) is situated within the
TM5 domain; a PS1 mutation linked to FAD at the homologous site (M233T)
also was documented (38), and substitution of Met for Val was observed
in multiple residues in the TM2 domain of PS1 (i.e. M139V
and M146V) (8), suggesting that Met to Val substitution may cause some
common structural changes in the TM domains of PS1 or PS2. Our
observation that the N141I/M239V double mutation did not have additive
effects on the increase in the levels or percentages of secreted A
42 suggests that A
42-promoting capacities of mt PS2 proteins reach the
maximum level with a given single amino acid substitution (i.e. N141I or M239V). This contrasts with the recent
observation that the A
42-promoting effects of M146L/L286V double
mutant PS1 were additive (39) and also with the clinical observation
that FAD patients with PS1 mutations develop AD at a uniformly early age, whereas the age of onset in Volga German families with the N141I
PS2 mutation is variable and relatively late (40). The reason for these
discrepancies is not clear at present. However, one should consider the
differences in the protein levels of endogenous PS1 versus
PS2 in the brains of FAD patients (10, 18). For example, it may be that
the changes in A
42-promoting effects of PS2 caused by a given single
mutation per molecule is stronger than those with mt PS1,
whereas the overall pathogenic effects of mt PS1 become more intense
than those of mt PS2 because the total amount of PS1 proteins in
neurons or brain tissues is higher than that of PS2.
NTFs or CTFs of PS2 of various sizes predominantly localized to ER.
Recently, it has been shown that the NH2-terminal 166 residues, but not 138 residues, of PS2 are sufficient for the ER
targeting, suggesting that the initial two transmembrane domains are
necessary for ER localization (41). Our findings confirmed these
observations with respect to the NTFs and further extended these data
by showing that CTFs of PS2, including those corresponding to the
COOH-terminal 103 amino acids (ALG-3) also localize to ER. Although the
precise orientation of the membrane insertion of these CTFs is yet to
be determined, the occurrence of "caspase-type" cleavage (32) of
these CTFs in similar patterns to those observed in cells expressing fl
PS2 suggests that the NH2-terminal portions (i.e. loop region of PS2) of these CTFs are properly
oriented to the cytoplasmic side. Moreover, these CTFs harbor two
transmembrane domains (i.e. TM7 and -8). Taken together, the
COOH-terminal region may harbor other ER-targeting signal sequences
besides those in the NH2-terminal region, or alternatively,
the presence of multiple (i.e. more than two) TM domains,
but not particular subregions, of PS2 may determine its ER
localization.
The most unexpected, yet intriguing, finding in this study was that
cells expressing COOH-terminally truncated N141I mt PS2 that are
equivalent to or longer than the endoproteolytic
NH2-terminal fragment did not overproduce A
42. This was
surprising because most of the PS proteins in native cells or tissues
(including brains) exist as NTF and CTF forms, and the NTFs contain six
of the eight TM domains of PS molecules. Recently, it has been
suggested that the levels of PS within cells are strictly regulated by
competition for limiting cellular factors (29). Moreover, it was shown
that NTF and CTF of PS1 or PS2 remain noncovalently bound to each other after cleavage forming a very stable complex (25, 42, 43) and that they
may form a 100-150-kDa molecular mass complex (43). Our finding that
NTF of mt PS2 or CTF alone does not promote A
42 overproduction
supports the notion that the stable complex forms of PS NTF and CTF
constitute the functional units under biological as well as
pathological conditions. An alternative possibility would be that the
nascent, full-length form of PS is functional and the cleavage at loop
domain is a switch-off phenomenon. However, the observations that
full-length PS is short-lived (44) and not readily incorporated into
the stable complex (29, 43) and that full-length PS is rare in native
cells (27) render this possibility rather unlikely. The COOH-terminally
truncated forms of PS1 (29) or PS2 (i.e. PS2/388stop in this
study), which contain the proteolytic cleavage sites, were not cleaved,
and the truncated PS1 did not influence the stable complex formation in
stably transfected N2a cells (29). Taken together with our results that
mt PS2/388stop did not promote overproduction of A
42, it is highly
conceivable that the condition under which nascent PS proteins are
stabilized and properly cleaved to produce NTF and CTF and form a
stoichiometric stable complex is the prerequisite for the normal or
pathological function of PS (29, 44). Our data that co-expression of mt
PS2/303stop together with PS2/304ctf did not reconstitute
overproduction of A
42 further support the notion that the
heterodimeric complex of NTF and CTF of PS derived from a full-length
PS molecule is required for the pathological effects of PS
mutations.
What is the nature of the limiting factor(s) that determines the
integrity of the functional complex of PS molecules? Our finding that
mt PS2/388stop, which comprises the NH2-terminal 87% of
the entire length of PS2 as well as 7 of the 8 putative TM domains,
failed to overproduce A
42 strongly suggests that some critical
subdomain in the COOH-terminal portion of PS2, or an as yet
unidentified binding protein(s) that interacts with this domain, should
play key roles in the formation of functional complex of PS. An
alternative possibility would be that the integrity of the whole PS
molecule, not particular subdomains, is required for the formation of
the complex. Further efforts to define this subdomain of PS2 or
identify binding proteins that are essential for the function as well
as stabilization of PS should facilitate our understanding of the
mechanisms whereby mt PS proteins influence the
-cleavage of
APP
to overproduce A
42, thereby leading to AD.
We thank A. Koyama and N. Takasugi for
skillful technical assistance, G. Thinakaran for anti-PS2loop
antiserum, J. Q. Trojanowski for helpful comments, D. J. Selkoe
for making available preprints of manuscripts in press, and Takeda
Chemical Industries for continuous support.