Increased Production of
-Amyloid and Vulnerability to
Endoplasmic Reticulum Stress by an Aberrant Spliced Form of
Presenilin 2*
Naoya
Sato
§,
Kazunori
Imaizumi
§¶,
Takayuki
Manabe
§,
Manabu
Taniguchi
§,
Junichi
Hitomi
§,
Taiichi
Katayama
§,
Takunari
Yoneda
§,
Takashi
Morihara
§,
Yuichi
Yasuda
§,
Tsutomu
Takagi
,
Takashi
Kudo
,
Takehide
Tsuda**,
Yasuto
Itoyama**,
Takao
Makifuchi
,
Paul E.
Fraser§§,
Peter
St George-Hyslop§§, and
Masaya
Tohyama
§
From the Departments of
Anatomy and Neuroscience and
Clinical Neuroscience, Psychiatry, Graduate School of Medicine,
Osaka University, Osaka 565-0871, Japan, ** Department of Neurology,
Tohoku University School of Medicine, Sendai 980-8574, Japan,

Department of Clinical Research, Saigata
National Hospital, Niigata 949-3193, Japan,
§§ Center for Research into Neurodegenerative
Diseases, Department of Medicine (Neurology), University of Toronto,
Toronto, Ontario M5S 1A8, Canada, and § CREST, Japan
Science and Technology Corporation, Osaka 565-0871, Japan
Received for publication, August 1, 2000, and in revised form, October 5, 2000
 |
ABSTRACT |
An alternative spliced form of the presinilin 2 (PS2) gene (PS2V) lacking exon 5 has previously been reported to
be expressed in human brains in sporadic Alzheimer's disease (AD).
PS2V encodes the amino-terminal portion of PS2, which contains residues
Met1-Leu119 and 5 additional amino acid
residues (SSMAG) at its carboxyl terminus. Here we report that PS2V
protein impaired the signaling pathway of the unfolded protein
response, similarly to familial AD-linked PS1 mutants and caused
significant increases in the production of both amyloid
40 and
42. Interestingly, PS2V-encoding protein was expressed in neuropathologically affected neurons of the
hippocampal CA1 region and temporal cortex in AD patients. These
findings suggest that the aberrant splicing of the PS2 gene may be
implicated in the neuropathology of sporadic AD.
 |
INTRODUCTION |
Alzheimer's disease
(AD)1 is a neurodegenerative
disorder clinically characterized by progressive loss of memory and
other cognitive abilities. Pathologically, severe neuronal loss, glial proliferation, extracellular deposition of senile plaques composed of
amyloid
protein (A
), and intraneuronal neurofibrillary tangles are found in the AD brain (1). Direct relationships, however, between
these morphological changes and the molecular mechanisms of AD onset
have not been established. Familial forms of AD (FAD) have been linked
to mutations in three different genes; the amyloid precursor protein
(APP) gene on chromosome 21 (2), the presenilin 1 (PS1) gene on
chromosome 14 (3), and the presenilin 2 (PS2) gene on chromosome 1 (4,
5). Because the pathological features of both FAD and sporadic AD
brains are thought to be identical or quite similar, genes mutated in
FAD are considered to be logical candidates for further investigation
of the etiology of sporadic AD.
Alternative splicing represents a typical mechanism underlying
regulation of gene expression in eukaryotic cells (6, 7). Exon
selection results in the production of different protein isoforms from
the same gene, isoforms that may share functions with the original
form. Alternatively, variant protein isoforms may either lack function
or confer novel characteristics on their cellular environment. In fact,
two splicing defects lacking exon 4 (8, 9) and exon 9 (10) of the PS1
transcript have been identified in FAD, and tau splicing mutations that
increase four-repeat isoforms containing exon 10 of tau were found in
frontotemporal dementia and Parkinsonism linked to chromosome 17 (11).
In addition, aberrant transcripts of the excitatory amino acid
transporter-2 gene are commonly present in sporadic amyotrophic
lateral sclerosis patients (12).
Recently, we found an alternatively spliced form of the PS2 gene, which
leads to generation of mRNA lacking exon 5 in sporadic AD brains,
and this product (PS2V) was preferentially expressed in AD brains
compared with those of age-matched controls (13). From in
vitro experiments, the aberrant splicing was demonstrated to be
induced in cultured cells under hypoxia. The neuroblastoma lines that
were stably transfected with PS2V were shown to be susceptible to
various cell stresses. However, the mechanisms by which PS2V sensitizes
cells to various stresses have been unknown, and it also has been
unclear whether PS2V is implicated in the neuropathology of sporadic
AD.
We report here that PS2V protein affected the unfolded protein response
(UPR) and also caused an increase in production of both A
x-40 and
A
x-42. Furthermore, PS2V-encoding protein was indeed translated from
the aberrant spliced form of the PS2 gene in neuropathologically
affected neurons of the hippocampus and the temporal cortex in sporadic
AD patients.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
An antibody to PS2V was obtained by serial
immunization of rabbits with the synthetic peptide SSMAG. The
anti-SSMAG antibody was then purified using a peptide affinity column
(Multiple Peptide Systems). An antibody recognizing the PS2 amino
terminus has been described before (13). The anti-A
x-42 monoclonal
antibody was obtained by serial immunization of mice with the synthetic
peptide MVGGVVIA. The anti-A
x-42 antibody was then purified using a
peptide affinity column. The anti-TAU2 monoclonal antibody,
anti-
-actin monoclonal antibody, and anti-Bcl-xl antibody
were purchased from Sigma, Stressgen, and MBL, respectively.
Immunoprecipitation and Ire1 Phosphorylation
Assay--
Immunoprecipitation was performed on 20 µg of crude
lysate from HEK293T cells transfected with flag-tagged wild PS2 or PS2V using anti-PS2N or anti-SSMAG polyclonal antibodies. Western blotting analysis was performed with an anti-flag monoclonal antibody. For
analysis of Ire1 phosphorylation, mock, wild PS2, or PS2V and Ire1,
which is tagged with flag epitope sequence at its carboxyl terminus,
were transiently cotransfected into HEK293T cells. Cells were incubated
for 2 h with 32P, extracted by Nonidet P-40 lysis
buffer (1% Nonidet P-40, 10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin), and
immunoprecipitated using anti-flag antibody. Quantitative analysis of
intensities of phosphorylated Ire1 was performed on a Power Macintosh
G3 computer using the public domain NIH Image program.
Immunohistochemistry and Electron
Microscopy--
Immunohistochemical processing was performed using
free-floating sections and the immunoperoxidase method. The sections
were fixed for 2 h and then processed for PS2V
immunohistochemistry. The anti-SSMAG and anti-PS2N antibodies were used
at a dilution of 1:500. Biotinylated anti-rabbit IgG (Vectastain Elite)
was used as a secondary antibody. Immunoreactivity was visualized with
0.05% diaminobenzidine and 0.01% hydrogen peroxide in 50 mM Tris, pH 7.6. After postfixation with 1%
OSO4 for 1 h and dehydration, they were
flat-embedded in Epon. Ultrathin sections were viewed without uranyl
acetate or lead citrate staining using an H-7000 electron microscope (Hitachi).
Mammalian Cell Culture and Analysis of Cell Viability--
Human
neuroblastoma SK-N-SH cells and mouse Neuro 2a cells were cultured in
-minimal essential medium and Dulbecco's modified Eagle's medium
supplemented with fetal calf serum, respectively. When cells achieved
confluence, the calcium ionophore A23187 (0.5 µM)
or 0.5 µg/ml tunicamycin (Tm) was added. Cell viability under stress
conditions was determined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay after 20 h of treatment with Tm or A23187 (14).
Preparation of Total RNA and RNA Blot Hybridization--
Total
RNA was extracted and purified from SK-N-SH cells under both normal and
stress conditions using an RNeasy total RNA kit (Qiagen). Aliquots of
20 µg of RNA were applied to formaldehyde-formamide gels,
electrophoresed, and blotted onto nylon filters. RNA was hybridized
with human GRP78/BiP cDNA labeled using a random-labeling kit
(Takara). The autoradiograms were quantified by densitometry to
determine the relative levels of GRP78 mRNA.
Semliki Forest Virus (SFV) Expression System--
Human GRP78
cDNA was subcloned into the pSFV1 expression plasmid (Life
Technologies). The GRP78 protein could then be expressed by infection
of SK-N-SH cells with the packaged SFV recombinant viral particles.
Control packaged virus was prepared from pSFV3-lacZ plasmid. Treatment
with Tm or A23187 was performed 12 h after infection. GRP78
expression was detected with anti-KDEL antibody (Stressgen).
A
Enzyme-linked Immunosorbent Assay and Metabolic Labeling of
APP--
Neuro 2a cells were plated on six-well dishes, and the medium
was changed the next day. Then, the culture media was collected 24 h after the medium change and subjected to sandwich enzyme-linked immunosorbent assays as developed by Takeda Chemical Industries, Ltd.
(15). For metabolic labeling of APP, stable transformants of each PS2
construct were incubated for specified times with [35S]methionine. The cells were extracted by
Nonidet P-40 lysis buffer and immunoprecipitated using anti-APP
antibody (22C11; Roche Molecular Biochemicals).
Brain Samples--
Brains from sporadic AD patients, age-matched
controls, and diseased controls were used for this study. Each brain
had a histopathologically confirmed diagnosis. Some of these human
brain samples were obtained from The Netherlands Brain Bank, autopsied
between 4 and 8 h postmortem, and stored at
80 °C until use
(Table I). The other brains of sporadic AD patients and diseased
controls were from the Department of Clinical Research, Saigata
National Hospital.
 |
RESULTS |
In Vitro Expression of PS2V Protein--
Recently, we found an
alternatively spliced form of the PS2 gene, which leads to generation
of mRNA lacking exon 5 in sporadic AD brains, and we found that
this product was preferentially expressed in AD brains compared with
those of age-matched controls (13). The lack of exon 5 causes a
frameshift in exon 6. This product (PS2V) encodes the amino-terminal
portion of PS2, which contains residues
Met1-Leu119 and an additional 5 amino acid
residues (SSMAG) at its carboxyl terminus (Fig.
1A). We generated a polyclonal
antibody specific for this "SSMAG" (anti-SSMAG) and a polyclonal
antibody specific for the PS2 amino-terminal portion (anti-PS2N). The
specificities of these were checked as shown in Fig. 1B.
Immunofluorescence assay showed that wild PS2 protein was localized to
intracellular membrane compartments, especially the endoplasmic
reticulum (ER) and Golgi apparatus (data not shown). These findings
were consistent with those of previous studies (16). PS2V
immunoreactivities were also found in the ER and Golgi as well as
in wild PS2 (data not shown).

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Fig. 1.
Expression of PS2V in vitro and
in vivo. A, structure of protein that was
generated from the PS2 gene. Because of the lack of exon 5, a frameshift occurs in exon 6. PS2V encods the amino-terminal portion
of PS2 and an additional 5 amino acid residues (SSMAG) at its carboxyl
terminus. PS2V has only one transmembrane region. B,
immunoprecipitation was performed with anti-PS2N or anti-SSMAG
polyclonal antibodies on 20 µg of crude lysate from flag-tagged wild
PS2- or PS2V-transfected HEK293T cells followed by Western blotting
analysis with an anti-flag monoclonal antibody. The arrow
and arrowhead show wild PS2 and PS2V proteins, respectively.
C, expression of PS2V protein under hypoxic conditions.
Immunoprecipitation with anti-PS2N antibody was followed by Western
blotting with anti-SSMAG antibody. PS2V protein was detected in SK-N-SH
cells exposed to hypoxia. N, normoxia; H,
hypoxia. Preabsorption of anti-SSMAG antibody was performed with 10 µM synthetic peptide (SSMAG) antigen (+Abs).
D, PS2V protein expressed in sporadic AD brain.
Immunoprecipitation was performed on 20 µg of crude lysate from the
temporal cortex of sporadic AD (AD#9) or control
(C#1) cases with anti-SSMAG antibody. Western
blotting analysis was performed with anti-PS2N antibody.
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We examined whether the PS2 splice variant could actually be translated
to the PS2V protein under conditions of hypoxia, because the aberrant
splicing of PS2 was induced by hypoxic stress in cultured cells (13).
Human neuroblastoma SK-N-SH cells were exposed to hypoxia for 20 h, and immunoprecipitation experiments were performed using an
anti-SSMAG antibody. PS2V protein, the molecular mass of which
was ~15 kDa, was detected in extracts of hypoxic cells by Western
blotting using an anti-PS2N antibody (Fig. 1C).
PS2V Affects the UPR Signaling and the Sensitivity to ER
Stress--
Previously, missense mutations in PS1 were reported to
downregulate the UPR and to lead to vulnerability to ER stress (17). To
examine whether the alternative spliced form of PS2 sensitizes cells to
ER stress similarly to PS1 mutants, SK-N-SH cells, which were stably
transfected with PS2V expression plasmids, were stimulated by ER
stressors such as Tm, which induces ER stress by preventing protein
glycosylation, and the calcium ionophore A23187, which depletes
intracellular calcium stores. The cells expressing PS2V were more
susceptible to ER stresses (Fig.
2A). The expression levels of
PS2V in the stable transfectants were very low, and exogenous
expression of PS2V did not affect the expression of endogenous
presenilins; i.e. we could not observe that PS2V replaces the endogenous presenilins (data not shown).

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Fig. 2.
Sensitivity to ER stress and effects on the
UPR in SK-N-SH cells expressing PS2V. A,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay. Stable transfectants of wild PS2 (w14, w17, w20, and
w23) and PS2V (dEX5-1,-3,-4,-5,-6) were treated
with 0.5 µg/ml tunicamycin (left) or 0.5 µM
A23187 (right). MTT reduction was quantified as a percentage
of the cells at 0 h. *, p < 0.01 compared with
corresponding values for wild PS2-transfected cells (n = 4; mean ± S.D. is shown). B, after treatment of
SK-N-SH cells stably transfected with wild PS2 or PS2V with Tm (0.5 or
1 µg/ml) or A23187 (0.5 or 1 µM). A human GRP78
cDNA fragment was used as a probe (upper panel). A
-actin cDNA fragment was used as a control probe (lower
panel). N, samples before treatment with Tm or
A23187.
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When misfolded proteins accumulate in the ER lumen from ER stress, gene
expression of ER molecular chaperones such as GRP78/BiP and GRP94 is
known to be immediately induced to refold the unfolded proteins (18,
19). Before and after treatment with Tm or A23187, total RNAs were
isolated from each stable transfectant. Under nonstress conditions, the
levels of GRP78 mRNA in cells expressing PS2V were reduced to
~50% compared with those of the transfectants of mock or wild PS2
(Fig. 2B). When cells were treated with 0.5 µg/ml Tm for
6 h, GRP78 mRNA was induced ~20-fold in mock or wild PS2
transfectants. In contrast, it was markedly inhibited in PS2V transfectants to ~10% of that in the controls (Fig. 2B).
Treatment of each cell line with 0.5 µM A23187 for 6 h led to the same results (Fig. 2B). Decreased induction of
GRP78 mRNA in cells expressing PS2V was caused by the impaired
phosphorylation of Ire1, an ER stress sensor, which is known to
oligomerize and be autophosphorylated by its own kinase domain on
accumulation of unfolded protein in the ER (Ref. 20 and Fig.
3A). In PS2V transfectants, the levels of autophosphorylation of Ire1 were decreased to ~50% compared with the mock-transfected cells. Phosphorylation of tau or
GSK-3
was not changed in any of these cell lines (data not shown).

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Fig. 3.
Effects of PS2V on phosphorylation of Ire1
and direct binding of PS2V and Ire1. A, phosphorylation
of Ire1 in each cell. Human Ire1 tagged with the flag epitope and wild
PS2 or PS2V were transiently coexpressed in HEK293T cells. Upper
panel, phosphorylated Ire1 levels (P-Ire1);
middle panel, Western blotting of Ire1 in each cell.
Although Ire1 was expressed to an equivalent extent, the level of
phosphorylated Ire1 was only reduced in cells expressing PS2V.
Lower panel, Ire1-phosphorylation levels were analyzed
quantitatively and normalized to expression levels of Ire1 protein.
Values are arbitrary intensities and represent the mean ± S.D. of
four analyses. B, interaction of PS2V and Ire1. HEK293T
cells were transiently cotransfected with the indicated plasmids.
Upper panel, lysates or immunoprecipitates (IP)
with anti-PS2N antibody were immunoblotted with anti-flag antibody.
Lower panel, lysates or immunoprecipitates with anti-flag
antibody were immunoblotted with anti-PS2N antibody. C,
Western blotting for Bcl-xl. HEK293T cells were transiently
cotransfected with Ire1-flag and Bcl-xl. Cell lysates or
immunoprecipitates with anti-flag antibody were immunoblotted with
anti-Bcl-xl antibody. Note that Bcl-xl is not coimmunoprecipitated with
Ire1-flag.
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We examined whether PS2V protein binds to Ire1 as well as PS1 (17).
HEK293T cells were transiently cotransfected with expression plasmids
for PS2V and Ire1-flag. Lysates were immunoprecipitated with anti-PS2N
antibody and then blotted with anti-flag antibody. PS2V was
coprecipitated with 140-kDa Ire1-flag (Fig. 3B). As the reverse experiments, we prepared immunoprecipitates using the anti-flag
antibody and blotted with anti-PS2N antibody. The PS2V protein was
coprecipitated with Ire1 (Fig. 3B). Bcl-xl, which is a
transmembrane protein of the ER and mitochondria, did not coimmunoprecipitate with Ire1 (Fig. 3C). These results
indicate that PS2V directly binds to Ire1 on the membrane of the ER.
However, we could not ascertain the cause of the decrease in
phosphorylated Ire1 by PS2V in the present study.
To confirm that vulnerability to ER stress by the repression of GRP78
mRNA induction in PS2V transfectants, SK-N-SH cells stably
transfected with PS2V were infected with recombinant GRP78 using
SFV-GRP78, and sensitivity to various ER stresses was examined. Increased sensitivity to ER stressors in SK-N-SH cells expressing PS2V
was reversed by the overexpression of GRP78 (Fig.
4).

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Fig. 4.
Effects of exogenous GRP78 expression on
sensitivity to ER stress. Quantitative analysis of the protective
effects of infection with recombinant SFV-GRP78 on the vulnerability to
ER stress in SK-N-SH cells expressing PS2V. Human GRP78 was transiently
expressed in cells stably transfected with wild PS2 or PS2V using the
SFV system (white columns). *, p < 0.01 compared with corresponding values for LacZ-expressing cells
(black columns; n = 8; mean ± S.D. is
shown). Left panel, results of Western blotting with
anti-KDEL antibody, which detects GRP78 (arrow).
+LacZ, LacZ-infected cells; GRP78, GRP78-infected
cells.
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Effects of Production of A
--
One of the most common
pathological features in AD brain is the deposition of A
. FAD-liked
PS1 mutants are known to affect production of A
(21, 22). Therefore,
it is of interest to determine whether PS2V enhances secretion of
endogenous A
into the cultured media. Twenty-four hours after
changes of conditioned media of Neuro 2a cell lines, which stably
expressed mock, wild PS2, PS2V, or FAD-linked PS2 mutant (Volga German
type, N141I), we collected those and measured the amounts of released
A
x-40 and A
x-42 by enzyme-linked immunosorbent assay (15).
Secreted A
x-40 and A
x-42 were both significantly increased by
~1.3-fold in stable transfectants of PS2V compared with the cells
expressing the mock or wild PS2 (Fig.
5A). However, the ratios of
A
x-42/A
x-40 plus A
x-42 were nearly equal to those of the
control cells. FAD-linked PS2 mutant specifically led to elevation of
A
x-42 secreted into the media but did not affect secretion of
A
x-40.

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Fig. 5.
Released A and
maturation of APP in Neuro 2a. A, A x-40
(left) and A x-42 (center) released into the
media for 24 h were determined in stable Neuro 2a transfectants of
each construct, and the ratios of A x-42/A x-40 plus A x-42
(right) were calculated. *, p < 0.01 compared with corresponding values for mock transfected cells
(n = 8; mean ± S.D. is shown). VG,
Volga German-type PS2 mutants (N141I). Lower panel, Western
blotting of APP using 22C11 monoclonal antibody. B, released
A from transformants of Ire1-derivative constructs. A x-40
(left), A x-42 (center), and the ratios of
A x-42/A x-40 plus A x-42 (right) released into the
media for 24 h are shown. Values represent means ± S.D. of
eight independent experiments. The expression levels of APP were
equivalent in each line (lower panel). C, Cells were labeled
with [35S]methionine for 10 min and chased for the
indicated time. Lysates were immunoprecipitated with anti-APP
monoclonal antibody (22C11). N- and
O-glycosylated (N+O) forms increased
with the indicated time course in transformants of wild PS2. In
contrast, maturation of APP was inhibited in transformants of PS2V.
D, quantitative analysis of the intensities of
autoradiographs was performed on a Power Macintosh G3 computer using a
public domain NIH Image program. Values are arbitrary intensities of
each band in C. Circle, N-glycosylated
APP; square, N- and O-glycosylated
APP.
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Having demonstrated that PS2V affects the function of Ire1, we examined
whether dominant negative Ire1 causes an increase in A
production,
as well as PS2V. This Ire1 derivative
Ire1 has a truncated
cytoplasmic region and therefore lacks the kinase and RNase L domains,
and we have confirmed that cells expressing
Ire1 showed
down-regulation of GRP78 induction and significantly increased
vulnerability to ER stress (17). Stable Neuro 2a transformants of
Ire1 showed that secreted A
x-40 and A
x-42 were significantly increased by ~1.3-fold compared with the cells expressing the mock
(Fig. 5B). The ratio of A
x-42/A
x-40 plus A
42 was
not changed in these cells.
We speculated that PS2V affects the correct folding and maturation of
APP, because PS2V down-regulates the UPR signaling mediated by
disturbed Ire1 and also decreases the expression levels of GRP78 in a
steady-state condition. Therefore, we examined the process of
biosynthesis of APP in stable transformants of wild PS2 and PS2V. The
cells were labeled for 10 min with [35S]methionine and
chased for 0-50 min. As shown in Fig. 5C,
immunoprecipitates at 0 min of chase showed a single band of 95 kDa
that apparently corresponds to N-glycosylated APP in both
cells. In wild PS2-expressing cells, two new N- and
O-glycosylated forms at 105 and 130 kDa were shown within a
20-min chase period. At the 50-min chase time, the bands of the matured
forms of APP reached maximum intensity. In contrast, N- and
O-glycosylated forms of APP were not detected within the
20-40-min period, and small amounts of those were observed at 50 min
in PS2V-expressing cells (Fig. 5, C and D). These
results indicate that PS2V caused inhibited intracellular maturation of APP, involving the retention of immature forms of APP in the ER.
Expression of PS2V Protein in Sporadic AD Brains--
To confirm
that PS2V was translated in sporadic AD brains, we carried out
immunoprecipitation followed by Western blotting. PS2V protein was only
detected in extracts of AD brain (AD 9; Table II) by Western blotting
(Fig. 1D). On immunohistochemical analysis using the
anti-SSMAG and anti-PS2N antibodies, PS2V-immnnoreactive cells were
observed in the CA1 region of the hippocampus (Fig. 6, B-F) and the temporal
cortex (data not shown). Immunoreactivity was completely abolished by
the preabsorption of the antibodies with an excess of synthetic peptide
antigen (10 µM; Fig. 6C). In the hippocampus,
the cells expressing PS2V protein were pyramidal neurons and were
scattered throughout the CA1 region. Judging from the morphology,
PS2V-immunoreactive cells could be divided into two cell types. The
first type had neurons showing moderate immunoreactivitiy for PS2V and
were localized to perinuclear regions (Fig. 6, B and
D). This type of cell was diffusely distributed throughout
CA1 of the hippocampus, and some of these cells exhibited degenerative
changes such as shrinkage and loss of neurites. The other type of cell
had apoptotic neurons containing PS2V-immunoreactive inclusion bodies
in the cytoplasm (Fig. 6, E, F, H, and I) and occasionally also contained neurofibrillary tangles (Fig.
6H). These neurons were sparsely distributed in the CA1
regions. Double labeling of PS2V and A
showed that some of the
PS2V-immunoreactive neurons lay closely adjacent to extracellular
amyloid deposits (Fig. 6G).

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Fig. 6.
PS2V immunoreactivity in sporadic AD
brain. A, staining with cresyl violet in
hippocampal sections of the human AD brain. B, staining with
anti-SSMAG antibody in the boxed area in A. The
arrowhead shows the PS2V-immmunoreactive structures.
C, absorption of the antibody by excessive amounts of
synthetic peptide (SSMAG, 10 µM). Morphologically intact
neurons (B) and degenerative neurons (D) show
PS2V immunoreactivities stained by anti-SSMAG antibody. E,
PS2V-immunoreactive inclusion bodies stained by anti-SSMAG antibody
(arrowhead). F, PS2V-immunoreactive inclusion
body stained by anti-PS2N antibody. G, double staining of
PS2V (anti-SSMAG) and A x-42. The stars and
arrowheads show amyloid plaques (blue) and
PS2V-immunoreactive structures (brown), respectively.
H, double staining for PS2V (anti-SSMG) and tau.
Colocalization of PS2V and a neurofibrillary tangle in single neurons
is shown (brown, PS2V; blue, neurofibrillary
tangle). I, electron microscopy of a PS2V-immunoreactive
neuron in sporadic AD brain (anti-SSMAG). Deposition of DAB reaction
product within a single neuron (arrowhead) is shown.
Scale bar, 4 µm.
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To quantify the number of neurons bearing the PS2V protein in sporadic
AD (10 cases), age-matched controls (nonneurological diseases, 6 cases), and control diseased brain (7 cases; Table I), immunohistochemical analysis was
performed on three sections of the hippocampal CA1 region in each case.
PS2V immunoreactivity was observed in all specimens from AD brains, and
the number of positive neurons in AD brains was almost more than
approximately ~100 per field (Table
II). In contrast, only one specimen from the age-matched control group (specimen 3; Table II) showed a small
number of PS2V-immunoreactive neurons. The other age-matched control
and diseased control specimens had no positive neurons in any of the
sections. The number of neurons containing PS2V protein in this control
case (specimen 3) was extremely low, and the neurons were located only
in the CA1 region. This case showed no clinical symptoms of dementia,
but a small number of amyloid plaques were pathologically observed in
the CA1 region and cortex.
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Table II
Expression of PS2V in AD brains
The PS2V was significantly expressed more in sporadic AD patients than
in controls. AD, sporadic AD patient; C, age-matched control. Age shown
is that at death. +, ~30 positive cells/specimen; ++, 30~100
positive; +++, >100 positive.
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DISCUSSION |
Effects of PS2V Protein on UPR Signaling--
SK-N-SH cells stably
transfected with PS2V exhibited increases in susceptibility to various
ER stresses. Overexpression of GRP78 almost completely restored
resistance to some ER stresses up to the level of that of wild PS2
transfectants, suggesting that PS2V protein increases vulnerability to
ER stress because of the inhibition of GRP78 mRNA induction. The
decreased induction of GRP78 mRNA was attributable to the
disturbance of UPR signaling caused by the reduction of Ire1
phosphorylation. Because PS2V directly binds to Ire1 on the membrane of
the ER, the binding might affect phosphorylation of Ire1. On the basis
of its structural features, it is unlikely that PS2V protein directly
dephosphorylates Ire1 molecules. If PS2V protein directly interacts
with Ire1, one possible mechanism responsible for the decrease in
phosphorylated Ire1 is that PS2V may bind to the kinase domain of Ire1
or a particular region that is important for modulating its activity,
and this causes inhibition of Ire1 phosphorylation. Alternatively, PS2V might inhibit the oligomerization of Ire1 and block
transautophosphorylation by neighboring Ire1 (19).
A
Production in PS2V-expressing Cells--
Secretion of both
A
x-40 and A
x-42 was increased in stable transformants of PS2V
compared with that in the controls. Recently, it was reported that
secreted A
levels were reduced by transfection of GRP78, and it was
suggested that GRP78 modulates A
secretion (23). Because basal
levels of GRP78 expression were decreased in PS2V-expressing cells
under the non-ER stress conditions, the increased secretion of A
could be associated with the down-regulation of GRP78. Stable
transformants of dominant negative Ire1 also showed significantly
increases of both A
x-40 and A
x-42 released into media under
steady-state conditions. Therefore, it is possible that the cause of
increased A
in PS2V expressing cells is based on the dysfunction of
Ire1 under non-ER stress conditions. Furthermore, N- and
O-glycosylated forms of APP in the transformants of PS2V were reduced compared with those of cells expressing wild PS2 at 20-50
min chase time after the pulse. Alternatively,
N-glycosylated forms of APP were accumulated and could be
considered to be retained in the ER, suggesting that correct folding of
APP is probably impaired in cells expressing PS2V. Increase in total
antibody could be associated with accumulation of unfolded APP in
the ER or delay of sorting of APP from ER to Golgi. However, in this study, we could not measure the intracellular A
pool in PS2V- or
Ire1-expressing cells. Therefore, to clarify the detailed mechanisms responsible for increased A
production in these cells, further analyses are needed.
Expression of PS2V Protein in Sporadic AD Brains--
PS2V protein
was detected in the cortex and hippocampus of all AD patients. In
age-matched control and diseased control brains, PS2V was found in only
one case. The number of neurons containing PS2V protein in this control
case was extremely low, and they were located only in the CA1 region.
The control case showed no clinical symptoms of dementia, but a small
number of amyloid plaques were pathologically observed in the CA1
region and cortex. These findings suggest a close correlation between
expression of PS2V protein and deposition of A
. At present, it is
unknown whether aberrant splicing of the PS2 gene is a trigger for
development of sporadic AD or whether it occurs as a result of
neuronal damage in AD brains. However, it could be a factor that
compromises neuronal viability in sporadic AD brains, because PS2V
causes vulnerability to ER stress and increases in A
production.
Indeed, immunohistochemically, cells showing intense immunoreactivities
for PS2V were morphologically degenerative or apoptotic, suggesting
that excessive production of PS2V protein may cause neuronal damage in
sporadic AD brain.
In summary, our results indicate that PS2V, which is expressed in
neuropathologically affected neurons of brains in sporadic AD patients,
affects both UPR signaling and A
production. Because both FAD-linked
PS1 mutants and the PS2V protein caused down-regulation of the UPR
pathway and resultant vulnerability to ER stress, regulation of
components of the UPR pathway could provide an opportunity for the
development of therapeutic strategies for AD.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H. Fukumoto (Takeda Chemical
Industries) for the A
enzyme-linked immunosorbent assay. We are
grateful to the support of Tanabe Seiyaku Co., Ltd., for the dispatch
of researchers K. I., N. S., and T. K.
 |
FOOTNOTES |
*
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 should be addressed: Dept. of Anatomy
and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3221; Fax:
81-6-6879-3229; E-mail: imaizumi@anat2.med.osaka-u.ac.jp.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006886200
 |
ABBREVIATIONS |
The abbreviations used are:
AD, Alzheimer's
disease;
FAD, familial AD;
APP, amyloid precursor protein;
PS, presenilin;
PS2V, an alternative spliced form of the PS2 gene;
A
, amyloid
protein;
ER, endoplasmic reticulum;
UPR, unfolded protein
response;
GRP78/BiP, glucose-regulated protein/immunoglobulin-binding
protein;
Tm, tunicamycin;
SFV, Semliki Forest virus.
 |
REFERENCES |
1.
|
Selkoe, D. J.
(1994)
Annu. Rev. Neurosci.
17,
489-517
|
2.
|
Goate, A.,
Chartier-Harlin, M. C.,
Mullan, M.,
Brown, J.,
Crawford, F.,
Fidani, L.,
Giuffra, L.,
Haynes, I. N.,
James, L.,
et al..
(1991)
Nature
349,
704-706[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Sherrington, R.,
Rogaev, E. I.,
Liang, Y.,
Rogaeva, E. A.,
Levesque, G.,
Ikeda, M.,
Chi, H.,
Lin, C.,
Li, G.,
Holman, K.,
et al..
(1995)
Nature
375,
754-760[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Rogaev, E. I.,
Sherrington, R.,
Rogaeva, E. A.,
Levesque, G.,
Ikeda, M.,
Liang, Y.,
Chi, H.,
Lin, C.,
Holman, K.,
Tsuda, T.,
et al..
(1995)
Nature
376,
775-778[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Levy-Lahad, E.,
Wasco, W.,
Poorkaj, P.,
Romano, D. M.,
Oshima, J.,
Pettingell, W. H., Yu, C. E.,
Jondro, P. D.,
Schmidt, S. D.,
Wang, K.,
et al..
(1995)
Science
269,
973-977
|
6.
|
Smith, C. W.,
Patton, J. G.,
and Nadal-Ginard, B.
(1989)
Annu. Rev. Genet.
23,
527-577
|
7.
|
Maniatis, T.
(1991)
Science
251,
33-34
|
8.
|
Tysoe, C.,
Whittaker, J.,
Xuereb, J.,
Cairns, N. J.,
Cruts, M.,
VanBroeckhoven, C.,
Wilcock, G.,
and Rubinsztein, D. C.
(1998)
Am. J. Hum. Genet.
62,
70-76[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Jonghe, C. D.,
Cruts, M.,
Rogaeva, E. A.,
Tysoe, C.,
Singleton, A.,
Vanderstichele, H.,
Meschino, W.,
Dermant, B.,
Vanderhoeven, I.,
Backhovens, H.,
et al..
(1999)
Hum. Mol. Genet.
8,
1529-1540[Abstract/Free Full Text]
|
10.
|
Perez-Tur, J.,
Froelich, S.,
Prihar, G.,
Crook, R.,
Baker, M.,
Duff, K.,
Wragg, M.,
Busfield, F.,
Lendon, C.,
Clark, R. F.,
et al..
(1995)
NeuroReport
7,
297-301[Medline]
[Order article via Infotrieve]
|
11.
|
Hutton, M.,
Lendon, C. L.,
Rizzu, P.,
Baker, M.,
Froelich, S.,
Houlden, H.,
Pickering-Brown, S.,
Chakraverty, S.,
Isaacs, A.,
Grover, A.,
et al..
(1998)
Nature
393,
702-705[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Lin, C. L.,
Bristol, L. A.,
Jin, L.,
Dykes-Hoberg, M.,
Crawford, T.,
Clawson, L.,
and Rothstein, J. D.
(1998)
Neuron
20,
589-602[Medline]
[Order article via Infotrieve]
|
13.
|
Sato, N.,
Hori, O.,
Yamaguchi, A.,
Lambert, J. C.,
Chartier-Harlin, M. C.,
Robinson, P. A.,
Delacourte, A.,
Schmidt, A. M.,
Furuyama, T.,
Imaizumi, K.,
et al..
(1999)
J. Neurochem.
72,
2498-2505[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Mattoson, M. P.,
Barger, S. W.,
Begley, J. G.,
and Mark, R. J.
(1995)
Methods Cell Biol.
46,
187-216[Medline]
[Order article via Infotrieve]
|
15.
|
Okada, A. A.,
Ishibashi, Y.,
Kikuchi, T.,
Kitada, C.,
and Suzuki, N.
(1995)
Biochemistry
34,
10272-10278[Medline]
[Order article via Infotrieve]
|
16.
|
Walter, J.,
Capell, A.,
Grunberg, J.,
Pesold, B.,
Schindzielorz, A.,
Prior, R.,
Podlisny, M. B.,
Fraser, P.,
Hyslop, P. S.,
Selkoe, D. J.,
and Haass, C.
(1996)
Mol. Med.
2,
673-691[Medline]
[Order article via Infotrieve]
|
17.
|
Katayama, T.,
Imaizumi, K.,
Sato, N.,
Miyoshi, K.,
Kudo, T.,
Hitomi, J.,
Morihara, T.,
Yoneda, T.,
Gomi, F.,
Mori, Y.,
et al..
(1999)
Nat. Cell Biol.
1,
479-484[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Sidrauski, C.,
Chapman, R.,
and Walter, P.
(1998)
Trends Cell Biol.
8,
245-249[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Welihinda, A. A.,
Tirasophon, W.,
and Kaufman, R. J.
(1999)
Gene Expr.
7,
293-300[Medline]
[Order article via Infotrieve]
|
20.
|
Tirasophon, W.,
Welihinda, A. A.,
and Kaufman, R. J.
(1998)
Genes Dev.
12,
1812-1824[Abstract/Free Full Text]
|
21.
|
Duff, K.,
Eckman, C.,
Zehn, C., Yu, X.,
Prada, C.-M.,
Perez-tur, J.,
Hutton, M.,
Buee, L.,
Harigaya, Y.,
Yager, D.,
et al..
(1996)
Nature
383,
710-713[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Borchelt, D. R.,
Thinakaran, G.,
Eckman, C. B.,
Lee, M. K.,
Davenport, F.,
Ratovitsky, T.,
Prada, C.-M.,
Kim, G.,
Seekins, S.,
Yager, D.,
et al..
(1996)
Neuron
17,
1005-1013[Medline]
[Order article via Infotrieve]
|
23.
|
Yang, Y.,
Turner, R. S.,
and Gaut, J. R.
(1998)
J. Biol. Chem.
273,
25552-25555[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.