Neurobiology of Aging 21 (2000) 829 – 836 www.elsevier.

com/locate/neuaging

Familial Alzheimer’s disease presenilin-1 mutants potentiate cell

cycle arrest
Susan M. Janicki, Stacy M. Stabler, Mervyn J. Monteiro*
Medical Biotechnology Center and Department of Neurology, & Division of Human Genetics, University of Maryland, 725 West Lombard Street,
Baltimore, Maryland 21201, USA

Received 18 May 2000; received in revised form 23 August 2000; accepted 28 August 2000

Abstract
We previously reported that overexpression of presenilin 1 and 2 (PS1 and PS2) in HeLa cells leads to cell cycle arrest, and that the
PS2(N141I) FAD mutant potentiates cell cycle arrest compared to wild-type PS2. Using similar BrdU incorporation studies we now report
that three different PS1 FAD mutants also increase cell cycle arrest compared to wild-type PS1when overexpressed in either HeLa cells or
an ATM deficient cell line. We detected reproducible differences in the degrees to which these FAD mutants induced arrest. PS1(P117L)
reduced BrdU incorporation the most (13 to 14%) followed by PS1(P267S) (7.5 to 9%), with the PS1(E280A) mutant inhibiting BrdU
incorporation the least (6 to 7%), compared to wild-type PS1. The degree to which the different mutants inhibited cell cycle progression
correlates somewhat with the age of AD onset induced by the mutations in carriers. Immunoblot analysis of protein extracts from
presenilin-overexpressing cells indicates that the cell cycle-regulated cytoplasmic pool of ␤-catenin is dramatically reduced, whereas the
insoluble ␤-catenin pool remains essentially unaffected. We discuss the implications of these findings in relationship to cell cycle arrest,
apoptosis and AD. © 2000 Elsevier Science Inc. All rights reserved.

Keywords: Presenilins; Alzheimer’s disease; Cell cycle; S phase; Apoptosis; ␤-catenin

1. Introduction somehow interconnected in causing an increase in accumu-

Alzheimer’s disease (AD), the most common cause of
dementia in the elderly, is characterized by progressive loss
of memory and a decline in cognitive function. The mech-
anisms that cause AD pathogenesis are unknown although
there is strong evidence that the disease is caused by the
interplay of several factors including age, genes, and the
environment. The majority of AD cases are late-onset, ap-
pearing in people over that age of 65 years, however, a
small percentage of cases (⬃5%), are early onset with
inception of disease occurring between the ages of 20 to 65
years. Many of the early onset cases have been traced to
familial autosomal dominant inheritance of mutations in
three genes: the ␤-amyloid precursor protein (APP) and two
homologous genes, presenilin-1 and 2 (PS1 and PS2) [8].
The precise mechanisms by which mutations in these three
genes cause AD are not known, although they all appear
* Corresponding author. Tel.: ⫹1-410-706-8132; fax: ⫹1-410-706-1732.
E-mail address: monteiro@umbi.umd.edu (M.J. Monteiro)
lation of the amyloidgenic A␤ fragment [8].
PS1 and PS2 are multi-pass trans-membrane proteins
which share 67% sequence identity, however, there is de-
bate regarding their precise topology. A commonly drawn
model depicts a polypeptide with eight trans-membrane
domains, such that the N- and C-termini and a large “loop”
between trans-membrane domains 6 and 7 are all oriented
toward the cytoplasm [9]. Although PS1 and PS2 share
extensive sequence identity along their entire lengths, their
N-terminal domains and the second half of their loops are
highly divergent, suggesting that these unique regions could
modulate potentially different functions of the two preseni-
lins. Examination of presenilin-linked familial AD (FAD)
mutations reveals that most are missense mutations in res-
idues conserved between the two polypeptides, suggesting
that these conserved residues are important for proper func-
tion.
Studies of presenilin homologs in a number of species
have indicated that the presenilin genes are required for
proper development. Mutation of the Caenorhabditis el-
egans presenilin gene, sel-12, produce defects in cell sig-

0197-4580/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 1 9 7 - 4 5 8 0 ( 0 0 ) 0 0 2 2 2 - 0
830 S.M. Janicki et al. / Neurobiology of Aging 21 (2000) 829 – 836
naling involving Notch-based receptors [9,16]. Disruption
of the Drosophila melanogaster presenilin gene is an em-
bryonic lethal with defective embryos displaying severe
Notch-like phenotypes [31,40]. Mammalian presenilins also
play important roles in early development, as disruption of
the mouse PS1 gene leads to death shortly after birth with
embryos displaying central nervous system defects together
with abnormal patterning of the axial skeleton and spinal
ganglia [28,39]. Interestingly, PS1⫺/⫺ mice can be rescued
by human transgenes containing FAD-linked mutations, in-
dicating that the FAD mutations do not affect presenilin
functions related to embryo development [4,26]. In contrast,
disruption of the mouse PS2 gene produces no obvious
defects, but PS1/PS2 double knock-out mice die earlier at
embryonic day 9.5 and, like PS1⫺/⫺ mice, display severe
misexpression of proteins involved in Notch signaling
[6,10]. These results indicate that there is functional redun-
dancy between PS1 and PS2; PS1 can compensate for PS2,
but PS2 cannot compensate for PS1, at least during early
mouse development.
A number of studies have indicated that presenilins are
involved in the regulation of apoptosis, and that cells ex-
pressing AD associated mutations in either PS1 or PS2 die
faster than cells expressing wild-type presenilins [3,5,7,37,
38,42]. Consistent with these findings we previously
showed that when overexpressed in epithelial HeLa cells the
PS2(N141I) FAD mutant induces increased apoptosis com-
pared to wild-type PS2 [12]. By BrdU incorporation studies
we further demonstrated that presenilin overexpression in
exponentially growing cultures leads to arrest in the G1
phase of the cell cycle, and that the PS2(N141I) mutant
potentiates the cell cycle arrest [13]. In this report we
demonstrate that cell cycle arrest is also potentiated by PS1
FAD mutants and that ␤-catenin levels are altered upon
presenilin overexpression.
2. Materials and methods
2.1. Construction of PS expression plasmids
The cloning of wild-type PS1 and PS2, and the FAD
PS2(N141I) mutant into a CMV expression plasmid was
previously described [12,13]. The wild-type PS1 expression
construct was used to prepare mutants corresponding to
FAD PS1(P267S), PS1(E280A) and PS1(P117L) mutations
by PCR modification using appropriately designed primers
as described previously [30].
2.2. Cell lines, cell culture
HeLa cells were grown in DMEM supplemented with
10% FBS [20], and an ataxia-telangiectasia (AT) trans-
formed cell line (GMO9607A, NIGMS Human Genetic
Mutant Cell Repository) was grown in MEM supplemented
with 10% FBS.
2.3. Immunofluorescence and BrdU staining
Exponentially growing cells were plated on glass cover-
slips, and transfected with plasmid expression constructs
using the CaPO4 coprecipitation method [12]. BrdU was
added to the media immediately after the transfection. At
19h post-transfection the cells were fixed in 50 mM Gly-
cine/70% ethanol at ⫺20°C for 20 min, rehydrated in PBS,
blocked in 0.8% BSA, and stained for expressed proteins.
Cells were next stained for BrdU incorporation (5-Bromo-
2⬘-deoxy-uridine Labeling and Detection Kit I, Boehringer
Mannheim), and with 1 ␮g/ml 4⬘6-diamidino-2-phenylin-
dole (DAPI) as previously described [13].
Antibodies used for the immunofluorescence staining of
PS proteins were described previously [13]. The stained
coverslips were viewed under a Leica DRB microscope
using a double FITC/Rhodamine cube to simultaneously
view PS staining (Rhodamine) and BrdU staining (FITC).
By scanning the slides with the double FITC/Rhodamine
cube the percentage of PS transfected cells that stained
positive for BrdU was determined. At least three popula-
tions of cells on independent coverslips were counted for
each construct evaluated. P values were calculated by com-
paring means in an unpaired two-tailed t test with the
Statview 512 program (Brain Power Inc.).
2.4. Fractionation of cells into soluble and insoluble
protein pools
HeLa cells transiently transfected with presenilin con-
structs were fractionated into soluble and insoluble protein
pools [19]. Dishes of transfected cells were washed two
times in ice-cold PBS, and the cells were scraped from the
dishes in 600 ␮l of PBS, spun down at 15,000 g for two
minutes (4°C), and resuspended in 150 ␮l of TEA buffer 1
(10 mM Triethanolamine-HCl (TEA), pH 7.4, 10 mM KCl,
1.5 mM MgCl2, 5 mM Iodoacetamide, and 1 mM Pefabloc).
Cells were left on ice for 10 min and then homogenized
15–20 times. 150 ␮l of TEA buffer 2 (10 mM TEA pH 7.4,
270 mM KCl, 1.5 mM MgCl2, 5 mM Iodoacetamide, and 1
mM Pefabloc) was then added, and the cells were vortexed
and spun at 100,000 g for 45 min in the ultracentrifuge at
4°C. The supernatants were removed from the pellets, and
the pellets were suspended in 300 ␮l of lysis buffer and
sonicated. BCA protein assays were used to measure the
protein concentration of each fraction, and equal amounts of
protein from each fraction were run on 8.5% SDS-PAGE
gels and transferred to nitrocellulose membranes.
2.5. Protein preparation, SDS electrophoresis, and
immunoblotting
Cells were lysed in a buffer containing protease inhibi-
tors [20], and protein concentration was determined by the
BCA protein assay (Pierce). For immunoblot analysis of PS
expression, lysates were mixed with Laemmli buffer con-
 S.M. Janicki et al. / Neurobiology of Aging 21 (2000) 829 – 836 831

taining 8 M urea and 15 mM DTT and electrophoresed

unboiled on 8.5% SDS-polyacrylamide gels. Lysates used
for analysis of ␤-catenin and ␤-tubulin were boiled in Lae-
mmli buffer and electrophoresed on 8.5% gels. The frac-
tionated proteins were transferred onto 0.2 ␮m nitrocellu-
lose membranes by electroblotting and processed as
described previously [12]. Antibodies used were the follow-
ing: a goat N-terminal specific anti-PS1 antibody (Santa
Cruz Biotechnology), a goat anti-␤-catenin antibody (Santa
Cruz Biotechnology) and a mouse monoclonal anti-␤-tubu-
lin antibody (Sigma).

3. Results

3.1. Transient overexpression of PS1 blocks cell cycle

progression: PS1 FAD mutants potentiate cell cycle
arrest

Our previous studies had indicated that the overexpres-

sion of presenilin 1 and 2 genes in HeLa and ATM⫺/⫺
cells induced cell cycle arrest [13]. Moreover, we demon-
strated by BrdU incorporation studies that cell cycle pro-
gression was more inhibited by PS2 proteins containing the
PS2(N141I) FAD mutation compared to the wild-type pro-
tein. In this study we investigated whether overexpression
of PS1 FAD mutants also potentiates cell cycle arrest. Three
different PS1 mutants PS1(P267S), PS1(E280A) and
PS1(P117L), which cause early onset AD (1,11,15,36; http:
molgen-www.uia.ac.be/ADMutations/) were studied. Of the
three mutations, the PS1(P117L) mutation is one of the
most aggressive FAD mutations described so far, causing
disease as early as the third decade of life [36]. Transient
transfection of wild-type PS1 and the three PS1 FAD mu-
tants in HeLa cells followed by immunoblotting of protein
lysates indicated that all constructs were expressed at sim-
ilar levels (Fig. 1). The full-length PS1 polypeptides mi-
grated as an approximately 45 kDa band (Fig. 1, lanes 2–5;
indicated by an arrow) and several high molecular weight
bands, the latter probably corresponding to PS1 complexes.
As expected these bands were not detected in untransfected
HeLa cells (Fig. 1, Lane 1).
To determine the effects of overexpressing PS1 on cell
cycle progression, HeLa cells exponentially growing on
coverslips were transfected with equivalent amounts of
plasmid DNA encoding either wild-type or mutant PS1
proteins. Immediately following the transfection, BrdU was
added to the medium and at 19 h the coverslips were fixed
and double stained for PS1 and BrdU [13]. The stained
coverslips were then examined by immunofluorescence mi-
croscopy and the percentage of PS1 expressing cells that
had incorporated BrdU into their genomes was determined.
As shown in Figs. 2 and 3 overexpression of wild-type PS1
significantly reduced BrdU labeling (53.1% ⫾ 3.2) com-
pared mock-transfected control cells (92.3% ⫾ 3.0), which
is consistent with our previous findings [13]. More interest-
Fig. 1. Immunoblot analysis of PS1 transfected HeLa cell lysates. Equiv-
alent amounts of protein lysates from PS1 transfected cells immunoblotted
with a PS1 specific antibody. Lysates were from cells transfected with the
following constructs: Lane 1, mock-transfected (control); lane 2, wild-type
PS1; lane 3, PS1(P267S); lane 4, PS1(E280A); and lane 5, PS1(P117L).
Full-length uncleaved PS1 is indicated by the arrow.
ingly, however, was that BrdU incorporation was reduced to
a greater extent upon overexpression of the three PS1 FAD
mutants compared to the wild-type protein (Fig. 3). BrdU
labeling was reduced 6.7% ((46.9 –53.1)/92.3 ⫻ 100) for the
PS1(E280A) mutant, 9.5% for the PS1(P267S) mutant, and
13.0% for the PS1(P117L) mutant compared to BrdU label-
ing obtained with wild-type PS1. Collectively, these results
indicate that the PS1 FAD mutants potentiate cell cycle
arrest in exponentially growing HeLa cells.
Previously we had demonstrated that cell cycle arrest due
to overexpression of presenilins in dividing cells also man-
ifests in ATM⫺/⫺ cells, which lack functional ATM pro-
tein, a kinase induced by double-stranded DNA breaks and
which activates cell cycle check-point [13,34]. To deter-
mine if ATM protein kinase is involved in presenilin in-
duced cell cycle arrest we studied the consequence of over-
expression of PS1 FAD mutants in the ATM⫺/⫺ cell line.
Fig. 4 demonstrates that BrdU labeling was reduced 6.1%
((36.4 – 41.8)/87.5 ⫻ 100) for the PS1(E280A) mutant,
7.5% for the PS1(P267S) mutant, and 14.2% for the
PS1(P117L) mutant in comparison to wild-type PS1 in the
ATM⫺/⫺ cell line. When taken together these results dem-
832 S.M. Janicki et al. / Neurobiology of Aging 21 (2000) 829 – 836

Fig. 2. Overexpression of PS1 in HeLa cells inhibits cell cycle progression. HeLa cells were mock-transfected (a-c) or transiently transfected with PS1 (d-f)
and evaluated for BrdU incorporation. Left panels show transfected protein, middle panels are BrdU labeling, and right panels are DAPI DNA images. Only
few of the PS overexpressing cells replicated their DNA as revealed by BrdU labeling while non-PS1 overexpressing cells were not inhibited in DNA
replication. Bar: 10 ␮m.

onstrate that PS1 FAD mutants cause increased cell cycle
arrest when overexpressed in two different cell lines and
that ATM protein kinase is not necessary for inducing
arrest.
3.2. Overexpression of presenilins proteins in HeLa cells
reduces cytoplasmic ␤-catenin levels
␤-catenin is a 92–97 kDa protein that is present in cells
in two forms, cytoplasmic (soluble) and a membrane-asso-
ciated form that have separate functions; signaling and ad-
hesion, respectively. As ␤-catenin has been shown to inter-
act with the presenilins [21,23,41,44], and because
cytoplasmic ␤-catenin levels oscillate during the cell cycle
[25], increasing dramatically during the G1 to S phase
transition, we investigated whether presenilin-induced cell
cycle arrest is correlated with changes in ␤-catenin accu-
mulation. Thus wild-type and mutant presenilin constructs
were overexpressed in HeLa cells and the level of ␤-catenin
accumulation were evaluated by immunoblot analysis. Im-
munofluorescence staining of the transiently transfected
cells indicated that approximately 60 –70% of the cells were
expressing presenilin proteins, similar to the transfection
efficiencies obtained previously [13]. The level of ␤-catenin
in mock-transfected HeLa cells were compared to those in
cells over-expressing wild-type PS1 and PS2 and the two
FAD mutants, PS1(P117L) and PS2(N141I). Although we
were unable to detect any significant difference in the levels
of ␤-catenin when we compared whole cell lysates of un-
transfected and presenilin-transfected cells (data not
shown), when soluble and insoluble protein fractions were
evaluated, cytoplasmic ␤-catenin levels were significantly
reduced (40 to 60%) in the soluble fraction of presenilin-
transfected cells compared to mock-transfected control cells
(Fig. 5, lanes 1–5). In contrast, there was not a dramatic
difference between the levels of ␤-catenin in the insoluble
fraction (Fig. 5, lanes 6 –10). We confirmed that equivalent
amounts of protein were loaded in the two sets of fractions
by evaluating ␤-tubulin immunoreactivity on the same blot;
the levels appeared to be approximately the same in all of
the lanes (Fig. 5).
Although all four presenilin constructs had reduced lev-
els of soluble ␤-catenin compared to the mock-transfected
cells (Fig. 5, lanes 1–5) we were unable to detect any
reproducible difference between the wild-type and corre-
sponding FAD presenilin mutants using this fractionation
assay. We believe that this due to technical difficulties
associated in measuring small differences (presumably a
6 –13% difference based on the cell cycle inhibition data)
between wild-type and mutant proteins by immunoblotting
of the transiently transfected lysates. The ␤-catenin immu-
noreactivity was detected as a doublet band (approximately
 S.M. Janicki et al. / Neurobiology of Aging 21 (2000) 829 – 836
Fig. 3. Quantification of BrdU incorporation of exponentially growing
HeLa cells transfected with wild-type and PS1 FAD mutants. (A) Wild-
type PS1 and three FAD mutants, PS1(P267S), PS1(E280A), and
PS1(P117L), were transiently transfected into exponentially growing HeLa
cells. BrdU was added to the media at the time of transfection, and cells
were fixed 19 h post-transfection. Compared to mock-transfected control
cells (92.3% ⫾ 3.0, n ⫽ 3), BrdU labeling was significantly reduced by
PS1 (53.1% ⫾ 3.2, n ⫽ 8; p value ⬍ 0.0001). When compared to wild-type
PS1 the three FAD mutants significantly reduced BrdU incorporation;
PS1(P267S) (44.3% ⫾ 1.4, n ⫽ 3), PS1(E280A) (47.0% ⫾ 0.9, n ⫽ 3) and
PS1(P117L) (41.1% ⫾ 1.6, n ⫽ 8), p values of 0.0016; 0.0111, and 0.0001,
respectively).
92–97 kDa) in the soluble fraction whereas only one band
was seen in the insoluble fraction. The higher of the two
␤-catenin bands probably corresponds to ubiquitinated
␤-catenin [24]. These results suggest that overexpression of
presenilins correlates with a reduction in the soluble pool of
␤-catenin which is involved in signaling.
4. Discussion
Previously we demonstrated that, when compared to the
wild-type protein, PS2 proteins containing the N141I FAD
mutation cause increased cell cycle arrest and apoptosis
833
Fig. 4. Quantification of BrdU incorporation of exponentially growing
ATM deficient cells transfected with wild-type and PS1 FAD mutants. (A)
Wild-type PS1 and three FAD mutants, PS1(P267S), PS1(E280A), and
PS1(P117L), were transiently transfected into exponentially growing HeLa
cells. BrdU was added to the media at the time of transfection, and cells
were fixed 19 h post-transfection. Compared to mock-transfected control
cells (87.5% ⫾ 0.9, n ⫽ 3), BrdU labeling was significantly reduced by
PS1 (41.8% ⫾ 0.8, n ⫽ 6). When compared to wild-type PS1 the three
FAD mutants significantly reduced BrdU incorporation; PS1(P267S)
(35.2% ⫾ 3.6, n ⫽ 3), PS1(E280A) (36.4% ⫾ 1.7, n ⫽ 6) and PS1(P117L)
(29.3% ⫾ 5.6, n ⫽ 4), p values of 0.0027; 0.0001, and 0.0006,
respectively).
when overexpressed in HeLa cells [12,13]. Here we dem-
onstrate that like PS2, three different PS1 FAD mutants also
increase cell cycle arrest as assayed by reduced incorpora-
tion of BrdU in transfected HeLa cells. Potentiation of the
cell cycle arrest by the PS1 FAD mutants was also observed
in ATM⫺/⫺ cells, indicating that the effects are indepen-
dent of ATM protein, a kinase that is involved in cell cycle
check-point control and which is activated in response to
double-stranded DNA breaks [34]. The increased cell cycle
arrest potentiated by FAD mutants may represent a funda-
mental mechanism by which the mutant proteins act in cells.
Interestingly the three FAD mutants inhibited BrdU labeling
to different extents. Despite inherent experimental errors in
834 S.M. Janicki et al. / Neurobiology of Aging 21 (2000) 829 – 836

Fig. 5. Immunoblot analysis of ␤-catenin levels in soluble and insoluble fractions of presenilin transfected cells. Equal protein amounts of soluble (lanes 1–5)
and insoluble fractions (lanes 6 –10) prepared from mock-transfected (lane 1 and 6, respectively) and PS transfected (lanes 2–5 and 7–10, respectively) were
immunoblotted with ␤-catenin and ␤-tubulin antibodies. Presenilin overexpression decreases the amount of ␤-catenin in the soluble cytoplasmic pool but does
not affect ␤-catenin levels in the insoluble fraction, whereas ␤-tubulin remained relatively unchanged. The band intensities of ␤-catenin and tubulin in PS
transfected cells were determined and expressed as a ratio of ␤-catenin to tubulin after normalization to the ratio determined for mock transfected cells:
Soluble fraction, Control mock-transfected ⫽ 1.0, PS1 ⫽ 0.57, PS1(P117L) ⫽ 0.48, PS2 ⫽ 0.45, PS2(N141I) ⫽ 0.40; Pellet fraction, Control mock-
transfected ⫽ 1.0, PS1 ⫽ 0.85, PS1(P117L) ⫽ 0.85, PS2 ⫽ 1.0, PS2(N141I) ⫽ 0.94.

normalizing expression levels between different constructs
in transiently transfected cells, we found reproducible dif-
ferences by which each of the mutants inhibited BrdU
incorporation when assayed in multiple experiments and
utilizing two different cell lines. Of the three PS1 mutants
studied, the PS1(P117L) reduced BrdU incorporation the
most (13 to 14%) followed by PS1(P267S) (7.5 to 9%), and
the PS1(E280A) mutant inhibiting BrdU incorporation the
least (6 to 7%). It is noteworthy that the different degree by
which the three mutants potentiated arrest correlates with
the approximate age of AD onset in patients carrying these
mutations [32]. PS1(P117L) which inhibited BrdU labeling
the most is one of the most aggressive FAD mutations
described, causing disease at early as 27–28 years of age
[36]. The two other mutations PS1(P267S) and PS1(E280A)
have been documented to cause disease at a slightly later
age, 35 and 45–54 years, respectively [32]. Thus it is con-
ceivable that the degree of cell cycle inhibition by the
different PS1 FAD mutants and the age of AD onset may be
related. However, since our studies are based on only a
small number of mutants, and since we do not know what
role these cell cycle changes play in AD pathogenesis, it is
not possible at this time to conclude with any certainty
whether the two processes are linked. Interestingly, Li et al.
[17] localized presenilins to centrosomes and centromeres
in fibroblasts, and have proposed that presenilins are in-
volved in chromosome segregation, and that FAD presenilin
mutations cause increased chromosome missegregation
leading to inappropriate apoptosis. Further studies will be
needed to determine whether all FAD mutations affect cell
cycle progression and whether cell cycle changes in divid-
ing cells can be extrapolated to loss of cells, including
terminally differentiated neurons in AD.
Our results showing presenilin overexpression halts the
cell cycle may be physiologically relevant to the normal
function of presenilins in dividing cells. Recent evidence in
transgenic mice has indicated that presenilins are involved
in regulating cellular proliferation of keratinocytes. Thus,
mice disrupted of the murine presenilin 1 (PS1) gene die at
day 1 after birth, but are rescued and survive to adulthood
upon expression of human PS1 transgene under the control
of the Thy-1 promoter [26]. However, these rescued mice
develop extensive epidermal hyperplasia and neoplasia
[43]. The abnormalities appear to arise due to the restricted
expression of the Thy-1 driven PS1 transgene to neurons
and its failure, unlike the normal murine PS1 gene, to be
expressed in skin. These results are consistent with our
findings, that increased expression of presenilins halts the
cell cycle, and conversely we presume loss of presenilin
expression leads to cellular proliferation. In may be fortu-
itous that the presenilin effects on the cell cycle we describe
were obtained utilizing HeLa cells, an epithelial derived cell
line.
In an effort to determine the mechanism by which pre-
senilin overexpression induces cell cycle arrest, we used
immunoblot analysis to determine if the expression profile
of a number of known cell cycle regulated proteins were
altered. As documented previously, presenilin overexpres-
 S.M. Janicki et al. / Neurobiology of Aging 21 (2000) 829 – 836
sion in HeLa cells leads to arrest of cells in the G1 phase of
the cell cycle. However, despite arrest at this phase of the
cell cycle, we did not detect any noticeable changes in the
expression patterns of p21, 27, pRb, c-myc, and p53 pro-
teins in cell lysates of untransfected and presenilin overex-
pressing cells [13]. Here we demonstrate that the overex-
pression of wild-type and mutant presenilin proteins causes
a specific and marked decrease in cytoplasmic (soluble)
pool of ␤-catenin. The decrease in the cytoplasmic pool of
␤-catenin upon presenilin overexpression could be respon-
sible for the cell cycle arrest, since ␤-catenin plays an
important role in cell cycle progression [25]. In an elegant
study, Orford et al. [25] demonstrated that the cytoplasmic
pool of ␤-catenin oscillates, increasing rapidly between G1
and S phase of the cell cycle. Furthermore, they demon-
strated that the elevation in cytoplasmic ␤-catenin is impor-
tant for driving cells from G1 into S phase. Interestingly,
they also showed that ␤-catenin can function as a potent
inhibitor of apoptosis since transgenic expression of ␤-cate-
nin inhibited apoptosis in their model system. Based on their
results they proposed that loss of cytoplasmic ␤-catenin
leads to cell cycle arrest at G1/S phase and under certain
circumstances this leads to apoptosis. Our findings are con-
sistent with this hypothesis, as we have shown presenilin
expression in HeLa cells causes cell cycle arrest at G1 and
increased apoptosis [12,13]. The decrease in ␤-catenin due
to presenilin overexpression may prevent cells from reach-
ing a critical threshold level of ␤-catenin that is necessary
for progressing into S phase, leading to G1 arrest that
culminates in apoptosis.
There is considerable support for the idea that presenilins
may be involved in modulating ␤-catenin stability [14,21,
23,35,42]. Due to technical reasons we were unable to
detect any reproducible differences in ␤-catenin levels in
cells overexpressing wild-type and FAD presenilin proteins.
However, Zhang et al. [42] and Weihl et al. [35] have
provided strong evidence that PS1 FAD mutations destabi-
lize ␤-catenin to a greater extent than the wild-type protein,
and that this destabilization potentiates apoptosis in trans-
fected neuronal cells.
Our observations that FAD mutations in presenilin 1 and
2 genes compromise cell cycle progression in dividing cells
needs to be integrated with the now well-document evi-
dence for misexpression of cell cycle proteins in AD af-
fected brains [2,18,22,27,29,33]. As discussed previously
[13], the misregulation of cell cycle proteins in AD may
reflect an abortive attempt of the cells to reenter the cell
cycle, or it could represent a protective response of the cells
to death stimuli. With regard to these possibilities, it will be
important to determine whether autosomal dominant muta-
tions in presenilin genes cause increased apoptosis in the
brains of affected individuals and whether cell cycle pro-
teins are misexpressed in these individuals. A better under-
standing of the mechanisms that lead to the upregulation of
these cell cycle proteins may eventually lead to better in-
tervention strategies in the effort to prevent and/or cure AD.
835
Acknowledgments
We thank Dr. Ann Pluta for critical comments on the
manuscript. Supported by NIA grant AG16839 to MJM.
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