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The Journal of Neuroscience, May 1, 2002, 22(9):3445-3453
Neuronal Deficiency of Presenilin 1 Inhibits Amyloid Plaque
Formation and Corrects Hippocampal Long-Term Potentiation But Not a
Cognitive Defect of Amyloid Precursor Protein [V717I] Transgenic
Mice
Ilse
Dewachter1,
Delphine
Reversé2,
Nathalie
Caluwaerts1,
Laurence
Ris2,
Cuno
Kuipéri1,
Chris
Van den
Haute1,
Kurt
Spittaels1,
Lieve
Umans1,
Lutgarde
Serneels1,
Els
Thiry1,
Dieder
Moechars3,
Mark
Mercken3,
Emile
Godaux2, and
Fred
Van
Leuven1
1 Experimental Genetics Group, Katholieke
Universiteit Leuven, B-3000 Leuven, Belgium,
2 Laboratory of Neuroscience, University of Mons-Hainaut,
7000 Mons, Belgium, and 3 Janssen Research Foundation, 2340 Beerse, Belgium
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ABSTRACT |
In the brain of Alzheimer's disease (AD) patients, neurotoxic
amyloid peptides accumulate and are deposited as senile plaques. A
major therapeutic strategy aims to decrease production of amyloid peptides by inhibition of  -secretase. Presenilins are polytopic transmembrane proteins that are essential for -secretase activity during development and in amyloid production. By
loxP/Cre-recombinase-mediated deletion, we generated mice with
postnatal, neuron-specific presenilin-1 (PS1) deficiency, denoted
PS1(n /), that were viable and fertile, with normal brain
morphology. In adult PS1(n/) mice, levels of endogenous brain
amyloid peptides were strongly decreased, concomitant with accumulation
of amyloid precursor protein (APP) C-terminal fragments. In the
cross of APP[V717I]xPS1 (n/) double transgenic mice, the
neuronal absence of PS1 effectively prevented amyloid pathology, even
in mice that were 18 months old. This contrasted sharply with
APP[V717I] single transgenic mice that all develop amyloid pathology
at the age of 10-12 months. In APP[V717I]xPS1 (n/) mice,
long-term potentiation (LTP) was practically rescued at the end of the
2 hr observation period, again contrasting sharply with the strongly
impaired LTP in APP[V717I] mice. The findings demonstrate the
critical involvement of amyloid peptides in defective LTP in APP
transgenic mice. Although these data open perspectives for therapy of
AD by -secretase inhibition, the neuronal absence of PS1 failed to
rescue the cognitive defect, assessed by the object recognition test,
of the parent APP[V717I] transgenic mice. This points to potentially
detrimental effects of accumulating APP C99 fragments and demands
further study of the consequences of inhibition of -secretase
activity. In addition, our data highlight the complex functional
relation of APP and PS1 to cognition and neuronal plasticity in adult
and aging brain.
Key words:
PS1; Alzheimer's disease; neuronal plasticity; cognition; amyloid pathology; mouse model
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INTRODUCTION |
Accumulation and deposition of
neurotoxic amyloid peptides in vasculature and brain parenchyma as
senile plaques is an important pathological hallmark of Alzheimer's
disease (AD). The amyloid peptides, A 40 and A42, are generally
believed to play a central but still poorly understood role in AD and
are formed by sequential endoproteolytic cleavages of the amyloid
precursor protein (APP) by - and -secretases, hence representing
important therapeutic targets (Vassar and Citron, 2000 ; Golde and
Younkin, 2001; Selkoe, 2001).
Presenilin-1 (PS1) is critical for the generation of amyloid
peptides from APP (De Strooper et al., 1998), either by exerting -secretase activity itself or by controlling trafficking of
-secretase and its substrates (for review, see Esler and Wolfe,
2001). Presenilins are polytopic transmembrane proteins that are
proteolytically processed into N- and C-terminal fragments that form
biologically active complexes in combination with other proteins, e.g.,
nicastrin and others (Haass and De Strooper, 1999; Yu et al., 2000;
Esler and Wolfe, 2001). PS1 is involved not only in APP processing, but
also in the processing of other integral membrane proteins, among which
Notch1 is most critical for embryonic development (Naruse et al., 1998;
Song et al., 1999; Struhl and Greenwald, 1999). The pleomorphic effects
of PS1 are best illustrated by the phenotype of the "classic" PS1
knock-out mice. Severely impaired mouse embryogenesis is characterized
by disturbed somitogenesis, cranial hemorrhages, impaired neurogenesis
with thinning of the ventricular zone, bilateral cerebral cavitations
resulting in malformation of brain, and late embryonic lethality (Shen
et al., 1997; Wong et al., 1997; Hartmann et al., 1999).
Although PS1 plays a critical role in the generation of amyloid
peptides, its potential as a therapeutic target for AD remains to be
scrutinized, especially regarding its role in adult brain. We have now
generated mice with a restricted postnatal and neuron-specific deletion
of PS1, to circumvent the embryonic lethality of the classically
induced, complete PS1 deficiency. This approach demonstrated that
PS1(n/) mice were viable and fertile and had normal brain morphology, indicating that neuronal deficiency of PS1 is well tolerated in adult mice.
The PS1(n/) mice were further crossed with our APP[V717I] mice,
which display some phenotypic changes that are highly reminiscent for
AD and shared by other APP transgenic models (Games et al., 1995; Hsiao
et al., 1996, 1998; Holcomb et al., 1998, 1999; Calhoun et al., 1999;
Moechars et al., 1999; Dewachter et al., 2000; Van Dorpe et al., 2000;
Duff and Rao, 2001), including impaired long-term potentiation (LTP)
and cognitive deficits with progressive development with age of typical
amyloid plaques and vascular pathology.
In this model we studied whether phenotypic changes in APP[V717I]
mice could be alleviated by neuronal deficiency of PS1, not only
because this is highly relevant to validate therapeutic strategies
based on -secretase inhibition, but also because it is very
informative for our fundamental understanding of the etiology of AD.
The exact contribution of the different biochemical APP derivatives,
i.e., the amyloid peptides 40/42 and the C-terminal stubs, still
remains to be established, as well as in which form, i.e., soluble or
precipitated, they exert or cause neurotoxicity (Nalbantoglu et al.,
1997; Suh, 1997; Chapman et al., 1999; Holcomb et al., 1999; Hsia et
al., 1999; Dewachter et al., 2000; Suh et al., 2000).
The results that are presented highlight the complex functional
relation of APP and PS1 and of their metabolites to cognition and
neuronal plasticity. This includes data supporting a critical role for
amyloid peptides in impairing LTP in APP transgenic mice. In addition,
our data demonstrate that neuronal deficiency of PS1 inhibited amyloid
peptide formation, prevented plaque formation, and corrected deficits
in LTP in APP transgenic mice, hence supporting the use of
-secretase inhibition as therapy for AD. However, the current data
also underline the potential detrimental effects of the accumulation of
APP -stubs or C99 fragments, which necessitate further detailed
study of the use of therapeutic strategies aimed at inhibition of
-secretase activity.
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MATERIALS AND METHODS |
Generation of transgenic mice. A PS1 allele was
constructed to contain three loxP sites and a neomycin resistance
cassette (neoR) by gene targeting in embryonic stem cells. This
allele contains two loxP sites flanking exon 7 and a third loxP site downstream of neoR (see Fig. 1). Thy-1 Cre-recombinase transgenic mice
were generated essentially as described for other neuron-specific expression constructs (Moechars et al., 1996, 1999; Dewachter et al.,
2000; Spittaels et al., 2000; Tesseur et al., 2000). Southern blotting
and PCR analysis of DNA isolated from tail biopt was used for
genotyping of all mice. Thy-1-driven Cre-recombinase activity was
assessed by crossing with LacZ reporter mice, demonstrating prominent
-gal activity in neurons of the cortex and hippocampus of adult mice
(see Fig. 1), consistent with our previous observations in transgenic
mice overexpressing transgenes under control of the mouse thy-1 gene promoter.
Analysis of behavior and cognition. The object recognition
task was essentially performed as described (Tang et al., 1999; Rampon
et al., 2000). Briefly, the mice were habituated for 1 hr to a
Plexiglas open-field box (52 × 52 × 40 cm) with black vertical walls and a translucent floor dimly illuminated by a lamp
placed underneath the box. The next day the animals were placed in the
same box and submitted to a 10 min acquisition trial. During this trial
mice were placed individually in the open field in the presence of
object A (marble or dice), and the time spent exploring object A (when
the animal's snout was directed toward the object at a distance <1
cm) was measured. During a 10 min retention trial (second trial), which
was performed 3 hr later, a novel object (object B: marble or dice) was
placed together with the familiar object (object A) in the open field.
The time (tA and
tB) the animal spent exploring the two
objects was recorded. The recognition index (RI), defined as the ratio
of the time spent exploring the novel object over the time spent
exploring both objects
[(tB/(tA + tB)) × 100] was used to
measure nonspatial memory. Statistical analysis was done by using ANOVA
single factor as described (Moechars et al., 1996, 1999).
Western blotting analysis. Biochemical analysis of
APP-processing intermediates has been described (Dewachter et al.,
2000). Briefly, mouse brains were homogenized in 6.5 vol of ice-cold buffer containing 20 mM Tris-HCl, pH 8.5, and a
mixture of proteinase inhibitors (Roche, Darmstadt, Germany). After
centrifugation at 135,000 × g at 4°C for 1 hr, the
supernatant was centrifuged again for 2 hr at 200,000 × g before analysis of soluble amyloid peptides by specified
ELISA. The pellets from the first centrifugation were resuspended in
TBS with proteinase inhibitors and used for analysis of membrane-bound
proteins. Biochemical analysis of intact membrane-bound APP and of the
C-stubs was done as described (Moechars et al., 1999; Dewachter et al.,
2000). -C-stubs derived from human APP in APP transgenic mice and
APPxPS1(n/) mice were measured by immunoblotting with antibody WO2
(Ida et al., 1996); C-stubs derived from murine APP were measured by
immunoblotting with polyclonal B10/4. For detection of PS1 and PS2
protein, brains were homogenized in sucrose buffer [5
mM Tris, 250 mM sucrose, 1 mM EGTA, pH 7.4, and a mixture of proteinase
inhibitors (Roche, Darmstadt, Germany)] with a potter homogenizer,
spun at 12,000 × g at 4°C for 10 min. The
supernatant was taken and further centrifuged for 30 min at 100,000 × g at 4°C. Proteins were denatured and
reduced in sample buffer containing a final concentration of 2% SDS,
1% 2-ME separated on 4-12% Nu-Page gels (Novex, San Diego, CA). PS1
and PS2 were detected with the mouse monoclonal antibody Ab 5232 (Chemicon, Temecula, CA), recognizing C-terminal fragments of PS1, and
the goat polyclonal antibody sc-1456 (Santa Cruz Biotechnology, Santa Cruz, CA), recognizing C-terminal fragments of PS2. After incubation with appropriate secondary antibodies, all Western blots were developed
with the ECL detection system and photographically recorded. Densitometric scanning of films and calculation and normalization were
performed as described (Moechars et al., 1999; Dewachter et al., 2000)
using a flatbed optical density scanner and dedicated software for
analysis and measurement (Image Master; Pharmacia, Uppsala, Sweden).
ELISA of amyloid peptides. Protein extracts were applied on
reversed-phase columns (C18-Sep-pack cartridges; Waters Corporation, Milford, MA) and washed with increasing concentrations of
acetonitrile (5, 25, and 50%) containing 0.1% trifluoroacetic
acid. The last fraction contained the amyloid peptides and was
dried in vacuo overnight and dissolved for measurements in
ELISA. Sandwich ELISA for human A40 and A42 peptides was
performed using the capture antiserum JRF/cA40/14 and 21F12,
respectively, and they were developed with monoclonal antibodies
JRFcAtot/14 and 3D6, respectively (Vanderstichele et al., 2000).
Sandwich ELISA for murine A40 and A42 peptides was performed
using the capture antibodies JRF/cA40/14 and JRF/cA42/17,
respectively, and they were developed with the HRP-coupled monoclonal
antibody JRF/A1-15/2.
Northern blotting. Total RNA was isolated from mouse brain,
separated by electrophoresis, and transferred by capillary transfer to
nylon membranes. Filters were prehybridized for 6 hr at 42°C in
SSPE (150 mM sodium chloride, 20 mM sodium phosphate, 5 mM EDTA),
Denhardt's solution, 0.5% SDS, 50% deionized formamide, 100 µg/ml
denatured sperm DNA, and 50 µg/ml heparin and hybridized in the same
solution supplemented with 10% dextran sulfate at 42°C overnight
with addition of a radiolabeled probe. Membranes were washed in 0.3×
SSPE, 0.5% SDS at 60°C for 1 hr. Specified DNA probes were generated
by PCR using mouse brain cDNA as template, with specified primers as
follows: for Hes5, 5'-CCAAGTACCGTGGCGGTGGA and 5'-GAGATGGCCGTCAGCTACCT;
for Dll1, 5'-CTCCTTCAGCCTGCCTGATG and 5'-AGGCACCTCACTGTGGGAGA.
Histology and quantitative analysis. Brains were dissected,
and the left cerebral hemisphere was snap frozen and stored at 70°C. The right cerebral hemisphere was immersion fixed in 4% paraformaldehyde in PBS overnight and used for histological and quantitative analyses. Coronal vibratome sections (40 µm) were cut
from the occipital two-thirds of the right hemisphere. Thioflavin-S staining was performed on vibratome sections according to standard protocols. Immunohistochemistry with monoclonal antibody JRF/AN25 specific for A was done on free-floating vibratome sections
according to previously published protocols, using diaminobenzidine as
chromogen (Van Dorpe et al., 2000). Quantitative analysis of amyloid
plaque load in the brain was performed on Thioflavin-S-stained and
immunostained coronal vibratome sections. Well defined coronal sections
at bregma 2.54 mm were selected for quantification of amyloid load
(Franklin et al., 1997). For quantification of amyloid plaque load in
the subiculum, two serial sections were used for Thioflavin-S staining and immunostaining with JRF/AN25, respectively. Images (200× magnification) from these sections were collected from a 3CCD color video camera and analyzed with appropriate software (AIS/C; Imaging Research, St. Catherine, Ontario, Canada). The surface of
individual amyloid deposits and the surface of the subiculum were
measured. The total amyloid plaque load was expressed as a percentage
of the total surface of the subiculum. Classic cresyl violet staining
was performed on paraffin sections prepared as described (Dewachter et
al., 2000).
In situ hybridization. In situ hybridization
was performed on paraffin sections (6 µm) of mouse brain placed on
silylated glass slides, dewaxed, and rehydrated through an ethanol
series. Sections were digested with proteinase K (20 µg/ml),
post-fixed in 4% paraformaldehyde, and treated with 0.25% acetic
anhydride in 0.1 M triethanolamine. Sections were
hybridized overnight in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, and
5 mM EDTA, pH 8.0, with 10% dextran sulfate, 1×
Denhardt's solution, 0.5 mg/ml yeast RNA, and 10 mM dithiothreitol, supplemented with the
appropriate radiolabeled riboprobe. After stringency washes and
ribonuclease A treatment, sections were dehydrated and dipped in
photographic emulsion (LM-1, Amersham Biosciences) and exposed for 1 week. For synthesis of the sense and antisense PS1 RNA, a 221 bp PCR
product from exon 7 of the presenilin-1 gene was cloned in a pGEM-T vector (Promega, Madison, WI). The plasmid was
linearized with either NotI or SphI and
transcribed with T7 and SP6RNA polymerase, respectively, in the
presence of [33P]UTP. In brain of
PS(+)/(+) mice, PS1 was readily detected in neurons from the pyramidal
layers of the hippocampus and in neurons from the cortex. In neurons
from PS1(n/) brain, staining was practically absent, comparable to
staining intensity in control mice with a sense probe (results not shown).
Electrophysiology. Hippocampal slices were bathed in
artificial CSF containing (in mM ): 124 NaCl, 5 KCl, 26 NaHCO3, 1.24 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 10 glucose, aerated with 95% O2 and 5%
CO2. Mice were anesthetized with ether and
decapitated, and transverse slices (400 µm thick) were cut in cold
artificial CSF using a vibratome and kept at room temperature until
placed in the interface recording chamber at 30°C.
Electrophysiological recordings were begun not earlier than 3 hr after
dissection to allow recovery of the slice. The chamber was perfused
with artificial CSF (1 ml/min). Bipolar tungsten microelectrodes (World
Precision Instruments, Sarasota, FL) were used to stimulate Schaffer's
collaterals, although evoked field EPSPs (fEPSPs) were recorded
in the stratum radiatum of the CA1 region with low resistance (2 M )
glass microelectrodes filled with 2 M NaCl. Test
stimuli were 0.1 msec constant-voltage pulses delivered every 30 sec at
an intensity sufficient to evoke an ~33% maximal response. LTP was
induced by an electrical high-frequency stimulation at an intensity
evoking a 50% maximal response. The slope of the field EPSP
(millivolts per millisecond) was measured from the average wave from
four consecutive responses.
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RESULTS |
Generation of mice with postnatal neuron-specific ablation
of PS1
Total inactivation of the PS1 gene severely impaired mouse
embryogenesis by disturbed somitogenesis, cranial hemorrhage, impaired neurogenesis with thinning of the ventricular zone, bilateral cerebral
cavitations resulting in malformation of brain, and late embryonic
lethality (Shen et al., 1997; Wong et al., 1997; De Strooper et al.,
1998; Hartmann et al., 1999). To circumvent the embryonic lethality, we
have used the Cre/loxP gene targeting system to generate mice with a
postnatal, neuron-specific deficiency of PS1, denoted PS1(n/).
These mice were generated by crossing two transgenic mouse strains: (1)
mice with a targeted PS1 gene to introduce loxP sites (PS1-flox mice)
and (2) mice expressing Cre-recombinase under control of the mouse
thy1-gene promoter (Fig. 1). The
characteristics of the thy1-gene promoter have been amply documented by
us and by others to yield postnatal expression of the transgene in
central neurons only (Moechars et al., 1996, 1999; Dewachter et al.,
2000; Spittaels et al., 2000; Tesseur et al., 2000; Van Dorpe et al., 2000).
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Figure 1.
Generation of PS1(n/) mice. A,
Overall strategy to generate mice with a conditional inactivation of
PS1, denoted PS1(n/), and APP transgenic mice with a neuronal PS1
deficiency. B, Schematic representation of the targeting
vector, the wild-type, and the targeted PS1 gene. Lox P sites are
represented by open triangles flanking the neomycin
cassette and exon 7. Arrowheads represent synthetic
primers used for genotyping by PCR. C, -Galactosidase
staining of brain section of the selected thy1-Cre-recombinase
transgenic mouse line after crossing with the LacZ reporter mouse
(Akagi et al., 1997) demonstrating neuronal staining in cortex
and hippocampus. D, Genotyping by PCR analysis of DNA
isolated from tail biopts using primers NE277 and NE278
(B) to identify the wild-type and targeted PS1
genes in mice with the indicated genotypes. E, In
situ hybridization of brain sections of PS1(n/) mice
(left panel) and of mice with a floxed but active
PS1 gene (right panel) with RNA probes specific
for PS1 exon 7, which is deleted by Cre-recombinase. F,
Western blotting for the C-terminal fragment of mouse PS1 (~20 kDa)
in total brain extracts from four individual PS1(n/) mice
[lanes marked PS1(n/)] and from
four individual mice with floxed but active PS1 genes
(lanes marked Control), and
diluted sample (1, 0.5, 0.25). Quantitation revealed levels of
100.0 ± 5.5 and 16.0 ± 0.7% in control mice and
PS1(n/) mice, respectively. Western blotting with antibodies
against APP and LRP was used as a loading control.
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In addition, the specificity of the selected thy1-Cre-recombinase
transgenic mouse strain was demonstrated in a cross with a reporter
transgenic mouse strain that carries a constitutionally inactive
reporter gene construct, i.e., an actin promoter and LacZ reporter gene
separated by a "floxed" spacer (Akagi et al., 1997). Prominent
-galactosidase staining was evident in nearly all neurons in cortex
and hippocampus of double transgenic mice, i.e., thy1-Cre recombinase x
LacZ-reporter mice, proving the specific neuronal expression of the
Cre-recombinase in our selected transgenic mouse strain (Fig.
1C).
The resulting PS1(n/) mice were genotyped by four independent PCR
reactions (Fig. 1D). The expression of PS1 was
demonstrated to be decreased specifically in cortical and hippocampal
neurons as assayed by in situ hybridization with RNA probes
specific for PS1 exon 7 (Fig. 1E), as opposed to
normal expression of PS1 in the parent transgenic mice with a
"floxed" PS1 gene. These had normal expression of PS1 and were used
throughout this study as "control" mice in all experiments, unless
indicated otherwise. Western blotting established strongly decreased
levels of PS1 protein in total brain extracts of PS1(n/) mice (84%
decrease) (Fig. 1F), with the very low residual
expression likely caused by glial and other non-neuronal cells, as
judged from in situ hybridization.
Decreased -secretase cleavage of APP in PS1(n/) mice
PS1 is critical for generating the amyloid peptides from APP in
primary cultures of neurons (De Strooper et al., 1998), either by
exerting -secretase activity itself or as an essential substrate binding adaptor protein or chaperone in the -secretase complex (for
review, see Esler and Wolfe, 2001).
In the brain of PS1(n/) mice, the levels of murine A40 and
A42 derived from endogenous APP were dramatically decreased, as
expected (Fig. 2). Concomitantly, the
C-terminal fragments of mouse APP accumulated, whereas the level of
membrane-bound full-length mouse APP remained unchanged (Fig. 2).
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Figure 2.
APP processing in brain of PS1(n/) mice.
Western blotting (left panel) of mouse brain extracts
demonstrating very similar levels of membrane-bound APP
(APPm; ~110 kDa) (top) and accumulation
of the APP C-terminal fragments (~10-12 kDa) (bottom)
in PS1(n/) mice. Results shown are from three individual
PS1(n/) mice (three right lanes) and from
three individual mice with floxed but active PS1 genes
(control, three left lanes). Antiserum
B10/4 was used that is specific for the C-terminal domain of human and
mouse APP (Dewachter et al., 2000). Levels of soluble amyloid peptides
(right panel) (nanograms per gram brain tissue)
extracted from the brain of mice with a floxed but active PS1 gene
(control) and from PS1(n/) mice, as measured
with specific ELISA for murine A40 (black bars) and
A42 (gray bars) (mean with SEM,
n = 6 for each genotype).
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The combined data proved that the neuronal deficiency of PS1 was
reflected in the expected biochemical effects on APP-processing, i.e.,
decreased levels of amyloid peptides and accumulation of C-terminal
fragments of APP. This established the pronounced inhibition of
-secretase processing of APP in the brain of PS1(n/) transgenic mice.
Unaltered expression of Notch ligand Dll1 and Hes5, an effector
gene downstream of Notch
PS1 is also involved in processing of other integral membrane
proteins, among which Notch1 is crucial for embryonic development (Naruse et al., 1998; De Strooper et al., 1999; Song et al., 1999; Struhl and Greenwald, 1999). In mouse brain in vivo, the
very low levels of the Notch1 intracellular domain are beyond detection limits, which precludes direct in vivo analysis of this
metabolite of Notch cleavage. We therefore analyzed whether Notch
signaling was affected in brain of adult PS1(n/) mice by analysis
of the expression level of Hes5, a gene affected downstream of Notch (Ohtsuka et al., 1999; Handler et al., 2000). In addition, we measured
the mRNA levels of the Notch ligand Dll1. Northern analysis demonstrated no significant differences in the mRNA levels of Hes5 and
Dll1 in brain of PS1(n/) mice (Fig.
3). This confirmed, in addition to the
normal phenotype of the adult PS1(n/) mice, that Notch signaling is
unaltered, in contrast to the severe embryonic phenotype of the
complete PS1 knock-out, in which Hes5 levels were reduced and Dll1
expression was increased (Handler et al., 2000).
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Figure 3.
Analysis of Notch signaling and PS2 expression.
Northern blotting analysis of RNA extracted from brain of mice with
floxed but active PS1 genes (lanes 1-3) and of
PS1(n/) mice (lanes 4-6) demonstrates no
changes of mRNA levels of Hes5 (top) and Dll1
(middle). Western blotting for the C-terminal fragment
of mouse PS2 (~20 kDa) (bottom), in total brain
extracts from four individual PS1(n/) mice [lanes
marked PS1(n/)] and from four individual mice with
floxed but active PS1 genes (lanes marked
Control), and diluted sample (1, 0.5, 0.25).
Quantitation revealed levels of 100 ± 2 and 127 ± 11% in
control mice and PS1(n/) mice, respectively.
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We further measured the expression of PS2 mRNA and protein levels,
demonstrating no change of PS2 mRNA levels, and a small increase (27%)
in the levels of the PS2 C-terminal fragment in brain of PS1(n/)
mice. Although this might compensate for the PS1 deficiency,
further study is required to establish the actual effect of this minor change.
No effect of postnatal neuron-specific PS1 deletion on brain
morphology and behavior
The strategy to generate mice deficient postnatally in PS1 in
neurons only, as outlined in Figure 1, proved successful in circumventing developmental problems and the lethality of the complete
PS1 deficiency. PS1(n/) mice were born at the expected mendelian
frequency and were viable and fertile. They did not show any
morphological anomaly in brain sections examined by cresyl violet
staining (Fig. 4A) and
by immunostaining with different antibodies, i.e., GAP43, MAP2, APP,
and synaptophysin (Fig. 4; and results not shown). No cerebral
hemorrhages, cavities, or tumors were found associated with the
neuronal PS1 deficiency in mice up to 2 years old.
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Figure 4.
Morphological analysis of brain of PS1(n/)
mice. A, Cresyl violet staining of paraffin-embedded
sections from brain of mice with floxed but active PS1 genes
(left panel) and from PS1(n/) mice
(right panel), aged 6 months, demonstrating
normal brain architecture in PS1(n/) mice. B,
Immunohistochemical staining for GAP43 of vibratome sections from brain
of mice with floxed but active PS1 genes (left
panel) and PS1(n/) mice (right
panel), demonstrating normal morphology of hippocampus.
C, D, Higher magnification of neurons in
hippocampal CA1 region (C) and cortex
(D) after immunohistochemical staining for MAP2
of vibratome sections from brain of mice with floxed but active PS1
genes (left) and PS1(n/) mice
(right).
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PS1(n/) mice did not behave differently from wild-type mice during
normal handling by caretakers and displayed no cognitive deficit in the
object recognition test, as detailed further. Taken together, these
data demonstrated that the postnatal neuronal deficiency of PS1 was
tolerated very well, and we conclude therefore that neuronal PS1 is not
essential for maintaining gross neuronal architecture and cognitive
functioning in the object recognition test in adult or aging mouse brain.
Generation and characterization of
APP[V717I]xPS1(n/) mice
The validation of the PS1(n/) transgenic mice by the
functional and biochemical characteristics described, i.e., the
effective inhibition of -secretase cleavage of APP, which
drastically inhibited amyloid peptide formation without causing major
abnormalities in brain morphology and behavior, allowed us to ask the
primary question: whether neuronal absence of PS1 is sufficient to
rescue the AD-related phenotypic defects of the APP[V717I] transgenic mice. These transgenic mice progressively and robustly recapitulate several of the most intrinsic features of AD pathology.
To experimentally address this question, PS1(n/) mice were crossed
with APP[V717I] transgenic mice to obtain transgenic mice that were
(1) homozygous for the targeted floxed PS1 gene and expressed (2) the
thy1-Cre-recombinase transgene and (3) the thy1-APP[V717I] transgene.
These mice, further denoted here as APPxPS1(n/), thereby expressed
the human APP[V717I] mutant protein in precisely the same neurons
that lack PS1 resulting from expression of Cre-recombinase,
because both are controlled by the same mouse thy1-gene promoter (Fig.
1) (Dewachter et al., 2000). Offspring with the APPxPS1(n/)
genotype, identified with four independent PCR reactions, were born at
the expected mendelian frequency, which again proved the absence of
major embryonic and developmental defects.
Amyloid plaque formation is prevented in APPxPS1(n/) mice
The levels of the human amyloid peptides, derived from the human
mutant APP[V717I] transgene, were dramatically lower in the brain of
APPxPS1(n/) mice relative to the parent APP[V717I] transgenic
mice (Fig. 5C), similar as
noted for endogenous mouse A (Fig. 2). Although the levels of
full-length transgene APP remained unchanged (Fig. 5A), the
human APP -C-stubs also accumulated as expected (Fig.
5B).
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Figure 5.
Inhibition of amyloid peptides and pathology in
brain of APPxPS1(n/) mice. A, B,
Western blotting of membrane proteins extracted from brain of three
individual APP[V717I] transgenic mice (lanes marked
APP) and from three individual APPxPS1(n/)
transgenic mice. The Western blots were stained with monoclonal
antibody 1G5 for full-length transgene membrane-bound APP
(APPm) (~110 kDa) (A) and with
monoclonal antibody WO2 for human APP -C-stubs (~12 kDa)
(B). C, Levels of soluble amyloid
peptides (nanograms per gram brain tissue) in brain of APP and
APPxPS1(n/) transgenic mice as measured with specific ELISA for
human A40 (red bars) and A42 (blue
bars) (mean with SEM, n = 6 for each
genotype). D, E, Staining with
thioflavinS (D) and immunostaining
(E) of amyloid deposits in brain of
APPxPS1(n/) (middle panels) and APP[V717I]
transgenic mice (right panels). Note the absence of
thioflavin-S staining and of immunoreaction in the subiculum of
APPxPS1(n/) mice in contrast to the parent APP[V717I] transgenic
mice. The left panels are a higher magnification of the
areas selected in the middle panels. F,
G, Amyloid load, expressed as relative surface area
occupied by thioflavin-S-positive plaques (F) and
of immune-positive plaques (G) in APP[V717I]
and APPxPS1(n/) mice, all 16-18 months old (mean with SEM,
n = 6).
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In the brain of all APP[V717I] transgenic mice aged 10-12 months and
older that we have examined to date, diffuse and senile amyloid plaques
were invariably present and increased exponentially with age (Moechars
et al., 1999; Dewachter et al., 2000; Van Dorpe et al., 2000). In
contrast, however, no thioflavin-S-reactive amyloid plaques nor diffuse
amyloid deposits could be detected in the brain of APPxPS1(n/)
transgenic mice, even at the age of 18 months (Fig.
5D-G).
The neuronal ablation of PS1 that decreased -secretase activity with
concomitant inhibition of amyloid peptide production thereby also
prevented development of the amyloid pathology, an important
neuropathological hallmark of AD, in the APPxPS1(n/) mice.
Long-term potentiation in CA1 of PS1(n/), APP[V717I], and
APPxPS1(n/) mice
Having demonstrated inhibition of the amyloid pathology, we
addressed the question whether typical hippocampal defects, previously demonstrated in the parent APP[V717I] transgenic mice, were
alleviated in the APPxPS1(n/) transgenic mice. Hippocampal LTP is a
favored model of training-dependent enhancement of synaptic efficacy
that can be induced experimentally by brief, high-frequency activation of specified synapses (for review, see Martin et al., 2000).
We measured LTP at the level of synapses between Schaffer's
collaterals and CA1 pyramidal cells in brain slices from groups of
transgenic mice with the four genotypes. Tetanic stimulation triggered
a significantly impaired LTP in APP[V717I] transgenic mice (Fig.
6A), confirming our
previous findings (Moechars et al., 1999). The pattern of LTP induction
in APPxPS1(n/) mice (Fig. 6C) was strikingly different
as compared with single APP transgenic mice (Fig.
6A). After an initial slight decrease, the slope of
fEPSP approached the level measured in control mice, sharply
contrasting with the progressively decreasing LTP measured in single
APP[V717I] transgenic mice.
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Figure 6.
Long-term potentiation in hippocampal slices of
APP[V717I], PS1(n/), and APPxPS1(n/) transgenic mice. The
slope of field Schaffer's EPSP recorded before and after tetanic
stimulation of Schaffer's collaterals in brain sections from
APP[V717I] (A), PS1(n/)
(B), and APPxPS1(n/) (C) mice
in each panel compared with mice with floxed but active PS1 genes
( ). Each data point shown is the mean ± SD of results from six
individual mice of each genotype. A statistically significant decrease
was evident for the fEPSP in APP[V717I] mice
(p < 0.05; indicated by
asterisk in A), as described previously
(Moechars et al., 1999; Schneider et al., 2001). The final LTP
after 2 hr was not significantly different in PS1(n/) mice or in
APPxPS1(n/) mice (NS; p > 0.05)
(B, C). Note the initial decrease 15 min
after tetanic stimulation that is borderline statistically significant
in the PS1(n/) mice (see Discussion).
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It is interesting to note that the pattern of LTP induction in
PS1(n/) mice was different from that of wild-type mice (Fig. 6B). In the initial phase the slope of the fEPSP was
lower in sections from PS1(n/) mice relative to control mice, i.e.,
168 ± 33% versus 221 ± 27%, 15 min after tetanic
stimulation. Subsequently, and opposite to the monotonous decrease in
APP[V717I] transgenic mice, the slope of fEPSP increased
progressively in sections of PS1(n/) transgenic mice to attain a
mean level that approached that of wild-type mice 2 hr after the
tetanic stimulus (Fig. 6B).
These results indicated that the neuronal absence of PS1 might
initially affect some mechanism(s) involved in the induction of
hippocampal LTP. At the same time, the data demonstrate the restoration, at the end of the 2 hr observation window in this experimental model, of hippocampal LTP in the APPxPS1(n/)
transgenic mice and hence strongly support the hypothesis that amyloid
peptides are responsible for impaired LTP in single APP[V717I]
transgenic mice.
Impaired object recognition of APP[V717I] and APPxPS1(n/)
transgenic mice
The cognitive capacity of transgenic mice with the four genotypes
was assessed in a paradigm of nonspatial visual recognition memory, by
subjecting them to a "novel object" recognition task that is known
to depend on hippocampal activity (Tang et al., 1999; Rampon et al.,
2000). Basically, after training to familiarize all mice with a given
object, they were tested for retention by confronting them with a novel
object, next to and in addition to the familiar one.
The retention measured after 1 hr was similar for all four
genotypes, displaying a similar increased preference for the novel object (results not shown). This proved that all the mice recognized and remembered the original object for at least 1 hr , thereby establishing that their motivation and their exploration capacity were
intact. Testing after 3 hr demonstrated that the retention of the
PS1(n/) mice was not significantly different from the control mice,
whereas the APP[V717I] mice and the APPxPS1(n/) transgenic mice
were both highly significantly impaired relative to the control group
(p < 0.05 and p < 0.001, respectively) (Fig. 7). We therefore must
conclude that the evident and documented impaired cognition of the
APP[V717I] mice is not alleviated by the neuronal PS1 deficiency.
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Figure 7.
Analysis of cognition of wild-type, PS1(n/),
APP[V717I], and APPxPS1(n/) transgenic mice in the object
recognition task. In the novel-object recognition task, recognition
memory is expressed as exploratory preference in the retention test.
The recognition index = tB/(tA + tB) × 100, with
A and B, respectively, representing the
familiar and the novel object. Exploratory preference for control mice,
i.e., mice with floxed but active PS1 genes (bar marked
CO; n = 22), PS1(n/) mice
(n = 17), APP[V717I] mice (n = 24), and APPxPS1(n/) (n = 18) transgenic
mice. All mice were 3-6 months old. Retention was measured at 1 and 3 hr after training (mean ± SEM). At 3 hr after training, the
APP[V717I] and APPxPS1(n/) mice were significantly impaired
compared with control mice (respectively, p < 0.05, p < 0.001; determined by ANOVA
analysis).
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DISCUSSION |
We report the generation and characterization of adult and aging
mice with a neuron-specific deletion of PS1 in their brain. The
successful generation of a "conditional" postnatal neuron-specific knock-out was evidenced by viable, fertile PS1(n/) mice that survived and were observed for up to 2 years. Thereby, we demonstrate that neuronal deficiency of PS1 is well tolerated in adult mouse brain,
without any major effects on brain architecture and morphology. Evidently, the activity of PS2 might contribute to this apparent redundancy of PS1 in neurons of adult mouse brain. Although PS2 mRNA
levels were unchanged, only a small increase in the level of the PS2
C-terminal fragment was observed, further supporting findings that
demonstrated competition between PS1 and PS2 for (unknown) limiting
factors (Thinakaran et al., 1997; Yu et al., 2000). Although it remains
to be defined whether and how this minor increase in PS2 could account
for the absent Notch-related phenotype in adult brain, it was clearly
not able to compensate for the PS1 deficiency and the ensuing inhibited
-secretase cleavage of APP.
We demonstrate a dramatic reduction of amyloid peptides, the primary
index for the activity of PS1 as -secretase, in brain of adult
PS1(n/) mice, with the concomitant expected accumulation of APP
C-terminal fragments, extending and confirming in vivo, original observations in primary hippocampal neurons (De Strooper et
al., 1998). The strongly decreased amyloid peptide production was
evident in brain of both PS1(n/) and APPxPS1(n/) transgenic mice. The inhibition of amyloid peptide production completely prevented
the late amyloid pathology, even at the age of 16-18 months. At this
age, all parent APP[V717I] transgenic mice that we have analyzed to
date contained, without exception, prominent brain amyloid plaque
pathology (Moechars et al., 1999; Dewachter et al., 2000; Van Dorpe et
al., 2000). Therefore, we confidently conclude that inhibition of PS1
is very efficient to prevent the typical amyloid plaque pathology that
develops in aging APP transgenic mice. Given the demonstrated and
documented neuronal specificity of the mouse thy-1 gene promoter in the
selected transgenic mouse strains, this outcome also confirmed that the
amyloid pathology is strictly neuronal in origin (Van Dorpe et al.,
2000).
In our APP[V717I] transgenic mice, the pathological hallmark of AD,
i.e., senile plaques, is preceded by earlier phenotypic changes that
comprise impaired LTP and cognitive defects as early as age 4-6 months
(Moechars et al., 1999; Schneider et al., 2001). Learning and memory
are processes encoded in the brain by long-lasting modification of the
efficacy of synaptic transmission, and hippocampal LTP is one model of
this type of plasticity. The defect in LTP that was characteristic for
the parent APP[V717] mice (Moechars et al., 1999; Schneider et al.,
2001) was now observed to be practically corrected at the end of the 2 hr measuring window in brain slices from APPxPS1(n/) mice. These
data support a critical role for amyloid peptides in the deficit in LTP
in APP[V717] transgenic mice because it is almost completely rescued
by the PS1 deficiency and the concomitant decreased -secretase
activity. This is in agreement with the recent demonstration that
intracerebroventricular injections of amyloid peptide fragments block
LTP in the CA1 region of the rat hippocampus in vivo (Freir
et al., 2001).
We and others have demonstrated that mutant PS1 affected calcium
homeostasis and LTP (Guo et al., 1999; Leissring et al., 1999, 2000;
Yoo et al., 2000; Schneider et al., 2001), whereas the current results
demonstrated that the absence of PS1 also, directly or indirectly,
affected the time course of LTP, especially the initial phase of the
evoked fEPSP immediately after tetanic stimulation. This effect is
likely to be linked to altered calcium ion homeostasis, although the
precise nature of this effect or defect needs further study (Schneider
et al., 2001). A possible explanation would be that the onset of LTP
immediately after the short-lasting initial post-tetanic potentiation
could be delayed, whereas alternatively unscheduled opening of
Ca2+-activated channels could decrease the
amplitude of the fEPSP (Nishiyama et al., 2000).
The failure of the PS1 deficiency to rescue or alleviate the impaired
object recognition of the parent APP[V717I] transgenic mice was
unexpected in light of the LTP results. Such a discrepancy between
in vitro hippocampal LTP and in vivo cognitive
performance has been noted before, however, and in either direction:
impaired LTP but normal behavior (Huang et al., 1995; Schurmans et al., 1997; Meiri et al., 1998; Okabe et al., 1998; Zamanillo et al., 1999;
Ho et al., 2000) or impaired behavior with normal LTP (Rosahl et al.,
1993; Silva et al., 1996; Matilla et al., 1998). This dissociation can
be ascribed to contributions of other types of LTP or synaptic
plasticity, or to the contribution of other parts of the hippocampus or
the brain, to preserve particular forms of cognition. Alternatively,
the mechanisms of memory consolidation could be affected, which would
explain the persisting memory defect in APPxPS1(n/) mice despite
the near rescue of LTP. In view of these results, it must be noted that
besides the evident amyloid peptides, A40 and A42, our current
data point to the C-terminal fragments of APP as additional
contributors to neuropathological deficits. This being the case, the
development and use of -secretase inhibitors as a therapy for AD
needs to be considered carefully.
In conclusion, the neuronal deficiency of PS1 was well tolerated in
PS1(n/) mice, yielding viable and fertile mice that behaved
apparently normal. Most importantly, we demonstrated that the neuronal
deficiency of PS1 inhibited amyloid peptide production, prevented
plaque formation, and practically restored impaired LTP. Moreover, the
data indicated a crucial role for the amyloid peptides in causing the
deficit in LTP in the APP[V717I] transgenic mice. These results
support the use of -secretase inhibitors as a therapy for AD.
However, because the neuronal deficiency of PS1 and the consequent
decrease of amyloid peptides failed to rescue the cognitive deficit in
the APPxPS1(n/) transgenic mice, we believe that the use of
-secretase inhibitors must be scrutinized further and also consider
potential neurotoxic effects of accumulating C-terminal fragments of
APP (Nalbantoglu et al., 1997; Suh, 1997; Chapman et al., 1999;
Dewachter et al., 2000; Suh et al., 2000).
Note added in proof. After submission of our manuscript, Yu
et al. (2001) reached similar conclusions as far as the PS1(n/) are
concerned. In this manuscript, we extend this research with the
in-depth analysis of the cross of PS1(n/) mice with mutant APP
transgenic mice.
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FOOTNOTES |
Received Sept. 17, 2001; revised Jan. 22, 2002; accepted Jan. 28, 2002.
This investigation was supported by the Fonds voor
Wetenschappelijk Onderzoek (FWO)-Vlaanderen, by the
Inter-University Attraction Pole program, by Vlaams Institut
Biotechnologie, by European Economic Community (EEC)-Biotech,
by EEC-5FP, by the Rooms-fund, by KULeuven Research Fund and by
KULeuvenR&D, and by the Queen Elisabeth Fund for Medical
Research. L.R. is a post-doctoral Researcher at the Belgian National
Fund for Scientific Research, and I.D. is a post-doctoral fellow at Vlaams Institut Biotechnologie-FWO-Vlaanderen. The
intellectual, technical, and material contributions of the
following scientists are gratefully acknowledged: H. Van der Putten, P. Krimpenfort, A. Berns, U. Betz, K. Rajewsky, P. Saftig, K. Von Figura,
E. Van Mechelen, H. Vanderstichele, G. Multhaup, and K. Beyreuther.
Correspondence should be addressed to Dr. Fred Van Leuven,
Experimental Genetics Group (LEGT-EGG), Department of Human
Genetics, K. U. Leuven, Campus Gasthuisberg, O&N
06, B-3000 Leuven, Belgium. E-mail:
fredvl{at}med.kuleuven.ac.be.
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TOP
ABSTRACT
INTRODUCTION
