From the Experimental Genetics Group, Center for Human Genetics,
Flemish Institute for Biotechnology, Katholieke Universiteit
Leuven, B-3000 Leuven, Belgium, ¶ Scios Inc., Sunnyvale,
California 94086, IPMC/CNRS, UPR411, Valbonne 06560, France,
and the § Laboratory of Neuroscience, University of
Mons-Hainaut, B-7000 Mons-Hainaut, Belgium
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ABSTRACT |
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Transgenic mice overexpressing different forms of
amyloid precursor protein (APP), i.e. wild type or clinical
mutants, displayed an essentially comparable early phenotype in terms
of behavior, differential glutamatergic responses, deficits in
maintenance of long term potentiation, and premature death. The
cognitive impairment, demonstrated in F1 hybrids of the different APP
transgenic lines, was significantly different from nontransgenic
littermates as early as 3 months of age. Biochemical analysis of
secreted and membrane-bound APP, C-terminal "stubs," and A The central role of the amyloid precursor protein
(APP)1 in the pathogenesis of
Alzheimer's disease (AD) was identified by distinct mutations in the
APP gene that cause early onset familial AD (for review see Ref. 1).
The Swedish (APP/Sw) (2) and London mutation(s) (3) alter APP
processing, causing increased production of the A We have generated additional transgenic mouse strains, expressing human
APP, either wild type or the London or Swedish clinical mutations, from
the neuron-specific mouse thy-1 gene promoter. Their
phenotype was analyzed by biochemical, histochemical, behavioral, electrophysiological, and pharmacological methods. Measurements of
different APP metabolites in brain demonstrated that increased A Generation of Transgenic Mice--
cDNA coding for human
wild type APP (695 isoform), the Swedish (K670N,M671L) mutant (770 isoform), and the London (V642I) mutant (695 isoform) were cloned in
the pTSC vector in the mouse thy-1 gene (16). The purified,
linearized minigenes were microinjected into prenuclear embryos from
superovulated FVB/N females.
Antibodies--
Rabbit antisera B11/4 and B12/4, generated
against a synthetic peptide representing the 20 C-terminal residues of
APP, react equally well with human and mouse APP C-terminal stubs (20). Antibodies FCA18, FCA3340, and FCA3542 were generated against synthetic
peptides (21). The following commercially available antibodies were
used: SMI31 (Affiniti); anti-GFAP, anti-synaptophysin, and
anti-ubiquitin (Dakopatts, Glostrup, Denmark); mAb 22C11 and anti-MAP-2
(Boehringer Mannheim); and mAb 1G5 (Athena Neurosciences, San
Francisco, CA).
Analysis of APP Transgene RNA and Protein--
Mouse brain RNA
was analyzed by Northern blotting (16). Relative mRNA levels of
transgene versus endogenous APP were quantified densitometrically after autoradiography. APP protein was analyzed by
Western blotting and immunoprecipitation with the antibodies indicated.
Total brain was homogenized in 15 volumes of 50 mM Tris, pH
7.4, 150 mM NaCl, and a mixture of proteinase inhibitors (Boehringer Mannheim) and cleared by centrifugation (100,000 × g, 1 h, 4 °C). The pellet was re-extracted with the
same buffer containing 2% Triton X-100, 2% Nonidet P40 and
centrifuged as before. Proteins were denatured and reduced (2% SDS,
1% 2-mercaptoethanol, 95 °C, 5 min), separated on polyacrylamide
gels (4-20% Tris-glycine), and transferred to nitrocellulose filters
(Hybond-C, Amersham) for Western blotting (16). C-terminal fragments
were immunoprecipitated with rabbit antiserum B11/4, separated on
10-20% Tris-tricine gels, and detected by Western blotting with B12/4
(1/1000) and ECL detection.
Soluble A Behavioral Tests--
Behavioral tests were performed conforming
with the Society of Animal Experimentation policy. Corner crossing and
aggression tests were described (16). Statistical analysis was by
Student's t test ( Morris Water Maze Test--
F1 offspring of matings of
transgenic FVB/N males with C57/Bl6 females were tested. The pool (a
white, circular vessel 1 m in diameter) contained water at
20 °C with titaniumoxide as an odorless, nontoxic additive to
hide the escape platform. Swimming of each mouse was videotaped and
analyzed (Poly-Track, San Diego Instruments, CA). Prior to place
navigation tests all mice were allowed a forced swim test for a
120 s period. For place navigation tests, mice were trained to
locate the hidden platform in seven blocks of three trials over 4 consecutive days. For different subjects the location of the platform
was changed between the four quadrants. 24 h after the last
training, each animal was tested (probe trial) with the platform
removed. Mice were allowed to search for 60 s and quadrant search
time and crossings of the original platform position were measured. The
same mice were tested 18 days later for cue navigation tests (platform
marked with visible cylinder), and platform location was changed over
the four quadrants. Statistical analysis of variance was by one- and
two-way analysis of variance with repeated measures and post-hoc
analysis for variable escape latency by Student's t test.
Electrophysiology--
Hippocampal slices were studied in an
artificial cerebrospinal fluid containing 124 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.24 mM KH2PO4, 2.4 mM
CaCl2, 1.3 mM MgSO4, and 10 mM glucose and bubbled with 95% O2 and 5%
CO2. Slices were cut transversally (400 µm) in cold
artificial cerebrospinal fluid and kept at room temperature till placed
in the interface recording chamber at 30 °C. Experiments started
3 h 30 min after dissection to allow recovery of the slice, and
the chamber was perfused with artificial cerebrospinal fluid (1 ml/min). A monopolar platinum microelectrode was used to stimulate Schaffer collaterals, and evoked field excitatory postsynaptic potentials were recorded in the stratum radiatum of the CA1 region with
low resistance (2 megohm) glass microelectrodes filled with 2 M NaCl. Test stimuli were biphasic pulses of 0.1 ms every
30 s. Tetanus-induced long term potentiation was by two pulse
trains of 1 s at 100-Hz, separated by 20 s with each pulse of
0.2 ms. Intensity was adjusted to obtain responses with amplitudes of about 30% of maximal response. The slope of the excitatory
postsynaptic potential (mV/ms) was measured from the average wave form
from four consecutive responses.
Glutamate Analogs--
NMDA and KA, dissolved in pyrogen free
0.9% sodium chloride solution, were injected intraperitoneal in
3-4-month-old mice. The mice were observed for 2 h to determine
LD50 by NMDA, while seizures induced by KA were scored in
seven stages: lethargy, rigid posture, head bobbing or circling, clonic
seizure, rearing alone or with falling, tonic-clonic seizures, and
death (16).
Histology and Immunohistochemistry--
Mice were perfused
transcardially with 4% paraformaldehyde. Brains were fixed overnight
at 4 °C, rinsed in phosphate-buffered saline, dehydrated, embedded
in paraffin, and sectioned. Histological staining by hematoxylin-eosin,
cresyl-violet, or silver impregnation were by routine procedures (24).
For immunohistochemistry, sections were dewaxed, rehydrated, and
endogenous peroxidase quenched with hydrogen peroxide (1% in 50%
methanol). After 1 h in blocking buffer (10% goat serum in 10 mM Tris, pH 7.4, 0.15 M NaCl, 0.1% Triton
X-100), appropriately diluted primary antibody was applied (overnight
at room temperature). Following rinsing, peroxidase or
biotin-conjugated secondary antibody was applied (1:300, 1 h), and
the latter was followed by streptavidin-peroxidase. Immunoprecipitates were stained with diaminobenzidine/H2O2. For
immunostaining for APP and synaptophysin, silanated slides were
microwaved in 10 mM sodium citrate, pH 6 (8 min, 450 W).
AT-8 staining was performed on free floating vibratome sections.
Microglia staining was performed by incubation with biotinylated tomato
lectin, followed by streptavidin-peroxidase and staining with
diaminobenzidine/H2O2.
Transgene Expression--
Wild type APP (APP/Wt) and London and
Swedish mutant APP (APP/Ld and APP/Sw, respectively) were expressed in
the brain of transgenic FVB/N mice by the mouse thy-1 gene
promoter (16). The 13 independent transgenic lines expressed transgene
mRNA at moderate to high levels (Fig.
1). In all lines the human APP protein was expressed in hippocampus and in parietal and frontal cortex (levels
two to five times higher than endogenous APP), with lower levels in
olfactory bulb and thalamus (about 65 and 30%) and very low in
cerebellum (results not shown).
Biochemical Analysis of APP Protein Intermediates--
APP
intermediates analyzed in the brain of heterozygous transgenic mice,
aged between 2 and 4 months, included the integral membrane-bound APP,
the secreted Premature Death--
The APP transgenic mice died prematurely (16)
as reported by others (17, 14). We monitored 136 heterozygous mice from six APP transgenic strains and 46 nontransgenic littermates for 1 year.
This revealed that only 4.3% of nontransgenic mice died, as opposed to
between 21 and 72% of APP transgenic mice (Table I). Mortality increased with increased
APP expression as reported before (16). Premature death was absent in
other transgenic strains housed in identical conditions and
overexpressing transgenes unrelated to APP at comparable protein levels
using the same promoter.
Spontaneous Behavior--
Typical for all APP transgenic mice were
alternating episodes of hyperactivity, anxiety, and aggression in the
home cage. Offspring developed and behaved normally for 6-8 weeks
after birth. APP/Ld transgenic mice with the highest expression levels
began to show increased agitation from 8 weeks onwards. Progressively, more mice of both sexes in this and other APP transgenic strains displayed this behavior, which was absent in nontransgenic littermates housed and handled in identical conditions. Indications for increased incidence of spontaneous seizures were also noted in the current human
APP transgenic mice (16). Spontaneous seizures are clearly not an early
phenomenon and were observed in less than 15% of APP transgenic mice
older than 6 months. Systematic observation and attempts to obtain
encephalographic recordings of epileptic activity proved the rare
nature of this
phenomenon.2
Open Field-Corner Crossing Test--
Many APP transgenic mice
showed transient cessation of locomotion upon routine transfer to a new
cage. This behavior was systematically approached by measuring motor
activity in a corner-crossing variant of the open field test (14, 16,
25) at three different ages. In nontransgenic mice, independent of age,
corner crossing behavior was distributed in a gaussian fashion around a
mean of about seven crossings in 30 s (Fig.
3 and Table
II). All APP transgenic mice were
significantly reduced in ambulation, which became progressively more
evident with age. Statistical analysis of the most extensively studied
APP/Sw/1 mice (Fig. 3 and Table II) was typical for the other
transgenic strains; the decrease in ambulation is related to genetic
status and age, with a significant interaction of both independent
variables (see legend to Fig. 3). Age was determinant in all transgenic
strains, except in the highest expressing APP/Ld/2 transgenic mice,
which showed already minimal ambulation even before 3 months old (Table
II). In these young APP/Ld/2 transgenic mice, also the second index of
motor activity measured, i.e. posture freezing induced by
the new environment, was already maximal at 3 months, as opposed to all
other APP transgenic mice, in which posture freezing frequency
increased with age (Table II). Reduced ambulation reflected a neophobic
response and not a motor impairment or lack of exploratory motivation,
because this behavior was transient and the mice were normally active
in the cued variant of the water maze paradigm and the forced swim test
(see below).
Morris Water Maze Test--
The functional repercussion of
expression of the APP transgenes on cognitive processes was measured in
the Morris water maze paradigm. Because FVB/N mice are not optimal for
this test (Ref. 23 and references therein), we generated F1 hybrid mice
from male transgenic mice and female C57/Bl6 mice. This offered the important additional advantage that the genotyped nontransgenic and
transgenic littermates constitute perfectly matched experimental groups. Data for heterozygous transgenic mice, aged between 3 and 6 months, of lines APP/Wt/4 and APP/Ld/2 were most intensely studied and
are presented, because both of these transgenic lines presented similar
early phenotypic traits but differed most prominently in amyloid plaque
formation later in life (see "Brain Histology and
Immunohistochemistry"). Both in place and in cue navigation tests,
nontransgenic F1 mice learned to rapidly locate and escape upon the
platform with stable terminal acquisition latency of less than 12 s (place navigation: F(6,497) = 48.3, p < 0.001; cue
navigation: F(6,497) = 14.18, p < 0.001). Transgenic
littermates of lines APP/Wt/4 and APP/Ld/2 were less apt, and although
they improved with training (APP/Wt/4: F(6,392) = 20.0, p < 0.001; APP/Ld/2: F(6,476) = 20.0, p < 0.001), they remained significantly impaired in
the place navigation test. The overall escape latency, an estimate of
spatial learning and memory capacity, was significantly longer for
transgenic as opposed to nontransgenic littermates (Fig.
4A). Statistical comparison by
two-way analysis of variance of the performances of APP/Wt/4 and of
APP/Ld/2 versus their respective nontransgenic F1
littermates explained the difference in escape latency by genetic
status (APP/Wt/4: F(1,127) = 26.7, p < 0.001; APP/Ld/2: F(1,139) = 87.21, p < 0.001) and by blocks
(F(6,123) = 42.9, p < 0.001 and F(6,135) = 22.4, p < 0.001) with a significant interactive effect of
these independent variables (APP/Wt/4: F(6,123) = 2.5, p < 0.05; APP/Ld/2: F(6,135) = 3.4, p < 0.01). The impairment in escape latency was not observed when the
same transgenic mice were tested to locate the platform visually (cued)
rather than from spatial memory (Fig. 4A). This important
demonstration of normal motivation to escape and of swimming ability
was independently confirmed by a forced swim test (Fig. 4B).
The magnitude of the spatial deficit was assessed in a probe test,
conducted 24 h after the place navigation test. Nontransgenic F1
mice (F(1,94) = 104.4, p < 0.001) and transgenic
APP/Wt/4 mice (F(1,69) = 14.5, p < 0.001) and APP/Ld/2
mice (F(1,90) = 23.9, p < 0.001) searched the correct quadrant (Fig. 4C), confirming that all mice exhibited
spatial learning capabilities. The spatial deficit was confirmed by the significant decreased time spent in the correct quadrant by the transgenic APP/Wt/4 mice (F(1,39) = 3.0, p < 0.01) and
APP/Ld/2 mice (F(1,45) = 3.5, p < 0.05) relative to
their nontransgenic F1 littermates. A more refined parameter for the
spatial bias in the place navigation test is the number of crossings
over the exact former location of the platform (26). Nontransgenic F1 mice crossed the correct site significantly more often than their transgenic littermates of line APP/Wt/4 (F(1,41) = 15.75, p < 0.001) and APP/Ld/2 (F(1,45) = 18.52, p < 0.001) (Fig. 4D).
In conclusion, these combined results obtained in the Morris water maze
paradigm demonstrated unequivocally that the APP transgenic mice were
markedly cognitively impaired already early in life.
Electrophysiology--
Memory and learning is dependent on changes
in the efficiency of synaptic transmission. An excellent and widely
used model for this type of plasticity is long term potentiation.
Synaptic potentiation was induced in the CA1 in hippocampal slices of
5-7-month-old APP/Ld/2 mice and age-matched nontransgenic littermates,
by applying a tetanic stimulus, activating the Schaffer collaterals.
There was no significant modification of the degree of short term
potentiation in the first 2 min following the stimulation in transgenic
mice (207.7 ± 26.5 S.E.) compared with nontransgenic littermates
(236.1 ± 20.3). However, with time there was a progressive decay
of long term potentiation in the transgenic mice. In nontransgenic mice potentiation was 172 ± 9.6, 161.1 ± 11.4, 156.5 ± 5.8, and 150.9 ± 4.8 after 30, 60, 90, and 120 min, respectively.
In transgenic mice potentiation was significantly decreased to
133.8 ± 6.8 (p < 0.01, Student's t
test), 118.2 ± 6.9 (p < 0.01), 116.2 ± 5.7 (p < 0.001), and 109.9 ± 3.3 (p < 0.001) 30, 60, 90, and 120 min after stimulation (Fig.
5C). The degree of paired
pulse facilitation measured in transgenic APP/Ld/2 mice was not
significantly altered compared with nontransgenic mice (Fig.
6), indicating that presynaptic function
is not altered in transgenic mice.
Reactivity to Glutamate Analogs--
The present human APP
transgenic mice behaved very similar to the previously reported mutant
mouse APP (APP/RK) transgenic mice that reacted differentially to
glutamate analogs NMDA and KA (16). In nontransgenic FVB/N mice of 3-4
months, the LD50 of NMDA was about 60-70 mg NMDA/kg. All
age-matched APP transgenic mice were less sensitive, with
LD50 values that ranged between 100 and 140 mg NMDA/kg
(results not shown). Sensitivity to KA (cumulative score of seven
stages of seizures; see under "Experimental Procedures") was
maximal in nontransgenic mice at 32 mg/kg (mortality of 15%). All APP
transgenic mice were more sensitive to KA; maximal cumulative scores
were obtained for APP/Wt/4 and APP/Ld/6 mice following doses of 28 and
24 mg KA/kg, respectively, resulting in a mortality of 43 and 29%,
respectively (results not shown).
Brain Histology and Immunohistochemistry--
Histological
analysis was performed on the brain of 60 representative heterozygous
transgenic mice, i.e. 20 APP/Ld/2 mice (2-18 months), 4 APP/Ld/6 mice (4-14 months), 20 APP/Sw/1 mice (3-25 months), and 6 APP/Wt/4 mice (2-19 months). All were sacrificed when healthy and of
normal body weight. Hematoxylin-eosin and cresyl-violet staining did
not indicate overt neuronal loss or degeneration, with the exception of
one APP/Ld/2 mouse (6 months) and two APP/Sw/1 mice (aged 12 and 25 months), in which some neurons were detected with a condensed nucleus
surrounded by an halo of clear cytoplasm. This morphology and location
at the border of the granular layer and the hilus of the dentate gyrus
is reminiscent of transgenic mice that overexpress the amyloid peptide
(17) or APP/RK (16, 25, 27). Immunohistological staining for human APP
demonstrated that the mouse thy-1 gene construct steered expression of the transgene predominantly to neurons, resulting in
robust staining of large pyramidal neurons in all cortical layers, in
hippocampus, and in amygdala. Diffuse amyloid deposits and compact
neuritic plaques were detected by silver staining and by
immunohistochemistry in all old APP/Ld/2 mice (13-18 months) and in
six of ten old APP/Sw mice (18-25 months) (Fig.
7) and were, particularly in the old
APP/Ld/2 mice, most abundant in hippocampus and cortex. Occasional
deposits were observed in thalamus and fimbria, external capsule,
pontine nuclei, and white matter. In the APP/Sw transgenic mice, the
amyloid deposits were less frequent but much larger (up to 130 µm)
and were located in the primary olfactory cortex and in the amygdala.
All amyloid deposits reacted with six different antibodies specific for
the amyloid peptide (results not shown). Differential staining of
adjacent sections with antibodies specific for A Transgenic mice have been reported that overexpress wild type or
mutant APP (9-14, 16), the A The senile plaques in the old APP/Ld transgenic mice were
authenticated by silver and thioflavin-S staining,
immunoreactivity for A The observation of amyloid plaques in transgenic mice older than 12 months especially when producing measurable A Correlation of these observations to precise biochemical intermediates
or end products of APP remained elusive, with the exception of amyloid
plaque formation. Besides confirming the convincing role of A To explain the additional phenotypic traits, the glutamate
neurotransmitter system was advanced as a prime candidate in accord with previous reports (16, 19). Glutamate is implicated in phenomena of
learning and memory, as well as in hyperactivity, seizures, neophobia,
and many other phenomena, underlined by many reports and beyond the
present discussion. The impaired learning and memory convincingly
demonstrated in the APP transgenic mice and underlined by the
electrophysiological data presented might be relevant for the central
problem in AD. Possible dysfunction of NMDA signaling pathways, as long
term potentiation, is likely to affect memory directly (34-36).
Seizures of variable intensity are a clinical feature of advanced AD,
although the reported incidence varies widely, i.e. from 6 to 64% in late onset AD patients but as high as 70% in early onset
familial AD caused by Presenilin-1 mutations, as discussed before (Ref.
16 and references therein).
The presented data are evidence that the phenotype results from
overexpression of APP and that it is a functional physiological disturbance and not the consequence of a developmental problem, because
expression of APP controlled by the thy-1 gene promoter construct is immediately after birth about 2 orders of magnitude lower
than in adults (results not shown). As already indicated, aspects of
the phenotype have been studied and reported by others in different
transgenic mouse strains overexpressing APP (9-12, 14, 16).
Differences among these studies include the APP isoform or mutant, the
expression levels, and the mouse host strain. The current study
demonstrates that with the exception of amyloid plaques, the
qualitative differences between transgenic lines were minimal. The
recipient mouse genetic background continues to remain an issue (37).
The very similar phenotype of transgenic mice that overexpress either
the A(40)
and A
(42) peptides in brain indicated that no single intermediate
can be responsible for the complex of phenotypic dysfunctions. As
expected, the A
(42) levels were most prominent in APP/London
transgenic mice and correlated directly with the formation of amyloid
plaques in older mice of this line. Plaques were associated with
immunoreactivity for hyperphosphorylated tau, eventually signaling some
form of tau pathology. In conclusion, the different APP transgenic
mouse lines studied display cognitive deficits and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models for both early and late
neuropathological and clinical aspects of Alzheimer's disease.
INTRODUCTION
Top
Abstract
Introduction
References
peptide of 42 amino acids (4), hypothesized to be pivotal in AD pathology (1, 5).
Early onset familial AD caused by mutations in the presenilin genes
supports this hypothesis, because they increase production of A
(42)
peptide (6, 7) due to the gain of an unknown function (8). The
extensive cell biological definition of the metabolic effects of the
different mutations in APP in vitro requires matching
analysis of their physiological impact in vivo. Transgenic
mice with wild type and different mutant forms of APP have been
generated and the original, most wanted end point, i.e.
AD-like amyloid plaques in mouse brain, was obtained (9, 10),
accompanied by cognitive deficits (11) and by hyperphosphorylation of
protein tau (12). In other transgenic mouse strains overexpression of
APP caused behavioral, synaptotrophic, and neurodegenerative effects,
accelerated senescence, and premature death, in the absence of amyloid
deposits (13-16). Intracellular expression of the A
peptide yielded
mice with extensive neuronal loss but no amyloidosis (17).
Overexpression of the C-terminal domain of APP caused neuronal
degeneration (18), whereas in another model, pre-amyloid
deposits, hippocampal cell loss, and cognitive deficits were
documented (19).
(42)
levels correlated with the formation of amyloid plaques in the brain of
old APP/London transgenic mice. The plaques were extensively
characterized immunohistochemically and displayed many aspects
typically observed in the brain of AD patients. As opposed to plaques
that developed only after at least 12 months of age, other deficits
were observed from 3 months onwards and included cognitive impairment,
decreased long term potentiation, differential glutamatergic responses,
aggression, and neophobia, among others. These signs were largely
independent of the actual isoform or mutant of APP that was expressed,
were not correlated with a single APP metabolite, and are dissociated
in time from plaque formation. These mice will be good models to study
both early and late, neuropathological, and clinical aspects related to
Alzheimer's disease.
EXPERIMENTAL PROCEDURES
Peptides--
Mouse brain was homogenized in 6.5 volumes of 20 mM Tris-HCl, pH 8.5, with 5 mM
EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml
leupeptin, 0.7 µg/ml pepstatin, 0.1 mg/ml phenanthroline, and 0.1 mg/ml benzamidine. The homogenate was cleared (135,000 × g, 1 h, 4 °C and 220,000 × g,
2 h, 4 °C) and concentrated on Sepak C18 cartridges (Waters,
Milford, MA). Sandwich enzyme-linked immunosorbent assay for A
(40)
and A
(42) peptides used mAb 1101.1 to capture and rabbit antiserum
BA#1 and mAb 108.1, respectively, as detecting antibodies (22).
= 0.05) and two-way analysis of variance.
RESULTS
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Fig. 1.
Generation of APP transgenic mice.
A, schematic representation of the mouse thy-1
gene promoter construct. B, Northern blot of brain total
RNA. Lane 1, APP/Wt/4; lane 2, APP/Wt/2;
lane 3, APP/Sw/1; lane 4, APP/Sw/3; lane
5, APP/Ld/2; lane 6, APP/Ld/6; lane 7,
nontransgenic FVB/N. The 3.5-kilobase endogenous APP transcript
(arrow) and the larger transgenic mRNA
(arrowhead).
-cleaved and total secreted APP, the residual
C-terminal fragments (stubs), and the soluble A
(40) and A
(42)
peptides. In APP/Sw transgenic mice,
-cleaved APP was 5-fold lower
than in APP/Wt mice (Fig. 2). The
-cleaved C-terminal fragments were most pronounced in APP/Sw and
APP/Ld transgenic mice (Fig. 2). When normalized for expression level, a 3-5-fold overproduction of
-cleaved C-terminal fragments was evident in line APP/Sw/1 relative to all other transgenic lines (Fig.
2, right column). Absolute levels of soluble A
(40)
peptide correlated most closely with levels of
-cleaved C-terminal
stubs (Fig. 2), whereas normalization highlighted their preponderant formation in APP/Sw mice. Measurements by specific enzyme-linked immunosorbent assay indicated that A
(42) peptide levels were high
only in the brain of APP/Ld mice (Fig. 2). These data essentially confirm the known metabolic effects of mutations in APP,
i.e. the Swedish mutation specifically favored
-cleavage,
resulting in increased concentrations of
-cleaved C-terminal
fragments and of A
(40) peptide, whereas the London mutation not only
produced
-cleaved C-terminal stubs but produced most conspicuously
A
(42) peptides.
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Fig. 2.
Analysis of APP and its processing
metabolites. A, Western blotting with mAb 1G5 of
membrane associated human APP. B, Western blotting with mAb
1G5 of soluble human - plus
-cleaved APP. C, Western
blotting with antiserum R1736 of
-cleaved soluble APP. D,
immunoprecipitation of
- and the larger
-cleaved C-terminal APP
stubs with antiserum B11/4 followed by Western blotting with antiserum
B12/4. E and F, levels of A
(40) and A
(42)
peptides in brain determined by enzyme-linked immunosorbent assay.
Quantitative analysis of Western blots was normalized (middle
panels) and corrected for expression levels per transgenic line
(right panels). Western blots shown are representative
examples, whereas histograms represent the means ± S.E. of four
to six mice per transgenic strain.
Overview of different transgenic APP mouse lines with some
characteristics
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Fig. 3.
Corner crossing test. Shown is the
relative number of mice (abscissa) that crossed a given
number of corners (ordinate) in 30 s. Nontransgenic
FVB/N mice of the different age groups reacted similarly: A,
4-9 weeks (n = 133); B, 12-17 weeks
(n = 48); and C, 20-52 weeks
(n = 39). Three age groups of APP/Sw/1 transgenic mice
reacted differently with age: D, 4-9 weeks
(n = 76); E, 12-17 weeks (n = 50); and F, 20-52 weeks (n = 73). Two-way
analysis of variance (genetic status and age) analysis of corner
crossing revealed significant effects of genetic status (F(1,413) = 27.3, p < 0.001), age (F(2,413) = 15.1, p < 0.001) and genetic status combined with age
(F(2,413) = 10.1, p < 0.001) indicative of a neophobic
reaction that progresses with age.
Corner crossing test
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Fig. 4.
Morris water maze of transgenic and
nontransgenic F1 (FVB/N × C57/Bl6) littermates.
A, mean escape latency in the different blocks of the place
and cue navigation tests. Asterisks denote significant
differences of transgenic with nontransgenic littermates (** for
p < 0.001). Student t testing of
nontransgenic versus transgenic mice showed significant
differences in all blocks of the place navigation test apart from the
first block but not in cue navigation tests. B, average swim
velocity in a forced swim test. Student t testing revealed
no significant differences between nontransgenic and transgenic mice.
C, average time spent in each quadrant in probe test.
Dwelling in the target quadrant is significantly lower for transgenic
mice relative to nontransgenic littermates (* for p < 0.05). D, number of platform crossings is significantly
lower for transgenic mice relative to nontransgenic littermates (** for
p < 0.001).
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Fig. 5.
Long term potentiation in hippocampal
slices. Samples of traces obtained from a nontransgenic
(A) and an APP/Ld/2 mouse (B). Either field
excitatory postsynaptic potential recorded 10 min after (left
panels) or 1 h 30 after (right panels) tetanic
stimulation are superimposed on a same control trace obtained before
stimulation. C, field excitatory postsynaptic potential
slopes recorded before and after tetanic stimulation in slices from
APP/Ld/2 mice (open circles) or nontransgenic mice
(filled circles). Each point shown is the mean ± S.D.
(n = 6 animals, 6 slices for APP/Ld/2 and nontransgenic
mice).
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Fig. 6.
Paired pulse facilitation in hippocampal
slices. Paired pulse facilitation is not altered in APP/Ld/2 mice.
Traces showing typical paired pulse facilitation (50-ms interval) in an
APP/Ld/2 (A) and in a nontransgenic mouse (B).
C, paired pulse facilitation as a function of the interpulse
interval. Points are the means ± S.D. (n = 6 animals, 6 slices).
(40) or A
(42)
(21) showed preferential staining for A
(42) of the plaques in APP/Ld mice, whereas plaques of APP/Sw mice reacted prevalently with A
(40)
antibodies (Fig. 7). Amyloid deposits were absent in the brains of
APP/Wt mice and in all mice analyzed when younger than 12 months. The
deposits were also readily detected by silver and thioflavin-S
staining, resulting in patterns that were reminiscent of AD brain with
wisps of fibers radiating from a central mass (Fig. 7). In addition,
amyloid deposits were immunoreactive for the ectodomain of APP, for
ubiquitin, and for cathepsin D, and they were surrounded by reactive
astrocytes and microglia (Fig. 8).
Astrogliosis was further evident in the hippocampus and cortex of many
APP transgenic mice from all lines, being more widespread and intense
in older mice. Neuritic pathology, i.e. dystrophic neurites
associated with thioflavine-S-positive amyloid deposits were visualized
by immunostaining for APP, ubiquitin, and synaptophysin. Staining with
the monoclonal antibody AT-8 recognizing hyperphosphorylated tau and
with antibody SMI31, revealing an epitope shared by phosphorylated tau,
neurofilament proteins, and MAP-1 detected structures resembling distorted neurites surrounding amyloid plaques, but comparing adjacent
sections, less plaques were immunoreactive for AT-8 than for A
.
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Fig. 7.
Immunohistochemical and histological
characterization of amyloid plaques. Overview (A) and
detail (B) of an immunostaining for A with FCA18 in the
brain of an APP/Ld/2 mouse (18 months). Extensive plaque formation is
visible in the hippocampus and entorhinal cortex. C, Garvey
silver impregnation of the piriform cortex of an APP/Sw/1 mouse (20 months). D, thioflavin S staining of an amyloid deposit in
the thalamus of an APP/Ld/2 mouse (16 months). Also shown is
immunostaining of adjacent sections of an APP/Ld/2 mouse (E
and F) (16 months) and of an APP/Sw/1 mouse (G
and H) (18 months) with FCA3340 antibodies specific for
A
(40) (E and G) and with FCA3542 specific for
A
(42) (F and H). The plaques in APP/Ld mice
show preferential staining for A
(42), in contrast with prevalent
A
(40) immunoreactivity in APP/Sw mice. Scale bars:
A, 1 mm; B-D, = 20 µm; E-H, 10 µm.
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Fig. 8.
Immunostaining for different
plaque-associated proteins. Immunostaining of adjacent sections of
an APP/Ld/2 mouse (16 months) for A with FCA 18 (A) and
anti-GFAP (B) demonstrated reactive astrocytes infiltrating
and surrounding the plaques. Microglial staining with tomato lectin
also showed positive reaction around plaques (C).
Immunostaining with antibody SMI31 (D), AT8 (E),
and APP (mAb 1G5) (F) in APP/Ld/2 transgenic mice revealing
dystrophic neurites and associated hyperphosphorylated tau.
Antibodies against synaptophysin (G), cathepsin D
(H), and ubiquitin (I) also reacted with plaques
in APP/Ld/2 mice (13-18 months). Scale bars:
A-H, 20 µm; I, 10 µm.
DISCUSSION
peptide (17), or the C-terminal domain
of APP (18, 19). Amyloid plaques, regarded as the first or ultimate
goal, are an acquired phenotypic trait in transgenic mice that
overexpress human APP mutants (Refs. 10-12 and this study). These
models allow us to test the long standing question in AD, i.e. if and how amyloid plaques play a role in cognitive
decline and dementia. We have experimentally addressed part of this
problem and observed that formation of typical amyloid plaques is a
late process, correlated with age and increased levels of A
(42)
peptide. Conversely, the earlier phenomena of behavioral and cognitive defects that we have observed and extensively documented as shared by
the different APP transgenic mouse lines occur before and
independent of amyloid plaques.
(40) and A
(42) peptides, APP,
ubiquitin, cathepsin D, and many additional other antigens.
The plaques appear very similar to plaques in AD brain, but no
indications for neurofibrillary tangles were observed. An epitope
typical for hyperphosphorylated tau was, however, detected in
dystrophic neurites associated with amyloid plaques, which might be
related to similar early neurofibrillary changes in AD (28). Additional
comprehensive quantitative analysis of all these parameters in a large
number of transgenic mice at different ages is ongoing. The preliminary
results already confirm that amyloid plaques in the brain of transgenic
mice are a late phenomenon in the overall phenotype, whereas the data
presented here clearly dissociate plaque formation in brain from other
phenotypic traits, in time but not necessary in mechanism.
(42) in brain confirms
previous reports (11, 29). The documented lesions were not paralleled
by neuronal loss in mice from either strain (30, 31). In a third model,
plaque formation was observed without reference to A
levels or
behavioral or cognitive defects (12). In the present study, the
disturbed behavior and cognitive defects occurred before plaque
formation, and the reduced water maze performance and deficits were
common to all transgenic lines that overexpressed wild type or mutant
APP, including those that did not develop amyloid plaques. The defects
were never observed in other transgenic mouse strains overexpressing
proteins unrelated to APP from a similar recombinant thy-1
promoter construct. The consistent common early defects in all APP
transgenic mouse lines, presented here and previously documented in two
different mouse genetic backgrounds (16), are the strongest evidence
for the direct role of APP, although it leaves open questions as to
which of its intermediates are involved. Differences among the APP
transgenic mouse strains in intensity or age of onset of phenotypic
characteristics appeared related to the level of the APP transgene. In
addition, many of the symptoms intensified and progressively worsened
with age, as revealed by the results presented and by research in
progress, but signs were already evident in young animals.
(42) in
amyloid plaque formation at late age, the data further suggest
important molecular pathological aspects of APP in brain. The
functional neuronal disturbances can be mediated by the amyloid
peptides but in either a molecular form or conformation that is
different from that needed for plaque formation (32). In addition, they
could exert their effects in combination with the amyloidogenic
C-terminal fragments (19, 33). Whereas high levels of A
(42) peptide
were confined to APP/Ld transgenic mice, production of A
(40) peptide
was general in all transgenic lines and closely linked to levels of the
-cleaved C-terminal stubs of APP, their obligate immediate
precursor. The early increased levels of both these APP intermediates
correlate with the early occurrence of phenotypic traits common to all
APP transgenic lines.
peptide intracellularly (17), wild type or mutant APP (14),
our mouse mutant APP/RK (16), or the human APP transgenic mice
presented here indicate that the FVB/N strain presents the lowest
threshold to physiological effects of APP and its metabolites.
Nevertheless, it remains a fact and very important to note that this is
not unique for the FVB/N background because the previously generated
APP/RK transgenic mice in the C57/Bl6 and the FVB/N genetic background
presented a phenotype that overlapped almost entirely (Ref. 16 and
results not shown). Behavioral abnormalities have been reported in
other APP overexpressing mice generated on very different genetic
backgrounds (15, 38). Moreover, there is an intriguing phenotypic
overlap (impairment of learning and long term potentiation) between the presented APP transgenic mice and the transgenic mice expressing a
C-terminal 104-amino acid fragment of APP, generated on an outbred background (B6xC3H) (19). Insight into the genetic aspects of the
different mouse strains is likely to bring forward important information on fundamental issues of AD. In this respect, the F1 hybrid
transgenic mice presented in this study, constitute a major advantage
in studying the cognitive early deficits in comparison to perfectly
matched littermates arising from the same matings. This consideration
is very important for behavioral and cognitive studies, and the APP
transgenic mice offer models allowing studies of the early and the late
phase of APP induced AD-like pathology.
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ACKNOWLEDGEMENTS |
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The intellectual, technical, and material contributions of the following scientists are gratefully acknowledged: M. Gilis, C. Kuipéri, I. Laenen, L. Serneels, L. Stas, M. Crauwels, S. Van Gestel, K. Meurrens, B. De Strooper, L. Hendrickx, C. Van Broeckhoven, K. von Figura, B. Greenberg, and H. Van der Putten. We thank the K. U. Leuven for continuous support.
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FOOTNOTES |
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*
This work was supported by the Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen, by National Fonds Wetensomappelk
Onderzoek-Lotto, by the Action Program for Biotechnology of the Flemish
government (VLAB, COT-008/IWT), by the 4th Framework
European Economical Commission-Biotechnology program, by the
Rooms-fund, and by Leuven Research and Development.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.
Recipient of a doctoral scholarship and the Katholieke
Universiteit Leuven Research Fund for a post-doctoral fellowship.
** To whom correspondence should be addressed: Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology (VIB), K. U. Leuven-Campus Gasthuisberg ON 06, B-3000 Leuven, Belgium. Tel.: 32-16-345888; Fax: 32-16-345871; E-mail: fredvl{at}med.kuleuven.ac.be; www.med.kuleuven.ac.be/legtegg.
2 J. Noebbels, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer's disease; mAb, monoclonal antibody; NMDA, N-methyl-D-aspartic acid; KA, kainic acid.
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REFERENCES |
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