From the a Centre for Research in Neurodegenerative Diseases, the g Department of Laboratory Medicine and Pathobiology, the i Department of Medical Biophysics, the j Department of Medicine (Division of Neurology), h University Health Network, University of Toronto, Toronto, Ontario M5S 3H2, Canada, c McLaughlin Research Institute, Great Falls, Montana 59405-4900, d Schering-Plough Research Institute, Milan, Italy, and e Neurochem Inc., Ville St.-Laurent, Montreal, Quebec H4S 2A1, Canada
Received for publication, January 25, 2001, and in revised form, March 9, 2001
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ABSTRACT |
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We have created early-onset transgenic (Tg)
models by exploiting the synergistic effects of familial Alzheimer's
disease mutations on amyloid Alzheimer's disease, the most common cause of dementia, has a
complex etiology involving both genetic and environmental determinants. It is characterized by cerebral amyloid deposits formed from the amyloid Since there are no naturally occurring rodent forms of AD, there has
been great interest in creating accurate transgenic facsimiles of this
disease. Such disease models have the potential to stratify pathogenic
events and practical utility for testing interventions directed against
synthesis or deposition of the A Construction and Analysis of Tg Mice--
The APP695 cDNA
cassette was based upon an isolate of the cDNA clone described by
Kang et al. (26). A SmaI to SpeI
fragment including 90 and 269 base pairs of the wt APP cDNA 5'- and
3'-untranslated region was cloned into the plasmid vector pUC19 (27).
This clone was subjected to mutagenesis using the "transformer"
protocol (CLONTECH) to convert the 5'
SmaI site to a SalI site, with a second
SalI site deriving from the pUC19 polylinker. The
~2.4-kilobase pair SalI fragment was excised and inserted
into the SalI site of pBR322 (28), to exclude extraneous
polylinker sites and thereby facilitate swapping of internal APP
restriction fragments containing either Swedish (KM670/671NL) or
Swedish plus Indiana (V717F) mutations ("Quick-change", Stratagene
Inc.). Completed plasmids were sequenced in their entirety with a total
of 12 sequencing primers covering the APP coding region, to exclude the
possible presence of erroneous mutations either present in the starting
plasmids or introduced during in vitro manipulations.
SalI fragments of APP or XhoI fragments of PS1
(9) were then cloned into cos.Tet (29). NotI transgene fragments excised from this cosmid vector were purified and injected into oocytes of different genetic backgrounds as noted, and founder animals were identified by dot-blot hybridization analysis of genomic
DNA using a probe within the hamster PrP gene 3'-untranslated region as
described previously (9, 30). Double transgenic mice deriving from
crosses of transgene heterozygotes were identified by dot-blot
hybridization analysis using human APP or PS1 cDNA probe fragments.
Protein Analysis--
Western blots were performed by enhanced
chemiluminescence as described previously (9), except ECL-Plus
(Amersham Pharmacia Biotech) was used in conjunction with a "Storm"
imaging system (Molecular Dynamics) for quantitative analyses. For
ELISA analysis, C3H/B6 × FVB/N mice at 4, 6, 8, 10, and 26 weeks
of age were transcardially perfused with cold saline. The entire brain
was removed and snap-frozen until analysis. Cerebral A Survival Census--
All pups were weaned at an age between 21 and 24 days. The identification of pups genotypes was carried out
between 23 and 26 days of age. Therefore, the reliable estimation of Tg
mice survival is limited to their post-weaning age. Statistical
analysis of survival as a function of genetic background was carried
out on three cohorts of TgCRND8 mice as follows: (C57) × (C3H/C57), (FVB) × (C3H/C57), and (C3H/C57/129SvEv/Tac) × (129SvEv/Tac). For comparative purposes the survival of non-Tg
littermates was included, and the analysis was performed for the 1st
year of life. Since the mortality of non-Tg mice was minimal (1 non-Tg
mouse out of the total of 158 included in the analysis died at the age of 26 days), non-Tg mice were pooled across their genetic backgrounds for graphical presentation and statistical analyses. The probability of
survival was assessed by the Kaplan-Meier technique (32), computing the
probability of survival at every occurrence of death, thus making it
particularly suitable for small sample size cases with variable event
intervals. The comparisons of cumulative survival curves for each
genetic background of mice were performed using Tarone-Ware test, which
weighs early events less than log rank or Breslow tests.
Histology and Immunohistochemistry--
Mice were anesthetized
and perfused with saline in accordance with The Canadian Council for
Animal Care guidelines. Generally, brains were removed and bisected in
the mid-sagittal plane. One-half was snap-frozen, and the portion for
immunohistochemistry was fixed in 10% neutral buffered formalin for a
minimum of 48 h. These specimens were then batch-processed on an
automatic tissue processor overnight with vacuum on each station to aid
penetration. Paraffin sections were cut at 5 microns and affixed to
Fisher brand Superfrost/Plus slides to ensure adhesion. Sections were stained by Bielschowsky's silver impregnation, cresyl violet, thioflavine S, and Luxol Fast Blue combined with hematoxylin and eosin,
as noted in the figure legends. For general morphological characteristics, 12 TgCRND8 mice from the hybrid C3H/B6 background, age
43-440 days, were studied. Additionally, 5 TgCRND8 mice
(n = 2, 213 days; n = 3, 282 days) and
4 non-transgenic controls (n = 3, 213 days;
n = 1, 282 days) were sectioned coronally to investigate hippocampal morphology. The percent volume occupied by the
dorsal hippocampus within the surrounding brain regions was estimated
using the Cavaleri point counting method. Paraffin-embedded brains were
serially sectioned on a rotary microtome at a thickness of 10 µm (as
described above) and stained with either hematoxylin and eosin or
cresyl violet to delineate the hippocampal borders. The hippocampus was
defined to include all regions of the hippocampus proper, hilus, and
dentate gyrus. To represent the dorsal hippocampal region a total of 6 serial sections were collected for analysis (every 15th section,
starting with the first section in which the hippocampus was visible).
Brain sections were visualized using a video microscopy system (Zeiss
Axoplan, using a lens for × 4 magnification) and a superimposed
point grid (680 µm spacing). Points falling over the hemisphere and
those falling over the hippocampus were tallied. Volumes were then
estimated using the formula: V =
For immunohistochemistry, all sections were blocked in dilute (3%)
hydrogen peroxide and non-immune goat serum. Epitope retrieval, in the
form of a 5-min immersion in formic acid, was carried out prior to
demonstration of amyloid and synaptophysin immuoreactivity. In all
cases the primary antibody was left to react overnight at 4 °C. The
remaining steps using the Dako StreptABC complex-horseradish peroxidase-conjugated "Duet" anti-mouse/rabbit antibody kit were completed according to the protocols provided by the manufacturer. End
products were visualized with diaminobenzidine. Sections were lightly counterstained with hematoxylin and were resin-mounted. The
sources of the antibodies used were as follows: 369 from Sam Gandy; 4G8
versus A Behavioral Tests and Data Analysis--
Experimentally naive
TgCRND8 mice were tested at 11 weeks of age in two cohorts
(NTg = 10, Nnon-Tg = 7 in
total) in the reference memory version of Morris water maze test. The
water maze apparatus, mouse handling, and general testing procedure are
described elsewhere (1, 33). Prior to the spatial learning training,
all mice underwent non-spatial pre-training (NSP), to assess swimming
abilities and familiarize mice with the test (1). Two days following the NSP phase, all mice underwent a reference memory training with a
hidden platform placed in the center of one quadrant of the pool
(northeast) for 5 days, with 4 trials per day. After the last trial of
day 5, the platform was removed from the pool and each mouse received
one 60-s swim "probe trial." Escape latency (in seconds), length of
swim path (centimeter) and swim speed (cm/s), were recorded using an
on-line HVS image video tracking system (33). For the probe
trials, an annulus crossing index was calculated, which represents the
number of passes over the platform site minus the mean of passes over
alternative sites in other quadrants. The index expresses the spatial
place preference and controls for alternative search strategies without
place preferences, such as circular search (34, 35). Behavioral data
was analyzed using a mixed model of factorial analysis of variance.
Degrees of freedom were adjusted by Greenhouse-Geisser epsilon
correction for heterogeneity of variance.
Creation of TgCRND8 Mice Expressing Mutant APP
Previous experiments have indicated that overexpression of APP
above a threshold of ~4× endogenous is a prerequisite for deposition of amyloid plaques in the central nervous system (18, 22). To avoid the
toxic effects associated with these levels of APP overexpression (22,
23, 36, 37), we exploited (i) permissive strain backgrounds and (ii)
APP cassettes, including multiple mutations, to maximize production of
A Expression of APP and A APP-specific antibodies were used to establish transgene
expression from founder lines, with previously characterized
TgAPPwt6209 transgenic mice providing a point of reference (22). Use of the N-terminal antibody 22C11, which reacts with mouse and human APP,
demonstrated overexpression of the full-length mature form of APP of
~120 kDa and different lower molecular mass species of 100 kDa
(which are not resolved in this gel system), including immature APP,
and APP cleaved at the -peptide (A
) biogenesis. TgCRND8
mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter.
Thioflavine S-positive A
amyloid deposits are present at 3 months,
with dense-cored plaques and neuritic pathology evident from 5 months
of age. TgCRND8 mice exhibit 3,200-4,600 pmol of A
42 per g brain at
age 6 months, with an excess of A
42 over A
40. High level
production of the pathogenic A
42 form of A
peptide was associated
with an early impairment in TgCRND8 mice in acquisition and learning
reversal in the reference memory version of the Morris water maze,
present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against A
42 (Janus, C., Pearson,
J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D.,
Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser,
P. E., St. George-Hyslop, P., and Westaway, D. (2000)
Nature 408, 979-982). Amyloid deposition in TgCRND8 mice
was enhanced by the expression of presenilin 1 transgenes including
familial Alzheimer's disease mutations; for mice also expressing a
M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to
investigate aspects of Alzheimer's disease pathogenesis, prophylaxis,
and therapy within short time frames.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-peptide (A
),1
neuronal loss, and intracellular deposits denoted neurofibrillary tangles (NFTs), aggregations of hyper-phosphorylated forms of the
microtubule-associated protein tau (
). Genetic analyses of diverse
familial Alzheimer's disease (FAD) kindreds indicate biosynthesis of
the amyloid
-peptide (A
), generated by secretase-mediated endoproteolysis of the amyloid precursor protein (APP), is a common denominator in inherited forms of the disease. In the case of chromosome 21-linked FAD kindreds, mutations in APP are found in close
proximity to the endoprotease sites where A
is excised by the action
of
- and
-secretases (2-6). Mutations in presenilins 1 and 2 are
thought to enhance cleavage of APP at the
-secretase site (7-9).
Finally, the
4 allele of the ApoE gene, which is correlated with
increased susceptibility to late-onset AD (10), is found to enhance the
formation of mature plaques in certain APP transgenic mice (11). These
genetic data indicate elevated A
biogenesis or accumulation is
likely a crucial pathogenic event in all forms of AD (i.e.
both familial and sporadic AD). This conclusion finds a parallel in
studies indicating that A
is neurotoxic (12-14).
peptide. However, despite intense
effort, remarkably few models exist (reviewed in Ref. 15). Some models
fail to produce APP and/or its metabolites by physiologically
appropriate pathways, and in cases where this caveat does not apply,
the transgenic animals may display only facets of the AD phenotype
(16). With regard to neuropathology, the phenotypes created thus far
include amyloid deposits that closely resemble those seen in AD,
selective neuronal loss (in one instance), some hyperphosphorylation of
tau, but no deposition of NFTs (17-20). Neuropathological
abnormalities in singly transgenic mice may not appear until 6-9
months of age and may not be robust until animals are well in excess of
1 year of age (see "Discussion"). Other complications encountered
in these models include hippocampal atrophy (21), neonatal lethality
attributed to overexpression of APP (22, 23), and complex and variable
relationships between cognitive dysfunction and transgene expression
(18, 21, 23-25). Here we describe a new line of transgenic mice that
exhibits deposition of A
-amyloid and robust cognitive deficits by
the age of 3 months. These mice have a demonstrated utility for
assessing procedures that interfere with amyloidogenesis (1) and may
serve as a platform to create more sophisticated models of AD.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was
solubilized in a 5 M guanidine HCl, 50 mM
Tris-HCl, pH 8.0 buffer (31), agitated, aliquoted, and stored at
80 °C. Thawed aliquots were diluted 10-fold or more and assessed
for A
40 or A
42 using commercially available enzyme-linked
immunosorbent assays (ELISAs) specific for either A
40 or A
42 and
calibrated with synthetic A
peptides (BIOSOURCE International). The A
40 ELISA does not display any cross-reactivity with A
42 or A
43, and the A
42 ELISA does not react with either A
40 or A
43. Each brain was analyzed in duplicate or triplicate, with the average value reported for each brain.
points·area per point·section thickness·section spacing. Student t tests were performed to compare mean
volume data.
residues 17-24 from Richard Rubenstein; A
42, 3542 from Frédéric Checler; 6F/3D versus A
residues 8-17, synaptophysin, and anti-ubiquitin from Dako Inc.; 6E10
versus A
residues 1-16 from Senetek Inc.; glial
fibrillary acidic protein monoclonal from Roche Molecular Biochemicals;
NF200 from Novo Castra Laboratories; CD11b from Chemicon Labs; and
hyperphosphorylated tau, AT8, from Innogenetics, Gent, Belgium. The
6F/3D antibody was of particular use for quantitative studies using
image analysis software as it visualized structures (i.e.
dense-cored plaques) with sharp boundaries.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
for a given level of APP expression. Transgene constructs were
based upon a cDNA cassette encoding the major APP isoform in human
brain, APP695. This cassette was modified to include either one or two
FAD mutations: the "Swedish" mutation (K670N, M671L) and the
"Indiana" mutation (V717F), lying adjacent to the N- and C-terminal
boundaries of the APP A
domain, respectively. APPSwe and APPSwe+717
cDNAs were introduced into cos.Tet (29), a cosmid-based expression
vector derived from the Syrian hamster prion protein gene. This vector
directs position-independent transgene expression in central nervous
system neurons and, to a much lesser extent, astrocytes (38-41).
Microinjections into C3H/HeJ × C57BL/6J or (C3H/HeJ × C57BL/6J) × C57BL/6J oocytes (the strains are hereafter referred
to as C57 and C3H) yielded a number of putative founders but just two
stable transgenic lines designated Tg CRND6 and TgCRND8. These lines
harbor APPSwe and APPSwe+V717F transgenes, respectively.
Peptide in TgCRND8 Mice
- and
-secretase sites, APPS
and APPS
(Fig.
1A). Overexpression in the
TgCRND8 line relative to mouse APP holoprotein detected in non-Tg
controls was estimated by quantitative image analysis at ~5-fold.
Similar results for high molecular weight APP species were obtained
with antibody 369, which reacts with an epitope close to the C terminus of APP shared by mouse and human APP (Fig. 1A). Lower
molecular weight species deriving from APP processing were also
observed in brain extracts of TgCRND8 and TgCRND6 mice analyzed with
the human APP-specific monoclonal antibody 6E10 antiserum (epitope positioned N-terminal to the
-secretase cleavage site) and antibody 369. These polypeptides represent APP C-terminal stubs arising from
cleavage at the
- and
-secretase sites, with antibody 369 recognizing both species and antibody 6E10 recognizing only the longer
-stubs. As anticipated, the APP6209 Tg line encoding wt human
APP does not exhibit
-stubs (as it lacks the Swedish mutation that
favors cleavage at this position) but exhibits
-stubs with a reduced
electrophoretic mobility due to the inclusion of a c-Myc epitope tag
within the C terminus of the APP cDNA cassette (22).
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Fig. 1.
Western blot analysis of transgene
expression. 10% brain homogenates made in 0.32 M
sucrose were diluted with Laemmli buffer, sonicated, and run on
10-20% Tricine gradient gels (NOVEX). Following transfer to
nitrocellulose, human APP and PS1 were detected using C- and
N-terminal-specific mAbs and developed by ECL (Amersham Pharmacia
Biotech). A, comparison of APP expression levels in Tg
lines. 1st lane, Non-Tg; 2nd
lane, Tg CRND6; 3rd lane, Tg CRND8;
and 4th lane, Tg APP6209. Proteins were detected
with human APP-specific C-terminal mAb 6E10 (left panel),
C-terminal antibody 369 reactive against mouse and human APP
(right panel), and N-terminal antibody 22C11, also reactive
against mouse and human APP (lower panel). Positions of high
molecular weight APP holoprotein and holoprotein derivatives ("APP
holoprotein"), and C-terminal stubs are indicated. B, time
course of A accumulation in Tg CRND8 mice: 1st
lane, 60 days; 2nd lane, 120 days;
3rd lane, 180 days; 4th
lane, 240 days; and 5th lane, 300 days. Detected with human-specific APP C-terminal mAb 6E10.
C, comparison of protein expression levels in Tg mice
expressing single and double mutants of PS1. Normalized samples of
brain homogenates are presented. Detection is with the human-specific
PS1 N-terminal mAb NT-1 (72). Lane 1, Non-Tg; lane
2, TgPS1(L286V)1274; lane 3, TgPS1(WT)1098; and
lanes 4 and 5 represent samples from
TgPS1(L286V+M146L)6500 mice.
In TgCRND8 mice, increasing levels of a 4-kDa species (but not
-stubs) were detected by Western blot analysis as the animals aged
(Fig. 1B). To investigate the composition of these 4-kDa A
peptide species, we performed ELISAs specific for A
40 and A
42. Both human A
40 and A
42 were detected in the brains of Tg
CRND8 mice. No signals above background were detected in non-Tg animals. Levels of both peptides increased with age, although in
different fashions (Table I). Thus A
40
levels were stable between 4 and 10 weeks of age. A
42 increased
slowly between 4 and 8 weeks, with a potent increase at age 10 weeks,
such that it predominated over A
40 by a ratio of ~5:1. There was
considerable spread in A
40 and A
42 levels in 10-week-old mice,
with levels of A
40 varying from 25 to 234 ng/g of brain and A
42
ranging from 115 to 728 ng/g of brain. The increase in A
42 and the
sample-to-sample variation between mice at age 10 weeks likely
represents a transition point as A
42, first present in soluble form,
begins to assemble into insoluble amyloid deposits. Measured at 6 months of age, levels of both A
42 and A
40 were enormously
increased and were ~510 and 190 times, respectively, the levels
observed in 4-week-old mice (which are free of amyloid plaque deposits;
Fig. 5A).
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Postnatal Lethality in TgCRND8 Mice
To gain insight into the ability of genetic backgrounds to
modulate lethality associated with APP overexpression (and, from a
practical point of view, to preempt premature extinction), the newly
established TgCRND8 line was bred to different strain backgrounds. Progeny of an F1 cross to the C3H/HeJ ("C3H") strain were bred to
mice derived from FVB/N and 129SvEv/Tac backgrounds. Estimated Kaplan-Meier cumulative survival curves for TgCRND8 mice and their littermates during post-natal development are presented in Fig. 2. Inspection of the curves clearly
indicates improved survival of mice with the APP transgene expressed on
the (C57) × (C3H/C57) genetic background. In this cohort of Tg
mice (n = 52), 20 mice died before the age of 120 days,
decreasing their survival to 60% and with three deaths at 130 days and
three further deaths after 250 days. When the APP transgene was
expressed on either (C3H/C57/129SvEv/Tac) × (129SvEv/Tac) or
(FVB) × (C3H/C57) genetic backgrounds (n = 12 and
n = 41 respectively), survival dropped rapidly to
25-40% within the first 120 days of their post-natal life (Fig. 2).
After this time point, survival with the (C3H/C57/129SvEv/Tac) × (129SvEv/Tac) genetic background dropped slightly to 33% (one death at
159 days) with only 25% (3 mice) of the cohort surviving until 365 days. Similarly, the survival of the TgCRND8 mice with (FVB)×(C3H/C57)
background dropped rapidly within the first 120 days of post-natal age
(Fig. 2) with 17% of mice (n = 7) surviving until 365 days of age. The survival of mice with the (C57) × (C3H/C57) was
significantly better than survival of Tg mice with (FVB) × (C3H/C57) or (C3H/C57/129SvEv/Tac) × (129SvEv/Tac) backgrounds (Tarone-Ware statistics: 5.13, p < 0.05, and 19.01, p < 0.001, respectively). The survival curves of the
latter two genetic backgrounds did not differ significantly from each
other (Tarone-Ware statistics: 0.42, p > 0.05), and
the survival of TgCRND8 mice with the three genetic backgrounds was
significantly different from the survival of non-Tg littermates
(Tarone-Ware statistics >50, all p values < 0.001.
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Although these data suggest significantly increased mortality of TgCRND8 mice as a consequence of a genetic contribution of 129SvEv/Tac or FVB mouse strains, some caveats have to be taken into consideration. First, the relatively small sample sizes of the studied cohorts, especially with the 129SvEv/Tac strain, where only a few mice survived for a long period, may not reliably reflect survival rates at later stages of life. The comparisons of larger cohorts should provide better estimation of survival curves. Second, future survival censuses must be extended to the pre-weaning developmental stage. The selective survival of pups before weaning, or for that matter at the pre-natal stage of development, may cause a bias of a particular cohort entering post-weaning stage. Also, the genetic composition of the particular outbred TgCRND8 parent might be a greater contributor to the differences in survival among the crosses than the composition of the inbred parent. Nonetheless, the major finding of the survival analysis, that the (C57) × (C3H/C57) genetic background significantly reduces mortality in TgCRND8 mice, is in accord with our starting hypothesis. Fifty percent of the studied cohort of 52 mice survived for a year with minimal mortality observed after the first 3 months of age. As shown in previous investigations, the cause of post-natal lethality was not obvious (22). No overt changes were revealed by routine histopathology, although it should be noted that seizures were observed in a small fraction of TgCRND8 animals, and APP transgenes have been correlated with altered vascular responses (37).
Cognitive Changes in TgCRND8 Mice
Non-spatial Pre-training-- Partial results related to the impaired acquisition of spatial information, as measured by the escape latency, were reported previously for a small cohort of mice (1). Here we present a characterization of a larger cohort of mice, and we include their behavioral analysis during NSP. The analysis showed that during NSP at age 10.5 weeks TgCRND8 mice performed comparably to non-Tg littermates when randomly searching the pool for a hidden platform. Escape latencies and lengths of search paths in the last trial of NSP for the groups were not significantly different (50.0 ± 7.3 versus 57.6 ± 8.8 s for latency and 961.1 ± 165.0 versus 1351 ± 247.4 cm for path-length, for the non-Tg and Tg mice, respectively). A "visible platform trial" administered during NSP, where the position of the submerged platform was marked by a striped beacon, also failed to reveal differences in performance between non-Tg and Tg groups. Average latencies to reach the cued platform were 9.9 ± 2.0 and 9.1 ± 1.6 s for non-Tg and Tg mice, respectively. The swim paths were 167.3 ± 16.1 cm for non-Tg and 153.1 ± 14.2 cm for Tg mice, and both groups had comparable swim speeds of 21.6 ± 1.4 and 20.2 ± 1.7 cm/s for non-Tg and Tg mice, respectively. In conclusion, these analyses showed TgCRND8 mice performed a random search comparable to non-Tg littermates when presented with the submerged platform and had similar swim paths to a visible platform when extra-maze distal spatial cues were occluded by a curtain.
Water Maze, Reference Memory Test--
TgCRND8 mice at 11 weeks
showed impairment in the acquisition of spatial information during
place discrimination training. They had significantly longer escape
latencies to reach the escape platform (Fig.
3A; F(1,15) = 17.98, p < 0.001) and longer search paths (F(1,15) = 15.91, p < 0.001). Both, Tg and non-Tg groups significantly improved during training (F(4,60) = 3.29, p < 0.02; F(4,60) = 3.33, p < 0.02, the latency and path, respectively), and no significant
interaction between the groups and sessions was found in both measures.
The concordance between measures of latency and search path is not
surprising, because the TgCRND8 mice did not differ significantly from
the non-Tg littermates in their swim speed during the test
(F(1,15) = 2.53, p > 0.05). The pronounced
spatial learning impairment of Tg mice was confirmed during the probe
trial administered after the completion of training. They showed lower
(t(15) = 2.99, p = 0.01) annulus
crossing index (Fig. 3B), searching the pool in a circular
fashion and frequently crossing the centers of alternative quadrants
(with an annulus crossing index approaching a zero value).
|
Neuropathology in TgCRND8 Mice
TgCRND6 mice expressing the Swedish mutant form of APP (see Fig. 1) on a B6 × C3H background failed to exhibit cerebral amyloid at ages up to 450 days. Similar results were obtained for two other Tg lines resulting from the microinjection of the same DNA construct into a FVB/N × 129SvEv/Tac background (not shown). On the other hand, hemizygous TgCRND8 mice expressing the double mutant form of APP exhibited potent deposition of cerebral amyloid, present in all animals by 90 days of age. In contrast to PDAPP mice expressing a V717F APP transgene (21), no difference in the volume of the dorsal hippocampus (or in the surrounding regions) was detected between Tg and non-Tg mice (p < 0.05 for all values). In TgCRND8 mice, the dorsal hippocampus occupied ~11.0 ± 0.3% of the volume of coronal sections (means ± S.E. of the mean), whereas in the non-Tg mice this value was 10.3 ± 0.2%. Also, the neuronal cytoarchitecture of the TgCRND8 mice appeared normal.
Amyloid deposits in TgCRND8 mice were successfully stained with
A42-specific antibodies, as anticipated from prior studies of the
mechanism of action of the V717F mutation (5). In addition to
confirming dense-cored deposits in aged TgCRND8 mice, the human APP-specific monoclonal antibody 4G8, and to a lesser extent monoclonal antibody 6F/3D, also detected diffuse immuno-staining in the neuropil (using formic acid-treated sections: Fig.
4B). These findings were
compatible with both the previously described specificity of the 4G8
antibody (42, 43), and the propensity of other mice expressing APP
codon 717 mutations to generate "diffuse" amyloid deposits (19, 21,
23). Although human APP was also expressed systemically in TgCRND8
mice, in accord with the tropism of the PrP gene promoter (44), amyloid
deposits were not apparent by immunostaining in the kidney, lung,
skeletal, and cardiac muscle of aged animals (not shown). These
observations suggest a role for tissue-specific factors affecting
amyloid deposition and/or APP processing.
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Plaque Ontogeny-- A single plaque was noted in one TgCRND8 animal at 43 days. Plaque load increased with age and spread to more regions of the brain such that multiple plaque deposits were present per sagittal section in most mice at 65 days of age, and in all mice by 90 days of age. Although studies on large cohorts of mice are in progress, analyses of representative mice revealed between 11-35 and 22-43 plaques in a mid-sagittal section of the cortex at the ages of 90 and 150 days, whereas burdens were greatly increased at 240 and 350 days of age (127-416 and 528-1024 plaques, respectively). A similar pattern of increase pertained to the hippocampal formation with 1-5 and 11-12 plaques at 90 and 150 days and 39-65 and 123-147 plaques at 240 and 360 days, respectively. Thus, the time period for a rapid increase in amyloid burden assessed histologically lags slightly behind that determined biochemically by ELISA assays (Table I).
Plaque Distribution-- The plaque present at 43 days was located in the subiculum. The frontal cortex of TgCRND8 mice often had several plaques by 65 days, whereas plaques were rare or absent in the white matter tracts (corpus callosum and alveus) and CA1 region of the hippocampus at this time. By 101 days plaques were widespread in many regions of the cortex as well as in the hippocampus proper, the dentate gyrus, the olfactory bulb, and within the pial vessels. The thalamus (111 days), then the cerebral vasculature and striatum (196 days), followed by the cerebellum and brain stem (243 days) all became progressively burdened by plaques. This pattern of deposition, with cortex and hippocampus affected early on and the cerebellum spared until a late stage in disease, is similar to that seen in AD (45).
Plaque Morphology-- The first plaques observed were small, cored deposits, with no radiating amyloid surrounding them (65 days). By 101 days, the plaques varied in size, with some larger plaques having haloes of radiating amyloid. By 131 days the plaques became more heterogeneous in nature. Plaques varied greatly in size as the age of the mice increased, with some being multicored in older animals. Diffuse amyloid deposits appeared in the olfactory bulb at an early stage (101 days). However, outside of the olfactory bulb, diffuse amyloid (i.e. amyloid not obviously associated with a cored plaque) did not appear until 243 days, as detected with the 6F/3D antibody. Diffuse amyloid was found primarily in the caudate, cerebellum, and molecular layer of the dentate gyrus (all at 243 days). By 315 days diffuse amyloid appeared throughout the cortex (see Fig. 4B).
The majority of amyloid plaque deposits in TgCRND8 mice, including
those first to appear at 65 days, stained positive for thioflavine S. These early deposits also revealed Congo Red birefringence. Together
these data indicate the deposited amyloid peptide adopts a -sheet
conformation. Amyloid plaques in TgCRND8 mice were associated with
dystrophic neurites, as indicated by a variety of histochemical and
immunohistochemical stains (Fig. 4). For example, Bielschowsky silver
impregnation revealed dystrophic neuritic processes around plaque cores
(Fig. 4E). Similar structures were imaged by antibodies raised against the 200-kDa isoform of neurofilament (NF-200; Fig. 4F), synaptophysin (Fig. 4G), and ubiquitin (Fig.
4H). The first enlarged plaque-associated neurites were seen
with Bielschowsky, synaptophysin, and NF-200 staining at 111 days of
age. Dystrophic pathology became more evident as the mice aged further
and the frequency of large, dense-cored plaques increased. Finally, it was noteworthy that dystrophic neurites were only observed in the
immediate vicinity of plaques, indicating these structures are a direct
consequence of amyloid deposition in the TgCRND8 mice.
Neuroinflammation-- Mature plaques in TgCRND8 mice were associated with a focal inflammatory response. Elongated cells in periphery of dense-cored amyloid deposits visualized by Luxol fast blue staining or focal staining with a Griffonia simplicifolia lectin I isolectin B4 stain (not presented) and staining with anti-CD11b antibody probe (Fig. 4I) were consistent with the presence of microglial cells. This microglial activation was accompanied by intense local astrocytic gliosis, illustrated by GFAP staining of adjacent sections encompassing a thioflavin-positive plaque (Fig. 4, K and J). This astrocytic response clearly exceeded a low basal level of staining of GFAP-positive astrocytes (mostly evident within white matter tracts) noted in both transgenic and non-transgenic mice.
Acceleration of Amyloid Deposition by Co-expression of Presenilin-1 Transgenes
Amyloid deposition in TgCRND8 mice was enhanced by mutant human
PS1 transgenes co-expressed with human APP via usage of the same
cos.Tet transgene vector (Fig. 5,
right-hand panels). This effect was evident in terms of a
potent increment in plaque burden over age-matched TgCRND8 single
transgenic controls. Conversely, expression of wt human PS1 had no
overt effect upon amyloid burden (not shown). With a PS1 transgene
incorporating two FAD mutations in cis (M146L and L286V
(46)), the potentiation was particularly remarkable. Here there was
robust deposition of plaques by 30-45 days of age (33 days of age
presented in Fig. 5B). Notably, the graded effects observed
for wild type, single, and double mutant transgenes upon amyloid
deposition cannot be attributed to different PS1 expression levels, as
these were very closely matched between the three selected TgPS1 lines
(wild type, mutant, and double mutant, Fig. 1C).
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DISCUSSION |
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Factors Affecting APP Transgenesis--
Although transgenesis is
generally regarded as a routine technique, the APP gene, first cloned
over a decade ago, can be seen to present particular challenges.
Difficulties encountered thus far include low expression levels in
first generation Tg mice, neonatal lethality associated with high level
expression in second generation mice, and physiological endoproteolysis
to generate multiple subfragments with diverse biological activities,
some of which (e.g. neuroprotection (reviewed in Ref. 47)
may confound the study of neuropathogenesis. We modified a number of
parameters in our experimental design in an effort to reduce the
"noise" from these confounding effects and thereby facilitate study
of the pathogenic attributes of the A peptide; such pathogenic
attributes are clearly suggested by genetic, neuropathological, and
toxicological studies (12-14, 48). Our strategy involved using the
following: (i) an expression cassette that retained ~90 nucleotides
of the APP mRNA 5'-untranslated region adjacent to the start codon,
as APP is expressed at high levels for a single copy gene and likely already contains optimized translational initiation signals; (ii) a
prion protein cosmid vector that can drive high level pan-neuronal expression in the central nervous system; (iii) two pro-endoproteolytic FAD mutations, to obtain a high level of A
peptide for a given level
of APP expression; and (iv) use of a genetic background that may offer
a degree of protection against high level APP expression favored by
parameters i and ii.
It is possible that genetic background may have been important for
establishing the TgCRND8 line. Our strategy was based upon the
hypothesis that dominant alleles protective against APP overexpression present in the C3H background (36) would facilitate the establishment of Tg lines with high levels of expression. As injections into C3H
oocytes are not usually attempted, the TgCRND8 line was established in
an outbred C3H/B6 background. Survival curves presented here document
that in TgCRND8 mice high levels of A peptide and modest levels of
APP
- and
-stubs can be tolerated in a C3H/B6 genetic background
without grossly compromising viability (Fig. 2). Whereas these data are
in accord with the starting hypothesis, they are incompatible with
facile genetic analysis, as confounding effects due to independent
segregation of alleles from the C3H and B6 strains conferring
protection or sensitivity to APP overexpression cannot be excluded.
Experiments to establish C3H congenic lines of TgCRND8 mice will lead
to more definitive information on this point. Furthermore, it is of
interest to note that the KM670/NL671+V717F construct injected into
oocytes from another potentially protective genetic background
(129SvEvTac × FVB/N (36)) yielded a transgenic line designated
Tg19959, which also exhibits cerebral plaques at 3 months of age (data
not shown). In sum, our results suggest manipulation of genetic
background may reduce postnatal death associated with APP
overexpression and should be considered in strategies for APP
transgenesis. However, insofar as parameters i-iv have been used
individually in the creation of APP Tg mice (8, 17-19, 22, 23), it is
plausible that a synergism between all four parameters contributes to
the desirable properties of TgCRND8 mice.
Biochemistry of TgCRND8 Mice--
With the possible exception of
the Tg19959 line, TgCRND8 mice are currently unique among single
transgenic APP mice with regard to the severity of A deposition.
Histological deposition of A
in amyloid plaques in TgCRND8 mice is
evident in 100% of animals by 3 months after birth (n = 28), earlier than in APP transgenic lines described previously
(17-19, 49). These results find a parallel in measurements of A
40
and A
42 derived from ELISA assays (Tables I and
II); for example, levels of A
42 in
6-month-old TgCRND8 mice approximate those seen in PDAPP mice at 16 months of age (31). Furthermore, two results emerging from these ELISAs show a direct parallel to AD pathogenesis and bear particular emphasis.
First, the levels of total A
in TgCRND8 mice at 6 months of age,
3,200-4,600 pmol/g brain (as determined by two independent ELISA
configurations employing different antibodies), fall into the range
observed for sporadic AD cases, 500-5000 pmol/g wet tissue (50).
Second, the ratio of A
42 to A
40 exceeds unity, as is also the
case in sporadic AD (50) (and in PDAPP mice expressing a V717F mutation
(31)). Insofar as A
42 is thought to be the most aggregation-prone
and toxic form of A
, the skew toward the production of A
42
apparent in these mice presumably contributes to the unusually early
onset of amyloid deposition.
|
Although the combined effect of the two FAD mutations affecting both
- and
-secretase processing of APP is presumed to be a powerful
determinant of this potent amyloidogenesis, it is notable that other
"double mutant" TgAPP mice only show 100% of animals positive for
plaques at 8-10, 18, or 21-25 months of age (19, 49, 51). Although
the degree of APP overexpression could also prove crucial in
distinguishing TgCRND8 mice from other double mutant TgAPP lines,
further variables include PrP, thy-1, or platelet-derived growth factor-
promoters with different tropisms and different APP
coding region cassettes (APP695, APP751, or intron-containing cassettes
capable of producing all three APP isoforms).
Finally, it is remarkable that even though TgCRND8 mice exhibit a high
basal synthesis of A, levels of this peptide can be elevated to yet
higher levels by mutant versions of PS1. Importantly, two PS1 mutations
in cis shown to act in an additive way in transfected cells
(46) behave in a similar fashion in vivo. Thus, double transgenic mice incorporating 4 FAD mutations can develop A
deposits by 1 month of age (Fig. 5B).
Neuropathology in Transgenic Models of Alzheimer's
Disease--
TgCRND8 mice exhibit AD-like amyloid plaque deposits with
a variety of morphologies. Dense-cored deposits are present from an
early stage, and the majority of these (>80%) can be stained with
either Congo Red or thioflavine S. With aging, the plaques become
larger and multicentric dense-cored deposits appear. Diffuse A
immunostaining is also apparent at later stages, being particularly prominent after formic acid pretreatment. Diffuse staining is evident
as a penumbra around large plaques and also in the form of isolated
deposits (i.e. not obviously associated with plaques) throughout the neuropil. From 5 months of age the mature plaques in
TgCRND8 mice exhibit neuritic changes strikingly similar to those seen
in AD (Fig. 4). Dystrophic neurites are revealed by silver impregnation
or NF-200 immunostaining, with dystrophic boutons visualized by
synaptophysin or ubiquitin antibodies. Astrocytes and microglial cells
often encircle dense-cored deposits, indicating the plaques are capable
of initiating an inflammatory response. The other pathological
hallmarks of AD are generally accepted to include neuronal loss and the
accumulation of NFTs. Focal neuronal loss has been reported in only one
Tg model of AD (20, 52, 53), and analogous studies in TgCRND8 mice are
underway. NFTs are absent in TgCRND8 mice, as indeed they are in other
TgAPP mice. It is possible coding sequence divergence between mouse and
human may be of importance, and expression of human tau and cognate tau
kinases, perhaps the p25 fragment of p35 (54, 55) or glycogen synthese
kinase 3
, may be required to fully recapitulate this pathology.
The Origins of Cognitive Dysfunction in Alzheimer's Disease-- In Alzheimer's disease patients, pathological changes detected post-mortem are foreshadowed in the clinical presentation by erosion of mental function leading to frank dementia. Therefore, plausible animal models of AD should develop cognitive deficits at least by the time of appearance of AD-related neuropathology. TgCRND8 mice fulfill this expectation. They represent an example of mice expressing full-length APP where deficits in acquisition of spatial reference memory are present at the onset of AD-related neuropathology (Fig. 3) (1) and thus exhibit some similarities to Tg2576 mice (18, 24). In studies of other TgAPP mice, impaired performance in the water maze test preceded neuropathological changes (23) or was not reported (19). Impaired performance in other testing paradigms has been reported in PDAPP V717F mice, but here a subset of cognitive deficits was better correlated to confounding neuroanatomical abnormalities than to AD-related pathologies (21, 25). In the most recent studies, an age-related deficit in learning capacity was detected in PDAPP mice using a new water maze testing regimen (56). Nonetheless, from a practical point of view, the cognitive deficits in TgCRND8 mice have three important attributes as follows: they occur early in life; they are easily detected in the conventional spatial reference memory version of the water maze; and they are sufficiently robust to be detected without recourse to large sample sizes.
Although overaccumulation of the A peptide is firmly implicated in
AD pathogenesis, the mechanisms leading to cognitive decline are not
clear. As attempts to correlate plaque burdens and cognitive deterioration have produced mixed results (see Refs. 57-65 and also
see Ref. 56), it is possible A
affects the central nervous system by
mechanisms other than (or in addition to) the toxicity of
extracellular, aggregated forms. Indeed, recent studies emphasize soluble forms of A
as crucial determinants of clinical outcome (50,
66, 67). Unfortunately, the molecular determinants for neurotoxicity
(for example, free radical generating capacity of metal-bound A
42
(68, 69) and perturbed signal transduction (70)) and the cellular
consequences thereof (altered synaptic function, excitotoxicity, and
induction of apoptosis (49, 66)) are undetermined. We suggest TgCRND8
mice comprise a useful and validated system to address these issues.
Amelioration of cognitive deficits by immunization against A
42
peptide (1) provides compelling evidence for a strong pathogenic role
for A
peptide in the TgCRND8 model of AD and indeed for the amyloid
cascade hypothesis. Parenthetically, these data effectively exclude the proposition that cognitive deficits in TgCRND8 mice derive from an
insertional mutation. Furthermore, synthesis of antisera in A
42-immunized mice that react strongly with the peptide presented in
a
-sheet conformation (1) suggests a potential to dissect the
mechanism of A
neuropathogenesis (for example, by passive immunization (71) with conformation-specific antibodies). In short, the
availability of a new AD model with robust cognitive deficits, levels
of A
peptide that equal those seen in AD cases, and AD-like
pathology may allow both an improved understanding of causal
relationships between these phenotypic traits and testing of candidate interventions.
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ACKNOWLEDGEMENTS |
---|
We thank Richard Rubenstein for generously supplying the 4G8 antibody, Frédéric Checler for the 3542 antibody, Sam Gandy for the 369 antibody, Julie Panakos for DNA microinjections, and Isabelle Aubert for discussions.
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FOOTNOTES |
---|
* This work was supported in part by the Canadian Institutes for Health Research, the Ontario Mental Health Foundation, The Howard Hughes Medical Institute, Natural Sciences and Engineering Research Council of Canada, The Scottish Rite Foundation, the Alzheimer Society of Ontario, the National Institute on Aging, and the Fraternal Order of Eagles.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.
b Supported by Canadian Institutes for Health Research post-doctoral fellowship.
f Supported by Natural Sciences and Engineering Research Council of Canada post-doctoral fellowships.
k To whom correspondence should be addressed: University of Toronto, Center for Research in Neurodegenerative Diseases, Tanz Neuroscience Bldg., 6 Queen's Park Crescent West, Toronto, Ontario M5S 3H2, Canada. Tel.: 416-978-1556; Fax: 416-978-1878; E-mail: david.westaway@utoronto.ca.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc. M100710200
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ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid
-peptide;
FAD, familial Alzheimer's disease;
AD, Alzheimer's
disease;
ELISA, enzyme-linked immunosorbent assay;
APP, amyloid
precursor protein;
NFTs, neurofibrillary tangles;
Tg, transgenic;
PS1, presenilin 1;
mAb, monoclonal antibody;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
wt, wild
type;
NSP, non-spatial pre-training;
PDAPP, APP transgenic mice
constructed using the platelet derived growth factor beta
promoter.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergereon, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982[CrossRef][Medline] [Order article via Infotrieve] |
2. | Citron, M., Oltersdorf, T., Haass, C., McConlogue, C., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Roher, A. E.,
Lowenson, J. D.,
Clarke, S.,
Wolkow, C.,
Wang, R.,
Cotter, R. J.,
Reardon, I. L.,
Zurcher-Neely, H. A.,
Heinrikson, R. L.,
Ball, M. J.,
and Greenberg, B. D.
(1993)
J. Biol. Chem.
268,
3072-3083 |
4. | Johnston, J. A., Cowburn, R. F., Norgren, S., Wiehager, B., Venizelos, N., Winblad, B., Vigo-Pelfrey, C., Schenk, D., Lannfelt, L., and O'Neill, C. (1994) FEBS Lett. 354, 274-278[CrossRef][Medline] [Order article via Infotrieve] |
5. | Suzuki, N., Cheung, T. T., Cai, X.-D., Odaka, A., Otvos, L., Eckman, C., Golde, T., and Younkin, S. G. (1994) Science 264, 1336-1340[Medline] [Order article via Infotrieve] |
6. |
Thinakaran, G.,
Teplow, D. B.,
Siman, R.,
Greenberg, B.,
and Sisodia, S. S.
(1996)
J. Biol. Chem.
271,
9390-9397 |
7. | Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C.-M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D., Morgan, D., Gordon, M., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., and Younkin, S. (1996) Nature 383, 710-713[CrossRef][Medline] [Order article via Infotrieve] |
8. | Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., Ratovitsky, T., Prada, C.-M., Kim, G., Seekins, S., Yager, D., Slunt, H. H., Wang, R., Seeger, M., Levey, A. I., Gandy, S. E., Copeland, N. G., Jenkins, N. A., Price, D. L., Younkin, S. G., and Sisodia, S. S. (1996) Neuron 17, 1005-1013[Medline] [Order article via Infotrieve] |
9. | Citron, M., Westaway, D., Xia, W., Carlson, G. A., Diehl, T., Levesque, G., Johnson-Wood, K., Lee, M., Seubert, P., Davis, A., Kholodenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R., Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St. George-Hyslop, and Selkoe, D. (1997) Nat. Med. 3, 67-72[Medline] [Order article via Infotrieve] |
10. | Corder, E. H., Saunders, A. M., Srittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Haines, J. L., and Pericak-Vance, M. A. (1993) Science 261, 921-923[Medline] [Order article via Infotrieve] |
11. |
Holtzman, D. M.,
Bales, K. R.,
Tenkova, T.,
Fagan, A. M.,
Parsadanian, M.,
Sartorius, L. J.,
Mackey, B.,
Olney, J.,
McKeel, D.,
Wozniak, D.,
and Paul, S. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2892-2897 |
12. | Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990) Science 250, 279-282[Medline] [Order article via Infotrieve] |
13. |
Lorenzo, A.,
and Yankner, B. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12243-12247 |
14. | Geula, C., Wu, C. K., Saroff, D., Lorenzo, A., Yuan, M., and Yankner, B. A. (1998) Nat. Med. 4, 827-831[Medline] [Order article via Infotrieve] |
15. | Hsiao, K. (1998) Exp. Gerontol. 33, 883-889[CrossRef][Medline] [Order article via Infotrieve] |
16. | Janus, C., Chishti, M. A., and Westaway, D. (2000) Biochim. Biophys. Acta 1502, 63-75[Medline] [Order article via Infotrieve] |
17. | Games, D., et al.. (1995) Nature 373, 523-527[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Hsiao, K. H.,
Chapman, P.,
Nilsen, S.,
Eckman, C.,
Harigawa, Y.,
Younkin, S.,
Yang, F. S.,
and Cole, G.
(1996)
Science
274,
99-102 |
19. |
Sturchler-Pierrat, C.,
Abramowski, D.,
Duke, M.,
Wiederhold, K. H.,
Mistl, C.,
Rothacher, S.,
Ledermann, B.,
Burki, K.,
Frey, P.,
Paganetti, P. A.,
Waridel, C.,
Calhoun, M. E.,
Jucker, M.,
Probst, A.,
Staufenbiel, M.,
and Sommer, B.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13287-13292 |
20. | Calhoun, M. E., Wederhold, K.-H., Abramowski, D., Phinney, A. L., Probst, A., Sturchler-Pierrat, C., Staufenbiel, M., Sommer, B., and Jucker, M. (1998) Nature 395, 755-756[CrossRef][Medline] [Order article via Infotrieve] |
21. | Dodart, J. C., Mathis, C., Saura, J., Bales, K. R., Paul, S. M., and Ungerer, A. (2000) Neurobiol. Dis. 7, 71-85[CrossRef][Medline] [Order article via Infotrieve] |
22. | Hsiao, K. K., Borchelt, D. R., Olson, K., Johannsdottir, R., Kitt, C., Yunis, W., Xu, S., Eckman, C., Younkin, S., Price, D., Iadecola, C., Clark, H. B., and Carlson, G. A. (1995) Neuron 15, 1203-1218[Medline] [Order article via Infotrieve] |
23. |
Moechars, D.,
Dewachter, I.,
Lorent, K.,
Reverse, D.,
Baekelandt, V.,
Naidu, A.,
Tesseur, I.,
Spittaels, K.,
Haute, C. V.,
Checler, F.,
Godaux, E.,
Cordell, B.,
and Van Leuven, F.
(1999)
J. Biol. Chem.
274,
6483-6492 |
24. | Chapman, P. F., White, G. L., Jones, M. W., Cooper-Blacketer, D., Marshall, V. J., Irizarry, M., Younkin, L., Good, M. A., Bliss, T. V., Hyman, B. T., Younkin, S. G., and Hsiao, K. K. (1999) Nat. Neurosci. 2, 271-276[CrossRef][Medline] [Order article via Infotrieve] |
25. | Dodart, J. C., Meziane, H., Mathis, C., Bales, K. R., Paul, S. M., and Ungerer, A. (1999) Behav. Neurosci. 113, 982-990[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987) Nature 325, 733-736[CrossRef][Medline] [Order article via Infotrieve] |
27. | Vieira, J., and Messing, J. (1982) Gene (Amst.) 19, 259-268[CrossRef][Medline] [Order article via Infotrieve] |
28. | Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., and Boyer, H. W. (1977) Gene (Amst.) 2, 95-113[Medline] [Order article via Infotrieve] |
29. |
Scott, M. R.,
Köhler, R.,
Foster, D.,
and Prusiner, S. B.
(1992)
Protein Sci.
1,
986-997 |
30. | Scott, M., Foster, D., Mirenda, C., Serban, D., Coufal, F., Wälchli, M., Torchia, M., Groth, D., Carlson, G., DeArmond, S. J., Westaway, D., and Prusiner, S. B. (1989) Cell 59, 847-857[Medline] [Order article via Infotrieve] |
31. |
Johnson-Wood, K.,
Lee, M.,
Motter, R.,
Hu, K.,
Gordon, G.,
Barbour, R.,
Khan, K.,
Gordon, M.,
Tan, H.,
Games, D.,
Lieberburg, I.,
Schenk, D.,
Seubert, P.,
and McConlogue, L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1550-1555 |
32. | Haccou, P., and Meelis, E. (1995) Statistical Analysis of Behavioural Data , pp. 120-186, Oxford University Press, Oxford |
33. | Janus, C., D'Amelio, S., Amitay, O., Chishti, M. A., Strome, R., Fraser, P. E., Carlson, G. A., Roder, J., St. George-Hyslop, P., and Westaway, D. (2000) Neurobiol. Dis. 21, 541-549 |
34. |
Gass, P.,
Wolfer, D. P.,
Balschun, D.,
Rudolph, D.,
Frey, U.,
Lipp, H. P.,
and Schutz, G.
(1998)
Learn. Mem.
5,
274-88 |
35. | Wehner, J. M., Sleight, S., and Upchurch, M. (1990) Brain Res. 523, 181-187[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Carlson, G. A.,
Borchelt, D. R.,
Dake, A.,
Turner, S.,
Danielson, V.,
Coffin, J. D.,
Eckman, C.,
Meiners, J.,
Nilsen, S. P.,
Younkin, S. G.,
and Hsiao, K. K.
(1997)
Hum. Mol. Genet.
6,
1951-1959 |
37. | Iadecola, C., Zhang, F., Niwa, K., Eckman, C., Turner, S. K., Fischer, E., Younkin, S., Borchelt, D. R., Hsiao, K. K., and Carlson, G. A. (1999) Nat. Neurosci. 2, 157-161[CrossRef][Medline] [Order article via Infotrieve] |
38. | Telling, G. C., Scott, M., Mastrianni, J., Gabizon, R., Torchia, M., Cohen, F. E., DeArmond, S. J., and Prusiner, S. B. (1995) Cell 83, 79-90[Medline] [Order article via Infotrieve] |
39. | Moser, M., Colello, R. J., Pott, U., and Oesch, B. (1995) Neuron 14, 509-517[Medline] [Order article via Infotrieve] |
40. | DeArmond, S. J., Sanchez, H., Yehiely, F., Qiu, Y., Ninchak-Casey, A., Daggett, V., Camerino, A. P., Cayetano, J., Rogers, M., Groth, D., Torchia, M., Tremblay, P., Scott, M. R., Cohen, F. E., and Prusiner, S. B. (1997) Neuron 19, 1337-1348[Medline] [Order article via Infotrieve] |
41. | Irizarry, M. C., McNamara, M., Fedorchak, K., Hsiao, K., and Hyman, B. T. (1997) J. Neuropathol. Exp. Neurol. 56, 965-973[Medline] [Order article via Infotrieve] |
42. | Kim, K. W., Miller, D. L., Sapienza, V. J., Chen, C. M. J., Bai, C., Grundke-Iqbal, I., Currie, J. R., and Wisnieski, H. M. (1988) Neurosci. Res. Commun 2, 121-130 |
43. | Spillantini, M. G., Goedert, M., Jakes, R., and Klug, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3947-3951[Abstract] |
44. | Oesch, B., Westaway, D., Wälchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, L. E., Prusiner, S. B., and Weissmann, C. (1985) Cell 40, 735-746[Medline] [Order article via Infotrieve] |
45. | Braak, H., and Braak, E. (1991) Acta Neuropathol. 82, 239-259[Medline] [Order article via Infotrieve] |
46. | Citron, M., Eckman, C. B., Diehl, T. S., Corcoran, C., Ostaszewski, B. L., Xia, W., Levesque, G., St. George-Hyslop, P., Younkin, S. G., and Selkoe, D. J. (1998) Neurobiol. Dis. 5, 107-116[CrossRef][Medline] [Order article via Infotrieve] |
47. | Storey, E., and Cappai, R. (1999) Neuropathol. Appl. Neurobiol. 25, 81-97[CrossRef][Medline] [Order article via Infotrieve] |
48. | Sherrington, R., Rogaev, E., Liang, Y., Rogaeva, E., Levesque, G., Ikeda, M., Chi, H., Lin, C., Holman, K., Tsuda, T., Mar, L., Fraser, P., Rommens, J. M., and St. George-Hyslop, P. (1995) Nature 375, 754-760[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Hsia, A. Y.,
Masliah, E.,
McConlogue, L., Yu, G. Q.,
Tatsuno, G.,
Hu, K.,
Kholodenko, D.,
Malenka, R. C.,
Nicoll, R. A.,
and Mucke, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3228-3233 |
50. | Wang, J., Dickson, D. W., Trojanowski, J. Q., and Lee, V. M. (1999) Exp. Neurol. 158, 328-337[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Mucke, L.,
Masliah, E., Yu, G. Q.,
Mallory, M.,
Rockenstein, E. M.,
Tatsuno, G.,
Hu, K.,
Kholodenko, D.,
Johnson-Wood, K.,
and McConlogue, L.
(2000)
J. Neurosci.
20,
4050-4058 |
52. |
Masliah, E.,
Sisk, A.,
Mallory, M.,
Mucke, L.,
Schenk, D.,
and Games, D.
(1996)
J. Neurosci.
16,
5795-5811 |
53. |
Irizarry, M. C.,
McNamara, M.,
Page, K. J.,
Schenk, D.,
Games, D.,
and Hyman, B. T.
(1997)
J. Neurosci.
17,
7053-7059 |
54. | Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P., and Tsai, L. H. (1999) Nature 402, 615-622[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Ahlijanian, M. K.,
Barrezueta, N. X.,
Williams, R. D.,
Jakowski, A.,
Kowsz, K. P.,
McCarthy, S.,
Coskran, T.,
Carlo, A.,
Seymour, P. A.,
Burkhardt, J. E.,
Nelson, R. B.,
and McNeish, J. D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2910-2915 |
56. | Chen, G., Chen, K. S., Knox, J., Inglis, J., Bernard, A., Martin, S. J., Justice, A., McConlogue, L., Games, D., Freedman, S. B., and Morris, R. G. (2000) Nature 408, 975-979[CrossRef][Medline] [Order article via Infotrieve] |
57. | Masliah, E., Terry, R. D., Alford, M., DeTeresa, R. M., and Hansen, L. A. (1991) Am. J. Pathol. 138, 235-246[Abstract] |
58. | Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., DeTeresa, R., Hill, R., Hansen, L. A., and Katzman, R. (1991) Ann. Neurol. 30, 572-580[Medline] [Order article via Infotrieve] |
59. | Arriagada, P. V., Growdon, J. H., Hedley-Whyte, E. T., and Hyman, B. T. (1992) Neurology 42, 631-639[Abstract] |
60. | Terry, R. D. (1996) J. Neuropathol. Exp. Neurol. 55, 1023-1025[Medline] [Order article via Infotrieve] |
61. | Cummings, B. J., Pike, C. J., Shankle, R., and Cotman, C. W. (1996) Neurobiol. Aging 17, 921-933[CrossRef][Medline] [Order article via Infotrieve] |
62. | Gomez-Isla, T., Hollister, R., West, H., Mui, S., Growdon, J. H., Petersen, R. C., Parisi, J. E., and Hyman, B. T. (1997) Ann. Neurol. 41, 17-24[Medline] [Order article via Infotrieve] |
63. | Bartoo, G. T., Nochlin, D., Chang, D., Kim, Y., and Sumi, S. M. (1997) J. Neuropathol. Exp. Neurol. 56, 531-540[Medline] [Order article via Infotrieve] |
64. | Crook, R., Verkkoneimi, A., Perez-Tur, J., Mehta, N., Baker, M., Houlden, H., Farrer, M., Hutton, M., Lincoln, S., Hardy, J., Gwinn, K., Somer, M., Paetau, A., Kalimo, H., Ylikoski, R., Poyhonen, M., Kucera, S., and Haltia, M. (1998) Nat. Med. 4, 452-455[Medline] [Order article via Infotrieve] |
65. | Beach, T. G., Kuo, Y. M., Spiegel, K., Emmerling, M. R., Sue, L. I., Kokjohn, K., and Roher, A. E. (2000) J. Neuropathol. Exp. Neurol. 59, 308-313[Medline] [Order article via Infotrieve] |
66. |
Lue, L. F.,
Kuo, Y. M.,
Roher, A. E.,
Brachova, L.,
Shen, Y.,
Sue, L.,
Beach, T.,
Kurth, J. H.,
Rydel, R. E.,
and Rogers, J.
(1999)
Am. J. Pathol.
155,
853-862 |
67. | McLean, C. A., Cherny, R. A., Fraser, F. W., Fuller, S. J., Smith, M. J., Beyreuther, K., Bush, A. I., and Masters, C. L. (1999) Ann. Neurol. 46, 860-866[CrossRef][Medline] [Order article via Infotrieve] |
68. | Huang, X., Atwood, C. S., Hartshorn, M. A., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Cuajungco, M. P., Gray, D. N., Lim, J., Moir, R. D., Tanzi, R. E., and Bush, A. I. (1999) Biochemistry 38, 7609-7616[CrossRef][Medline] [Order article via Infotrieve] |
69. |
Huang, X.,
Cuajungco, M. P.,
Atwood, C. S.,
Hartshorn, M. A.,
Tyndall, J. D.,
Hanson, G. R.,
Stokes, K. C.,
Leopold, M.,
Multhaup, G.,
Goldstein, L. E.,
Scarpa, R. C.,
Saunders, A. J.,
Lim, J.,
Moir, R. D.,
Glabe, C.,
Bowden, E. F.,
Masters, C. L.,
Fairlie, D. P.,
Tanzi, R. E.,
and Bush, A. I.
(1999)
J. Biol. Chem.
274,
37111-37116 |
70. | Harkany, T., Abraham, I., Konya, C., Nyakas, C., Zarandi, M., Penke, B., and Luiten, P. G. (2000) Rev. Neurosci. 11, 329-382[Medline] [Order article via Infotrieve] |
71. | Bard, F., Cannon, C., Barbour, R., Burke, R.-L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., and Yednock, T. (2000) Nat. Med. 6, 916-919[CrossRef][Medline] [Order article via Infotrieve] |
72. | Capell, A., Saffrich, R., Olivo, J.-C., Meyn, L., Walter, J., Grunberg, J., Dotti, C., and Haass, C. (1997) J. Neurochem. 69, 2432-2340[Medline] [Order article via Infotrieve] |
73. |
Dewachter, I.,
Van Dorpe, J.,
Smeijers, L.,
Gilis, M.,
Kuiperi, C.,
Laenen, I.,
Caluwaerts, N.,
Moechars, D.,
Checler, F.,
Vanderstichele, H.,
and Van Leuven, F.
(2000)
J. Neurosci.
20,
6452-6458 |