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Human apolipoprotein E4 accelerates -amyloid deposition in APPsw transgenic mouse brain.

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Human Apolipoprotein E4 Accelerates
␤-Amyloid Deposition in APPsw Transgenic
Mouse Brain
Donald B. Carter, PhD, Edwige Dunn, MS, Denise D. McKinley, BS, Nancy C. Stratman, MS,
Timothy P. Boyle, BS, Susan L. Kuiper, MS, Jo A. Oostveen, MS, Royal J. Weaver, PhD,
Jennifer A. Boller, BS, and Mark E. Gurney, PhD
The human apolipoprotein E4 (ApoE4) isoform is associated with genetic risk for Alzheimer’s disease. To assess the
effects of different ApoE isoforms on amyloid plaque formation, human ApoE3 and ApoE4 were expressed in the brains
of transgenic mice under the control of the human transferrin promoter. Mice were crossed with transgenic mice expressing human amyloid precursor protein containing the Swedish mutation (APPsw), which facilitates amyloid ␤ peptide (A␤) production. The following progeny were selected for characterization: APPswⴙ/– ⴛ ApoE3ⴙ/– and APPswⴙ/–,
APPswⴙ/– ⴛ ApoE4ⴙ/– and APPswⴙ/– littermates. All mice analyzed were wild type for the endogenous mouse APP and
ApoE genes. Mice expressing ApoE4 in combination with APPsw have accelerated A␤ deposition in the brain as assessed
by enzyme immunoassay for A␤40 and A␤42 extractable in 70% formic acid, by assessment of amyloid plaque formation
using thioflavin-S staining, and by immunohistochemical staining with antibodies specific for A␤40 or A␤42 and the 4G8
monoclonal or 162 polyclonal antibody. No difference in the rate of A␤ deposition in the brain was seen in mice
expressing ApoE3 in combination with APPsw. Thus, our data are consistent with the observation in Alzheimer’s disease
that ApoE4 is associated with increased accumulation of A␤ in the brain relative to ApoE3.
Ann Neurol 2001;50:468 – 475
Apolipoprotein E (ApoE) appears to be involved in the
pathogenesis of late-onset Alzheimer’s disease (AD) because of the acceleration of AD in ApoE4 carriers.1–3
Various hypotheses have been proposed to explain how
ApoE affects the relative risk and age at onset of AD.
One hypothesis asserts that ApoE4 is involved in either
the deposition or the clearance of amyloid ␤ (A␤) peptide.4 –7 Evidence in support of this hypothesis was demonstrated in a transgenic model where the ApoE–/–
knockout was crossed with transgenic mice overexpressing human APP (V717F).8,9 The brains of 6-month-old
mice with the APP⫹/⫹ ⫻ ApoE–/– genotype showed
neither Congo red birefringence nor thioflavin-S fluorescence, whereas APP⫹/⫹ mouse brains showed robust
thioflavin-S and Congo red birefringence. Sparse, diffuse
immunoreactive A␤ deposits were detected in the
APP⫹/⫹ ⫻ ApoE–/– mouse cerebral cortex, but they
amounted to 12% of those detected in the APP⫹/⫹
mouse cerebral cortex.8,9 Another murine model of human ApoE effects on A␤ accumulation in mouse brain
showed that up to 10 months of age, expression of human ApoE suppressed A␤ deposition.10 In this model of
AD, human ApoE expression was driven by the
astrocyte-specific promoter for glial fibrillary acidic protein on a murine ApoE null background. However, further research using this model showed that at 15
months, expression of human ApoE4 was associated
with significantly more A␤ accumulation than was human ApoE3 in the mouse brains.11 Also, fibrillar deposits in the brains of ApoE3 or ApoE4 expressors were
associated with neuritic dystrophy.11
To examine the effect of human ApoE on amyloid
deposition in vivo, we crossed transgenic mice expressing human ApoE3 and ApoE4 under the control of the
human transferrin promoter with the APPsw transgenic
mouse. ApoE transgenic mice were developed using a
670 bp human transferrin promoter12 to drive ApoE
cDNA. The transferrin promoter allows expression of
ApoE in astrocytes and possibly in microglial cells.13
Two lines of ApoE3 and ApoE4 transgenic mice with
similar levels of human ApoE expression in the brain
were chosen for crosses with APPsw mice. Comparisons of brain A␤ accumulation were made between the
APPsw⫹/– ⫻ ApoE3⫹/– and their APPsw⫹/– littermates, as well as between APPsw⫹/– ⫻ ApoE4⫹/– and
their littermates at time points between 5 and 12
From the Pharmacia Corporation, Kalamazoo MI.
Received Apr 6, 2001, and in revised form May 25. Accepted for
publication May 29, 2001.
Address correspondence to Dr Carter, Pharmacia Corporation, CNS
Research, 7251-209-505, Kalamazoo, MI 49007. E-mail: Donald.
B.Carter@am.pnu.com
Published online Aug 6 2001; DOI: 10.1002/ana.1134
468
© 2001 Wiley-Liss, Inc.
months. Brain content of A␤40 and A␤42 was measured by enzyme immunoassay after extraction with
70% formic acid. This was correlated with histological
assessment of amyloid plaque deposition as measured
by thioflavin-S staining and immunohistochemical
staining with antibody 4G8, which labels the A␤ peptide.
Materials and Methods
Development of Transgenic Mice
ApoE transgenic mice were developed using a 670 bp human
transferrin promoter12 to drive human ApoE3 and ApoE4
(hApoE) cDNA; and high, medium, and low ApoEexpressing strains for each transgene were selected for characterization. The APPsw⫹/–,14 hApoE3⫹/–, and hApoE4⫹/–
populations were kept heterozygous by mating mice expressing the genes with C57BL/6 ⫻ SJL F1 hybrids. Mice expressing hApoE3⫹/– and hApoE4⫹/– were mated with
APPsw⫹/– mice to introduce the ApoE gene into APPsw
transgenic mice. Offspring from all matings consisted of four
genotypes: hApoE⫹/– transgenic mice, APPsw⫹/– transgenic
mice, APPsw⫹/– ⫻ ApoE⫹/– double transgenic mice, and
nontransgenic littermate controls. Genotyping was performed by polymerase chain reaction (PCR) using a 7700
PRISM (Perkin-Elmer Applied Biosystems, Foster City, CA)
to quantitate the transgene copy number. Two lines in which
hApoE3 and hApoE4 protein levels from the transgene approximated those in human brain were closely matched to
each other and selected for cross-breeding with APPsw animals. A breeding colony of APPsw⫹/– with ApoE⫹/– transgenic lines was maintained until enough double heterozygotes having the APPsw⫹/– ⫻ ApoE⫹/– and APPsw⫹/–
littermates were generated to do comparative analysis of
A␤1– 40 and A␤1– 42 peptides by enzyme-linked immunosorbent assay (ELISA), plaque counts using immunohistochemistry with 4G8, and fluorescence detection of thioflavin-S
staining in compact plaques. About 300 of these animals
were generated and used from 5 to 12 months of age.
Enzyme-linked Immunoabsorbent Assay for
␤Amyloid1– 40 and ␤Amyloid1– 42
Tissue extract was
prepared according to Gravina and colleagues.15
phosphate-buffered saline (DPBS) without CaCl2 or MgCl2
(GIBCO, Gaithersburg, MD) in the presence of a Complete
protease inhibitor tablet (Boehringer-Mannheim, Mannheim,
Germany) at a concentration of 200mg wet weight tissue/ml.
Brain was homogenized with a sonicator and centrifuged at
105g for 1 hour at 4°C. ELISA 96-well one-half area highbinding plates (Corning Costar, Corning, NY) were coated
50␮l/well with the following mouse antihuman apoE MAbs
prepared in 100mM ammonium bicarbonate and incubated
overnight at 4°C: 3H1 (Dr Ross Milne, University of Ottawa Heart Institute, Ottawa, Canada; 1mg/ml) and
MAB1062 (Chemicon, Temecula, CA; 5mg/ml). Plates were
subsequently blocked 100␮l/well with 1% BSA in DPBS ⫹
0.05% Tween-20 (DPBST) for 75 minutes at room temperature. Recombinant hApoE2, hApoE3, or hApoE4 (Calbiochem, San Diego, CA) was used to generate standard curves
prepared in wild type mouse brain supernatant or pellet
preparations (50␮l/well), and antigens were incubated for 90
minutes at room temperature. The primary antibody utilized
was a goat polyclonal anticynomolgus monkey ApoE antibody (produced and characterized at Pharmacia, Kalamazoo,
MI) diluted in 1% BSA in DPBST (50␮l/well) and incubated for 60 minutes. The secondary antibody was a mouse
antigoat conjugated with biotin, B3148 (Sigma, St. Louis,
MO) used at 0.3ng/ml and prepared in 1% BSA in DPBST
(50␮l/well); it was incubated for 60 minutes at room temperature. Neutravidin-horseradish peroxidase (HRP; Pierce,
Rockford, IL) at 0.1␮g/ml (50␮l/well) prepared in 1% BSA
in DPBST was then added for 30 minutes at room temperature. Tetramethyl benzidine peroxidase substrate (Kirkegaard and Perry, Gaithersburg, MD; 50␮l/well) was used to
produce a colored end product, which was obtained within
10 to 15 minutes. Reactions were terminated with 1M
H3PO4 (25␮l/well). All samples were analyzed in triplicate,
and ELISAs performed in duplicate. Levels of human ApoE
were expressed in nanograms of ApoE per milliliter of supernatant or pellet homogenate, and values were normalized to
brain tissue protein determined with the bicinchoninic acid
protein assay (Pierce, Rockford, IL). Total human ApoE
brain levels were obtained by adding supernatant and pellet
ApoE levels.
BRAIN TISSUE EXTRACT PREPARATION.
Human A␤1– 40
or A␤1– 42 was measured using monoclonal antibody (MAb)
6E10 (Senetek, St. Louis, MO) and biotinylated rabbit antiserum 162 or 165 (New York State Institute for Basic Research, Staten Island, NY) in a direct sandwich ELISA. The
capture MAb 6E10 is specific for an epitope present on
N-terminal amino acid residues 1 to 16 of human A␤ (hA␤).
Conjugated detecting antibodies 162 and 165 are specific for
an epitope at C-terminal amino acid residues 32 to 40 and
33 to 42, respectively, of hA␤. Direct sandwich ELISA was
performed according to a modified protocol.16
DIRECT SANDWICH ELISA FOR HA〉1– 40/42.
ApoE ELISA
PREPARATION OF BRAIN FOR DETECTION OF HUMAN
APOE BY ELISA. Brain without cerebellum was frozen at
– 80°C and thawed in cold (4°C) 0.1% NP-40 in Dulbecco’s
PERCENTAGE OF BRAIN AREA OCCUPIED BY 4G8 IMMUNOSTAIN. The percentage of the area of sections occupied by
4G8-immunoreactive plaque was determined as follows:
brains were fixed in 4% paraformaldehyde for 48 hours at
4°C, immunostained with biotinylated mouse MAb 4G8,
and detected with streptavidin-HRP conjugate and diaminobenzidine. The 4G8- and thioflavin-S-stained plaque area
was quantified using Optimus v 6.1 image analysis software
(Optimus, Silver Spring, MD), divided by the total area, and
expressed as the percentage of brain region for each section.
Data were analyzed using Student’s t test on the raw data in
conjunction with Student’s t test on ranked data. R indicates
that ranks of data were analyzed. The entire cortex at the
level of the hippocampus (ie, approximately midway between
rostral and caudal hippocampus) and one section per mouse
was analyzed. Five to 12 mice per group were surveyed for
4G8, 162, and thioflavin-S stains. The 162 polyclonal antibody produced the same results as the 4G8 MAb.
Carter et al: ␤-Amyloid Deposition Accelerated by Human ApoE4
469
Table 1. ␤-Amyloid 1-40/42 Levels in 70% Formic Acid-Soluble Brain Extracts of Transgenic Mice from 7 to 12 Months
Age
(mo)
7
8
10
12
A␤ (1 to 40) pmol/g Tissue ⫾ SEM
APPsw⫹/⫺
51 ⫾ 8
69 ⫾ 10
242 ⫾ 27
1,187 ⫾ 146
A␤ (1 to 42) pmol/g Tissue ⫾ SEM
APPsw⫹/⫺ ⫻ ApoE3⫹/⫺
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 12)
(n ⫽ 6)
53 ⫾ 7
43 ⫾ 5
247 ⫾ 46
1,147 ⫾ 113
(n ⫽ 8)
(n ⫽ 9)
(n ⫽ 9)
(n ⫽ 8)
APPsw⫹/⫺
30 ⫾ 7
48 ⫾ 6
203 ⫾ 18
401 ⫾ 38
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 12)
(n ⫽ 6)
APPsw⫹/⫺ ⫻ ApoE3⫹/⫺
36 ⫾ 7
36 ⫾ 4
202 ⫾ 20
501 ⫾ 48
(n ⫽ 8)
(n ⫽ 9)
(n ⫽ 9)
(n ⫽ 8)
A␤ ⫽ amyloid ␤; SEM ⫽ standard error of the mean.
A soluble brain extract was
prepared by sonication of one-half of a sagittally cut hemibrain without cerebellum and olfactory bulb in 500␮l homogenization buffer (0.1M KCl, 1mM EDTA, 0.5% Triton
X-100, pH 7.0) and centrifugation for 20 minutes at 40,000g
in a bench ultracentrifuge. Extracts were pooled from two to
four brains per age group for average representation. Protein
concentration was determined using the BCA microwell
method. Total protein (50␮g) per sample was loaded on the
gels. Samples electrophoresed for human and mouse ApoE
were run according to the manufacturer’s protocol on 4% to
12% NuPAGE Bis-Tris gels (Novex, San Diego, CA) for
human APP on 4% to 20% MiniPlus Sepra gels and for the
low-density lipoprotein receptor–related protein (LRP) on
7.5% MiniPlus Sepra gels (Owl Separation Systems, Natick,
MA). Control protein or peptide (10ng) was added where
applicable as positive control. Proteins were transferred onto
nitrocellulose. The various blots were immunoprobed with
the following antibodies: for hApoE mouse MAb 2E1
(Boehringer-Mannheim, specific for hApoE), for mouse
ApoE goat antirat ApoE antibody (from Dr Paul Roheim,
Louisiana State University Medical Center, Baton Rouge,
LA; specific for mouse and rat), for human APP mouse MAb
6E10 (Senetek, Napa, CA; specific for human APP), and for
LRP mouse anti-LRP/A2MR clone 5A6 (Research Diagnostics, Flanders, NJ; specific for human LRP but cross-reacts
with mouse and rabbit). The following secondary antibodies
were used: sheep anti-mouse HRP conjugate (Amersham, Arlington Heights, IL) for mouse MAbs and rabbit antigoat
HRP conjugate (Dako, Carpinteria, CA) for the goat antibody. For detection of the signal, enhanced chemiluminescence reagent (Renaissance; NEN/DuPont, Boston, MA) was
used, according to the manufacturer’s instructions.
WESTERN IMMUNOBLOTS.
Results
Human ApoE Expression in Transgenic Mouse Brain
Lines of mice expressing the hApoE3 and hApoE4
transgenes were evaluated for levels of protein expression by ELISA; two lines were picked to cross with
APPsw mice based on the following criteria: levels of
hApoE in the range found in human brain
(0.5–1.0␮g/mg protein) and levels of hApoE3 matched
as closely as possible to levels of hApoE4.
ApoE4 Accelerates Accumulation of ␤-Amyloid40 and
␤-Amyloid42 Peptides Between 7 and 10 Months of
Age
A␤ deposition in the brains of TgN(PrP-APPsw)2576
mice increases rapidly between 8 and 12 months of
age. In the crosses with ApoE3 transgenic mice this
was unchanged, while in the crosses with ApoE4 transgenic mice deposition began by 7 months of age and
A␤ accumulated to higher levels at 7, 8, and 10
months of age. By 12 months of age, accumulation of
A␤40 was no longer statistically different in ApoE4/
APPsw mice versus APPsw littermate controls. Thus,
ApoE4 in this model accelerates the timing of A␤ peptide accumulation, but not its eventual extent.
Total (soluble and insoluble) human A␤1– 40 and
A␤1– 42 levels in the cortex and subcortical tissue without cerebellum and olfactory bulb (brain), measured by
direct sandwich ELISA at 7, 8, 10, and 12 months in
extracts from APP⫹/–sw ⫻ ApoE3⫹/– brains, did not
change significantly compared to APP⫹/–sw littermates
Table 2. ␤-Amyloid 1-40/42 Levels in 70% Formic Acid-Soluble Brain Extracts of Transgenic Mice from 5 to 12 Months
Age
(mo)
5
6
7
8
10
12
A␤ (1 to 40) pmol/g Tissue ⫾ SEM
APPsw⫹/⫺
26 ⫾ 1
37 ⫾ 5
29 ⫾ 2
50 ⫾ 13
339 ⫾ 104
1,254 ⫾ 231
A␤ (1 to 42) pmol/g Tissue ⫾ SEM
APPsw⫹/⫺ ⫻ ApoE4⫹/⫺
(n ⫽ 6)
(n ⫽ 6)
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 12)
(n ⫽ 9)
29 ⫾ 1
37 ⫾ 3
45 ⫾ 2a
138 ⫾ 26b
650 ⫾ 114c
1,662 ⫾ 292
(n ⫽ 6)
(n ⫽ 6)
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 9)
(n ⫽ 12)
p ⬍ 0.005; bp ⬍ 0.001; cp ⬍ 0.05.
a
470
Annals of Neurology
Vol 50
No 4
October 2001
APPsw⫹/⫺
6 ⫾ 0.2
13 ⫾ 3.0
11 ⫾ 1.0
11 ⫾ 2.0
157 ⫾ 28.0
400 ⫾ 53.0
(n ⫽ 6)
(n ⫽ 6)
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 12)
(n ⫽ 9)
APPsw⫹/⫺ ⫻ ApoE4⫹/⫺
6⫾1
11 ⫾ 2
26 ⫾ 2a
40 ⫾ 8b
251 ⫾ 36c
548 ⫾ 42c
(n ⫽ 6)
(n ⫽ 6)
(n ⫽ 8)
(n ⫽ 6)
(n ⫽ 9)
(n ⫽ 10)
Table 3. ␤-Amyloid 40 Levels and A␤ 40/42 Ratios in ApoE4 ⫻ APPsw Versus ApoE3 ⫻ APPsw
Age
(mo)
7
8
10
12
Cross
A␤ 1 to 40
(pmol/g)
A␤ 1 to 42
(pmol/g)
A␤ 1 to 40/A␤ 1 to 42
ApoE4 ⫻ APPsw
ApoE3 ⫻ APPsw
ApoE4 ⫻ APPsw
ApoE3 ⫻ APPsw
ApoE4 ⫻ APPsw
ApoE3 ⫻ APPsw
ApoE4 ⫻ APPsw
ApoE3 ⫻ APPsw
45 ⫾ 2
53 ⫾ 7
138a⫾ 26
43 ⫾ 5
649a⫾ 114
247 ⫾ 46
1,662 ⫾ 292
1,147 ⫾ 113
26 ⫾ 2
36 ⫾ 7
40 ⫾ 8
36 ⫾ 4
251 ⫾ 36
202 ⫾ 20
548 ⫾ 42
501 ⫾ 48
1.8 ⫾ 0.07
1.6 ⫾ 0.08
3.5 ⫾ 0.30b
1.3 ⫾ 0.30
2.5 ⫾ 0.20b
1.2 ⫾ 0.10
3.0 ⫾ 0.30
2.0 ⫾ 0.30
p ⬍ 0.05; bp ⬍ 0.0005.
a
(Table 1). In contrast, total hA␤1– 40 at 7, 8, and 10
months in the brain of APP⫹/–sw ⫻ ApoE4⫹/– mice
was significantly elevated compared to APP⫹/–sw littermates (Table 2). Total hA␤1– 42 followed the same pattern in the APP⫹/–sw ⫻ ApoE4⫹/– brains, and A␤1– 42
was elevated significantly above littermate control levels
at 12 months. Total hA␤1– 40 brain levels and the
A␤1– 40 to A␤1– 42 ratio at 8 and 10 months were significantly higher in APP⫹/–sw ⫻ ApoE4⫹/– compared
with APP⫹/–sw ⫻ ApoE3⫹/– mice (Table 3). In contrast, no significant changes were seen in total hA␤1– 42
brain levels between the hApoE3 and hApoE4 crosses
at 8 and 10 months, suggesting that the effect of hApoE4 was primarily on A␤1– 40 accumulation. That
ApoE4 greatly increases accumulation of A␤40, as
shown by the A␤40/A␤42 ratio (see Table 3), is an interesting and salient aspect of this model. The Swedish
mutation elevates production of both A␤40 and A␤42
compared with the effect of the London mutation,
which selectively elevates A␤42. A␤42 normally has
greater propensity than A␤40 to accumulate as
plaque,17,18 but that may be reversed in the presence of
ApoE4. Levels of ApoE3 and ApoE4 expression increased with age in ApoE3/APPsw and ApoE4/APPsw
mice but remained at comparable levels (Table 4).
Table 4. Levels of Human ApoE3 and ApoE4 in Double
Transgenic Mice
Genotype
APPsw ⫻ ApoE3
APPsw ⫻ ApoE4
Age
(mo)
No.
Animals
Mean ⫾ SE
(ng ApoE/␮g
Protein)
7
8
10
12
4.8
6
8
10
12
6
9
9
7
2
4
2
6
7
0.733 ⫾ 0.098
0.891 ⫾ 0.068
0.939 ⫾ 0.072
1.162 ⫾ 0.119a
0.505 ⫾ 0.050
0.670 ⫾ 0.080
0.943 ⫾ 0.372
0.573 ⫾ 0.078
0.683 ⫾ 0.036
p ⬍ 0.05 for 7 months versus 12 months.
a
ApoE4 Accelerates Deposition of Amyloid Plaque at
10 Months of Age
Measurements of amyloid plaque areal density of A␤
senile plaque (SP) deposits in cortex and hippocampus
agree with measurements of A␤ accumulation by enzyme immunoassay. No significant differences in the
total or compact plaques, visualized by 4G8/162 immunostaining or thioflavin-S fluorescent staining, respectively, were observed at 10 or 12 months in the
APP⫹/–sw ⫻ ApoE3⫹/– group (Fig 1). In contrast, significant increases of areal density of total and compact
SPs in the hippocampus of APP⫹/–sw ⫻ ApoE4⫹/–
compared with APP⫹/–sw littermates were observed at
10 and 12 months, and only at 12 months in the hippocampus, using 4G8 staining of amyloid deposits (Fig
2). The difference between levels of 4G8 plaque density in APP⫹/–sw mice derived from the ApoE3 and
ApoE4 littermates (see Figs 1 and 2) at 10 months of
age were not statistically significant. In addition, significant increases in compact plaques were seen at 10
months in cortex and at 12 months in hippocampus,
as evidenced by thioflavin-S fluorescent staining. The
differences between levels of thioflavin-S fluorescent
staining in APP⫹/–sw mice derived from ApoE3 and
ApoE4 littermates (see Figs 1 and 2) at 10 months of
age were not statistically significant.
Changes in Amyloid Accumulation Are Not Due to
Changes in APP, ApoE, or LRP Protein Expression
Protein levels (Fig 3) from the transgenes hApoE4,
hAPP, and endogenous mouse ApoE and LRP (lowdensity lipoprotein receptor for ApoE, APP, and ␣2macroglobulin) did not significantly change over the
time points analyzed in cortex or cerebellum for either
APP⫹/–sw ⫻ ApoE4⫹/– or APP⫹/–sw littermates. Levels of hApoE3 were on average about 30% higher than
those of hApoE4 during the course of the experiment.
Levels of hApoE3 showed an increasing trend from 7
to 12 months that was statistically significant only
when the 12-month level of ApoE3 was compared with
the 7-month level (see Table 4). Levels of hApoE4 in
Carter et al: ␤-Amyloid Deposition Accelerated by Human ApoE4
471
Fig 1. Immunoreactive plaque density of 4G8 (A) and
thioflavin-S fluorescent (B) staining of APPsw ⫻ ApoE3 versus APPsw littermates in cortex and hippocampus (⫾SEM).
solid bars ⫽ APPsw; striped bars ⫽ APPsw ⫻ APoE3.
the crosses at 6 months of age were 0.670 (⫾0.080,
n ⫽ 4) to 0.573 (⫾0.078, n ⫽ 6) ng ApoE/␮g protein
at 10 months of age, and there was no significant
change in steady-state levels of hApoE4 over the time
course of the experiment.
Discussion
The differences in deposition of A␤, evidenced by total
human A␤40 and A␤42 levels in brain and diffuse or
compact SPs of the APP⫹/–sw ⫻ ApoE3⫹/– transgenic
crosses versus APP⫹/–sw littermates compared with
APP⫹/–sw ⫻ ApoE4⫹/– versus APP⫹/–sw littermates,
demonstrate a human ApoE isoform-specific effect on
the initial rate of accumulation in the transgenic mouse
brain. The effect that endogenous mouse ApoE has on
deposition of A␤ is expressed in the littermate hApoE
control mice, against which the crosses were compared.
In addition, mouse ApoE promotes formation of A␤immunoreactive deposits in this mouse model of A␤
deposition. Holtzman and colleagues19 showed that the
lack of ApoE attenuates A␤ deposition in the cortex
and cerebral vasculature of 12-month-old APPsw mice
and that no fibrillar deposits of A␤ are evident without
the presence of ApoE. Since there is no evidence that
endogenous mouse ApoE differentially affects the dep-
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Annals of Neurology
Vol 50
No 4
October 2001
osition and clearance of A␤ in control littermate mice
versus crosses, additional hApoE in the brains of the
crosses should give insight to the role of transgenederived isoforms of hApoE in these processes. Coexpression of human APPsw and hApoE4 in transgenic
mouse brain accelerates the age-dependent accumulation of A␤ as measured by enzyme immunoassay, 4G8/
162 immunostaining, or thioflavin-S staining compared
with mice coexpressing APPsw and ApoE3. Levels of
deposition in the hApoE3 lines of mice were not detectably affected by levels of hApoE expressed since the
hApoE3 crosses did not show any differences from
their singly transgenic littermates at each time point,
while having more than double the amount of ApoE
(endogenous mouse ⫹ hApoE3) than the singly transgenic littermates. These data are consistent with the
neuropathological findings in postmortem brain tissue
of human AD patients, where statistically greater A␤
SP densities in brain of E4/E4 versus E3/E3 or E3/E4
patients have been reported.5,20 In addition, the significant increases in brain levels of total human A␤40 and
the A␤40/A␤42 ratio in APP⫹/–sw ⫻ ApoE4⫹/– compared with APP⫹/–sw ⫻ ApoE3⫹/– crosses correlate
with findings in postmortem brain tissue of human
AD patients.21,22 In human brain tissue from AD patients, increased A␤ SP frequency in E4 genotypes was
Fig 2. Immunoreactive plaque density of 4G8 (A) and
thioflavin-S fluorescent (B) staining of APPsw ⫻ ApoE4 versus APPsw littermates in cortex and hippocampus (⫾SEM).
solid bars ⫽ APPsw; striped bars ⫽ APPsw ⫻ APoE4.
Fig 3. Western blots of human apolipoprotein E (ApoE), mouse ApoE, human APP, and mouse low-density lipoprotein–related protein (LRP) in the brains of APPsw ⫻ ApoE3, Appsw ⫻ Apoe4, and littermate APPsw controls from 6 to 12 months over the experimental time course. (A) Time course of hApoE3 and hApoE4 protein levels. (B) Levels of endogenous mouse ApoE protein.
(C) Levels of hAPP expressed from the APPsw transgene. (D) Level of mouse LRP.
largely attributed to a significant increase in A␤1– 40immunoreactive plaques, in both number and ratio of
A␤1– 40 to A␤1– 42 plaques, in contrast to a lack of difference detected in A␤1– 42-immunoreactive plaques
with either E3 or E4 genotype.21 This study was done
with 68 AD brain samples, 10 of which were E4 homozygotes and 32 E3/E4. Another, smaller study with
28 AD brains showed by immunohistochemistry that
in humans the E4 genotype is associated with increases
in both A␤40 and A␤42.22 Using formic acid extraction
from 36 AD brains, A␤40 levels were found by ELISA
to be significantly higher in E3/E4 and E4/E4 cases
than in E3/E3 cases.23 The ApoE isoform differences
in amyloid accumulation between the ApoE3 and
ApoE4 crosses with the APPsw mice described above
can also be accounted for by an increase in the amount
of A␤40 species accumulated under the influence of the
hApoE4 protein, relative to hApoE3.
Age-dependent accumulation of A␤ in transgenic
mouse brain is not due to changes with age in the expression of APP, ApoE4, or LRP mRNA (data not
shown) or protein expression. Determination of levels
of protein and RNA (data not shown) from hAPPsw
and hApoE4 transgenes and endogenous mouse ApoE
or LRP in APP⫹/–sw ⫻ ApoE⫹/– and the APP⫹/–sw
littermates over the time course of the experiment
showed no significant changes in the transgene expression level for APP, nor in the endogenous expression of
murine ApoE or LRP. There was a statistically significant increase of hApoE3 protein level at the 12-month
time point when compared with the 7-month time
point. However, there were no statistically significant
differences in A␤ deposition measured between the single and double transgenic mice at 7 or 12 months of
age. These data suggest that changes seen in the deposition of A␤ do not occur because of varying levels of
RNA or protein encoded by either the transgenes or
the endogenous genes for APP, ApoE, or LRP.
Our data are consistent with other results demonstrating an isoform-specific effect of ApoE on A␤ accumulation in transgenic mouse crosses.11 Expression of
hApoE3 and hApoE4 in APPV717F transgenic, ApoE–/–
mice resulted in fibrillar (thioflavin-S-positive) A␤ deposits by 15 months of age. Substantially (⬎10-fold)
more fibrillar deposits were observed in ApoE4expressing APPV717F transgenic mice. Holtzman and
colleagues11 contend that ApoE protein plays a critical
and isoform-specific role in A␤ deposition and struc-
Carter et al: ␤-Amyloid Deposition Accelerated by Human ApoE4
473
ture in AD pathology because it facilitates fibrillar
amyloid (A␤) formation in vivo, which is toxic for
neurites, as evidenced by neuritic plaques at 15 months
in their ApoE transgenic cross. Comparison of the levels of A␤ measured in the APPV717F ⫻ ApoE4 versus
APPV717F ⫻ ApoE3 mice with the data from our
APP⫹/–sw ⫻ ApoE4⫹/– model presented here indicates
that the APPV717F ⫻ ApoE model produces a larger
ratio of thioflavin-S-stained plaques in ApoE4 mice relative to ApoE3 mouse brain. In APPsw ⫻ ApoE mice,
that ratio was approximately 2 to 1 at 12 months of
age, and in APPV717F ⫻ ApoE mice, it was 10 to 1 at
15 months of age. Data from human AD brain indicate that the ratio of neuritic plaques in ApoE4 genotypes relative to ApoE3 homozygotes is about 1.5 to
15. In addition, there is no ApoE isoform-specific effect on the length of the A␤ peptide, as no alteration
of the A␤42 to total A␤ ratio was observed.11 The differences between these models may be due to the different promoters used for transgene expression, mouse
genetic background differences, use of endogenous
ApoE knockout mice versus intact mice with endogenous ApoE, or the APPsw transgene versus the
APPV717F transgene. We conclude that ApoE protein
plays a critical and isoform-specific role in influencing
A␤ deposition and structure in AD pathology because
it facilitates fibrillar amyloid (A␤) formation in vivo,
which is toxic for neurites, as evidenced by neuritic
plaques at 15 months in their ApoE transgenic cross.
ApoE forms a complex not only with fibrillar A␤ but
also with soluble A␤ in human brain,10,24,25 supporting the notion that ApoE–A␤ interactions before A␤
deposition could regulate A␤ clearance. ApoEcontaining lipoproteins in the brain may sequester A␤
and facilitate its cellular uptake and degradation locally
by cells, or its removal from the brain into the systemic
circulation.
Our data suggest that the isoform-specific effects of
hApoE4 expressed from the transgene either do not
promote clearance of A␤ as efficiently as hApoE3, or
promote deposition relative to ApoE3, or both. The
considerable in vitro data concluding that native
ApoE2 and ApoE3 have a much higher binding affinity for A␤ than ApoE426 –28 suggest that the decreased
efficiency of ApoE4 protein to sequester A␤ relative to
the other isoforms of ApoE is one of the mechanisms
for involvement of ApoE in the pathogenesis of AD.
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