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The Journal of Neuroscience, January 15, 2001, 21(2):372-381
Age-Dependent Changes in Brain, CSF, and Plasma Amyloid  Protein in the Tg2576 Transgenic Mouse Model of Alzheimer's
Disease
Takeshi
Kawarabayashi1, 2,
Linda H.
Younkin1,
Takaomi
C.
Saido3,
Mikio
Shoji2,
Karen Hsiao
Ashe4, and
Steven G.
Younkin1
1 Mayo Clinic Jacksonville, Jacksonville, Florida
32224, 2 Department of Neurology, Gunma University, Gunma,
Japan 371-8511, 3 Proteolytic Neuroscience Laboratory,
RIKEN Brain Science Institute, Saitama, Japan 351-0198, and
4 Departments of Neurology and Neuroscience, Center for
Clinical and Molecular Neurobiology, University of Minnesota,
Minneapolis, Minnesota 55455
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ABSTRACT |
The accumulation of amyloid  protein (A) in the Tg2576 mouse
model of Alzheimer's disease (AD) was evaluated by ELISA,
immunoblotting, and immunocytochemistry. Changes in A begin at 6-7
months as SDS-insoluble forms of A42 and A40 that require formic
acid for solubilization appear. From 6 to 10 months, these
insoluble forms increase exponentially. As insoluble A appears,
SDS-soluble A decreases slightly, suggesting that it may be
converting to an insoluble form. Our data indicate that it is
full-length unmodified A that accumulates initially in Tg2576 brain.
SDS-resistant A oligomers and most A species that are
N-terminally truncated or modified develop only in older Tg2576
mice, in which they are present at levels far lower than in human AD
brain. Between 6 and 10 months, when SDS-insoluble A42 and A40
are easily detected in every animal, histopathology is minimal because
only isolated A cores can be identified. By 12 months, diffuse
plaques are evident. From 12 to 23 months, diffuse plaques, neuritic
plaques with amyloid cores, and biochemically extracted A42 and
A40 increase to levels like those observed in AD brains. Coincident with the marked deposition of A in brain, there is a decrease in CSF
A and a substantial, highly significant decrease in plasma A. If
a similar decline occurs in human plasma, it is possible that
measurement of plasma A may be useful as a premorbid biomarker for AD.
Key words:
Alzheimer's disease; neurodegeneration; Tg2576
transgenic animal model; amyloid protein; cerebrospinal
fluid; plasma
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INTRODUCTION |
Amyloid protein (A), the
principal protein in the senile plaques of Alzheimer's disease (AD),
is an ~4 kDa secreted polypeptide that is derived from several
isoforms of a large protein referred to as the amyloid protein
precursor (APP) (Glenner and Wong, 1984 ; Masters et al., 1985; Kang
et al., 1987). Secreted A is readily detected in CSF, plasma, and in
medium conditioned by a wide variety of cultured cells (Cai et al.,
1993; Citron et al., 1994, 1997; Suzuki et al., 1994; Scheuner et al.,
1996; Younkin et al., 1998). In each situation, most secreted A is
A1-40, but a small percentage (5-15%) is A1-42. A1-42 is
especially important in AD. Synthetic A1-42 forms amyloid fibrils
in vitro much more readily than A1-40 (Jarrett et al.,
1993), and there is good evidence that A1-42 is deposited early and
selectively in senile plaques (Iwatsubo et al., 1994).
Early onset Alzheimer's disease can be caused by mutations in the
APP (Chartier-Harlin et al., 1991; Goate et al.,
1991; Murrell et al., 1991; Mullan et al., 1992), presenilin 1 (PS1) (Sherrington et al., 1995), and presenilin 2 (PS2) (Levy-Lahad et al., 1995) genes. Studies of human
plasma, human fibroblasts, transfected cells, and transgenic mice have
shown that each of these genetic forms of AD either selectively
increases the extracellular concentration of A42 (Suzuki et al.,
1994; Borchelt et al., 1996; Duff et al., 1996; Scheuner et al., 1996;
Citron et al., 1997) or increases both A42 and A40 (Cai et al.,
1993; Citron et al., 1994; Scheuner et al., 1996). Thus, in all of
these genetic forms of AD, A metabolism is altered in a way that
fosters A aggregation and deposition.
The Tg2576 mouse model of Alzheimer's disease (Hsiao et al., 1996)
expresses the Swedish mutation of APP
(APPK670N,M671L) at high level under control of
the hamster prion protein (PrP) promoter. It is well established
that this mutation causes concomitant increases in secreted A42 and
A40 (Cai et al., 1993; Citron et al., 1994; Scheuner et al., 1996).
As Tg2576 mice age, classic neuritic plaques with Congo red-positive
amyloid cores appear that are similar to those seen in Alzheimer's
disease (Irizarry et al., 1997). In addition, Tg2576 mice develop
age-dependent behavioral deficits as assessed by Y maze, T maze, and
Morris water maze testing (Hsiao et al., 1996; Chapman et al., 1999; Westerman et al., 2000).
To exploit the Tg2576 model of AD, it is essential to obtain baseline
information on the amount and rate at which various forms of A are
deposited in the Tg2576 model compared with human AD. In this study, we
obtain this information using an analytic paradigm that combines
sandwich ELISAs, immunoblots, and immunocytochemistry based on
antibodies to specific domains in the various forms of A.
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MATERIALS AND METHODS |
Transgenic mice and extraction. Transgenic mice and
nontransgenic littermates, bred by mating Tg2576 males with C57B6/SJL F1 females, were killed at 1-25 months (M). Plasma was
collected in 0.1% EDTA, and CSF was obtained according to the method
of Carp et al. (1971). One hemibrain was frozen in liquid nitrogen, and
the other hemibrain was fixed in 4% paraformaldehyde with 0.1 M phosphate buffer, pH 7.6. Plasma, CSF, and
frozen brains were stored at  80°C. Frozen hemibrains were
sequentially extracted. At each step, sonication (35 sec at level 10;
XL-2000 Microson Ultrasonic Cell Disruptor; Misonix Inc., Farmingdale,
NY) in an appropriate buffer was followed by centrifugation at
100,000 × g for 1 hr at 4°C. The supernatant was
then removed, and the pellet was sonicated in the next solution used in
the sequential extraction process. For four-step extraction, sonication
of the frozen brain (150 mg/ml wet weight) began in Tris-buffered
saline (TBS) (20 mM Tris and 137 mM NaCl, pH 7.6), which contained protease
inhibitors (complete protease inhibitor cocktail, 1 tablet in 50 ml
solution; Boehringer Mannheim, Mannheim, Germany). The next three
sequential extraction steps used 1% Triton X-100 in TBS with protease
inhibitors, 2% SDS in water with the same protease inhibitors, and
70% formic acid (FA) in water. For two-step extraction, the initial
sonication of brain (150 mg/ml wet weight) took place in 2% SDS with
protease inhibitors, and the resultant pellet was then extracted with
70% formic acid in water.
Antibodies. The following antibodies to A were used:
monoclonal, BAN-50 (anti-A1-16), BA-27 (anti-A1-40), BC-05
(anti-A35-43), BNT-77 (anti-A11-28), 4G8 (anti-A17-24), and
6E10 (anti-A1-16); polyclonal, 3160 (anti-A1-40), Saeko
(anti-C-terminal 30 amino acids of APP) (Kawarabayashi et al., 1996),
and five antibodies described by Saido et al. (1995, 1996), which
specifically detect N termini of A, anti-AN1(D), for the
unmodified A N terminus (N1(D));
anti-L-iso-Asp for isomerized forms of AN1
(N1(iD)); anti-rectus Asp for stereoisomerized forms of AN1
(N1(rD)); anti-AN3-pyroglutamate (N3(pE)); and
anti-AN11-pyroglutamate (N11(pE)).
Sandwich ELISA for A. Brain extracts were
measured by sandwich ELISA as described previously (Suzuki et al.,
1994; Gravina et al., 1995). The following systems were used: (1)
BAN-50 capture and BC-05 or BA-27 detection or (2) 3160 capture and
BC-05 or BA-27 detection, both of which detect A1-42 and A1-40,
respectively, and (3) BC-05 or BA-27 capture and 4G8 detection, which
detect Ax-42 and Ax-40, respectively. Direct comparison of many
Tg2576 brains from mice of all ages showed that the amounts of A42
and A40 detected with 3160 capture ELISAs were essentially the same as when BAN-50 was used for capture. For measurement of plasma and CSF
A, BNT-77 capture and BC-05 detection was used for A42, and
BAN-50 capture and BA-27 detection was used for A40.
The 2% SDS extracts were diluted at least 1:40 so that A capture
took place in EC buffer [0.02 M phosphate buffer, pH 7, 0.4 M NaCl, 2 mM EDTA, 0.4% Block Ace
(Dainipponseiyaku, Suita, Osaka, Japan), 0.2% bovine serum albumin,
0.05% CHAPS and 0.05% sodium azide] containing 0.05% SDS. The TBS
(at least 1:10) and Triton X-100 (at least 1:20) extracts were also
diluted so that A capture took place in EC buffer containing 0.05%
SDS. Formic acid extracts were neutralized initially by 1:20 dilution
into 1 M Tris phosphate buffer, pH 11, and then diluted as
necessary in EC buffer. The program Softmax (Molecular Devices,
Menlo Park, CA) was used to calculate A concentration (in
picomolar) by comparing the sample absorbance with the
absorbance of known concentrations of synthetic A1-42 or A1-40
standards assayed identically on the same plate. Using the wet weight
of brain in the original homogenate, the final values of A in brain
were expressed as picomoles per gram wet weight. Nontransgenic tissues
were processed identically in parallel with the transgenic tissues.
Immunoblots. To detect SDS-soluble A around the critical
period (4M-10M) when deposition begins, SDS fractions were
immunoprecipitated with 3160 by diluting 40 µl of each extract
40-fold with RIPA buffer (150 mM NaCl, 1% Triton
X-100, 0.5% cholic acid, 0.1% SDS, and 50 mM
Tris, pH 8) containing protease inhibitors and immunoprecipitating with
protein G-agarose that had been incubated with 1 µl of 3160. To
detect SDS-soluble A in 21M transgenic and AD brain, SDS fractions were directly applied to the gel. The formic acid fractions were evaporated using a Speed-Vac concentrator (Savant, Holbrook,
NY), and dissolved in dimethyl sulfoxide. SDS and FA fractions
were separated on 10-20 or 16% Tricine SDS gels (Novex, Wadsworth, OH) and electrotransferred to Immobilon P (Millipore, Bedford, MA) at
100 V for 1.5 hr. Membranes were labeled with primary antibody (BAN50
or 4G8) overnight at 4°C, incubated with horseradish
peroxidase-linked secondary antibody (Amersham Pharmacia Biotech,
Arlington Heights, IL) for 1 hr, and detected using Supersignal
(Pierce, Rockford, IL). To detect full-length APP and C-terminal
fragments, SDS fractions by two-step extraction were separated on 10%
Tricine SDS gels and detected with Saeko anti-C-terminal antibody or 6E10.
Immunocytochemistry. Tissue samples were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer, pH
7.6, for 8 hr at 4°C. Paraffin sections (5 µm) were pretreated with
70% formic acid for 5 min and immersed in 0.5% periodic acid for 10 min to block intrinsic peroxidase. They were then incubated with 1.5%
blocking serum in PBS for 1 hr, with primary antibodies (BC-05, 0.1 µg/ml; BA-27, 0.4 µg/ml; or antibodies to specific N termini of
A, 2.5 µg/ml) overnight, and with horseradish
peroxidase-conjugated secondary antibody (1:100; Dako, High Wycombe,
UK) for 1 hr. Immunoreactivity was visualized by incubation with 0.03%
3,3'-diaminobenzidine, 0.065% sodium azide, and 0.02%
H2O2. To stain
anti-AN1(D), anti-N1(iD), and anti-N1(rD), PBS containing 500 nmol/l
NaCl was used to prevent cross-reaction. Methyl green was used for
nuclear staining. Sections from seven AD brains were stained in parallel.
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RESULTS |
Human APP expression in Tg2576 mice is not confined to
the brain
SDS extracts from Tg2576 brain were analyzed on immunoblots
labeled with anti-C (Saeko), a rabbit polyclonal antibody that recognizes the C terminus of both human and mouse APP (Fig.
1A), or with 6E10, a
mouse monoclonal antibody specific for human APP (Fig.
1B). The APP holoprotein and its 8-14 kDa C-terminal
fragments (CTFs) were elevated in the brains of Tg2576 mice as expected and showed no increase in older mice (Fig.
1A,B) as reported previously (Hsiao
et al., 1996). SDS extracts of other organs from Tg2576 mice (10.9M)
and their nontransgenic littermates (9.3M) were analyzed similarly
(Fig. 1C-F). In nontransgenic mice, the level of
endogenous mouse APP holoprotein was highest in brain and lung (Fig.
1E), and none of the endogenous proteins in any mouse
organ cross-reacted appreciably with the human-specific 6E10 antibody
(Fig. 1F). In Tg2576 transgenic mice, human APP was
present at high level not only in brain but also in spleen and lung
(Fig. 1D). Moderate levels of transgenic human APP
were present in Tg2576 heart, skin, bone, and muscle, and there was
some human APP in pancreas, stomach, and large intestine (Fig.
1D). Thus, transgenic APP expression is not confined
to the brain in the Tg2576 mouse model of AD. The A in various
organs of 10.9M Tg2576 mice was analyzed by immunoprecipitation
followed by immunoblotting. With this approach, A was detected only
in the brain (Fig. 1G). The more sensitive sandwich ELISA
assays (Fig. 1H) were able to detect small amounts of
SDS-extractable A in all systemic organs (10-24 pmol/gm), but the
amount of SDS-extractable A in the brain was much larger (400 pmol/gm). Analysis of SDS-insoluble A (formic acid extract of the
pellet left after SDS extraction) in various organs from 10.9M Tg2576
mice showed that insoluble A accumulates only in the brain (Fig.
1G,H).
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Figure 1.
Expression of APP, CTF, and A in
brain and systemic organs of Tg2576 and nontransgenic mice. Immunoblots
in A-F were labeled with anti-C (Saeko)
(A, C, E), which detects
both human and mouse APP, or with 6E10 (B,
D, F), which specifically detects
human APP. A, B, Immunoblots of APP
and CTF in transgenic and nontransgenic mice of various ages (months).
Immunoblots were prepared from 16% Tricine gels. C-F,
Immunoblots of SDS extracts from systemic organs of a 10.9 month Tg2576
mouse (C, D) and a 9.3 month
nontransgenic mouse (E, F).
Immunoblots were prepared from 10-20% Tricine gels loaded at 20 mg/lane total protein. Tg, Transgenic;
NTg, nontransgenic; Br, brain;
H, heart; Lg, lung; Lv,
liver; K, kidney; P, pancreas;
Sp, spleen; St, stomach;
Si, small intestine; Li, large intestine;
M, muscle; Bo, bone; Sk,
skin. G, Immunoblot of A in SDS and FA extracts of
systemic organs. The A in SDS extracts (40 µl) was analyzed by 4G8
immunoprecipitation followed by immunoblotting with 4G8; FA acid
extracts (40 µl), dried and resuspended, were also analyzed by
immunoblotting with 4G8. H, Total A (A42 plus
A40) in SDS and FA extracts of systemic organs. A42 and A40
were analyzed by 3160/BC-05 and 3160/BA-27 ELISAs, respectively.
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Cored plaques appear early in Tg2576 brain
At 7-8 months, dense cored plaques that contain both A40
(BA-27) and A42 (BC-05) appeared in the Tg2576 brain (Fig.
2M,N). These early cored plaques were stained by Congo red and showed green
birefringence with polarized light (data not shown). They increased
between 7 and 10 months but, even at 10 months, only a few cores were
present in each section (Fig.
2K,L). At 12-15 months, diffuse
plaques appeared that were labeled preferentially by the BC-05 antibody
to A42 (Fig. 2G-J). Between 15 and 23 months, A plaques in the Tg2576 brain (Fig. 2C-H)
accumulated to levels like those seen in AD brain (Fig.
2A,B) as reported previously (Irizarry et al., 1997). Both meningeal and parenchymal blood vessels
in the brain also showed progressive A accumulation. The A in
blood vessels of the aging Tg2576 brain was preferentially labeled by
the BA-27 antibody to A40 (data not shown).
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Figure 2.
Immunohistochemistry of AD (A,
B) and aging Tg2576 (C-P) brains.
Serial sections of temporal cortex from AD brain and Tg2576 brains were
labeled with BA-27, which is specific for A40, or BC-05, which is
specific for A42. The age (months) of the Tg2576 brains is shown
above the serial sections stained with BA-27 (top
panel) and BC-05 (bottom panel).
A40 (stained by BA-27) and A42 (stained by BC-05) are detected as
dense microdeposits from 8 (M, N)
to 12 (I, J) months. From 15 to 23 months (C-H), A deposits increase in number
and size and are detected both as cored plaques labeled by both BC-05
and BA-27 and as diffuse plaques, which are selectively labeled by
BC-05. Sections are 5-µm-thick. Scale bar, 15 µm.
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Most A in normal mouse brain requires SDS for extraction
The brains of Tg2576 mice and nontransgenic littermates were
extracted sequentially in TBS, 1% Triton X-100, 2% SDS, and 70% FA
as described in Materials and Methods. The A42 and A40 in the
supernatants produced by this four-step extraction were analyzed by
3160/BC-05 and 3160/BA-27 ELISAs, respectively (Table
1). In normal nontransgenic and young
transgenic mouse brains in which there is no A deposition, most A
required SDS for solubilization and surprisingly little was extracted
into TBS or Triton X-100. In 6 month Tg2576 mice, for example, the
percentages of total A extracted into TBS, Triton X-100, and SDS
were 5% (0.7% A42, 4% A40), 28% (7% A42, 21% A40),
and 67% (20% A42, 47% A40), respectively (Table 1). As
expected, there was essentially no insoluble A in normal mouse brain
that required formic acid for solubilization.
A that requires formic acid for extraction appears at 6-8
months in Tg2576 brain and increases with aging to a level like that
seen in AD brain
It is well established that much of the A deposited as amyloid
in AD brain is resistant to SDS extraction and requires formic acid for
solubilization (Roher et al., 1993). In the brains of 21 month Tg2576
mice, in which there are numerous amyloid-containing senile plaques
(Fig. 2), there also was abundant A that required formic acid for
solubilization (Table 1). To track the time course of formation of this
insoluble (formic acid-requiring) A in Tg2576 mice, we used a
simplified two-step extraction procedure in which brains were first
extracted in 2% SDS and then in 70% formic acid. The results of our
analysis of the resultant supernatants using 3160/BC-05 (A42) and
3160/BA-27 (A40) ELISAs are shown in Figure 3. A42 and A40 first appeared in
the FA fraction at ~7 months (Fig. 3C,D). By
8-9 months, FA-requiring A42 and A40 appeared unequivocally in
the brain of every Tg2576 mouse examined. Between 6 and 12 months,
FA-requiring A42 and A40 increased exponentially, and both forms
continued to increase substantially from 12 to 23 months, reaching
levels like those seen in the AD brain (Gravina et al., 1995).
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Figure 3.
A in aging Tg2576 brain. A42
(A, C) and A40 (B,
D) were analyzed in Tg2576 brains sequentially extracted
in 2% SDS (A, B) and 70% formic acid
(C, D). The ELISA assay was 3160/BC05 for
A42 and 3160/BA27 for A40. Note that the y-axes
are logarithmic and that there was no detectable A40 or A42 in
the formic acid extract of young (2-5 months) Tg2576 mice.
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Remarkably, the unequivocal biochemical change that occurred between 6 and 10 months was accompanied by minimal immunocytochemical evidence of
A deposition, although isolated, cored plaques were observed in
virtually every section on careful inspection (Fig. 2). Thus,
biochemical assessment of brain A is a sensitive way to quantitate
the early AD-like changes that occur in Tg2576 mice.
The A initially extracted into SDS decreases when FA-requiring
A first appears
The increase in FA-requiring A that occurred between 6 and 10 months was accompanied by a small decrease in the A extracted initially into SDS (Fig. 3A,B).
Both declines were significant when analyzed by Spearman's rank
correlation (A42, p = 0.02; A40,
p = 0.003). In the four-step extraction (Table 1), in
which TBS, Triton X-100, and SDS fractions were obtained before formic acid extraction, it was the TBS and Triton X-100 fractions that significantly decreased as FA-requiring A appeared (Mann-Whitney comparison of 5M vs 7M; p = 0.009 for TBS A42, TBS
A40, and Triton A40; p = 0.05 for Triton
A42).
The percentage of A in SDS and FA extracts of depositing Tg2576
and AD brain is influenced by the extraction procedure
The total amounts of A42 and A40 extracted by two-step and
four-step protocols are virtually identical, although slightly more
A is extracted with the four-step method. In Tg2576 and AD brains,
in which there is A deposition, the relative amounts of A
extracted into SDS and FA with the two methods are considerably different. A much higher percentage of total A is extracted into SDS
in the four-step method. The relevant percentages in 21 month Tg2576
are 66.2% SDS versus 33.1% FA with the four-step extraction and
10.6% SDS versus 89.4% FA with the two-step procedure, and in AD
brains, 81.5% SDS versus 18.2% FA with four-step and 13.8% SDS
versus 86.2% FA with two-step extraction.
Compared with AD, Tg2576 brain has much less A that is truncated
or modified at its N terminus
In our previous examination of insoluble A in 27 AD brains
(Gravina et al., 1995), A42 predominated in 70% and, in 33%, there
was essentially no A40 deposited. In the 30% in which A40 was
the predominant species deposited, there was typically prominent congophilic angiopathy. As shown in Table
2, the amounts of total A40 and A42
deposited in Tg2576 brain most closely resemble the AD brains in which
A40 deposition predominates. In our previous study (Gravina et al.,
1995), ELISAs for Ax-42 (BC-05/4G8) and Ax-40 (BA-27/4G8)
showed that most of the A in AD brain is N-terminally truncated or
modified. This is not the case in Tg2576 mice (Table 2). In the brains
of the oldest mice examined (21-23 month), the amount of insoluble
A detected by BAN-50 or 3160 capture was 95% of that detected with
the BC-05/4G8 and BA-27/4G8 assays that can detect additional
modified-truncated forms of A. This indicates that, in Tg2576
brain, only 5% of insoluble A is N-terminally truncated or
modified, whereas in AD brain, the corresponding percentage is 69-85%
(Table 2). Thus, it appears that N-terminally modified or truncated
forms of A, which predominate in AD brain (Saido et al., 1995, 1996;
Hosoda et al., 1998), are minor species in Tg2576 brain that only begin
to appear in very old mice.
The results obtained by sandwich ELISAs are confirmed
by immunoblotting
Immunoblotting of the SDS and FA fractions obtained by two-step
extraction (Fig. 4A-D)
gave results that were consistent with the results from sandwich ELISAs
performed on the same extracts (Fig. 3). SDS-insoluble 4 kDa A
labeled by both BAN-50 (Fig. 4B) and 4G8 (Fig.
4D) appeared at 6-8 months and increased
substantially by 10 months. To analyze the SDS-extractable A, the
SDS extracts were immunoprecipitated with 3160, a rabbit polyclonal
antibody to A1-40, and the immunoprecipitate was analyzed by
immunoblotting with BAN-50 (Fig. 4A) or 4G8 (Fig.
4B). As expected from ELISA analysis (Fig.
3A,B), the total 4 kDa A
extracted into SDS decreased slightly between 6 and 8 months before
beginning to increase substantially at 10 months (Fig.
4A,C, arrowhead). APP
CTF, which contains full-length A, was detected by both BAN-50
(Fig. 4A, arrow) and 4G8 (Fig.
4C, arrow) in the SDS extracts but not in the
formic acid extracts (Fig. 4B,D).
As expected, CTF in Tg2576 brain showed no change with aging.
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Figure 4.
Analysis by immunoblotting of early A
deposition in Tg2576 brain. A in the SDS (A,
C) and formic acid (B, D)
extracts from 4-10 month Tg2576 brains was analyzed on immunoblots
labeled with BAN-50 (anti-A1-16) (A,
B) and 4G8 (anti-A17-24) (C,
D). Proteins were separated on 10-20% Tricine gels,
and each lane shows the A in 40 µl of the formic
acid or SDS extract as described in Materials and Methods.
Immunoblotting was first performed with BAN-50. The blots were then
stripped and reblotted with 4G8. Note that SDS-soluble A decreases
transiently at 8 months, when SDS-resistant, formic acid-soluble A
appears. The arrows identify CTF, and the
arrowheads identify A.
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The full-length, unmodified A in AD compared with 21 month Tg2576
brain was analyzed by immunoblotting two-step SDS and FA extracts (Fig.
5A-D). In the FA extracts,
BAN-50 (anti-A1-16) detected abundant full-length 4 kDa A in the
three Tg2576 brains but almost nothing in any of the four AD brains
examined (Fig. 5B), a result that is in good agreement with
the ELISA data shown in Table 2. In contrast, 4G8 (anti-A17-24),
which recognizes both full-length A and A that is N-terminally
truncated or modified, detected substantial amounts of A in the FA
extract of both Tg2576 and AD brains (Fig. 5D). Similarly,
in SDS extracts, BAN-50 labeled far more 4 kDa A in Tg2576 than in
AD brains (Fig. 5A), whereas 4G8 labeled large amounts of
A in both Tg2576 and AD brains (Fig. 5C). It is
noteworthy that the A detected by 4G8 appeared to be slightly
smaller in AD than in Tg2576 brain (Fig. 5C, and to a lesser
extent D), consistent with the A in AD brain being
truncated at its N terminus.
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Figure 5.
Specific forms of A in Tg2576 and AD brains.
A-D, Immunoblot analysis of SDS (A,
C) and formic acid (B, D)
extracts of 21M Tg2576 mouse brains and AD brains labeled with BAN-50
(A, B) or 4G8 (C,
D). Two microliters of the SDS or FA extract (dried and
resuspended) were directly added to each lane; proteins
were separated on 10-20% Tricine gels. E,
F, Immunoblot analysis of SDS (E)
and formic acid (F) extracts of AD and 23M Tg2576
brains labeled with the following: 4G8, which detects both N-terminally
modified and unmodified A; anti-AN1(D), which detects the
unmodified N terminus; anti-N1(iD), which recognizes isomerized forms
(L-iso-Asp) of AN1; anti-N1(rD), which detects
stereoisomerized forms (rectus Asp) of AN1; anti-AN3(pE), which
detects forms beginning with pyroglutamate at position 3; or
anti-AN11(pE), which recognizes forms beginning with pyroglutamate
at position 11. The A in 10 µl of SDS or FA extracts was
examined on each lane. In the SDS extracts, A was
immunoprecipitated with the indicated antibody as described in
Materials and Methods before separation and immunoblotting with the
same antibody. In the FA extracts, A was dried and resuspended as
described in Materials and Methods before separation and
immunoblotting. Proteins were separated on 16% Tricine gels.
G-L, Time course of accumulation of formic acid-soluble
A in Tg2576 brains (8M-23M) and AD brains labeled with 4G8
(G), anti-N1(D)
(H), anti-N1(rD)
(I), anti-N3(pE)
(J), BA-27 (K), or BC-05
(L). The A in 10 µl of formic acid extract
was examined on each lane, and proteins were separated
on 16% Tricine gels.
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The immunoblots in Figure 5A-D show that 4G8 and BAN-50
label A oligomers that are present in both SDS and FA fractions from 21 month Tg2576 brains. Similar oligomers are observed in the SDS and
FA extracts of AD brains. As shown in Figures 4A-D
and 5G, these oligomers are not detectable at 8 months but
are definitely present by 12 months and increase substantially
thereafter. Even with prolonged exposure, we have been unable to
demonstrate oligomers in either SDS or FA extracts from brains in the
critical 6-8 month period when A aggregation begins.
BA-27, a monoclonal antibody that specifically detects the C terminus
of A40, and BC-05, which is specific for the C terminus of A42,
were used to analyze 6-23 month Tg2576 by immunocyochemistry (Fig. 2)
and immunobloting (Fig. 5K,L). This
analysis confirmed that As terminating at both A40 and A42
accumulate in aging Tg2576 brain with A40 predominating (Figs. 2,
5K,L). In contrast, virtually all
A terminated at A42 in the two AD brains examined (Fig. 5
K,L), as occurs in ~33% of AD
cases (Gravina et al., 1995).
N-terminally modified As accumulate in aging Tg2576 brain
Antibodies that detect specific N-terminal modifications of A
(Saido et al., 1995, 1996) were used to analyze 23 month Tg25676 brain
by immunoblotting (Fig. 5E-J) and
immunocytochemistry (Fig. 6). Comparison
of 4G8, which detects virtually all A regardless of modification,
and anti-N1(D), which specifically detects unmodified A, confirmed
that most A in AD brains is N-terminally modified or truncated (Fig.
5G,H), whereas most A in 8-23 month
Tg2576 brains is unmodified (Fig. 5G,H and 6,
N1(D) versus 4G8). Isomerized A (N1iD) was labeled well in FA
extracts of AD brain (Fig. 5F) and accumulated at a
low level in 8-23 month Tg2576 brain (Fig. 6), but the amount of A
in Tg2576 brain was far less than in AD brain (Fig.
5F). Stereoisomerized A (N1rD) accumulated with aging between 8 and 23 months in Tg2576 brain and, at 23 months, was
intensely labeled in Tg2576 brain (Figs. 5I, 6) as it was in
AD brain (Fig. 5I). AN3-pyroglutamate (N3(pE)) is
a major form of A in AD brain but a minor form in aged Tg2576 brain
(Fig. 5J). Histochemical analysis (Fig. 6) showed
that AN3-pyroglutamate (N3(pE)) appears late in Tg2576 brain,
accumulating between 16 and 23 months. AN11-pyroglutamate was not
detected in Tg2576 brains but was definitely detected in AD brain (Fig.
5F). In both Tg2576 and AD brains, the N-terminally
modified As were more evident in the SDS-insoluble A found in FA
extracts (Fig. 5F) than in SDS extracts (Fig.
5E). Collectively, these findings indicate that, with the
exception of AN11-pyroglutamate, N-terminal modifications or
truncations of A, which are thought to make A more insoluble in
human brain, also occur in the aging Tg2576 brain. With the exception
of stereoisomerized A, all of these modified forms are, however, far
less abundant in Tg2576 than in AD brain.
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Figure 6.
Immunohistochemical analysis of modified forms of
A in aging Tg2576 brain. Serial sections (5 µm) of Tg2576
cerebral cortex were labeled with anti-N1(D),
anti-N1(iD), anti-N1(rD), anti-N3(pE), and 4G8. The age of the mouse
brain analyzed is shown at the top of each set of serial
sections. Scale bar, 17 µm.
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CSF and plasma A decline as A is deposited in the
Tg2576 brain
It is well established that A42 declines in the CSF of patients
with typical late onset AD (Motter et al., 1995; Nitsch et al., 1995;
Kanai et al., 1998; Andreasen et al., 1999), and this decrease could
conceivably cause plasma A42 to decline because CSF A, which has
a high concentration relative to plasma A, is normally cleared into
blood (Ghersi-Egea et al., 1996). To determine whether CSF and plasma
A decline in Tg2576 mice as A42 and A40 are deposited in the
brain, we analyzed brain (Fig. 7A,B),
CSF (Fig. 7C, D), and plasma (Fig.
7E,F) A42 and A40 in
parallel. This analysis showed that, in aged Tg2576 brain, as in human
AD brain, there is a decline in CSF A42. This decline in CSF A42
(p = 0.03) occurred in parallel with the marked
increases in brain A42 and A40 that occur between 9 and 23 months, and it was accompanied by a decline in CSF A40, although
this decrease did not achieve significance. Remarkably, plasma A42
(p = 0.008) and A40 (p = 0.006) both showed highly significant decreases (Fig.
7E,F) that paralleled the
marked accumulation of brain A and the decline in CSF A that
occurred between 6 and 23 months. To be sure that the declines in CSF
and plasma A that occur in aging Tg2576 mice are linked to A
deposition and not to aging alone, A42 and A40 were analyzed in
CSF (Fig. 7G, H) and plasma (Fig.
7I, J) of aging
nontransgenic littermates (Fig. 7G, H). In
these nontransgenic mice, there was no suggestion of a decline in A
because A42 and A40 both showed a slight upward trend in CSF
(Fig. 7G,H) and plasma (Fig.
7I,J) with aging.
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Figure 7.
CSF and plasma A in Tg2576 mice decline as A
is deposited in the brain. Total brain A42 (A)
and A40 (B) were assayed by 3160/BC05 or
3160/BA27 ELISAs, respectively; see also Figure 2 and Table 2. Tg2576
CSF and plasma A42 (C, E) were assayed
by BNT77/BC05 ELISA, and Tg2576 CSF and plasma A40
(D, F) were assayed by BAN50/BA27
ELISA. Both nontransgenic (NTg) CSF and plasma A42
(G, I) and A40
(H, J) were assayed with BNT77
capture. The number of Tg2576 CSF samples assayed for the four time
groups are 9, 9, 16, and 11, totaling 45. The number of Tg2576 plasma
samples assayed for the four time groups are 30, 18, 64, and 19, totaling 131. The decline for CSF A42 is significant
(p = 0.02), and the declines for plasma
A40 and A42 are highly significant (A42, p = 0.008; A40, p = 0.006; Spearman's rank
correlation for the 6-23 month age range). The number of nontransgenic
CSF samples assayed for the three time groups are 2, 2, and 6, totaling
10. The number of nontransgenic plasma samples assayed for the four
time groups are 7, 6, 12, and 6, totaling 31.
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DISCUSSION |
As Tg2576 animals age, A is altered beginning at 6-7 months
with the appearance and subsequent increase of A in the FA-extracted fraction. As insoluble (FA-requiring) A appears at 6-9 months, the
remaining A in the brain (SDS-extracted in two-step, or TBS- and
Triton-extracted in four-step) decreases slightly, suggesting that it is converting to an insoluble form. The definite biochemical change that occurs by 10 months in Tg2576 mice is accompanied by
minimal histological evidence of A deposition, although single plaque cores can be observed in many sections. From studies of trisomy
21 brains (Iwatsubo et al., 1994), it is generally believed that, in
human AD, A deposition begins with the formation of diffuse plaques.
In Tg2576 brains, diffuse plaques are not observed in appreciable
number until 12 months, 4 months after biochemically detectable
alterations in A have begun in every animal. Thus, there is an early
period from 6 to 10 months in Tg2576 mice in which insoluble A
appears accompanied only by rare cored plaques.
Support for the view that there may be a similar early period in human
brain comes from a study by Funato et al.(1998), who used the same
ELISA system used here. They report that both SDS-dissociable and
-insoluble forms of A accumulate in human cortex, that insoluble A correlates with amyloid load, and that its biochemical detection precedes plaque formation. They calculate that immunodetection of A
in human cortex requires 400 pmol/gm insoluble A42 (or 200 pmol/gm
in hippocampus) (Funato et al., 1998). These values are similar to
those in 10-month-old Tg2576 mice, the time when plaques become evident.
Our immunohistochemical and biochemical analyses show that, from 10 to
21 months, there is a rapid increase in both diffuse and cored plaques
in Tg2576 brain and a coordinate marked increase in SDS-dissociable and
FA-requiring A, with both the biochemical and histological changes
rising to levels like those observed in human AD. In most AD patients,
very little A40 is deposited in the brain, but in ~33%,
extraordinary amounts of A40 are deposited, and most of these
patients show substantial amyloid angiopathy (Gravina et al., 1995).
The Tg2576 model is like this latter group of AD patients in that there
is marked congophilic angiopathy and the deposition of a large amount
of A40. It is not clear why large amounts of A40 are deposited in
the Tg2576 model. It may be that more than one factor contributes and
that some combination of species, strain, promoter, expression level,
and mutated transgene causes the large amount of A40 deposition in
this model.
Much of the A in AD brain is N-terminally truncated or modified
(Saido et al., 1995, 1996; Hosoda et al., 1998), and it has been
suggested that the formation of SDS stable A oligomers may be an
early event in AD (Enya et al., 1999). Because N-terminally truncated
or modified A [especially forms beginning with pyroglutamate at
position 3 (3pE)] and oligomers are resistant to proteolysis (Saido et al., 1996; Kuo et al., 1998), these modifications of A are
thought to be important for amyloid deposition. Our studies indicate
that, in Tg2576 brain, it is full-length, unmodified A that becomes
insoluble initially. After this early period, oligomers and most
modified forms (e.g., forms beginning at 3pE) appear, but at levels far
below those observed in AD brain. It is not clear whether this late
development and relative paucity of modified or oligomeric forms
reflects the much shorter time over which A aggregates in Tg2576
brain or is attributable to fundamental biochemical differences
between the human and mouse brain. One intriguing possibility is that
the A modifications observed in the AD brain may play an important
pathogenic role and that the relative paucity of these forms in Tg2576
brain may account for the minimal neurofibrillary pathology and
neuronal loss observed in Tg2576 brain.
In Tg2576 mice, the APPK670N,M671L transgene is
expressed at highest level in the brain, but there is also substantial
expression in other organs (Fig. 1), although expression is driven by
the hamster PrP promoter. Thus, in Tg2576 mice as in human subjects, plasma A is likely to be derived from both peripheral organs and
brain, where A may enter the bloodstream either through the normal
flow of CSF or by directly crossing CNS endothelium. In a previous
study of human plasma (Scheuner et al., 1996), we showed that the
Swedish APPK670N,M671L mutation increases both
A42 and A40 and that other FAD-linked APP, PS1, and PS2 mutations
selectively increase A42. In that report, we suggested that cerebral
A deposition in FAD occurs because of an increase in CNS A that
develops as part of a generalized genetic effect that also increases
plasma A. We emphasized that A deposition in FAD probably does
not occur as a direct effect of increased plasma A. We have reported previously a transgenic mouse line (NOR 0304) that expresses an A
containing CTF of APP (Kawarabayashi et al., 1996). Because expression
in NOR 0304 is driven by the -actin promoter, the transgenic APP
CTF is expressed in all organs, and expression is much higher in many
peripheral organs than in brain. Significantly, plasma A is even
higher in NOR 0304 (1400 pM A42; 5600 pM
A40) than in Tg2576, but NOR 0304 mice do not develop
age-dependent A deposition in brain. Thus, elevated plasma A can
be a good indicator that deposition will occur when it occurs as part
of a generalized response in human subjects or in transgenic mice in
which expression is under the control of a promoter such as PrP, which
causes expression to be highest in brain. Elevated plasma A does not
directly drive deposition, however, because NOR 0304 mice, which
have a -actin promoter that causes expression to be highest in the
periphery, show no deposition, although they have higher plasma A
levels than the Tg2576 line.
We have shown recently that human plasma A42 and A40 increase
with aging over age 65 and that plasma A42 and A40 are heritable traits that are increased in first degree relatives of patients with
typical late onset AD (Younkin et al., 1998). These findings suggest
that, in typical late onset AD as in early onset FAD, elevated
plasma A may be associated with the development of AD. If so, one
would expect plasma A to be elevated in typical late onset AD. Our
initial analysis of typical AD patients (Scheuner et al., 1996) showed,
however, that very few AD patients have high plasma A42 when
compared with age-matched controls. Other published studies of plasma
A in AD have shown no change or a slight increase in plasma A42
(Iwatsubo, 1998; Matsubara et al., 1999). Because it is well
established that CSF A42 decreases in AD, one way to account for
these negative results is to postulate that plasma A42 also declines
as AD develops. If so, then analysis of symptomatic late onset patients
could miss many patients whose disease was initiated by increased A
in the presymptomatic period.
To test the hypothesis that plasma and CSF A both decline as A is
deposited in the brain, plasma, CSF, and brain A were analyzed
coordinately in aging Tg2576 mice. This analysis showed that,
coincident with the marked deposition of A42 and A40 in brain,
there is not only a decline in CSF A but also a substantial, highly
significant decrease in plasma A42 and A40. If this also occurs
in human subjects, then declining plasma A could be a useful marker
for subjects in whom there is cerebral A deposition and who are,
therefore, at risk for AD. Similarly, elevated plasma A could be an
excellent premorbid biomarker for AD, although it is not useful as a
diagnostic marker, if it identifies those who are destined to deposit
A and those who are in the early stages of deposition.
| |
FOOTNOTES |
Received Sept. 5, 2000; revised Oct. 23, 2000; accepted Oct. 30, 2000.
This work was supported by National Institutes of Health Grant AG15453.
We thank Dennis Dickson for help with immunocytochemistry, Virginia
Phillips and Linda Rousseau for cutting sections, and Blaze Birinyi for
technical assistance.
Correspondence should be addressed to Steven G. Younkin, Mayo Clinic
Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224. E-mail:
younkin{at}mayo.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
