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The Journal of Neuroscience, March 1, 2001, 21(5):1444-1451
 -1-Antichymotrypsin Promotes  -Sheet Amyloid Plaque
Deposition in a Transgenic Mouse Model of Alzheimer's Disease
Lars N. G.
Nilsson1, 2, 3,
Kelly R.
Bales5,
Giovanni
DiCarlo4,
Marcia N.
Gordon4,
Dave
Morgan4,
Steven M.
Paul5, and
Huntington
Potter1, 2, 3
1 Suncoast Gerontology Center, 2 Department
of Biochemistry and Molecular Biology, 3 Moffitt Cancer
Center, and 4 Department of Pharmacology, College of
Medicine, University of South Florida, Tampa, Florida 33612, and
5 Neuroscience Discovery Research, Lilly Research
Laboratories, Indianapolis, Indiana 46285
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ABSTRACT |
 1-Antichymotrypsin
(ACT), an acute-phase inflammatory protein, is an integral component of
the amyloid deposits in Alzheimer's disease (AD) and has been shown to
catalyze amyloid  -peptide polymerization in vitro. We
have investigated the impact of ACT on amyloid deposition in
vivo by generating transgenic GFAP-ACT-expressing mice and
crossing them with the PDGF-hAPP/V717F mice, which deposit amyloid in
an age-dependent manner. The number of amyloid deposits measured by
Congo Red birefringence was increased in the double ACT/amyloid
precursor protein (APP) transgenic mice compared with transgenic
mice that only expressed APP, particularly in the hippocampus where ACT
expression was highest, and the increase was preceded by elevated total
amyloid -peptide levels at an early age. Our data demonstrate that
ACT promotes amyloid deposition and provide a specific mechanism by
which inflammation and the subsequent upregulation of astrocytic ACT
expression in AD brain contributes to AD pathogenesis.
Key words:
1-antichymotrypsin; Alzheimer's disease; amyloid
deposition; inflammation; transgenic mice; amyloid -peptide; Congo
Red
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INTRODUCTION |
1-Antichymotrypsin
(ACT), an acute-phase inflammatory protein, has been found to be an
integral component of the amyloid core filaments of Alzheimer's
disease (AD) brain and to be overexpressed in astrocytes surrounding
amyloid plaques (Abraham et al., 1988 ; Pasternack et al., 1989; Koo et
al., 1991). ACT has been shown to be only associated with amyloid
deposits containing amyloid -peptides (A),suggesting the presence
of a specific molecular interaction between ACT and AD plaque
components, likely the amyloid -peptides themselves (Abraham et al.,
1990). This idea has been further reinforced by in vitro
experiments showing high-affinity binding of ACT to amyloid
-peptides (Potter et al., 1992; Fraser et al., 1993; Hughes et al.,
1998). The term "pathological chaperone" has been coined to
describe amyloid-associated proteins, such as ACT or apolipoprotein E
(ApoE), that have the potential ability to favor conformational
transition of A peptides into a -pleated sheet structure and
thereby promote their deposition into senile plaques (Wisniewski and
Frangione, 1992).
In vitro studies have demonstrated that ACT and ApoE
promote the assembly of the A1-42 peptide into amyloid
filaments (Ma et al., 1994, 1996; Sanan et al., 1994; Wisniewski et
al., 1994; Janciauskiene et al., 1996, 1998). However, using the
A1-40 peptide or an equimolar ratio, ApoE and ACT have
also been reported to inhibit A filament formation (Fraser et al.,
1993; Eriksson et al., 1995; Evans et al., 1995). Although the
epidemiological evidence and their overexpression in AD brain suggest
that ACT and ApoE are more likely to be amyloid promoters, in
vivo experiments would greatly aid in clarifying their
pathophysiological role.
Recent studies have supported the hypothesis that ApoE is a
pathological chaperone. The frequency of thioflavine S-positive A deposits in the human amyloid precursor protein
(hAPP)/V717F overexpressing transgenic mouse strain has been
found to be ApoE gene dose-dependent, and no such deposition occurs in
the absence of the murine ApoE (Bales et al., 1997). Furthermore, human
ApoE4 has been shown to restore thioflavine S-positive A deposition and neuritic plaque formation at an accelerated rate compared with
human ApoE3 (Holzman et al., 2000), reflecting the epidemiological data
on neuritic plaque formation in AD brain (Rebeck et al., 1993;
Schmechel et al., 1993).
A similar set of in vivo experiments should clarify the
role of ACT in amyloid formation. Unfortunately, the knock-out approach is not possible, because mice lack an ACT homolog with respect to its
A peptide interacting ability. The human ACT gene shares the highest
sequence homology with the family of murine Spi-2 protease inhibitors
(Inglis and Hill, 1991). However, the reactive center loop of these
protease inhibitors, which is the likely A peptide interacting
region in the human ACT protein (Potter et al., 1992; Janciauskiene et
al., 1996, 1998), is hypervariable and differs substantially from that
of the human ACT protein (Hill and Hastie, 1987). Here we report on a
new transgenic mouse and on experiments pursued to determine the
function of ACT as an amyloid promoter in vivo.
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MATERIALS AND METHODS |
Construction of transgenic mice. A cDNA fusion
construct with a 6 kbp mouse glial fibrillary acidic protein
(GFAP) promoter and 100 bp of the 5'-end of the GFAP gene attached to
the human ACT cDNA clone was constructed to direct human ACT synthesis
in activated mouse astrocytes. The unique
ApaI-SalI fragment ( 107 to +87) in the
GFAP-secreted alkaline phosphatase expression vector (Sarid,
1991) was subcloned into the Litmus-38 vector, and the two existing ATG
start codons in the GFAP transcript were altered to ATC and AGG,
respectively, with PCR-based site-directed mutagenesis. The original
ApaI-SalI fragment was then replaced with the
mutant fragment. A 1.5 kbp full-length human ACT cDNA clone was
excised with EcoRI from the pGEM4 vector (Abraham et al.,
1988) and blunt-ended. SalI linkers were attached, and the
fragment was subcloned into the modified GFAP expression vector
construct. The noncoding 3'-untranslated region of the mRNA was
derived from the rat preproinsulin II gene, which provided a 3'
intronic region and a polyadenylation [poly(A)] site. The final
construct was sequenced to confirm that translation initiation should
occur solely at the start codon in the ACT cDNA. The DNA was linearized
with EcoRV and ScaI, and the 10 kbp fragment was
separated on a low melting point agarose gel, purified with -agarase according to manufacturer's instructions (FMC
Bioproducts, Rockland, ME), and injected into pronuclei of FVB/N
zygotes at the Core Transgenic Mouse Facility (Brigham and Women's
Hospital, Harvard Medical School, Boston, MA). Injected zygotes were
transferred to pseudopregnant females. Heterozygous ACT-transgenic mice
were identified with two PCR primer pairs located in different parts of
the construct. Mice from one of these founder lines (#8784) were
crossed with homozygous PDGF-hAPP/V717F mice (Games et al., 1995), and
all offspring were screened with PCR for the GFAP-ACT and the
PDGF-hAPP gene constructs. In addition, the presence of proper
ACT protein expression in astrocytes of the PDGF-hAPP/V717F (+/)ACT(+/) mice and its absence in the PDGF-hAPP/V717F
(+/)ACT(/) mice was verified by ACT immunohistochemistry in all
of the pathologically examined animals.
Northern blot. Total RNA was extracted with RNA
Stat-60-kit (Tel-Test), and poly(A+)
mRNA was selected with Dynabeads
Oligo(dt)25 (Dynal, Great Neck, NY). The RNA
samples were heat-denatured (70°C) in 50% formamide, 2.2 M formaldehyde, 1 M
4-morpholinepropanesulfonic acid buffer, pH 7.0, and separated
by electrophoresis on a 1.0% agarose-formaldehyde gel, blotted onto
Hybond-N+ filters, and baked for 2 hr at
80°C. Filters were prehybridized overnight and hybridized with
P32-labeled hACT and hGAPDH-cRNA probes
(Rogers et al., 1999) in 50% formamide, 120 mM
Tris-HCl, 8 mM EDTA, 600 mM
NaCl, 1% nonfat dry milk powder, and 1% SDS. The filters were
subsequently washed at a stringency of 0.1× SSC, 0.1% SDS at 85°C,
and exposed to autoradiographic Hyper-MP film (Amersham Pharmacia
Biotech, Arlington Heights, IL).
Immunoprecipitation and Western blot analysis. Brain tissue
from a transgenic mouse was homogenized (2 × 10 strokes on ice) in 10 (v/w) STEN/Lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.2% BSA,
50 µg/ml PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 2 µg/ml pepstatin A). The homogenate was centrifuged at 50,000 × g for 1 hr at 4°C. The supernatant was recovered, 2 µl
of polyclonal ACT antibody (AXL-145; Accurate Chemicals, Westbury, NY)
was added, and the mixture was allowed to incubate for 2 hr at 4°C.
We added 25 µl of equilibrated protein A-Sepharose slurry
(Pierce, Rockford, IL), and the mixture was allowed to incubate
overnight at 4°C. The antibody-antigen complex was retrieved by
centrifugation at 15,000 × g for 10 min and
subsequently washed six times (50 mM Tris-HCl,
150 mM NaCl, 6 mM EDTA, pH
7.4, and 2.5% Triton X-100) and finally twice with 50 mM Tris-HCl and 6 mM EDTA,
pH 7.4. The final pellet was resuspended in 20 µl of 2×
Laemmli's buffer and boiled for 10 min, centrifuged 15,000 × g for 1 min, and the supernatant was loaded onto a 8%
polyacrylamide gel (Novex). The samples were transferred to an
Immobilon-P filter (Millipore, Bedford, MA), blocked with 5% nonfat
dry milk powder in 1× TTBS buffer (1× TTBS is 0.1 M Tris-HCl, pH 7.5, 0.9% NaCl, and 0.1% Tween
20), incubated with primary monoclonal ACT antibody (dilution, 1:2000;
catalog #178218; Calbiochem, La Jolla, CA) for 1 hr at room temperature
(RT) in 1× TTBS buffer, washed three times for 10 min with TTBS
buffer, blocked with 5% nonfat dry milk powder in 1× TTBS, incubated
with secondary goat anti-mouse antibody (dilution 1:5000; catalog
#31434; Pierce) in blocking buffer solution for 30 min at RT, washed
four times for 10 min each in 1× TTBS buffer, incubated with
ECL reagents (Pierce) for 5 min, and finally exposed to ECL Hyperfilm
(Amersham Pharmacia Biotech).
Histology and immunohistochemistry. Mice were anesthetized
with Nembutal (1 mg/10 gm body weight) and intracardially perfused with
0.9% NaCl (25 ml) and then with 50 ml 4% paraformaldehyde in 1×
Sorenson's phosphate buffer. Dissected brains were allowed to immerse
in the same fixative solution overnight at 4°C and were further
cryoprotected by sequential overnight incubation in 10, 20, and 30%
(w/v) sucrose in 0.1× Sorenson's phosphate buffer. The brains were
then frozen on a temperature-controlled freezing stage, coronally
sectioned (25 µm) on a sliding microtome, and the sections were
stored in 1× PBS with 10 mM
NaN3 until mounted on slides. The mounted
sections were preincubated with 0.3%
H2O2 in 50% Dako-block
(catalog #X0909; Dako, Carpinteria, CA) for 15 min to block endogenous
peroxidase activity, permeabilized with 0.4% Triton X-100 in 1× PBS,
pH 7.4, and incubated with Dakoblock (10 min) to reduce nonspecific
antibody staining. Primary antibodies used were rabbit anti-ACT
(AXL-145; dilution 1:1000; Accurate), mouse anti-A (6E10; dilution
1:5000; Senetek), and mouse anti-GFAP (G-A-5; dilution 1:400; Sigma,
St. Louis, MO). Tissue sections were stained with the Vectastain ABC
Elite or MOM kits (Vector Laboratories, Burlingame, CA). Secondary
antibodies were anti-rabbit IgG (BA-1000; 1:300; Vector) or anti-mouse
IgG as provided with the kits (Vector). Incubations with primary
antibodies were for 1 hr at RT and with secondary antibodies for 30 min
at RT. We added 50% Dakoblock solution to the ABC reagent to lower the
nonspecific signal. The immunostaining was developed with DAB (0.4 mg/ml), 0.03% H2O2 in
NiSO4-acetate buffer, pH,6.0 (9 mg/ml with
respect to Ni2+), or with Vector DAB or
Vector SG substrate kits. Congo Red staining was accomplished by
initially hydrating the section in H2O for 30 min, incubating in saturated alcoholic sodium chloride solution [4%
(w/v) NaCl in 80% EtOH] that was alkalinized with 10 mM NaOH (final concentration) before use.
Sections were stained in a 0.2% Congo Red (Sigma) solution that had
been equilibrated in saturated alcoholic sodium chloride solution by
stirring overnight, filtered (Whatman1 filter), and alkalinized with 10 mM NaOH (final concentration) before use. To
induce gliosis and increase ACT expression, the same mice (3-month-old
mice) were inhalation-anesthetized with Isoflurane, placed in a
sterotactic instrument, and a Hamilton syringe was inserted (bregma
coordinates: posterior 2.7 mm, lateral ±2.5 mm, ventral 3 mm). The
animals were killed 3 d later.
Image analysis. Six equally spaced tissue sections (bregma
1.06 to 2.30 mm; Franklin and Paxinos, 1996) from each animal were
used for the quantitative evaluations. The part of cerebral cortex
examined was defined as that laterally extending to a perpendicularly drawn line from the apex of the hippocampal pyramidal cell layer in the
CA3 region of the hippocampus. Hippocampal area measurement was
according to regular anatomical definitions. The sections were examined
in a Nikon Microphot-FX-microscope at 200× magnification at a constant
predefined light setting, and video images were captured with a color
CCD camera. Each Congo Red-positive amyloid plaque was circled at 200×
magnification, and its location was noted onto an anatomic atlas. The
captured image was segmented with respect to threshold settings for
hue, saturation, and luma that had been specified before analysis to
distinguish specific signals from background. The area occupied by
amyloid within each circle, as defined by the image segmentation, was
then quantitated. This predefined segmentation was subsequently used
throughout the analysis without operator editing to determine the area
of each plaque structure (Oncor). The total measurement area was quantified at 20× magnification, and the amyloid load was expressed as
area fraction (= stained areatot/measured
areatot). The Congo-positive plaques counted were
defined as those objects displaying gold-green bifringence under
crossed polarized light and having their perimeter located outside one
radius from the center of any other adjacent Congo Red-positive plaque
structure. The complete plaque size distribution analysis were based on
(~300 and ~175 Congo Red-positive plaques/brain region) in the
PDGF-hAPP/V717F(+/)ACT(+/) and the PDGF-hAPP/V717F(+/)ACT(/)
mice, respectively.
A ELISA measurements. Hippocampi from
3-month-old PDGF-hAPP/V717F(+/)ACT(+/) mice (n = 10) and PDGF hAPP/V717F(+/)ACT(/) mice (n = 14)
were microdissected and quickly homogenized in 5.5 M guanidine buffer. Homogenates were diluted 1:10
with cold casein buffer (0.25% casein and 0.05% sodium azide)
followed by centrifugation for 20 min at 4°C at 10,000 × g. Total amyloid -peptide (A) and A1-42 were
measured by sandwich ELISA, as previously described (Bales et al.,
1999).
Statistical analysis. The data samples collected were
analyzed for deviation from Gaussian distribution using
Kolgomorov-Smirnov test, and group comparison was evaluated with
unpaired t test. The plaque size data (in square
micrometers) was logarithm transformed (with a logarithmic base
of 10) and analyzed in relative frequency histograms with bins of 0.2. The logarithmic transformation was made for each individual animal
separately, so that each animal would have an equal impact on the
Gaussian distribution curve within its experimental group. The
distribution of the population of Congo-positive plaques, with respect
to plaque size, was fitted to a Gaussian distribution equation with
nonlinear regression analysis (GraphPad Prism, version 2.0).
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RESULTS |
Astrocyte-specific and inducible ACT expression in
transgenic mice
A novel transgenic mouse that synthesizes ACT in an
astrocyte-specific, inducible expression pattern that mimics ACT
expression in the human brain was desired. To this end, we designed a
novel astrocyte-specific GFAP (6 kbp) promoter construct extending 80 bp into a modified GFAP-coding region, to allow interactions between possible gene regulatory sequences within the GFAP coding region and
upstream enhancer elements (Nakatani et al., 1990). Start codons in the
GFAP segment were then altered before the introduction of the ACT cDNA
so that the construct directed the translation to begin at the ACT
start codon. The ACT transgenic animals resulting from the embryos
transfected with the GFAP-ACT construct were viable with no overt
pathological signs and expressed fully glycosylated ACT protein (~68
kDa) in the brain that closely comigrated with human plasma ACT protein
(Fig. 1a). Furthermore, the
animals displayed a restricted expression pattern of the chimeric
GFAP-ACT mRNA transcript that was limited to brain tissue (Fig.
1b). These experiments were all performed on GFAP-ACT
transgenic mice without stab wound injury.
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Figure 1.
Expression of ACT mRNA
and protein in ACT transgenic mice. a,
Immunoprecipitation and Western blot of brain protein extracts from a
nontransgenic mouse and ACT-transgenic founder lines (#8782, #8783, and
#8784) displaying a protein band (~68 kDa) that closely comigrates
with human serum ACT. b, GFAP/ACT mRNA and GAPDH mRNA
expression in brain of a nontransgenic mouse and various tissues of a
heterozygous ACT-transgenic mouse showing brain-specific expression of
ACT only in the transgenic mouse. These experiments (a
and b) were performed on singly ACT transgenic animals
without stab wound injury. c, Astrocyte expression and
secretion of ACT immunoreactivity was found in the hippocampal
formation of heterozygous ACT-transgenic mice
(d), whereas both astrocyte staining and the
diffuse ACT-immunostaining in the hippocampus seen in ACT transgenic
mouse was absent in nontransgenic mouse. e,
Colocalization of ACT (brown) and GFAP
(blue) immunoreactivity in astrocytes of the
stab-wounded ACT(+/) mice. f, High-power magnification
of the tissue section depicted in c showing the
morphology of ACT-immunopositive astrocytes. The ACT
immunostaining of brain sections from both of these animals was
performed 3 d after stab wound injury
(c-f). Sections were counterstained with Methyl
Green (c, d, f).
Scale bars: e, 7.8 µm; f, 12.5 µm.
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The animals were further investigated using stab-wound injury to
demonstrate additional desired expression characteristics such as
inducible synthesis of the ACT protein in ACT(+/) mice (Fig.
1c,f), but not in ACT(/) mice (Fig.
1d), and astrocyte specificity (shown by double
immunostaining for GFAP and ACT (Fig. 1e). Similar
immunostaining of astrocyte processes has been observed after
GFAP-driven expression of the secretory chaperone protein ApoE (Sun et
al., 1998). The diffuse ACT immunostaining seen in the hippocampus of
ACT(+/) mice (Fig. 1c,f) was very faint in the
cerebral cortex and completely absent in brain sections of a stab
wound-injured ACT(/) sibling mouse that was processed in parallel
throughout the immunostaining procedures (Fig. 1d). We
suggest that the diffuse ACT immunostaining likely represents secretion
of ACT from astrocytes, which would be expected of a secretory acute
phase protein such as ACT. The validity of the double-staining protocol
was established by processing tissue sections from animals that either
expressed ACT or did not express ACT in the absence of one or both of
the primary ACT and GFAP antibodies and finding no labeling (data not shown).
ACT in astrocytes and amyloid plaques of APP/ACT
transgenic mice
Mice from the founder line (#8784) were crossed with homozygous
PDGF-hAPP/V717F mice because this founder mouse easily transferred the
transgene to its offspring (~50%; Games et al., 1995). At the
earliest time point investigated (4 months of age) ACT-immunopositive astrocytes were apparent in the PDGF-hAPP/V717F(+/)ACT(+/) mice in
the absence of any treatment such as stab wound, and were mostly restricted to astrocytes in white matter areas such as the anterior commissure, the cingulum, and the corpus callosum and close to the
ventricles. This expression pattern represented the basal expression of
the ACT transgene and was identical to that of singly ACT transgenic
mice and also similar to the expression of ACT in normal human brain
(Abraham et al., 1988; Pasternack et al., 1989; Koo et al., 1991).
At 6 months of age, when amyloid plaques were sparsely distributed in
the hippocampus, ACT-immunostained astrocytes were clearly visible
along the hippocampal fissure (Fig.
2a). By 10 months, when
amyloid deposition was more pronounced, the ACT-immunostained astrocytes were more widespread and often located around Congo Red
birefringent plaques in the PDGF-hAPP/V717F(+/) ACT(+/) mice (Fig.
2b,d), but were absent in PDGF-hAPP/V717F(+/)ACT(/) mice of the same age (Fig. 2c). Furthermore, polyclonal ACT
antibody not only stained the surrounding astrocytes but also Congo Red birefringent plaques, as depicted in the hippocampus of a 10-month-old PDGF-hAPP(+/)ACT(+/) mouse (Fig. 2e,f).
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Figure 2.
Astrocyte-specific expression and plaque
association of ACT protein in double APP/ACT transgenic mice.
a, Astrogliotic ACT immunostaining along the hippocampal
fissure in 6-month-old mouse. b, ACT-immunopositive
astrocytes were present in a 10-month-old
PDGF-hAPP/V717F(+/)ACT(+/) mouse (c)
but absent in a 10-month-old PDGF-hAPP/V717F(+/)ACT(/)
mouse. d, High-power magnification of ACT-immunopositive
astrocytes close to Congo Red-positive amyloid plaques in a hippocampal
subregion marked by an arrow in b.
e, High-power magnification of an ACT-immunopositive
Congo Red-positive amyloid plaque in the hippocampus of a
PDGF-hAPP/V717F(+/) ACT(+/) mouse at 10 months of age
(f) displaying birefringence under polarized
light. Scale bars: a, e,
f, 7.8 µm; d, 12.5 µm.
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Higher amyloid plaque load and frequency in APP/ACT
transgenic mice
Amyloid burden, as measured by Congo Red staining, was quantified
using high-power microscopy and image analysis in 10-month-old PDGF-hAPP/V717F(+/) ACT(+/) (n = 7) and
PDGF-hAPP/V717F(+/)ACT(/) (n = 6) animals.
Amyloid burden (area fraction) was significantly increased in the
hippocampus (0.0136 ± 0.0019 vs 0.0088 ± 0.0008%; +55%;
p < 0.05) and modestly increased in the cerebral
cortex (0.0155 ± 0.0038 vs 0.0136 ± 0.0012%; +14%; NS) of
the PDGF-hAPP/V717F(+/) ACT(+/) animals compared with the
PDGF-hAPP/V717F(+/)ACT(/) animals (Fig.
3a). The increased amyloid
burden was largely attributable to an increased numerical density of
Congo Red-positive plaques both in the hippocampus (+85%;
p < 0.05) and the cerebral cortex (+58%;
p < 0.05; Fig. 3b). The plaque density data
were then stratified according to plaque size (Fig.
4). The increased numerical density was
most pronounced among small size plaques (<50
µm2) both in the hippocampus (+170%;
p < 0.05; Fig. 4a) and the cerebral cortex
(+85%; p < 0.05; Fig. 4b). The density of
larger plaques (>50 µm2) was also
increased, but to a lower extent (+43% in the hippocampus, +39% in
the cerebral cortex) that was not statistically significant.
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Figure 3.
Increased total Congo Red-positive amyloid plaque
load (a) and numerical plaque density
(b) in the hippocampus and the cerebral cortex of
10-month-old PDGF-hAPP/V717F(+/)ACT(+/) mice (n = 7; solid bar), compared with age-matched
PDGF-hAPP/V717F (+/)ACT(/) mice (n = 6;
open bar). The results are expressed as percentage
of the control group (the PDGF-hAPP/V717F (+/)ACT(/) genotype
mice) and represent mean ± SEM.
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Figure 4.
Plaque density analysis. Congo-positive amyloid
plaques in the hippocampus (a, c) and the
cerebral cortex (b, d) were stratified
according to their sizes. There was an increased numerical density of
small plaques (<50 µm2) in the 10-month-old
PDGF-hAPP/V717F(+/) ACT(+/) mice (n = 7;
solid bar) compared with the age-matched
PDGF-hAPP/V717F(+/) ACT(/) mice (n = 6;
open bar) in the hippocampus (a)
and cerebral cortex (b). Shown below bar graphs
are the corresponding relative frequency histograms of plaque size
distribution in mice of the PDGF-hAPP/V717F(+/) ACT(+/)
(solid bar) and the PDGF-hAPP/V717F(+/), ACT(/)
genotypes (hatched bar) in the hippocampus
(c) and the cerebral cortex
(d). Superimposed is the best fit of our data to
a Gaussian distribution for mice of the PDGF-hAPP/V717F(+/)ACT(+/)
(solid line) and the PDGF-hAPP/V717F(+/) ACT(/)
genotypes (broken line). The center of the Gaussian
distribution curve for the PDGF-hAPP/V717F(+/)ACT(+/) mice was
shifted toward a smaller average plaque size in the hippocampus
(c) as well as the cerebral cortex
(d). The results displayed are expressed as
percentage of the control group [the
PDGF-hAPP/V717F(+/)ACT (/)] genotype and represent mean ± SEM.
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The plaque size distribution was more completely analyzed using
relative frequency histograms. We found that the linear histograms were
substantially skewed to the right (data not shown) and that a
logarithmic transformation of the plaque size data generated a very
good fit of the sizes of the Congo Red-positive plaques to a Gaussian
distribution (Fig. 4c,d). The correlation coefficient was
 0.94 for all analyses when using a Gaussian distribution equation for
the nonlinear regression analysis. This approach has been shown to
successfully describe the senile plaque size distribution in human AD
brain (Hyman et al., 1995). The center of the Gaussian distribution
curve for the PDGF-hAPP/V717F(+/)ACT(+/) mice was significantly
shifted toward a smaller average plaque size in the cerebral cortex
(56 ± 3 vs 70 ± 7 µm2;
p < 0.05; Fig. 4d) compared with
PDGF-hAPP/V717F(+/) ACT(/) mice, and a similar not statistically
significant trend was apparent in the hippocampus (47 ± 3 vs
56 ± 5 µm2; p < 0.1; Fig. 4c).
Increased amyloid -peptide levels in young APP/ACT
transgenic mice
Amyloid -peptides levels in the hippocampus of 3-month-old mice
were examined to determine whether ACT influenced amyloid -peptide metabolism. There was a significant increase in total amyloid -peptide levels (0.55 ± 0.05 vs 0.43 ± 0.03 ng/mg protein; +27% p < 0.05; n = 10 and n = 14, respectively) in the hippocampus of
3-month-old PDGF-hAPP/V717F(+/)ACT(+/) mice compared with age-matched PDGF-hAPP/V717F(+/)ACT (/) mice. In addition, a similar not statistically significant trend toward increased
hippocampal amyloid -peptide(1-42) levels in the
PDGF-hAPP/V717F(+/)ACT(+/) mice compared with age-matched
PDGF-hAPP/V717F(+/)ACT(/) mice was apparent (0.11 ± 0.01 vs
0.09 ± 0.01 ng/mg protein; +22%; p < 0.1; Fig.
5). However, the amyloid
-peptide(1-42)/total amyloid -peptide ratio was identical
(0.21 ± 0.01 vs 0.21 ± 0.01).
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Figure 5.
Increased total amyloid -peptide (A) and
A1-42 levels in the hippocampus of 3-month-old
PDGF-hAPP/V717F(+/)ACT(+/) mice (n = 10;
solid bar) compared with age-matched PDGF
hAPP/V717F(+/)ACT(/) mice (n = 14;
open bar).
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DISCUSSION |
In the present transgenic mouse model, we have been able to
reproduce several of the previous findings with respect to ACT expression in normal and Alzheimer's disease-afflicted human brain. Specifically, basal ACT expression in human brain is very low (Abraham
et al., 1988), as is the expression in the transgenic mice at an early
age before amyloid deposition. The singly ACT transgenic mice express
fully glycosylated ACT protein (~68 kDa) that closely comigrates with
human plasma ACT protein. We cannot exclude a subtle size difference
between mouse brain and human plasma ACT because of the extent of
glycosylation. Indeed human liver ACT (66-75 kDa), the source of human
plasma ACT, and hippocampal ACT (60-65 kDa) differ slightly in
molecular size (Hwang et al., 1999). The basal expression of transgene
ACT in singly ACT transgenic mice as well as in crossed APP/ACT
transgenic mice is restricted to astrocytes in white matter areas and
close to the ventricles. These findings are consistent with strong GFAP
immunostaining of white matter astrocytes in nontransgenic mice, as
well as the distribution of ACT-immunostained astrocytes in human brain
without pathological changes (Abraham et al., 1988; Pasternack et al., 1989; Koo et al., 1991). The ACT immunostaining of astrocyte processes likely represents intracellular ACT that is about to be secreted as
well as extracellular ACT in the immediate vicinity of the astrocyte
processes. Immunostaining of astrocyte processes of the secretory
chaperone protein ApoE has previously been described after GFAP
promoter-driven transgene expression (Sun et al., 1998).
As the amyloid formation proceeds, the astrocytic ACT expression
gradually increases, primarily in the hippocampus but also in the
cerebral cortex of the PDGF-hAPP/V717F (+/)ACT(+/) transgenic mice. ACT also becomes a constituent of the Congo Red-positive amyloid
plaque structure itself, similar to previous observations in AD brain
(Abraham et al., 1988). Most importantly there is an increased amyloid
burden in 10-month-old PDGF-hAPP/V717F(+/)ACT(+/) mice compared
with PDGF-hAPP/V717F(+/)ACT(/) mice, particularly in the
hippocampal formation where ACT expression in astrocytes is observed
early on as the amyloidosis develops. We suggest that the stronger
amyloid-promoting effect of ACT in the hippocampus, compared with the
cerebral cortex, is attributable to the more extensive astrogliotic
reaction and GFAP induction in the hippocampus, which in turn will lead
to the secretion of higher concentrations of transgene ACT in the hippocampus.
These findings suggest that the ACT protein not only becomes
synthesized and secreted as a secondary response to ongoing cerebral amyloidosis, but also further promotes amyloid deposition and formation
of the -sheet structure of mature plaques, likely by a direct
molecular interaction with A. This increased amyloid formation
caused by ACT is over and above that attributable to endogenous ApoE
expression, which is present in all of the mice analyzed. Furthermore,
because the increased amyloid burden was largely attributable to
increased plaque density, it is likely that ACT either initiates
amyloid filament formation or catalyzes other early amyloid filament
processes by interacting with A peptides or intermediate oligomeric
A structures. The more numerous Congo Red-positive plaques were also
of a smaller size in the ACT-expressing mice, again consistent with ACT
functioning at an early, rate-limiting step in plaque development, but
also with ACT either retarding further growth of plaques or altering
their structure so that they become more dense. The increased plaque density, the shift in senile plaque size distribution, and the ACT-immunopositive staining of Congo Red-positive plaques together strongly suggest that ACT is directly involved in amyloid plaque formation at the molecular level, likely by promoting filament formation. It is possible that the amyloid-promoting effect by ACT
would have been less with the weaker human ACT promoter. The choice of
GFAP as a surrogate promoter was based on previous findings that
ACT-mRNA and GFAP-mRNA expression correlate well in both healthy and
human brain tissue afflicted by various neuropathological conditions
(Koo et al., 1991). It was also unclear whether the human ACT promoter
would have been functional in mouse brain astrocytes with respect to
cytokine responsiveness and therefore might not have responded to
astrogliosis (Das and Potter, 1995).
Finally, the increased steady-state levels of amyloid -peptide
observed in the brains of 3-month-old ACT-expressing mice before
amyloid deposition might also contribute to the increase in amyloid
deposition observed in the animals at 10 months of age. We suggest that
the increased steady state levels of A in the hippocampus are caused
either by ACT/A peptide complex formation, which will tend to
unspecifically retard A peptide clearance in the extracellular
space, or by inhibition of a specific amyloid -peptide degrading
enzyme by ACT, as previous in vitro studies have suggested
(Yamin et al., 1999).
Our findings provide further evidence that amyloid-associated proteins,
induced by inflammatory cytokines, act as pathological chaperones and
play an essential role in the AD disease pathogenesis (for review, see
Nilsson et al., 1998; Wisniewski et al., 1998; Bales et al., 2000). The
results also suggest that ACT may exert an effect on APP processing or
more likely A peptide clearance.
The overexpression of ACT in astrocytes and of interleukin-1
(IL-1) in microglia in regions of AD brain with frequent
dense-core senile plaques first suggested that these inflammatory
proteins might be involved in senile plaque formation or maturation
(Abraham et al., 1988; Griffin et al., 1989; Das and Potter, 1995;
Sheng et al., 1995). IL-1 is a powerful inducer of APP-mRNA translation (Rogers et al., 1999) as well as ACT-mRNA expression (Das and Potter,
1995) in primary human astrocytes, and glial activation is accompanied
by increased expression of ApoE both in vitro and in AD
brain (Diedrich et al., 1991; Bales et al., 2000). Indeed, the finding
of pronounced astrogliosis and microgliosis in
PDGF-hAPP/V717F(+/)ApoE(/) mice in the absence of -pleated
sheet amyloid formation (Bales et al., 1999) suggests that the
importance of gliosis with respect to amyloid formation in AD is that
it leads to the production and release of specific amyloid-promoting
factors, such as ACT and ApoE.
Additional support for the involvement of an inflammatory cascade
leading to increased amyloid formation comes from the discovery of
polymorphisms that confer increased risk of developing AD or amyloid
angiopathy. Such polymorphisms have been identified in the IL-1
promoter (Du et al., 2000; Grimaldi et al., 2000; Nicoll et al., 2000),
the ACT (Kamboh et al., 1995; Yamada et al., 1998), and the ApoE coding
regions (Corder et al., 1993; Strittmatter et al., 1993) as well as the
ApoE promoter (Bullido et al., 1998).
In light of the current findings, it is interesting and important that
retrospective, and more recently, prospective pharmaco-epidemiological studies on certain populations have concluded that inflammation plays a
role in AD (McGeer et al., 1990; for review, see Nilsson et al., 1998;
Akiyama et al., 2000). Together with these results, our data support
the hypothesis that specific inflammatory processes accelerate the
progression of AD as a part of a "pathogenic pathway" (Fig.
6). Each step in the pathway discussed
here for example the overexpression and release of IL-1 in microglial
cells, the induction of ACT-mRNA expression in astrocytes, and the
molecular interactions between ACT (and ApoE) and A peptides and
aggregates to promote amyloid formationconstitutes a specific
therapeutic target at which new AD drug discovery may be directed.
Note added in proof. While this paper was
being reviewed, Mucke et al. (2000) published a related study using
hACT transgenic mice that had been created using a different GFAP-hACT
expression vector construct. The authors showed, similar to our
findings, that hACT increased amyloid deposition in the PDGF-hAPP/V717F transgenic mice.
| |
FOOTNOTES |
Received Oct. 2, 2000; revised Nov. 27, 2000; accepted Dec. 15, 2000.
The research was supported by National Institutes of Health Grant
AG09665 to H.P. L.N.G.N. is presently supported by a fellowship from the John Douglas French Alzheimer's Foundation and previously by
the Stiftelsen für Internationalisering au högre utbildning och forskning foundation and Riksbankens Jubileumsfond (after a
donation from Erik Rönnberg). H.P. occupies the Eric Pfeiffer Chair for Research in Alzheimer's disease at the Suncoast Gerontology Center at the University of South Florida.
Correspondence should be addressed to Dr. Huntington Potter or Dr. Lars
Nilsson, Department of Biochemistry and Molecular Biology, College of
Medicine, MDC07, University of South Florida, Tampa, FL 33612. E-mail: hpotter{at}hsc.usf.edu and lnilsson{at}hsc.usf.edu.
| |
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ABSTRACT
INTRODUCTION
