mcgowan 1999 amyloid phenotype ch
TRANSCRIPT
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
1/14
Amyloid Phenotype Characterization ofTransgenic Mice Overexpressing both MutantAmyloid Precursor Protein and Mutant Presenilin 1
Transgenes
E. McGowan,* S. Sanders,* T. Iwatsubo, A. Takeuchi, T. Saido,
C. Zehr,* X. Yu,* S. Uljon, R. Wang, D. Mann, D. Dickson,* andK. Duff,1
*M ayo Clinic, Jacksonv ille, Florida 32224; Department of N europathology and N euroscience,
University of Tokyo, Tokyo, Japan; Laboratory for Proteolyt ic S cience, R IKEN Brain Science
Inst itu te, Japan; Laboratory for M ass Spectrometry, R ockefeller Univ ersity, N ew York, N ew
York 10021; Department of Pathological Sciences, U niv ersity of M anchester, M 13 9PT, U nit ed
Kingdom; and N eurotransgenics Lab, N athan Kline Inst itu te, O rangeburg, N ew York 10962
Received November 6, 1998; revised March 4, 1999; accepted for publication March 11, 1999
Doubly transgenic mice (PSAPP) overexpressing mutant APP and PS1 transgenes were examinedusing antibodies to A subtypes and glial fibrillary acidic protein (GFAP). Visible A deposition began
primarily in the cingulate cortex of PSAPP mice at approximately 10 weeks of age. By 6 months, the
mice had extensive amyloid deposition throughout the hippocampus and cortex as well as otherregions of the brain. Highly congophilic deposits consisting of N-terminal normal and modified forms
of A were identified, reminiscent of those found in human AD brain. Both immunohistochemistry and
mass spectrometry showed that A42 forms were underrepresented relative to A40, and A43 wasundetectable. Deposits were associated with prominent gliosis which increased with age, but in
14-month-old PSAPP mice, GFAP immunoreactivity in the vicinity of amyloid deposits was substan-
tially reduced compared to APP littermates. These mice have considerable utility in the study of theamyloid phenotype of AD. 1999 Academic Press
INTRODUCTION
Alzheimer s disease (AD) is characterized by the
presence in the brain of neurofibrillary tangles and A
containing plaqu es (dep osits) that contain mu ltiple A
isoforms (Iwa tsubo et al., 1996). These isoforms in clud e
A starting at N1 (L-asp) together with the racemizedand isomerized forms (D asp and L-iso-asp, respec-
tively), A containing p yroglutam ate m odified resi-
du es at positions N3 and N11, and A starting at N17,
w hich is especially prevalent in diffuse d eposits (Gow -
in g et al., 1994). A C terminal heterogeneity is also a
feature of AD, as A isoforms terminating at residues
39, 40, and 42 have been identified in p laques from
patients with typical late-onset AD (Iwatsubo et al
1996). Patients with PS1 mutations harbored a highe
burden, or number, of A42 senile plaques compared
with patients with sporadic AD (Mann et al., 1996
most likely due to the effect of mutant PS1 on APP
metabolism wh ich leads to specific elevation of thhighly fibrillogenic A42 peptide (Scheuner et al., 1996
Jarrett et al., 1993). The identification of AD causing
mutations in APP, PS1, and PS2 that affect A level
suggests that A plays a critical role in AD pathogen
esis. Transgenic mice that model the deposition pro
cess have therefore been generated to study the pro
cess of A accumulation and deposition.
Transgenic mice that overexpress m utant and wild
type PS1 cDNA s have been assayed for A levels (Duf
et al., 1996). Mutant PS1 transgenic m ice h ad elevate
1To w hom correspondence should be add ressed at Nathan Kline
Institute, 140 Old Orangebu rg Road, Orangebu rg, N ew York 10962.
Fax: 914 398 5422. E-mail: d uff@nk i.rfm h.or g.
Neurobiology of Disease6, 231244 (1999)
Article IDnbdi.1999.0243, available online at http://www.idealibrary.com on
231
0969-9961/99 $30.00
Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
2/14
levels of endogenous A42, but not A40, whereas
overexpression of wild-type PS1 led to marginally
elevated A42 levels that did not reach significance
compared with nontransgenic littermates (Duff et al.,
1996). Although A42 levels are significantly elevated
in the mutant PS1 mice, the mice do not form amyloid
deposits upon aging, presumably because the levels of
A do n ot reach the level required to start the aggrega-
tion p rocess in mice. Transgenic m ice th at overexpress
mu tant hu man APP at high levels show elevated levels
of human A40 and/ or A42 depending on the APP
mutation used. These animals d eposit am yloid be-
tween 6 and 18 months depending on the transgenic
line (Games et al., 1995; Hsiao et al., 1996; Stu rchler-
Pierrat et al., 1997; Borch elt et al., 1997). The Tg2576 line
that normally forms A deposits between 9 and 12
months of age (Hsiao et al., 1996) showed greatly
accelerated deposition of am yloid (Holcomb et al.,
1998) wh en crossed w ith a mu tant PS1 line (Duffet al.,
1996). The deposition event in the doubly transgenicmice (PSAPP) has now been characterized further as
these m ice have great utility for the study of AD-
related amyloidosis due to their fast and predictable
pathology developm ent.
METHODS
Transgenic Mouse Generation
Hem izygous transgenic mice that expresses mu tanthuman APPK670N,M671L (Hsiao et al., 1996) (line Tg2576)
and hemizygous lines of PS1 mice (Duff et al., 1996)
wh ich express either hu man mu tant PS1M146V (line 8.9)
or PS1M146L (line 6.2) were crossed t o gener ate offspr ing
with four possible genotypes: PSAPP, APP, PS1, and
nontransgenic (non tg). Hem izygotes w ere used, as it
has not been possible to generate homozygote lines of
Tg2576. The singly transgenic mutant PS1 and APP
offspring, together with non tg littermates, were used
as controls for the d ouble m utan t PSAPP mice.
Tissue Processing (in Situ Hybridization)
Transgenic m ice from both the PS1M146V line and the
PS1M146L line together with non tg littermates were
cervically dislocated and the brains were rapidly
removed and snap frozen on dry ice. Sets of cryostat
sections (15 m) were cut at approximately 300-m
intervals in the coronal plane through the forebrain
and cerebellum and thaw mounted onto Plus micro-
scope slides (Fisher Scientific).
Tissue Processing (Immunohistochemistry)
Transgenic mice at various ages (8, 10, 12, 2832, and
4359 weeks) were analyzed in this stud y. At leas
three double mutant mice and two mice from each o
the other genotypes (PS1, APP, and non tg) wer
examined at each time point. Tissue was prepared in
one of two ways:
(1) Mice were anesthetized with sodium pentobarbital and perfused transcardially w ith ice-cold salin
followed by 4% paraformaldehyde in 0.1 M phosphat
buffer. Brains were removed and placed in the fixativ
overnight at 4C. After fixation brains were cryopro
tected in 30% sucrose/ 0.1 M phosphate-buffered salin
(PBS) for at least 24 h. Serial coronal sections (30 m
were cut through the forebrain and cerebellum using
freezing sledge microtome (Leica) and stored at 4C in
2 mM sod ium azide/ 0.1 M PBS solution.
(2) Mice were cervically dislocated and the brain
were removed and immersion fixed in 70% ethanol/150 m M Na Cl a n d e m be d d ed in p a ra ffin wa x a
described previously (Iwatsubo et al., 1996). Seria
sections (6 m ) were cut on a rotary m icrotome.
Tissue Processing (Mass Spectrometry)
Double transgenic PSAPP m ice at 6, 16, and 3
weeks (n 2 at each age) were cervically dislocated
and the brains were removed, hemisected, and snap
frozen on dr y ice.
In Situ Hybridization
A synthetic oligomer designed to the 5-end of th
hum an PS1 mRNA corresponding to bases 5 to 4
(5-TgT gCA TTC Tgg AAg TAg gAC AAC ggT gCA
ggT AAC TCT g-3) w a s 3-end labeled to a specifi
activity greater than 1 108 dpm/ g. Slides wer
quickly warmed to room temperature, fixed in neutral
buffered p araformald ehyd e (Sigma) for 15 min, rinse
in 0.1 M PBS, and sequentially dehyd rated through
graded ethanol (70, 80, 90, 100%) and allowed to aidry. Hybridizations w ere standardized such that each
slide (46 sections/ slide) was hybridized with 12 ng
of the labeled oligonucleotide in 300 l of hybridiza
tion buffer (50% (v/ v) deionized formamide, 4 SSC
1 Denhardts solution, 10% (w/ v) dextran sulfate
0.3% -mercapthoethanol, and 200 g/ ml sonicate
denatured salmon-tested DNA). Sections were hybrid
ized overnight in a moist chamber at 37C. Contro
sections were hybridized in the presence of a 50-fold
molar excess of the unlabeled oligonucleotide. Afte
232 McGowan et a
Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
3/14
hybridization, slides were stringently washed (3) in
1 SSC plu s 0.001% -mercaptoethanol for 30 min,
prior to dehyd ration in 70% ethanol containing 300
mM ammonium chloride, followed by 100% ethanol.
Air-dried slides w ere exposed to Hyperfilm -Max
(Amersham ) at room temp erature for 12 weeks.
Immunohistochemistry (Free-Floating Sections)
Sets of serial 30-m sections at app roximately 300-m
intervals through the forebrain and cerebellum were
immunostained with an A1-12 antibody (1/ 100 mou se
monoclonal, Grant et al., 1997). Briefly, end ogeno us
peroxidase activity w as inhibited by incubating the
tissue in 20% methanol/ 1.5% H 2O2 diluted in 0.1 M
PBS for 30 min . Sections were r insed in 0.1 M PBS pr ior
to permeabilization and blockade of nonspecific bind-
ing by incubation in 0.2% Triton/ 100 mM lysine/ 5%
normal serum diluted in 0.1 M PBS. Sections wereincubated in primary antiserum diluted in 5% normal
serum/ 0.1 M PBS overnight at room temperature.
Sections were extensively washed in 0.1 M PBS and
incubated in anti-mouse biotinylated IgG (1/ 1000,
Vector Labs) diluted in 5% normal serum/ 0.1 M PBS
for 2 h at room temp erature, w ashed in 0.1 M PBS, and
incubated in streptavidinhorseradish peroxidase (1/
1000, Vector Labs) d iluted in 0.1 M PBS for 1 h at room
temperature. The sections were washed repeatedly
and the color reaction was developed using either a
0.05% solution of 3,3-diaminobenzidine tetrahydro-chloride (DAB)/ 0.03% H 2O2 for 510 min or the a bove
chromogen with the addition of 2.5% nickel ammo-
nium sulfate. The free-floating sections were then
washed extensively in 0.1 M PBS, mou nted on Plus
slides (Fisher), dehydrated, cleared in xylene, and
cover-slipp ed w ith E-Z mou nt (Shan don ).
Fo r t h e A glial fibrillary acidic protein (GFAP)
double labeling, the tissue was permeabilized as de-
scribed previously, th en incubated in GFAP (1/ 1000
mouse monoclonal, Boerhinger Mannh eim, Indianapo-
lis, IN) d i lu t ed in 5% n or m al se ru m / 0.1 M P BSovernight at room temperature. Standard streptavidin
biotin complex p rocedure was used to detect the
primary antiserum, which was visualized with DAB,
as described above. Sections were then washed exten-
sively in 0.1 M PBS, incubated in methanol/ H 2O2 to
inhibit residual peroxidase activity, and incubated in
A1-12 ant isera (1/ 100 mou se mon oclonal, Grant et al.,
1997) and detected using a standard streptavidin
biotin complex procedure. Specific immunoreactivity
was visualized using Vector SG (Vector Labs) or
nickel-enhanced DA B as the chrom ogen. Sections w er
then mounted on slides, air-dried, dehydrated i
graded ethanols, cleared in xylene, and cover-slipped
with E-Z mount (Shandon).
Immunohistochemistry (Paraffin-Embedded
Sections)
After dep araffinization, serial sections were immu no
stained overnight at room tem perature w ith a pan el o
A antibodies raised against different portions of th
A peptide (Saido et al., 1995, 1996) (see Table 1 fo
antibody epitope d etails). This was followed by stan
dard avidinbiotin complex procedure using Tris
buffered saline (50 mm ol/ L Tris/ HCl, pH 7.6) fo
dilution of the antibodies and washing the slides, an
visualization w ith 3,3-diaminobenzidine in 150 or 50
mM NaCl as the chromogen. Sections were stained
with and without formic acid pretreatment (Iwatsubet al., 1996). Tissue sections were counterstained with
hematoxylin.
TABLE 1
Relative Levels of Immunoreactivity for A , Thioflavin S,
and GFAP in PSAPP Mice and Singly Transgenic APP and
PS1 Littermates
Genotyp e Age A Th io fla vin S G FA P
P SAP P 23 m on th s
36 mon ths
6 months
8 months
12 months
APP 6 m onths
812 months / / /
1218 months
PS 014 m onths
N ote. A n a rbit rary u n it syste m w a s u sed t o cl assi fy t h e A
deposition and inflammatory response in double transgenic PSAP
mice and their singly transgenic littermates. Animals w ith lownumbers (515) of A/ th iofl av in S p osit ive d ep osit s in th e cor te
and hippocampus or a slight astrocytic response were assigned on
().An imals with intermed iate num bers (50100) of A/ th iofl av in
deposits per section or marked astrocyte activation surroundin
deposits were assigned (). Mice with extensive (100300) A
thioflavin S positive deposits per section were given ( ). Intens
GFAP immunoreactivity clustered around plaques and present i
the neuropil between plaques was assigned (). () wer
also assigned to mice which had both diffuse (thioflavin S negative
amyloid and extensive compact thioflavin S positive deposits pe
section. Mice with an astrocytic response which was no longe
closely associated with the A deposits were given ()*.
PSAPP Mouse Characterization 23
Copyright 1999 by Academic PreAll rights of reproduction in any form reserve
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
4/14
Mass Spectrometry
Soluble A and deposited A in brain tissue was
isolated by sequential extraction from each individual
mouse brain. Hemispheres (approximately 0.2 g) were
thaw ed and wa shed three times in ice-cold TBS. Brain
tissues w ere homogen ized in 1.0 ml of homogen ization
buffer consisting of 150 mM NaCl, 50 mM TrisHCl,
pH 8.0, and protease inhibitors (EDTANa2, 2 m M ;leupeptin, 10 M; pepstatin, 1 M; PMSF, 1 mM;
TLCK, 0.1 m M; TPCK, 0.2 mM). The homogenates
w ere centrifuged at 100,000g RCF for 1.5 h and the
supernatants (TBS extract) collected for the water-
soluble A assay. For detection of deposited A, the
brain tissue pellets were washed three times with
ice-cold TBS and then extracted using 1.0 ml of
hexafluoroisopropanol (HFIP) by sonication and vor-
texing for 2 h at 4C. The HFIP extracts were centri-
fuged at 100,000g RCF for 2 h and the HFIP layers
carefully collected a nd speed -vacuu m d ried. The HFIPextracts were resuspended by TBS containing 1%
CHAPS and protease inhibitors overnight. Both the
TBS extracts and the HFIP extracts (0.5 ml) were
imm un oprecipitated w ith 1.0 l of mon oclonal anti-A
antibody, either 4G8 or 6E10 (Senetek, Maryland
Heights, MO) and 3 l of p rotein G Plus/ protein
Aagarose bead s (Oncogene Science, Inc., Cam bridge,
MA) at 4C for 3 h. Immu noprecipitated A peptides
w ere analyzed u sing a matrix-assisted laser desorption
ionization time-of-flight mass spectrometer (Voyager-
DE STR BioSpectrom etry Worksta tion, Per Sept ive Bio-
system) as d escribed p reviously (Wang et al., 1996).
RESULTS
Regional Expression of Mutant PS1 Transgenes
The gene expression pattern for the 8.9 (PS1M146V)
and 6.2 (PS1M146L) lines was regionally similar, but in
general the hybridization signal was greater in the 8.9
line. The highest transgene expression was in the CA3
region of the h ipp ocamp us (Fig. 1B). There w as little orno transgene expression in the other hippocampal
subfields (CA1 and dentate gyrus). A robust laminar
expression pattern was present in the cortex, with the
highest cortical gene expression in the cingulate area
(Figs. 1A and 1B). Lower transgene expression was
observed in th e entorhinal and piriform cortices. A
strong hybridization signal was detected in the major-
ity of thalamic nuclei but was minimal in the hypotha-
lamic region an d the striatum (Figs. 1A and 1B). Little
or no transgene expression was detected in white
matter suggesting predominant neuronal expression
of the transgene.
The 35S-labeled PS1 probe used was specific fo
human PS1 mRNA and did not hybridize to endog
enous mouse PS1 transcripts, therefore, no hybridization signal was observed in sections from nontrans
genic animals. Similarly, no specific hybridization signa
was detected on control sections from PSAPP m ic
hybridized in the presence of an excess of unlabeled
PS1 oligonucleotide.
Age-Dependent Increase in A Deposition
Both lines of PSAPP mice have been examined
primarily using an N-terminal A1-12 mon oclona
FIG. 1. Expression of the hum an PS1M146V transgene in the 8
transgenic mouse line. A shows the expression pattern of the PS
transgene in mouse forebrain; B shows the midbrain from the sam
animal. The strongest hybridization signal was detected in the CA
region, followed by the cortex, particularly the cingulate cortica
region, and thalamic nuclei. There was lower transgene expressio
in the entorhinal and piriform cortices. Minimal transgene expres
sio n w a s p resen t i n t h e C A1 reg io n , d e n ta te g yru s, stria t u m
hypothalamus, and white matter. Scale bar 1.5 mm. C, cingulat
cortex; CA3, CA3 region of Ammons horn; CPu, caudate putamen
E, entorhinal cortex.
234 McGowan et a
Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
5/14
antibody (Grant et al., 1996) to investigate where and
wh en amyloid deposits develop. Both lines of m ice
were examined at 8, 10, and 12 weeks of age to assess
when N-terminal A immu noreactivity could first be
identified (Figs. 2A2C). Results were essentially iden-
t ica l i n P SAP P m i ce o v er exp r e ss in g e it h er t h e
6.2PS1M146L or the 8.9PS1M146V transgene. A deposits
were not seen in 8-week-old doubly transgenic mice(Fig. 2A). A imm un oreactivity was first detected at 10
weeks of age (Fig. 2B). By 12 weeks of age small
numbers of deposits were consistently detected in
PSAPP mice derived from either the 6.2PS1 or the
8.9PS lines, in both the cingulate and the adjacent
motor cortex, and also in the hippocampus (Figs. 2C
and 2G). In the PSAPP d oubly transgenic mice, early
deposition occurs most prominently in the cingulate
cortex and adjacent motor cortex, in a pattern that
correlates with the expression pattern of the PS1
transgene. As the animals aged, A deposition becamemore widespread (Fig. 2D). Both the size and the
number of the deposits increased, in the hippocampus,
corpus callosum, and extensive areas of the cortex,
with deposition spreading longitudinally to the ento-
rhinal and piriform cortices in animals aged 2832
weeks (Figs. 3A and 3B). A deposits were scattered
throughout the polymorphic and molecular layers of
the hippocamp us. Deposits congregated in the molecu-
lar layer of the dentate gyrus and in areas adjacent to
the hippocampal fissure, generally sparing the pyrami-
dal and dentate granule cell layers of the h ippocam-pus.
By 14 months of age, a great number of compact
(thioflavin S positive) d eposits w ere p resent through-
out the cortex and hippocampal regions (Figs. 2E and
2H). Furthermore, diffuse (thioflavin S negative) A
immu noreactivity was now abund ant also throughout
the cingulate and motor cortex and hippocampus
(Figs. 2E and 2H). In the somatosensory cortices, there
was less diffuse A immunoreactivity present in la-
mima III/ IV, correlating with the expression pattern of
the PS1 transgene in the cortex. The pyramidal andgranule cell regions of the hippocampal formation in
old mice were relatively devoid of A deposition,
while the remainder of the hippocampal neuropil,
including the stratum oriens, stratum radiatum, the
polymorphic, and molecular layer of the dentate gyrus
was filled with diffuse and compact A immunoreac-
tivity (Fig. 2H).
In animals aged 2832 weeks, A deposition was
also d etected in the thalamus, striatum , septum, infe-
rior colliculus, and med ial geniculate nucleus (Figs.
3C3E). Microdep osits w ere detected, albeit rarely, i
the cerebellum in older mice (Fig. 3F). In 59-week-old
mice, d eposition was more prominent in the area
described above and was also p resent at low levels i
the hypothalamus; furthermore, some diffuse A im
munostaining in the striatum and thalamus was pre
sent also. Vascular amyloidosis was clearly visible in
the PSAPP mice, especially those older th an 12 mon th
of age and w as particularly of note in th e cerebellum .
Deposits were not routinely detected in the singly
t ra n sg en ic APP lit te rm a te s a t a ge s u p t o 1 y ea
consistent with previous published reports. In th
oldest mice examined (59 weeks), A deposition had
been initiated and was prominent in the entorhina
cortical areas with variable deposition in the cingulat
and motor cortices and hipp ocampu s (Figs. 2F and 2I
This pattern contrasts w ith that seen in the PSAP
animals suggesting that early deposition in PSAP
animals was influenced by the pattern of expression o
the PS1 transgene and the concommittant regiona
increases in the level of A42, or less likely PS1 protein
within different parts of the cortex (see Fig. 1). No A
immunostaining was detected in nontransgenic mic
at a ny age examined . Similarly, no A immunoreactiv
ity was observed in singly transgenic PS1 mice up to 1
months of age.
Multiple A Isoforms Are Present in the Deposits
in the Double Mutant Mice
Serial sections from the brains of PSAPP mice and
singly transgenic littermates at 6 months of age wer
examined using a panel of antibodies which recogniz
different A isoform s (see Table 1 for antibod y sp ecific
ties). These antibodies have been used previously t
examine the comp osition of A aggregates in human
brain tissue with senile (congophilic) and diffus
plaques (Saido et al., 1995, 1996; Iwatsubo et al., 1996
In cortical and hippocampal regions, the most exten
sive immu nostaining w as detected w ith an N -termina
antibody (9204) that recognizes the first L-aspartatresidue, AN 1 (L-Asp) (Fig. 4). Nu merou s d eposits o
varying sizes w ere observed throughout the corte
and the hipp ocamp al formation. A proportion of thes
A aggregates were also immunoreactive for race
mized (D-Asp) and isomerized (L-iso-Asp) forms o
the N1 aspartate (see Fig. 4). Most deposits wer
labeled with an antibody (Ban 50) that recognizes th
first 16 amino acids of A (A1-16) (Fig. 4). Fewe
deposits were immu noreactive for A starting at N1
co m p ar ed t o A1-16, suggesting that most of th
PSAPP Mouse Characterization 23
Copyright 1999 by Academic PreAll rights of reproduction in any form reserve
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
6/14
FIG. 2. A deposition becomes markedly more extensive with age in the double mutant mice. Coronal sections at the level of the striatum
(AF) or hippocamp us (GI) immu nostained with an N-terminal A1-12 antibod y. A immu noreactivity was not d etected at 8 weeks of age (A
Dark-stained cell bodies are n onspecific and d ue to nickel-DAB, which w as used as the chrom ogen. Similar artifacts were present in PS, non tg
and APP mice. A small number of A aggregates were present in the cingulate cortex by 10 weeks of a ge (B). By 12 weeks, more extensive A
236
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
7/14
FIG. 3. In older PSAPP mice (sections depicted are from a mouse aged 32 weeks), A deposition was detected throughout the neocorte
nclud ing the ectorhinal, entorhinal (A), and piriform cortices (A and B). Deposition w as not restricted to th e cortex and h ippocamp al formatio
alone in the old er PSAPP mice but w as also present in the striatum (C), thalamu s (D), medial geniculate nu cleus (E), the inferior colliculus, an
very rarely in the cerebellum (F). Scale bar for A 200 m, BE 100 m , F 50 m . aca, anterior commisure; AV, anteroventr al thalam
nucleus; CPu, caud ate p utam en; Ect, ectorhinal cortex; gr, granule cell layer of the cerebellum; MGN, m edial geniculate nu cleus; mol, molecula
cell layer of the cerebellum; Pir, piriform cortex; sm, stria medullaris.
FIG. 2Continued immunostaining was seen in the cingulate and frontal cortex (C and G) and deposits were present in the hippocampa
formation (G). At 32 weeks, A deposition was widespread and the deposits were more numerous and larger in size, and were also detected i
wh ite matter (D). As the mice age deposition becomes increasingly widesp read throu ghout th e forebrain; at 59 weeks of age the cortex (E and H
and hippocampus (H) were filled with both compact and diffuse A immunoreactivity. No A immunostaining was detected in singl
ransgenic APP litterma tes at 30 weeks of age. At 59 weeks of age variable A dep osition wa s present in the cortex (F and I) and hippocamp us (
of APP mice. Arrows indicate some of the A aggregates. Scale bar for AF 200 m and for GI 500 m.
PSAPP Mouse Characterization 23
Copyright 1999 by Academic PreAll rights of reproduction in any form reserve
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
8/14238
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
9/14
deposits were derived from A cleaved at the
secretase site.
The majority of deposits was immunoreactive for
AN3 (glutamate) and a similar number of deposits
were immu nostained with AN3-pyroglutam ate. Occa-
sional aggregates were labeled with an antibody ra ised
against mouse AN3-pyroglutamate (Fig. 4). No AN11
pyroglutamate immunoreactivity was detected. C-
terminal antibodies show ed the p resence of A termi-
nating at residues 39, 40, and 42. More deposits
contained A40 (identified with antibodies BA27 and
A36-40) than A42 (BC05 and A38-42) but aggre-
gates did not ap pear to contain A fragments terminat-
ing at A43, since no immunostaining was detected in
any region of the brain (Fig. 4). Similar levels of
immunostaining with all the antibodies w ere seen
with or without formic acid pretreatment. Similarly,
sections p rocessed in either 150 or 500 mM N aCl gave
identical results. A immunoreactivity was not ob-
served in singly transgenic PS1, APP, and nontrans-genic littermates at this age for any of the 15 A
isoforms (data not shown).
A Marked Astrogliotic Response Is Associatedwith A Deposition
Double imm unostaining with GFAP (Fig. 5, brown
reaction product) and an N-terminal A antibody
(purple/ black reaction produ ct) showed the extent of
astrocytosis in the double mutant mice. Some of the
astrocytic processes appeared to show positive immu-noreactivity w ith the A antibody bu t this was thought
to reflect incomplete quenching of peroxidase activity
after the first immunolabeling rather than A accumu-
lation in astrocytes as single labeling w ith A does not
label glial processes.
Gliosis is an invariant and early event associated
w ith amyloid d eposition, as mice as young as 12 w eeks
showed microdeposits associated with a few or single
reactive astrocytes (Fig. 5A). Astrogliosis was more
extensive in th e 28- to 32-week-old PSAPP m ice, w here
most of the cortex and hippocampus had a markedastrocytic response to A and clusters of GFAP immu-
noreactive astrocytes were seen closely associated with
A aggregate deposition (see Fig. 5B, which depict
the typical reactive astrocytic response in a 32-week
old PSAPP mouse). A deposition was not observed in
the APP mice at th is age and there was n o increase i
GFAP immunoreactivity. By 59 weeks of age, th
astrocytic profile had changed markedly in the PSAPP
m ice (Fig. 5B). Exten sive, d iffu se A as w ell as compac
amyloid was now present throughout the neocorte
and hippocampus at this age, but little or no GFAP
immunoreactivity surround ed the compact deposit
(Fig. 5C). GFAP immunoreactivity was present in th
neuropil between plaques but was not closely associ
ated with compact aggregates in contrast to that seen
in the 28- to 32-week-old anima ls (Fig. 5B). Althou gh
the A deposition in APP mice aged 59 weeks wa
variable, all deposits were associated with a heav
reactive gliosis (Fig. 5D), which was very similar t
that observed for the 28- to 32-week-old PSAPP mic
(Fig. 5B). Rarely, an increase in gener al ast rogliosis w aseen in the cortex of PS, young APP, or nontransgeni
littermates, but the reaction was never seen in foca
clusters and was thought to represent a nonspecifi
immune response. Table 1 summarizes the majo
pathological findings in the PSAPP and their singl
transgenic APP and PS littermates at all ages exam
ined.
Mass Spectrometry: Confirmation ThatHeterogeneous Carboxy-Terminal A Peptides ArPresent in the Deposits in the Double Mutant Mice
The peptide profile of both soluble and deposited
A peptides in mou se brains w as analyzed by sequen
tial extraction and immu noprecipitation/ mass spec
trometry (see Fig. 6) as d escribed in Wang et al. (1996
Soluble A peptides were extracted with TBS. A 1-4
and A1-42 were detected as two major A species i
the TBS extracts and they represent 48.9% (0.9%
n 2) and 33.6% (2.5%, n 2) of total A peptide
detected in the same measurement based on the pea
intensity (Fig. 6A). Several other A peptides, A1-41A1-39, and A1-38, were also d etected in TBS extract
FIG. 4. A isoform immunostaining in a 6-month-old PSAPP mouse brain. Serial sections were immunostained with a panel of antibodie
raised against different A species. AN1 (L-asp (9204)) immunolabeled the greatest number of A deposits throughout the neocortex an
hippocam pu s. The m ajority of these deposits were imm unoreactive for the racemized (D-Asp) and isomerized (L-iso-Asp) forms of AN1. Th
aggregates were immunopositive also, to varying degrees, for A116 (116/ Ban 50); A1724 (1724); AN3 (N3), both human and mous
AN3 pyroglutamate (N3p and N3p[m], respectively). There was little or no A N11 pyroglutamate (N11p) immunostaining. C-terminal A
species, including A39, A40, and A42, were present in the deposits. A 40 immunoreactive aggregates were more numerous than A 4
aggregates as detected by two d ifferent antibodies.A 43 imm unoreactivity was not observed in PSAPP mouse br ain. Scale bar 200 m .
PSAPP Mouse Characterization 23
Copyright 1999 by Academic PreAll rights of reproduction in any form reserve
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
10/14
w ith m uch less abund ance (5, 6, and 6%, respectively).
The composition of deposited A peptides was deter-
mined by mass spectrometric analysis of A in HFIP
e xt ra ct s in P SA PP m ice. A p ep t id e s A1-42
(7.7 0.7%), A1-40 (61.5 1.3%), as well as A1-39
(3.8 0.7%), A1-38 (17.6 0.8%), A1-37 (2.9 0.3%
A1-34 (1.5 0.7%), an d A1-33 (0.2 0.2%), w er
observed in the spectra (n 4) of 16-week-old PSAPP
mice resulting from antibody (4G8 and 6E10) immu no
precipitation (Figs. 6B and 6C). Furthermore, severa
FIG. 5. Early A deposition is associated with a marked and progressive astrocytic response as shown by d ouble immunostaining for A
(purp le/ black) and GFAP (brown) in the cortex of 12-week-old (A) and 32-week-old PSAPP mice (B). By 59 weeks of age GFAP im m un orea ctivity wamar kedly redu ced or absent aroun d the comp act deposits (C) although GFAP immu noreactivity was still visible in the neurop il between the A
dep osits (as ind icated by arrows). In contrast, singly transgenic APP littermates at 59 weeks of age exhibited a similar h eavy astrogliotic respons
o youn ger PSAPP mice, with focal clusters of GFAP imm unoreactivity being observed around A deposits (D). Scale bar in AD 200 m.
240 McGowan et a
Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
11/14
A N -terminal fragm ent p eptides (less than 5% of total
A pep tides based on th e peak intensity, n 2) includ -
ing A1-15, A1-17, A1-19, and A1-20 were d etected
using monoclonal antibody, 6E10 (Fig. 6C). The domi-
nant A isoform identified in the deposited fractions
w a s t h e A1-40 peptide. Less A 1-42 peptide was
detected in the deposited fractions and the p eptide
concentration ratio between A1-42 and A1-40 p ep
tides (A1-42/ A1-40) was estimated as approxi
mat ely 25% by the relative peak intensity and stan dard
qua ntitation curves (Wang et al., 1996). A1-43 pep tid
was not detected in the HFIP-extract. We were als
unable to detect AN3 (A3-40), AN3-pyro (Ap 3
40), or A11-40 in the spectra of HFIP extracts, mos
likely due to the low abundance of these isoforms. A
compar ison between 6-week-old PSAPP animals w ith
out visibly dep osited amy loid and 16-week-old PSAP
animals showed that both the A1-40 and the 1-4
levels were greatly elevated in the older animals
Although A1-40 levels remained higher than A 1-4
levels, the ratio of A1-42/ A1-40 was significantl
elevated in the older animals (data not shown). Be
cause nontr ansgenic, PS, and A PP mice have n o d etect
able A deposition at 16 weeks of age, HFIP fraction
w ere not examined by IP/ MS.
DISCUSSION
The current stud y d escribes the am yloid p henotyp
of mice overexpressing a mutant human APP trans
gene and one of tw o mutant presenilin transgenes
ELISA data h ave show n tha t the m utan t PS1 mice used
in the cross have an approximate twofold elevation i
endogenous A42 levels (but not A40) compared t
nontr ansgenic litterma tes (Duffet al., 1996), suggestin
that mutant PS1 selectively influences the metabolism
of APP to increase the levels ofA 1-42 (Duffet al., 1996Borchelt et al., 1996; Citron et al., 1997). The curren
study shows that the doubly transgenic mu tant PSAP
mice develop robust amyloidosis by 12 weeks of age
which is app roximately four times faster than thei
singly transgenic APP littermates. A deposits i
PSAPP mice derived from either mutant PS1 lines 8.
or 6.2 crossed with Tg2576 are first v isible at 101
weeks of age and they have been an invariant featur
in m o re t ha n 15 a n im a ls e xa m in ed in t he 10- t
12-week age group. At all ages examines, the path olog
cal features were essentially identical in PSAPP micderived from either the 8.9 PS1M146V or the 6.2PS1M146lines. Once deposition is initiated, the nu mber a nd siz
of the deposits in PSAPP mice increases steadily with
age. Deposition is first d etected in the cingu late cortex
but in general, d eposits are seen in the neocortex
hippocampus, and corpus callosum and extensiv
pathology develops throughout these regions of th
brain by 6 mon ths of age (see Fig. 2). There is ver y littl
detectable pathology in the singly transgenic APP mic
until at least 9 months of age and A deposition wa
FIG. 6. Mass spectra of soluble and deposited A peptide profiles
of 16-week-old PSAPP mouse brain. Soluble A peptides were
detected following immunoprecipitation with 4G8 (A) whereas
deposited A peptid es in the HFIP extract were imm unop recipitated
with 4G8 (B) and 6E10 (C). Peaks correspond ing to A peptides are
abeled with the A sequence nu mber. Peaks labeled as 1402, Ins.,
and 1228 (std) represent doubly protonated A140 peptide ions,
doubly p rotonated insulin m olecular ions (added for mass calibra-
ion), and A12-28 (added as internal standard) ions, respectively.
Several unidentified peaks are tagged with an asterisk.
PSAPP Mouse Characterization 24
Copyright 1999 by Academic PreAll rights of reproduction in any form reserve
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
12/14
variable even at 14 mon ths of age (see Fig. 2). To d ate,
no d eposition has been observed in m utant PS1 trans-
genic mice at 14 months of age, the oldest age exam-
ined. See Table 1 for a summary of the pathological
findings in the PSAPP mice compared to their singly
transgenic APP and PS litterm ates.
PSAPP m ice not only show accelerated pathology
development, but the distribution of deposits differs
between PSAPP m ice and their APP counterparts.
Singly transgenic APP (Tg2576) mice (and hum ans
with AD) dep osit preferentially in the entorhinal an d
piriform cortices whereas initial, visible deposition in
the PSAPP mice always begins in the cingulate and
adjacent motor cortices, with entorhinal and piriform
involvement becoming evident later. In situ analysis
shows that in most respects, the initial d istribution of
deposits follows the pattern of PS1 transgene mRNA
expression, which is high in the cingulate cortex and
relatively low in the entorhinal cortex (see Fig. 1).
Furthermore, in the older double m utants, a lower
amyloid burden was observed in layers III/ IV of the
somatosensory cortex, which is a region of low PS1
transgene expression (see Fig. 1). These data all sug-
gest that the mutant PS1 transgene contributes to
accelerated A accum ulation, with regions of high PS1
expression showing the earliest deposition. This is
most likely due to the local elevation of A42 in
response to mutant PS1, but the possible contribution
of mutant PS1 protein levels to deposition cannot be
discounted from this observation. One obvious excep-
tion to the correlation between PS1 levels and initialdeposition is in the CA3 pyramidal cell layer of the
hippocampu s. Expression of transgene-derived mu -
tant PS1 and APP is thought to colocalize in this region,
yet A immu noreactivity is rarely seen, even wh en
amyloidosis is extensive in other regions of the brain.
This is similar to the human AD brain where senile
plaques are underrepresented in the CA3 pyramidal
region and are usually only associated with very
severe hippocampal pathology (D. Mann, unpublished
data).
Immunohistochemistry has shown that, in general,the composition of deposits seen in humans with AD
(Iwatsubo et al., 1996) and the PSAPP tran sgenic mice
are very similar. A peptides deposited in the brains of
transgenic mice and humans are largely composed of
A species beginning at N1 (Asp) and include both
racemized and isomerized forms of the amino acid.
The deposits also contain A species beginning with
pyroglutamate at N3, and in addition to modification
o f t h e h u m a n A molecule, murine pyroglutamate-
modified A peptide is also sequestered into deposits
in the mou se. In contrast to hum an AD brain wh ere th
pyroglutamate N3 residue is highly represented rela
tive to other A peptides, in the mouse this species i
less well represented. Also, pyroglutamate N11 wa
not p resent in the m ouse plaques. A small proportio
of the deposits in the mou se contain A starting at Leu
N17, su g ge st in g t ha t m o st o f t h e a m ylo id is n o
generated by cleavage at the secretase cleavage sit
at N16 (Sisodia, 1992). This is most likely due to th
inclusion of pathogenic mutations at residues 670 and
671 of the APP transgene which have been shown t
enhance cleavage at the putative secretase site (a
position N-1) at the expense of secretase cleavag
(Cai et al., 1993; Citro n et al., 1992).
A g re at er n u m b er o f d e p osit s in t h e b ra in s o
6-month-old mice w ere imm unoreactive for A(1-40
compared to A(1-42). ELISA (Holcomb et al., 1998
and mass spectrometry (data not show n) have demon
strated that although young PSAPP mice initially hav
elevated levels of A1-42 relative to APP littermates
A40 levels w ere still twofold h igher than A42 level
and both A forms increase in the brain once deposi
tion begins. The correlation amon g imm un ohistochem
istry, ELISA, and mass spectrometry suggests that th
observed predom inance of d eposited A (1-40) versu
A(1-42) in the immu nohistochemistry stud y is no
due to an antibody artifact such as differences i
antibody sensitivity or epitope masking. In addition
two different sets of antibodies were used (A(36-40
a n d Ba n 27 fo r A (1-40); A(38-42) and BCO5 fo
A(1-42)) which show the same trend and thesantibodies are known to recognize A(1-40) and A(1
42) to a similar extent in human brain studies (Iwat
subo et al., 1996). It is u nlikely th at th e sensitivity of th
A(1-42) specific antibodies is reduced due to epitop
masking as there w as little d ifference in the intensity o
the immunostaining when the sections were pre
treated with formic acid which destabilizes aggregate
to make the peptide more accessible to the antibody
One possibility, however, is that the A (1-42) isofor m
could be more sensitive to degradation d uring samp l
preparation, leading to red uced signal. Overall, therwas good correlation between immunohistochemistr
and mass spectrometry with the exception that d epos
its were immunoreactive for both AN3 a n d AN3pyr
b u t n o AN3-40 or AN3pyro-40 species were de
tected by mass spectrometry. As mass spectrometr
relies on the competitive binding of immunoprecipi
tated peptides from a complex mixture and the level
of AN3 peptides were low even as judged by immu
nohistochemistry, it is likely that these isoforms fel
below detection of the mass spectrometry assay. I
242 McGowan et a
Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
13/14
general, however, the immunohistochemical data cor-
relate w ith the mass spectrometry analysis of deposit
composition, i.e., there is greater A 40 dep osition th an
A42, most species of A were represented, and
neither m ethod detected A species terminating at
residu e 43 in th e aggregates.
The majority of deposits in very young mice stained
positively with thioflavin S (Holcomb et al., 1998, and
data not shown) suggesting that even at this early
stage, the d eposits consist of fibrillar a myloid. They d o
not ap pear to p ass through a d etectable diffuse stage
on their way to becoming compact, senile plaques,
which correlates with data from other high-level-
expressing APP transgenic mice (Sturchler-Pierrat et
al., 1997). Diffuse plaques in humans are rich in A
starting at residue 17 (Gowing et al., 1994) an d the
observation of very little A17-x in the transgenic mice
confirms our observations that most of the dep osits are
compact. As the an imals age, however, diffuse (thiofla-
vin S negative) A imm un oreactivity begins to overlay
the compact deposits. The source of this diffuse A
and the reason w hy it does not form compact deposits
are unknown.
Double immu nostaining against A and GFAP indi-
cate that reactive gliosis is closely associated with A
pathology progression, as single reactive astrocytes
w ere observed in close proximity to dep osits in PSAPP
mice as young as 12 weeks of age (see Fig. 4). Further
work is required to d etermine whether amyloid depo-
sition or gliosis initiates the process. By 6 months, there
is an extensive astrocytic respon se aroun d A deposits(see Fig. 4 and Holcomb et al., 1998) which is not seen
in nondepositing controls suggesting that the reaction
is in direct response to the presence of the A aggre-
gates. Interestingly, old PSAPP mice show a different
pattern of GFAP immunoreactivity. At 14 months, the
PSAPP animals have extensive amyloidosis which
includes both compact and diffuse forms of A . GFAP
immunoreactive astrocytes are no longer closely asso-
ciated with the compact deposits, but it is unclear at
this stage whether they have stopped expressing GFAP,
migrated away from the deposits, or died. The situa-tion is, however, reminiscent of the transient gliosis
seen in certain injury paradigms such as excitotoxic
injury (Bjorklund et al., 1986) or deafferentat ion (And ers
& Johnson, 1990), but even in these situations, the
response of the astrocyte is not fully u nd erstood. There
are no reports of transitory gliosis in late-stage human
AD brain but humans have an overall lower amyloid
burden than mice at this stage. Mouse age does not
app ear to be a factor as focal clusters of GFAP imm un o-
reactivity were still concentrated around A deposits
(Fig. 4) in singly transgenic 14-month-old APP litter
mates in a similar manner to that seen in the 28- to
32-week-old double transgenic mice. The amyloi
burden in th e 14-month APP m ice is, however, lowe
than that of age-matched PSAPP mice and the APP
mice have had detectable deposited amyloid for a fa
shorter time. In addition, the diffuse amyloid tha
accumulates in the oldest PSAPP mice is not seen in
APP litterma tes or youn ger PSAPP anima ls.
A similar acceleration of A pathology and inflam
matory markers has been described by Borchelt et a
(1997) where HuPS1-A246E mutant mice were crossed
with mice expressing the APP Swedish mutatio
(K670N, M671L). A deposition was detected at
month s in their PSAPP mutants compared to 18 month
for singly transgenic APP mutant mice. The acceler
ated pathology described in this study is more aggres
sive and far earlier but is essentially similar in charac
ter, especially as both PSAPP mice have more A 4
immunoreactive profiles than A42 (Borchelt et al1997, and Fig. 4). Although patients with presenilin-
mu tations have m ore A42-containing dep osits (Iw at
subo et al., 1996), the inclusion of APP K670N, M671
mutations in the APP transgene in both sets of mice i
thought to account for the preponderance of A4
dep osits relative to those composed of A42.
In conclusion, because of their highly predictable
early, and robust amyloid phenotype, the PSAPP mic
are an excellent model in which to study amyloidosis
Given the central link between AD-causing mutation
in different genes and A accumu lation, it woulappear that A is central to the disease and that th
study of these types of model is justified. Unfortu
nately, mice do not always replicate the human condi
tion exactly, and the A depositing mice are n
exception. Despite the accumulation of large amount
of A in several d ifferent tran sgenic mou se m odel
(Games et al., 1995; Hsiao et al., 1996; Borchelt et al
1997; Holcomb et al., 1998), the mice do not show
uniform response that mimics the hallmark features o
hu man ADnamely amyloid deposition, tangle forma
tion, extensive cell loss, and cognitive impairment. Ifhow ever, as the genetic da ta suggest, A accumulation
is central to the d isease, then the PSAPP m ice w ill b
useful resources for the testing of therapeutic agent
against this feature, even if they are incomp lete mod el
ofAD.
ACKNOWLEDGMENTS
The au thors are grateful to Dr C. Cuello, McGill Universit
Canada, for the gift of the A1-12 antibody and to Dr. Karen Hsiao
PSAPP Mouse Characterization 24
Copyright 1999 by Academic PreAll rights of reproduction in any form reserve
-
7/29/2019 McGowan 1999 Amyloid Phenotype Ch
14/14
University of Minnesota, for the gift of the Tg2576 line. This work
was supported by an Alzheimers Association/ The Stasia Borsuk
Memorial Fund grant (RG1-96-070) to R.W. and an NIH program
grant (AG-146133) to K.D.
REFERENCES
Anders, J. J., & Johnson, J. A. (1990) Transection of the rat olfactorynerve increases glial fibrillary acidic protein immunoreactivity
from the olfactory bulb to th e piriform cortex. Glia 3(1), 1725.
Bjorklund, H., Olson, L., Dahl, D., & Schwarcz, R. (1986) Short- and
long-term consequences of intracranial injections of the excito-
toxin, qu inolinic acid, as eviden ced by GFA imm unoh istochemis-
try of astrocytes. Brain Res. 371(2), 267277.
Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Daven-
port, F., Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D.,
Slunt, H. H., Wang, R., Seeger, M., Levey, A. I., Gandy, S. E.,
Copeland, N. G., Jenkins, N . A., Price, D. L., Younkin, S. G., &
Sisod ia, S. S. (1996) Fam ilial Alzheim er s d isease-linked p resenilin
1 variants elevate Abeta1-42/ 1-40 ratio in vitro and in v ivo. Neuron
5, 10051013.
Borchelt, D. R., Ratovitski, T., van Lare, J., Lee, M. L., Gonzales, V.,
Jenkins, N. A., Cop eland , N. G., Price, D. L., & Sisodia, S. S. (1997)
Accelerated amyloid deposition in the brains of transgenic mice
coexpressing mutant presenilin 1 and amyloid precursor proteins.
N euron 19, 939945.
Cai, X. D., Gold e, T. E., & Youn kin, S. G. (1993) Release of excess
amyloid beta protein from a mutant amyloid beta protein precur-
sor. Science 259, 514516.
Citron, M., O ltersdorf, T., H aass, C., McConlogue, L., H ung, A. Y.,
Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., & Selkoe, D. J. (1992)
Mutation of the amyloid precursor protein in familial Alzhei-
mer s d isease increases protein production. Nature 360, 672674.
Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque,
G., John son-Wood, K., Lee, M., Seubert, P., Davis, A., Kholoden ko,D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R.,
Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St
George Hyslop, P., & Selkoe, D. J. (1997) Mutant presenilins of
Alzheimers disease increase prod uction of 42-residue a myloid
-protein in both transfected cells and transgenic m ice. Nature
M ed. 3, 6772.
Duff, K., Eckman , C., Zehr, C., Yu, X., Prad a, C.-M., Perez-tur, J.,
Hu tton, M ., Buee, L., Ha rigaya, Y., Yager, D., Morgan , D., Gordo n,
M. N ., Holcomb , L., Refolo, L., Zenk, B., Har dy, J., & Youn kin, S.
(1996) Increased amyloid-42(43) in brains of mice expressing
mutant presenilin 1. Nature 383, 710713.
Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P.,
Blackwell, C., Carr, T., Clemena, J., Donaldson, T., Gillespie, F.,
Guido, T., Hagopian, S., Johnson-Wood, K., Khan, K., Lee, M.,
Leibowitz, P., Leiberberg, I., Little, S., Masilah, E., McConlogu e, L.,
Montoya-Zavala, M., Mucke, L., Paganini, L., Penniman, E.,
Pow er, M., Schenk, D ., Seubert, P., Snyder, B., Soriano, F., Tan, H.,
Vitale, J., Wadsworth, S., Wolozin, B., & Zhao, J. (1995) Alzheimer-
type neuropathology in transgenic mice overexpressing V717F
b-amyloid precursor protein. Nature 373, 523527.
Gowing, E., Roher, A. E., Woods, A. S., Cotter, R. J., Chaney, M.,
Little, S. P., & Ball, M. J. (1994) Chemical characterization of the
A17-42 peptide, a component of d iffuse amyloid deposits o
Alzheimers d isease. J. Biol. Chem. 269, 1098710990.
Grant, S. M., Szyf, M., & Cuello, A. C. (1997) Ultrastructura
localization of am yloid beta p rotein in transfected embryona
carcinoma (P19) cells after neuroectodermal differentiation. So
N eurosci. 23, 540. (Abstr act)
Holcomb, L., Gord on, M. N., McGowan , E., Yu, X., Benkovic, S
Jantzen, P., Wright, K., Saad, I., Mueller, R., Morgan, D., Sanders
S., Zehr, C., OCampo, K., Hardy, J., Prada, C.-M., Eckman, C
Youn kin, S., H siao, K., & Du ff, K. (1998) Accelerated Alzheim etype phenotype in transgenic mice carrying both mutant amyloi
precursor protein and presenilin 1 transgenes. Nature Med. 4
97100.
Hsiao, K., Chapm an, P., N ilsen, S., Eckman, C., Harigaya, Y
Younkin, S., Yang, F., & Cole, G. (1996) Correlative mem or
deficits, A elevation, and amyloid plaques in transgenic mice
Science 274, 99102.
Iwatsubo, T., Saido, T. C., Mann, D. M. A., Lee, V. M.-Y., &
Trojanowski, J. Q. (1996) Full-length am yloid-(1-42(43) and amino
terminally modified and truncated amyloid-42(43) d eposit i
diffuse p laques. Am. J. Pathol. 149, 18231830.
Jarrett, J. T., Berger, E. P., & Lansbury, P. T., Jr. (1993) The carbox
terminus of the amyloid protein is critical for the seeding o
amyloid formation: Implications for the pathogenesis of Alzhei
mers disease. Biochemistry 32, 46934697.
Mann, D. M., Iw atsubo, T., Cairns, N. J., Lantos, P. L., Nochlin, D
Sumi, S. M., Bird, T. D., Poorkaj, P., Hardy, J., Hutton, M., Prihar
G., Crook, R., Rossor, M. N., & Haltia, M. (1996) Amyloid bet
protein (Abeta) dep osition in chromosome 14-linked Alzheimer
disease: Predominance of Abeta 42(43). Ann. Neurol. 40, 149156.
Saido, T. C., Iwatsu bo, T., Mann , D. M. A., Shimad a, H ., Ihara, Y., &
Kawashima, S. (1995) Dominant and differential deposition o
distinct beta-amyloid p eptide species, A-beta N3(pE), in seni
plaques. Neuron 14, 457466.
Saido, T. C., Yamao-Har igaya, W., Iwatsubo, T., & Kawashima, S
(1996) Amino- and carboxyl-terminal heterogeneity of beta
amyloid peptides deposited in human brain. Neurosci. Lett. 215173176.
Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuk
N., Bird, T. D., Hardy, J., Hutton, M., Kukull, W., Larson, E
Levy-Lahad, E., Viitanen, M., Peskind, E., Poorkaj, P., Schellen
berg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., & Youn kin
S. (1996) Secreted amyloid beta-protein similar to that in the senil
plaques of Alzheimers disease is increased in vivo by th
presenilin 1 and 2 and APP m utations linked to familial Alzhe
mers disease. Nat. Med. 2, 864870.
Sisodia, S. S. (1992) Beta-amyloid precursor protein cleavage by
membrane-bound protease. Proc. Natl. Acad. Sci. USA 89, 6075
6079.
Sturchler-Pierrat, C., Abramoski, D., Duke, M., Wiederhold, K.-H
Mistl, C., Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paga
netti, P. A., Waridel, C., Calhoun, M. E., Jucker, M., Probst, A
Staufenbiel, M., & Sommer, B. (1997) Two amyloid precurso
protein transgenic m ouse models with Alzheimer disease-lik
pathology. Proc. Natl. Acad. Sci. USA 94, 1328713292.
Wang, R., Sweeney, D., Gandy, S. E., & Sisodia, S. S. (1996) The profil
of soluble amyloid protein in cultured cell media: Detection an
quantification of amyloid protein and variants by immunopre
cipitationmass spectrometry. J. Biol. Chem. 271, 3189431902.
244 McGowan et a