mcgowan 1999 amyloid phenotype ch

7/29/2019 McGowan 1999 Amyloid Phenotype Ch

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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

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0969-9961/99 $30.00

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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



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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

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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



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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




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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

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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.




8/14238


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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 .




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



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.




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



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




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.

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