Leon 2010 JAD_Novel Transgenic Rat Model With AD Like Amyloid Pathology Displays Pre Plaque...

7/29/2019 Leon 2010 JAD_Novel Transgenic Rat Model With AD Like Amyloid Pathology Displays Pre Plaque Intracellular AB C

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Journal of Alzheimers Disease 20 (2010) 113126 113DOI 10.3233/JAD-2010-1349IOS Press

A Novel Transgenic Rat Model with a Full

Alzheimers-Like Amyloid Pathology

Displays Pre-Plaque Intracellular

Amyloid--Associated Cognitive Impairment

Wanda Carolina Leona,1

, Fabio Cannevaa,1

, Vanessa Partridgea

, Simon Allarda

, Maria Teresa Ferrettia

,Arald DeWildea, Freya Vercauterena,2, Ramtin Atifeha, Adriana Ducatenzeilera, William Kleinb,Moshe Szyfa, Leena Alhonenc and A. Claudio Cuelloa,d,e,aDepartment of Pharmacology and Therapeutics, McGill University, Montreal, QC, CanadabCognitive Neurology and Alzheimers Disease Center, Northwestern University Institute for Neuroscience,

Chicago, IL, USAcDepartment of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University

of Kuopio, Kuopio, FinlanddDepartment of Anatomy and Cell Biology, McGill University, Montreal, QC, CanadaeDepartment of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada

Accepted 2 December 2009

Abstract. Alzheimers disease (AD) is a neurodegenerative pathology in which amyloid-(A) peptide accumulates in different

brain areas leading to deposition of plaques and a progressive decline of cognitive functions. After a decade in which a number

of transgenic (Tg) mouse models mimicking AD-like amyloid-deposition pathology have been successfully generated, few rat

models have been reported that develop intracellular and extracellular Aaccumulation, together with impairment of cognition.

The generation of a Tg rat reproducing the full AD-like amyloid pathology has been elusive. Here we describe the generation and

characterization of a new transgenic rat line, coded McGill-R-Thy1-APP, developed to express the human amyloid- precursor

protein (APP) carrying both the Swedish and Indiana mutations under the control of the murine Thy1.2 promoter. The selected

mono-transgenic line displays an extended phase of intraneuronal Aaccumulation, already apparent at 1 week after birth, which

is widespread throughout different cortical areas and the hippocampus (CA1, CA2, CA3, and dentate gyrus). Homozygous Tg

animals eventually produce extracellular Adeposits and, by 6 months of age, dense, thioflavine S-positive, amyloid plaques are

detected, associated with glial activation and surrounding dystrophic neurites. The cognitive functions in transgenic McGill-R-

Thy1-APP rats, as assessed using the Morris water maze task, were found already altered as early as at 3 months of age, when no

CNS plaques are yet present. The spatial cognitive impairment becomes more prominent in older animals (13 months), wherethe behavioral performance of Tg rats positively correlates with the levels of soluble A (trimers) measured in the cortex.

Keywords: Alzheimers disease, amyloid-oligomers, cognitive impairment, intracellular amyloid-, transgenic rat

Supplementary data available at http://www.j-alz.com/issues/20/vol20-1.html#supplementarydata03

1WL and FC equally contributed to the experimental work.2Present affiliation: Sleep Research Centre, Universite de Mon-

treal, Faculty of Medicine, Sacre-Coeur Hospital, Montreal, QC,Canada.

Correspondence to: A. Claudio Cuello, 3655 Promenade SirWilliam Osler, H3G 1Y6, Montreal, QC, Canada. Tel.: +1 514 3983618; Fax: +1 514 398 8317; E-mail: [email protected].

ISSN 1387-2877/10/$27.50 2010 IOS Press and the authors. All rights reserved


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114 W.C. Leon et al. / A Novel Transgenic Rat Model with a Full Alzheimers-Like Amyloid Pathology Displays

INTRODUCTION

Alzheimers disease (AD) is a neurodegenerative

condition characterized by a progressive loss of cogni-

tive abilities associated with increased levels of brain

amyloid-peptide (A) that tends to deposit as extra-

cellular senile plaques, the presence of intraneuronal

neurofibrillary tangles composed of abnormally phos-

phorylated tau protein, the loss of synapses, and even-

tually neuronal death [1,2]. A peptide is detected in

healthy individuals [3,4], but its increased production,

or diminishedclearance, are consideredas the causative

agents of the disease. This view is supported by the

discovery of genetic forms of AD (familial AD, FAD)

in which variants of the amyloid- protein precursor

(APP) result in the elevation of A production [5,6].

Since the first successful transgenic (Tg) mouse

model of AD was created [7], a number of valuable

Tg mouse models have been described (for reviews

see [810]). However, the generation of a Tg rat re-

producingthe full AD-like amyloid pathologyhas been

elusive. The desirability of rat models is based on

the extensive Neuroscience Database and the richer be-

havioral display of rats as compared to mice [11]. In

addition, the larger brain size makes surgical proce-

dures and pharmacological manipulation easier to per-

form. In recent years, some Tg rat models have beendeveloped by us and by others, which failed, how-

ever, to produce extracellular deposition of amyloid

plaques [1214]. Nevertheless, intracellular A (iA)

accumulation has been shown in one of these mod-

els, to induce changes in sub-cellular organelles and

alterations in the hippocampal proteomic patterns, as

well as cognitive impairments [1517]. More recent-

ly Flood and colleagues [18] reported the successful

generation of Tg rats in which the co-expression of

three different transgenes (hAPPswe, hAPPswe,ind,

and hPS1finn) leads to deposition of mature plaquesat around 9 months of age. The behavioral characteri-

zation of these Tg rats [19] previously showed a signif-

icant cognitive impairment, as assessed by the Morris

water maze (MWM) task, as early as two months prior

to plaque deposition.

Since the first publication describing a Tg rat model

in 2004, our lab has continued to work on developing a

complete model of AD in this species, given the advan-

tages mentioned above. Here we report the generation

and characterization of a new Tg rat line (McGill-R-

Thy1-APP) in which the expression of a single trans-

gene coding for a modified variant of the human pro-

teinAPP751, containing both the Swedish andIndiana

mutations, results in the full AD-like amyloid pathol-

ogy, including the generation of mature plaques anddystrophic neurites. In homozygous Tg rats, the human

A peptide was found to accumulate intraneuronally

from one week postnatal, and the progressive extracel-

lular amyloid pathology was established at 6 months of

age. The earliest amyloid plaques were first detected in

the subiculum and later spread to the hippocampus and

cerebral cortex and some sub-cortical regions. Coinci-

dentally with the earlier phases of iA accumulation,

3 month-old homozygous Tg rats displayed a signifi-

cant cognitive impairment, as assessed by the MWM,

compared to wild type (wt) control animals, while the

behavior of hemizygous littermates was found to be in-

termediate between the two populations. Interestingly,

the levels of oligomeric A (trimers) measured in cor-

tical samples of homozygous Tg rats were found to be

around four-foldhigher than in hemizygouslittermates,

suggesting a direct effect of this species of the peptide

on the cognitive status of the McGill-R-Thy1-APPrats.

MATERIALS AND METHODS

Generation and selection of the transgenic rats

The McGill-R-Thy1-APP transgenic rats were gen-erated by the standard pronuclear injection tech-

nique [20] using HsdBrl:WH Wistar rats. The DNA

construct used was constituted by 5 and 3 regula-

tory elements of the murine thymocyte antigen pro-

moter (Thy1.2) [21,22], carrying the human APP 751(hAPP) cDNA containing theentire coding regionand

approximately 900 bp of 3 non-translated sequence,

including the Swedish double mutation (K670N,

M671L [23]) and the Indiana mutation (V717F [24]).

All the animals were genotyped by PCR amplifica-

tion of DNA extracted from the tails to assess the

presence of the hAPP transgene with the follow-

ing primers: 5-AAGAGGTGGTTCGAGAGGTG-3

and 5-CTGCATCCAGATTCAC-3. Throughout the

study period, pairs of animals were housed in a con-

trolled environment (temperature 22C, humidity 50

60%, 12 h light/12 h dark schedule) and efforts were

made to minimize discomfort and the number of ani-

mals used. The animal experimentation protocol was

approved by the McGill University Animal Care Com-

mittee and conducted in accordance with the Guide

to the Care and Use of Experimental Animals of the

Canadian Council on Animal Care and the National

Institutes of Health (NIH).


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W.C. Leon et al. / A Novel Transgenic Rat Model with a Full Alzheimers-Like Amyloid Pathology Displays 115

A

B C

Fig. 1. Generation and characterization of the transgenic animals. A) Thy1-APP construct showing the cloning of hAPP751 variant (containingthe Swedish and Indiana mutations) under the transcriptional control of the murine Thy1.2 promoter. The approximate location of the HindIIIrestriction sites used for digesting the genomic DNA is indicated by the arrows, and the approximate annealing site of the probe used in theSouthern blot experiment is also shown as a grey bar. B) Southern blot analysis of representative samples from hemi- (+/) and homozygous(+/+) Tg animals revealing the presence of one copy of the transgene per haploid genome set compared to the intensity of the calibration bands.No band was detected in samples from wt littermates (/). C) Analysis of hAPP expression in different tissues as revealed by the 6E10antibody. sAPP expression was used as an indicator of transgene expression given the antibody specificity for the human form. No signalwas detected in the sample from a wt rat, while an obvious expression of the transgene was specifically found in cortical samples from hemi- andhomozygous Tg rats. No signal was detectable in any other tissues tested: cerebellum (Cb), liver (L), heart (H), kidney (K), and thymus (T).

Southern blotting

Transgene copy number was determined on puri-

fied genomic DNA by Southern blotting according tostandard procedures. Briefly, 10 g of DNA were di-

gested with HindIII endonuclease (Fermentas Canada

Inc., Burlington, ON), excising a fragment of about

4000 bp from the transgenic construct, and separated

on a 0.9% agarose gel together with copy number con-

trols (from 1 to 16 copies). A digoxygenin-labelled

probe was obtained by PCR (Roche Applied Sciences,

Laval, Canada) from the Thy1.2-APP plasmid using

the primers 5-ATCCCACTCGCACAGCAG-3 and

5-GGAATCACAAAGTGGGGATG-3, to anneal on

the 5 region of the transgene (see Fig. 1A). Chemi-

luminescence detection was performed using the pro-

tocol for DIG-labeled probes according to the manu-facturers instructions (Roche Applied Sciences, Laval,

Canada).

Transgene expression analysis

Transgene mRNA expression was analyzed by re-

verse transcription-coupled quantitative real-time PCR

(qRT-PCR). Total RNA from the hippocampus of Tg

and wt rats was isolated and retro-transcribed into cD-

NA using a commercially available kit (Roche Applied

Sciences, Laval, Canada). Primers for the amplification

ofhAPP and rAPP mRNA were purchasedfrom Su-

perArray (SuperArray Bioscience Corporation, Fred-

erick, MD). Real-time PCR analysis was performed on

a Roche light cycler and 25

l reactions were preparedfollowing the manufacturers instructions.

The relative amount of each transcript was deter-

mined by plotting the cycle threshold (Ct) for that tran-

script, calculated on a six-point calibration curve of

the same cDNA (1:1, 1:10, 1:50, 1:100, 1:200, and

1:1000), against the log of the amount of cDNA used

for each reaction. A negative control (no cDNA) was

included for each reaction, and samples were amplified

in triplicate.

Fold changes in gene expression were obtained, nor-

malizing the values measured for the transcript of in-

terest on the relative expression of the housekeep-

ing gene glyceraldehyde-3 phosphate dehydrogenase(GAPDH).

Immunohistochemistry

Rats were deeply anaesthetized with Equithesin

(pentobarbital-based, 2.5 ml/kg, i.p.) and briefly per-

fused transcardially with saline solution to remove

blood content and facilitate post-fixation. The brain

was quickly removed, and divided into left and right

hemispheres. The left hemisphere was retained for bio-

chemistry, while the right hemisphere was post-fixed in


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4% paraformaldehyde in 0.1 M phosphate buffer (PB),

pH 7.4 for 24 h at 4

C, and finally transferred to asolution of 30% sucrose in 0.1 M PB for another 16

48 h, or until sectioned into 40 m coronal sections

with a freezing sledge microtome (SM 2000R, Leica)

at 20C.

The sections were then stained using a free-floating

immunohistochemistry(IHC) procedure. Briefly, PBS-

T (0.01 M phosphate-buffered saline containing 0.2%

Triton X-100) was used throughout all the passages,and

incubations were all performed at room temperature,

if not otherwise specified. Tissue was permeabilized

with PBS-T for 20 min and then incubated in 0.3% hy-

drogen peroxide for 20 min to quench endogenous per-

oxidases. After washing, tissue was blocked with 5%

normal goat serum (Invitrogen Canada, Toronto, ON)

for 30 min, and then incubated overnight at 4 C with

the primary antibody. The following day, sections were

washed, and then incubated with a secondary antibody

(goat-anti-mouse IgG, 1:100; MP Biomedicals, Irvine

California) for 1 h. Sections were washed again, and

then incubatedfor 1 h with a peroxidase-antiperoxidase

(PAP) monoclonalantibody complex (MAP/HRP com-

plex, Medimabs Canada Montreal QC [25]). The stain-

ing was developed with 0.6% DAB (Sigma-Aldrich

Canada, Oakville, ON) and 0.01% H2O2. After wash-

ing, sections were mounted on gelatin-coated glassslides, air-dried, dehydrated in ascending ethanol con-

centrations, cleared with xylene, and coverslipped with

Entellan (EM Science, NJ). Digital images were ac-

quiredon an Axioplan 2 Imaging microscope, equipped

with an AxioCam HRc digital camera, using Axiovi-

sion 4 Imaging program (Zeiss Canada, Toronto, ON).

Primary antibodies used in the study were: mouse

monoclonal McSA1 (1:4000 [26]), mouse monoclon-

al anti-MHCII (Major Histocompatibility Complex II)

(1:100, AbD Serotech, Raleigh,NC), mouse monoclon-

al Nu1 (1:3000 [27]), rabbit polyclonal anti-VAChT

(1:10000 [28]), guinea-pig polyclonal anti-VGluT1

(1:4000, MediMabs, Montreal, Canada [29]) and rabbit

polyclonal anti-GAD65 (1:1000, Millipore, Billarica,

MA). Thioflavin-S staining was performed as already

described [30].

Western blotting

Left brain hemispheres isolated after the perfusion

of the animals were dissected into hippocampus, cere-

bral cortex, and cerebellum, snap frozen in liquid ni-

trogen and then transferred to 80C. Soluble cortical

homogenates were obtained by sonication of the tis-

sue in lysis buffer (Cell Signaling, New England Bio-

labs, Pickering, ON) containing a complete protease in-hibitor cocktail (Roche Applied Sciences, Laval, Cana-

da) and centrifugation at 13,000 rpm for 45 min at 4C.

Supernatants were collected and protein concentration

was measured using a standard protein assay protocol

(BioRad, Mississauga, ON). To evaluate A oligomer-

ization in the samples, 250 g of proteins were loaded

on a Tris-Tricine gel (BioRad, Mississauga, ON) and

transferred to nitrocellulose membranes (BioRad, Mis-

sissauga, ON). Membranes were boiled for 5 min in

PBS, and blocked with 5% skim milk in tris-buffered

saline (TBS) containing 0.1% Tween-20 (TBS-T) at

room temperature for 2 h under constant agitation.

A mouse anti-A monoclonal antibody was applied

overnight at 4C (6E10, 1:1000 in 5% skim milk in

TBS-T, Signet laboratories, California, USA), and af-

ter washing in TBS-T buffer, the membranes were in-

cubated with a peroxidase-conjugated secondary anti-

body (1:5000, Promega, Madison, WI) for 1 h at room

temperature. Full length APP levels were also deter-

mined in the same samples using the monoclonal anti-

body 22C11 (1:1000, Roche Applied Sciences, Laval,

Canada). Immunoreactivity was visualized using an

enhanced chemiluminescence detection system (ECL,

GE Healthcare, Baie DUrfe, Canada). Band intensity

was quantified from film exposures (X-Omat LS, Ko-dak, Rochester, NY) using densitometry (MCID4 im-

age analysis system, USA).Group values wereobtained

simultaneously and normalized with respect to III-

tubulin immunoreactivity (1:50000, Promega, Madi-

son, WI).

Behavioral studies

Male and female rats were trained in the MWM

task [31], a hippocampus and cortex-dependent spatial

learning and memory test [3235]. The maze consisted

of a circular pool with a diameter of 1.75 m, filled with

water at 22C, which was made opaque by the addition

of non-toxic white paint, and contained a submerged

platform with a diameter of 10 cm, positioned 12 cm

under the level of the water. The experimental room

contained various distal visual cues. Each rat was test-

ed three times perday for fiveconsecutive days. During

each trial the rat was placed in the pool, facing the wall,

at one of the four starting points (North, South, East,

and West) and given a maximum of 120 s to locate the

hiddenplatform. When an animal failed the task, it was

placedon the platform by the experimenter and allowed

to explore for 10 s. Each performance was recorded


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Fig. 2. Temporal evolution of the A pathology in McGill-R-Thy1-APP rats. A mouse monoclonal antibody (McSA1) was used to detectA immunoreactivity in fixed tissue from Tg animals. The progression of the amyloid pathology is illustrated in hemi- (upper panels) and

homozygous (lower panels) Tg rats. Intraneuronal accumulation of A is well established by 3 months of age, and both lamina layers III andV of the cerebral cortex (ccx) and hippocampus appear intensely stained. The earliest mature amyloid plaques appear in the subiculum (S) ofhomozygous rats at 6 months of age. By 13 months of age, the extracellular amyloid deposition was found extended to most of the areas ofthe hippocampus and spreading to cortical areas. In 20 month-old Tg rats amyloid plaques and diffuse extracellular amyloid material are foundin most areas of the brain. In hemizygous Tg animals the phenotype was always found to be restricted to the intracellular accumulation of Apeptide, with virtually no hemizygous rat ever developing extracellular amyloid deposition. Scale bar = 500 m.

using a video camera connected to a tracking system

(HVS Image, Buckingham, UK), and the time spent by

the rats at each trial to find the platform (latency) was

also timed by the experimenter. Control (not trained)

animals were allowed to swim freely in absence of the

hidden platform.

To exclude visualimpairments,at theend of thetrain-

ing sessions the platform was raised above the surface

of the water and the latency measured again. The pos-

sibility of locomotorimpairments was also ruled out by

measuring the swimming speed during the training ses-

sions. Animals which floated or displayed swimming

impairments were excluded from the analysis.

Twenty four hours after completing the training

phase, memory recall was determined in a probe test.

The platform was removed and the animals were al-

lowed to swim freely for 60 s in the same context in

which they were trained. Scores of memory recall were

determined by measuring the percentage of time spent

by each animal in the target quadrant (the one contain-

ing the platform) as well as the number of times the rats

passed through the platform site.

RESULTS

Generation of McGill-R-Thy1-APP rats and transgene

expression

The McGill-R-Thy1-APP Tg rats were developed to

express the human APP containing both the Swedish

and Indiana mutations, under the control of the Thy1.2

promoter (Fig. 1A). Southern blot analysis revealed

the presence of 1 copy of the transgene per haploid

genomic set (Fig. 1B). The hAPP mRNA expression,

evaluated in brain samples by qRT-PCR, was detected

in all the Tg animals analyzed, while no signal was

revealed in samples from wt littermates. On the other


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Fig. 3. Intracellular and extracellular phases of A accumulation in McGill-R-Thy1-APP rats. Intracellular A is apparent in large pyramidalneurons of Tg animals as early as at 1 week of age (a) and the intensity of the A immunoreactivity is comparable to that observed in youngadult rats (d, 3 months old). Panels b and e describe the intra- versus extracellular A accumulation in the subiculum in 3 and 6 month-old tgrats, respectively. In c and f, extracellular (plaques) Adeposition is illustrated in the entorhinal cortex of 6 and 13 month-old rats, respectively.Scale bar = 20 m.

hand, the expression of endogenousrAPP mRNA was

unchanged in Tg rats compared to wt rats (data not

shown).

The regulation of transgene expression provided by

the Thy1.2 promoter resulted in a widespread expres-

sion of hAPP protein in the forebrain. 6E10 im-

munoreactivity (IR) was undetectable in lysates from

cerebellum, heart, liver, and thymus (Fig. 1C). As a

consequence of the transgene expression, average pro-

duction of full-length APP in the cortex of Tg an-

imals was doubled when compared to wt littermates

(Fig. 5A).

A immunoreactivity and amyloid pathology

A accumulation in the brains of Tg rats was stud-

ied by immunohistochemistry, using a monoclonal an-

tibody specific for human Apeptide (McSA1) (Figs 2

and 3). One week old Tg rats showed the accumulation

of iA in pyramidal neurons of the cerebral cortex and

hippocampus (Fig. 3 panel a), and this phenotype (iA

accumulation) was strongly established in 23 month-

old rats.

In the cortex, A-positive neurons were distribut-

ed throughout all the layers. Particularly intense A-

immunostaining was found in the entorhinal (Ent), piri-

form (Pir), and retrosplenial (RS) cortices (supplemen-

tary Fig. S1). In neurons of lamin III and V McSA1-

IR was localized in the soma and proximal dendrites

(Fig. 3 panel d). In the hippocampus, A-IR was ho-

mogeneously distributed to all the areas (CA1, CA2,

CA3, and subiculum) while granular neurons of the

dentate gyrus (DG) always appeared lightly immunos-

tained. This phenotype was common to hemi- and ho-

mozygous Tg rats, even if hemizygous animals always

showed lower levels of A-IR.

As the iA accumulation progresses with aging,

extracellular dense amyloid deposits (plaques) were

found in homozygous animals as young as 6 months,

usually starting in the subiculum area (Figs 2 and 3

panel e). Occasionally, Aaggregates were also found

in the entorhinal cortex, where they seem to derive from

leaking or bursting A-loaded neurons (Fig. 3 panel

c). By 13 months of age, both diffuse and dense (fib-

rillar) plaques were detected in most areas of the hip-

pocampal formation and started to appear in the cere-

bral cortex(Fig. 2). Finally, in 20 month-oldrats, dense


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Fig. 4. Amyloid plaque deposition and signs of neuroinflammation and neurodegeneration in the McGill-R-Thy1-APP rats. Sections from 13 (aand d) and 20 (b and e) month-old rats were stained for A and MHCII (respectively blue and brown reactions). The dense, fibrillar nature ofthese plaques was confirmed by thioflavine S staining (c and f). From g to i, the presence of neurodegeneration surrounding the sites of plaquedeposition (*) was investigated in the same animal (20 month-old): dystrophic cholinergic (VAChT-IR) (g) and glutamatergic (VGluT-IR) (h),but not GABA-ergic (GAD65-IR) (i) neurites were observed, confirming the differential vulnerability of different neurotransmitter systems toamyloid plaques deposition [29]. Scale bar = 500 m in panels a to c, and = 20 m in panels d to i.

amyloid plaques were found in most areas of the brain,and particularly in the hippocampus as well as in the

entorhinal and parietal cortices (Fig. 2 and S1, panels

g-i). The fibrillar nature of the plaques observed by A

immunostaining was confirmed by their fluorescence

following the application of thioflavin S (Fig. 4, panels

c and f). All the pathological features described were

equally present in male and female Tg animals, leading

us to conclude that no gender-associated difference ex-

ists in the amyloid distribution in our McGill-R-Thy1-

APP model.

Recruitment of activated microglia was evident

at the stage of extracellular A deposition. Thus

MHCII-positive cells were found surrounding ma-ture, fibrillar, amyloid plaques (Fig. 4), indicating an

overt inflammatory reaction. Staining with antibod-

ies specific for presynaptic markers (vesicular acetyl-

choline transporter protein [VAChT], vesicular gluta-

mate transporter 1 [VgluT1], or glutamate decarboxy-

lase [GAD65]) in 20 month-old rats revealed the pres-

ence of cholinergic and glutamatergic, dystrophic neu-

rites surrounding the sites of plaque deposition, while

no overt dystrophy of GABAergic neurites could be

detected (Fig. 4, panels g-i). In this Tg model, we

observed no A-immunoreactive material surrounding


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Fig. 5. Human APP expression and A oligomerization in Tg rats. A) Total (rodent and human) APP expression levels were revealed by

western blotting in cortical samples from wt and Tg rats (n = 7 for each group) at 6 months of age using the antibody 22C11, and they werefound significantly increased in Tg animals (p < 0.001, unpaired t-test). B) Aexpression and oligomerization as evaluated by western blottingusing the 6E10 monoclonal antibody. Cortical samples from 6 month-old Tg rats contained a greater abundance of A oligomers (trimers) inhomozygous animals as compared to hemizygous littermates. C) The oligomeric nature of intraneuronal A was also confirmed by IHC usingthe monoclonal antibody Nu1 that specifically recognizes soluble aggregates of A. The immunoreactive pattern revealed with Nu1 antibodywas very similar to that obtained with McSA1, and consistently reproduced both on PFA-fixed and unfixed tissue. The immunoreactivity ofundenatured, unfixed tissue confirmed that a large proportion of the iA material is indeed of oligomeric nature. Scale bar = 20 m.

CNS blood vessels which would indicate the presence

of vascular amyloidosis.

McSA1 specificity for A peptide

The possible cross-reactivity of the human A-

specific antibody used (McSA1) with human holo-

APP or N-terminal products thereof (e.g., soluble

APP sAPP) was investigated to better de-

fine the nature of the intracellular staining observed

(Fig. S2). Thus, the pre-absorption of the McSA1 anti-

body with 2.5 g/mL of synthetic A142 completely

abolished the intracellular immuno-staining, while the

same molar concentration of synthetic soluble sAPP

(50 g/mL) did not show anysignificant effect (Fig. S2,

panels a to c).

On the other hand, pre-absorption with the above

peptides had opposite effects on the immuno-staining

produced by the well-characterized monoclonal anti-

body 22C11, which recognizes both human and rodent

full length APP as well as N-terminal fragments there-

of. Thus, as shown in Fig. S2 (panels d to f), A pre-

absorption did not affect the 22C11 immunoreaction in

tissue sections, while as expected, the reactivity was

abolished by pre-absorption with equimolar sAPP.

These tests confirmed the selective specificity of the

two antibodies to differentially recognize A species

from full length APP and N-terminal fragments.

Finally, the distribution of McSA1-IR and 22C11-

IR inside the neurons was investigated by fluorescent

double-labeling with confocal microscopy. As shown

in panels g to i of Fig. S2, 22C11-IR (green) appeared

to be homogeneously diffused in the soma as compared

to McSA1-IR,whichappearedconcentrated in granule-

like bodies, without any noticeable overlapping be-

tween the two signals (panel i). The above-described


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A B

DC

Fig. 6. Cognitive impairment in the McGill-R-Thy1-APP rats appears at 3 months of age, coincidental with intracellular accumulation of Apeptide. A) Latency to locate the hidden platform during the learning phase of the MWM. Wt (n = 8) and Tg (n = 7) rats were trained in 3 trialsa day for 5 consecutive days and memory recall was assessed 24 h later. Homozygous Tg animals (n = 3) displayed an impaired performanceduring the learning phase of the task (p < 0.05, two-way ANOVA) when compared to wt controls, and a significant impairment in retrieving thespatial memory (panel B, p < 0.01, unpaired t-test), suggesting that the intracellular accumulation of A is sufficient to trigger an impairmentin spatial cognitive functions. A trimers levels were determined by western blotting after detection with the monoclonal antibody 6E10 (seeFig. 4), and normalized against the III-tubulin content of the samples. C) Relative amounts of soluble A trimers as measured in corticalsamples from Tg rats were found to be 4-fold increased in homozygous compared to hemizygous littermates (p < 0.0001, unpaired t-test). D)A trimers levels were plotted against the escape latency of Tg rats on the last day of MWM training. This revealed a clear trend towards apositive correlation.

analysis strongly supports the notion that the intraneu-

ronal material revealed with McSA1 in aldehyde-fixedbrain sections is indeed A.

A oligomerization in McGill-R-Thy1-APP rats

The nature of the A material that accumulates in-

side the neurons in Tg rats was investigated by west-

ern blotting and IHC. As shown in Fig. 5B, the main

species detected in soluble homogenates from the cor-

tex were A trimers, with monomeric A becoming

apparent in some old animals (13 to 25 months) with

a heavy A load phenotype (not shown). As expect-

ed, the levels of oligomeric A (trimers) measured by

western blotting were significantly higher in homozy-

gous Tg rats compared to hemizygous littermates (p


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A B

DC

Fig. 7. Pronounced cognitive impairment in the McGill-R-Thy1-APP rats at 13 months of age. 13 month-old rats (wt n = 12 and Tg n = 10)were trained in the MWM task in 4 trials/day for 5 consecutive days and memory recall was assessed 24 h later (A and B). Among Tg animals,homozygous rats (n = 6) showed a significant impairment throughout all the learning phase (p < 0.001, two-way ANOVA) as compared towt controls, and a significant deficit in memory recall (p < 0.05, one-way ANOVA). The performance of hemizygous tg rats was intermediatebetween homozygous Tg and wt. C) Aoligomers (trimers) levels in the cortex, as detected by WB, were found to be 3.5 0.5 (mean SEM)fold higher in homozygous animals compared to hemizygous littermates (p < 0.01, unpaired t-test). D) A significant (p = 0.0064) positivecorrelation was found in Tg rats between Aoligomer levels and the escape latency as an indicator of learning impairment.

Cognitive impairment in McGill-R-Thy1-APP rats

correlates with oligomeric A load

The cognitive abilities of McGill-R-Thy1-APP Tg

rats were tested in the MWM task. Three month-

old Tg rats were found to be already impaired in this

hippocampus-dependent behavioral test. In particular,

although both Tg and wt littermates improved their

performance throughout the five days of training, as

indicated by the decrease of the latency to locate the

submerged platform (Fig. 6A), the performance of ho-

mozygous Tg rats was significantly poorer than that

of wt, and on the last day of the training they spent a

significantly longer time to find the platform compared

to the control animals (p < 0.05, two-way ANOVA).

On the contrary, the learning curve of hemizygous Tg

rats was, at this stage, undistinguishable from that of wt

rats. Twenty four hours later, memory recall was mea-

sured with a probe test. Tg homozygous rats spent 12.9

4.7% of the time in the target quadrant (Fig. 6B), the

percentage being significantly lower compared to the

wt group (40.6 3.3%; p = 0.0056, one-way ANO-VA). The performance of Tg rats which spent 24.8

6.5% of the time in the target quadrant was interme-

diate between the other two groups. Also, wt animals

crossed the target platform site 3.4 0.5 times, com-

pared to 1 0.6 measured for homozygous Tg rats

(p < 0.05, one-way ANOVA).

When the amount of A oligomers (trimers) was

measured by western blotting in soluble extracts from


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the cerebral cortex of Tg rats (Fig. 6C), significant-

ly higher levels were detected in homozygous animalscompared to hemizygous animals (4.1 0.3 fold; p

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