Journal of Alzheimers Disease 20 (2010) 113126 DOI 10.

3233/JAD-2010-1349 IOS Press

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A Novel Transgenic Rat Model with a Full Alzheimers-Like Amyloid Pathology Displays Pre-Plaque Intracellular Amyloid- -Associated Cognitive Impairment
Wanda Carolina Leon a,1, Fabio Canneva a,1, Vanessa Partridge a , Simon Allarda , Maria Teresa Ferrettia , Arald DeWildea , Freya Vercauteren a,2, Ramtin Atifeha , Adriana Ducatenzeiler a, William Kleinb , Moshe Szyfa , Leena Alhonen c and A. Claudio Cuelloa,d,e,
a

Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada Cognitive Neurology and Alzheimers Disease Center, Northwestern University Institute for Neuroscience, Chicago, IL, USA c Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland d Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada e Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada
b

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 A accumulation, 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 (A PP) 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 A accumulation, 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 A deposits and, by 6 months of age, dense, thioavine S-positive, amyloid plaques are detected, associated with glial activation and surrounding dystrophic neurites. The cognitive functions in transgenic McGill-RThy1-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), where the 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

and FC equally contributed to the experimental work. afliation: Sleep Research Centre, Universit e de Montreal, Faculty of Medicine, Sacr e-Coeur Hospital, Montreal, QC, Canada.
2 Present

1 WL

to: A. Claudio Cuello, 3655 Promenade Sir William Osler, H3G 1Y6, Montreal, QC, Canada. Tel.: +1 514 398 3618; Fax: +1 514 398 8317; E-mail: claudio.cuello@mcgill.ca.

Correspondence

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

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INTRODUCTION Alzheimers disease (AD) is a neurodegenerative condition characterized by a progressive loss of cognitive abilities associated with increased levels of brain amyloid- peptide (A ) that tends to deposit as extracellular senile plaques, the presence of intraneuronal neurobrillary tangles composed of abnormally phosphorylated tau protein, the loss of synapses, and eventually neuronal death [1,2]. A peptide is detected in healthy individuals [3,4], but its increased production, or diminished clearance, are considered as 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 (A PP) result in the elevation of A production [5,6]. Since the rst 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 reproducing the full AD-like amyloid pathology has been elusive. The desirability of rat models is based on the extensive Neuroscience Database and the richer behavioral display of rats as compared to mice [11]. In addition, the larger brain size makes surgical procedures and pharmacological manipulation easier to perform. In recent years, some Tg rat models have been developed by us and by others, which failed, however, to produce extracellular deposition of amyloid plaques [1214]. Nevertheless, intracellular A (iA ) accumulation has been shown in one of these models, to induce changes in sub-cellular organelles and alterations in the hippocampal proteomic patterns, as well as cognitive impairments [1517]. More recently Flood and colleagues [18] reported the successful generation of Tg rats in which the co-expression of three different transgenes (hA PP swe , hA PPswe,ind , and hPS1 f inn ) leads to deposition of mature plaques at around 9 months of age. The behavioral characterization of these Tg rats [19] previously showed a significant cognitive impairment, as assessed by the Morris water maze (MWM) task, as early as two months prior to plaque deposition. Since the rst 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 advantages mentioned above. Here we report the generation and characterization of a new Tg rat line (McGill-RThy1-APP) in which the expression of a single transgene coding for a modied variant of the human protein A PP751 , containing both the Swedish and Indiana

mutations, results in the full AD-like amyloid pathology, including the generation of mature plaques and dystrophic neurites. In homozygous Tg rats, the human A peptide was found to accumulate intraneuronally from one week postnatal, and the progressive extracellular amyloid pathology was established at 6 months of age. The earliest amyloid plaques were rst detected in the subiculum and later spread to the hippocampus and cerebral cortex and some sub-cortical regions. Coincidentally with the earlier phases of iA accumulation, 3 month-old homozygous Tg rats displayed a signicant cognitive impairment, as assessed by the MWM, compared to wild type (wt) control animals, while the behavior of hemizygous littermates was found to be intermediate between the two populations. Interestingly, the levels of oligomeric A (trimers) measured in cortical samples of homozygous Tg rats were found to be around four-fold higher than in hemizygous littermates, suggesting a direct effect of this species of the peptide on the cognitive status of the McGill-R-Thy1-APP rats.

MATERIALS AND METHODS Generation and selection of the transgenic rats The McGill-R-Thy1-APP transgenic rats were generated by the standard pronuclear injection technique [20] using HsdBrl:WH Wistar rats. The DNA construct used was constituted by 5 and 3 regulatory elements of the murine thymocyte antigen promoter (Thy1.2) [21,22], carrying the human A PP 751 (hA PP) cDNA containing the entire coding region and 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 amplication of DNA extracted from the tails to assess the presence of the hA PP transgene with the following primers: 5-AAGAGGTGGTTCGAGAGGTG-3 and 5-CTGCATCCAGATTCAC-3. Throughout the study period, pairs of animals were housed in a controlled environment (temperature 22 C, humidity 50 60%, 12 h light/12 h dark schedule) and efforts were made to minimize discomfort and the number of animals used. The animal experimentation protocol was approved by the McGill University Animal Care Committee 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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Fig. 1. Generation and characterization of the transgenic animals. A) Thy1-A PP construct showing the cloning of hA PP751 variant (containing the Swedish and Indiana mutations) under the transcriptional control of the murine Thy1.2 promoter. The approximate location of the HindIII restriction sites used for digesting the genomic DNA is indicated by the arrows, and the approximate annealing site of the probe used in the Southern 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 hA PP expression in different tissues as revealed by the 6E10 antibody. sA PP expression was used as an indicator of transgene expression given the antibody specicity for the human form. No signal was detected in the sample from a wt rat, while an obvious expression of the transgene was specically found in cortical samples from hemi- and homozygous 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 puried genomic DNA by Southern blotting according to standard procedures. Briey, 10 g of DNA were digested 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 controls (from 1 to 16 copies). A digoxygenin-labelled probe was obtained by PCR (Roche Applied Sciences, Laval, Canada) from the Thy1.2-A PP plasmid using the primers 5-ATCCCACTCGCACAGCAG-3 and 5-GGAATCACAAAGTGGGGATG-3, to anneal on the 5 region of the transgene (see Fig. 1A). Chemiluminescence detection was performed using the protocol for DIG-labeled probes according to the manufacturers instructions (Roche Applied Sciences, Laval, Canada). Transgene expression analysis Transgene mRNA expression was analyzed by reverse transcription-coupled quantitative real-time PCR (qRT-PCR). Total RNA from the hippocampus of Tg and wt rats was isolated and retro-transcribed into cDNA using a commercially available kit (Roche Applied Sciences, Laval, Canada). Primers for the amplication

of hA PP and rA PP mRNA were purchased from SuperArray (SuperArray Bioscience Corporation, Frederick, MD). Real-time PCR analysis was performed on a Roche light cycler and 25 l reactions were prepared following the manufacturers instructions. The relative amount of each transcript was determined by plotting the cycle threshold (Ct) for that transcript, 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 amplied in triplicate. Fold changes in gene expression were obtained, normalizing the values measured for the transcript of interest on the relative expression of the housekeeping gene glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Immunohistochemistry Rats were deeply anaesthetized with Equithesin (pentobarbital-based, 2.5 ml/kg, i.p.) and briey perfused transcardially with saline solution to remove blood content and facilitate post-xation. The brain was quickly removed, and divided into left and right hemispheres. The left hemisphere was retained for biochemistry, while the right hemisphere was post-xed in

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4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 for 24 h at 4 C, and nally transferred to a solution 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-oating immunohistochemistry (IHC) procedure. Briey, PBST (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 specied. Tissue was permeabilized with PBS-T for 20 min and then incubated in 0.3% hydrogen peroxide for 20 min to quench endogenous peroxidases. 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 incubated for 1 h with a peroxidase-antiperoxidase (PAP) monoclonal antibody complex (MAP/HRP complex, Medimabs Canada Montreal QC [25]). The staining was developed with 0.6% DAB (Sigma-Aldrich Canada, Oakville, ON) and 0.01% H 2 O2 . After washing, sections were mounted on gelatin-coated glass slides, air-dried, dehydrated in ascending ethanol concentrations, cleared with xylene, and coverslipped with Entellan (EM Science, NJ). Digital images were acquired on an Axioplan 2 Imaging microscope, equipped with an AxioCam HRc digital camera, using Axiovision 4 Imaging program (Zeiss Canada, Toronto, ON). Primary antibodies used in the study were: mouse monoclonal McSA1 (1:4000 [26]), mouse monoclonal anti-MHCII (Major Histocompatibility Complex II) (1:100, AbD Serotech, Raleigh,NC), mouse monoclonal 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). Thioavin-S staining was performed as already described [30]. Western blotting Left brain hemispheres isolated after the perfusion of the animals were dissected into hippocampus, cerebral cortex, and cerebellum, snap frozen in liquid nitrogen and then transferred to 80 C. Soluble cortical homogenates were obtained by sonication of the tis-

sue in lysis buffer (Cell Signaling, New England Biolabs, Pickering, ON) containing a complete protease inhibitor cocktail (Roche Applied Sciences, Laval, Canada) and centrifugation at 13,000 rpm for 45 min at 4 C. Supernatants were collected and protein concentration was measured using a standard protein assay protocol (BioRad, Mississauga, ON). To evaluate A oligomerization in the samples, 250 g of proteins were loaded on a Tris-Tricine gel (BioRad, Mississauga, ON) and transferred to nitrocellulose membranes (BioRad, Mississauga, 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 4 C (6E10, 1:1000 in 5% skim milk in TBS-T, Signet laboratories, California, USA), and after washing in TBS-T buffer, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000, Promega, Madison, WI) for 1 h at room temperature. Full length A PP levels were also determined in the same samples using the monoclonal antibody 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 quantied from lm exposures (X-Omat LS, Kodak, Rochester, NY) using densitometry (MCID4 image analysis system, USA). Group values were obtained simultaneously and normalized with respect to IIItubulin immunoreactivity (1:50000, Promega, Madison, 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, lled with water at 22 C, 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 tested three times per day for ve consecutive 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 hidden platform. When an animal failed the task, it was placed on 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 detect A immunoreactivity in xed 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 and V of the cerebral cortex (ccx) and hippocampus appear intensely stained. The earliest mature amyloid plaques appear in the subiculum (S) of homozygous rats at 6 months of age. By 13 months of age, the extracellular amyloid deposition was found extended to most of the areas of the hippocampus and spreading to cortical areas. In 20 month-old Tg rats amyloid plaques and diffuse extracellular amyloid material are found in most areas of the brain. In hemizygous Tg animals the phenotype was always found to be restricted to the intracellular accumulation of A peptide, 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 nd 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 visual impairments,at the end of the training sessions the platform was raised above the surface of the water and the latency measured again. The possibility of locomotor impairments was also ruled out by measuring the swimming speed during the training sessions. Animals which oated 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 allowed 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 containing 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 A PP 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 hA PP 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 pyramidal neurons 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 young adult 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 tg rats, respectively. In c and f, extracellular (plaques) A deposition is illustrated in the entorhinal cortex of 6 and 13 month-old rats, respectively. Scale bar = 20 m.

hand, the expression of endogenous rA PP 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 expression of hA PP protein in the forebrain. 6E10 immunoreactivity (IR) was undetectable in lysates from cerebellum, heart, liver, and thymus (Fig. 1C). As a consequence of the transgene expression, average production of full-length A PP in the cortex of Tg animals was doubled when compared to wt littermates (Fig. 5A). A immunoreactivity and amyloid pathology A accumulation in the brains of Tg rats was studied by immunohistochemistry, using a monoclonal antibody specic for human A peptide (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 monthold rats.

In the cortex, A -positive neurons were distributed throughout all the layers. Particularly intense A immunostaining was found in the entorhinal (Ent), piriform (Pir), and retrosplenial (RS) cortices (supplementary Fig. S1). In neurons of lamin III and V McSA1IR was localized in the soma and proximal dendrites (Fig. 3 panel d). In the hippocampus, A -IR was homogeneously distributed to all the areas (CA1, CA2, CA3, and subiculum) while granular neurons of the dentate gyrus (DG) always appeared lightly immunostained. This phenotype was common to hemi- and homozygous 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, A aggregates 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 (brillar) plaques were detected in most areas of the hippocampal formation and started to appear in the cerebral cortex (Fig. 2). Finally, in 20 month-old rats, dense

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Fig. 4. Amyloid plaque deposition and signs of neuroinammation and neurodegeneration in the McGill-R-Thy1-APP rats. Sections from 13 (a and d) and 20 (b and e) month-old rats were stained for A and MHCII (respectively blue and brown reactions). The dense, brillar nature of these plaques was conrmed by thioavine S staining (c and f). From g to i, the presence of neurodegeneration surrounding the sites of plaque deposition (*) 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, conrming the differential vulnerability of different neurotransmitter systems to amyloid 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 brillar nature of the plaques observed by A immunostaining was conrmed by their uorescence following the application of thioavin 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 exists in the amyloid distribution in our McGill-R-Thy1APP model. Recruitment of activated microglia was evident at the stage of extracellular A deposition. Thus

MHCII-positive cells were found surrounding mature, brillar, amyloid plaques (Fig. 4), indicating an overt inammatory reaction. Staining with antibodies specic for presynaptic markers (vesicular acetylcholine transporter protein [VAChT], vesicular glutamate transporter 1 [VgluT1], or glutamate decarboxylase [GAD65]) in 20 month-old rats revealed the presence of cholinergic and glutamatergic, dystrophic neurites 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 A PP expression and A oligomerization in Tg rats. A) Total (rodent and human) A PP 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 were found signicantly increased in Tg animals (p < 0.001, unpaired t-test). B) A expression and oligomerization as evaluated by western blotting using the 6E10 monoclonal antibody. Cortical samples from 6 month-old Tg rats contained a greater abundance of A oligomers (trimers) in homozygous animals as compared to hemizygous littermates. C) The oligomeric nature of intraneuronal A was also conrmed by IHC using the monoclonal antibody Nu1 that specically recognizes soluble aggregates of A . The immunoreactive pattern revealed with Nu1 antibody was very similar to that obtained with McSA1, and consistently reproduced both on PFA-xed and unxed tissue. The immunoreactivity of undenatured, unxed tissue conrmed 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 specicity for A peptide The possible cross-reactivity of the human A specic antibody used (McSA1) with human holoA PP or N-terminal products thereof (e.g., soluble A PP sA PP) was investigated to better dene the nature of the intracellular staining observed (Fig. S2). Thus, the pre-absorption of the McSA1 antibody with 2.5 g/mL of synthetic A 142 completely abolished the intracellular immuno-staining, while the same molar concentration of synthetic soluble sA PP (50 g/mL) did not show any signicant 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 antibody 22C11, which recognizes both human and rodent full length A PP as well as N-terminal fragments thereof. Thus, as shown in Fig. S2 (panels d to f), A preabsorption did not affect the 22C11 immunoreaction in tissue sections, while as expected, the reactivity was abolished by pre-absorption with equimolar sA PP. These tests conrmed the selective specicity of the two antibodies to differentially recognize A species from full length A PP and N-terminal fragments. Finally, the distribution of McSA1-IR and 22C11IR inside the neurons was investigated by uorescent 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, which appeared concentrated in granulelike bodies, without any noticeable overlapping between the two signals (panel i). The above-described

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Fig. 6. Cognitive impairment in the McGill-R-Thy1-APP rats appears at 3 months of age, coincidental with intracellular accumulation of A peptide. 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 trials a day for 5 consecutive days and memory recall was assessed 24 h later. Homozygous Tg animals (n = 3) displayed an impaired performance during the learning phase of the task (p < 0.05, two-way ANOVA) when compared to wt controls, and a signicant impairment in retrieving the spatial memory (panel B, p < 0.01, unpaired t-test), suggesting that the intracellular accumulation of A is sufcient to trigger an impairment in spatial cognitive functions. A trimers levels were determined by western blotting after detection with the monoclonal antibody 6E10 (see Fig. 4), and normalized against the III-tubulin content of the samples. C) Relative amounts of soluble A trimers as measured in cortical samples 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 a positive correlation.

analysis strongly supports the notion that the intraneuronal material revealed with McSA1 in aldehyde-xed brain sections is indeed A . A oligomerization in McGill-R-Thy1-APP rats The nature of the A material that accumulates inside the neurons in Tg rats was investigated by western blotting and IHC. As shown in Fig. 5B, the main species detected in soluble homogenates from the cortex 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 expected, the levels of oligomeric A (trimers) measured by

western blotting were signicantly higher in homozygous Tg rats compared to hemizygous littermates (p < 0.0001, unpaired t-test). A further conrmation of this result was provided by the immunostaining of rat brain sections with the Nu1 antibody, which specically recognizes A oligomers [27] and not brils. As already found with the McSA1 antibody, many areas of the brain showed Nu1-IR, with particularly intense staining localized in the large pyramidal neurons of lamin III and V of the cerebral cortex, hippocampus and entorhinal cortex. In particular, Nu1-staining inside the cells appeared concentrated in granule-like bodies, probably cytoplasmatic vesicles containing the oligomeric aggregates (Fig. 5C).

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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 signicant impairment throughout all the learning phase (p < 0.001, two-way ANOVA) as compared to wt controls, and a signicant decit in memory recall (p < 0.05, one-way ANOVA). The performance of hemizygous tg rats was intermediate between homozygous Tg and wt. C) A oligomers (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 signicant (p = 0.0064) positive correlation was found in Tg rats between A oligomer 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 monthold 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 ve days of training, as indicated by the decrease of the latency to locate the submerged platform (Fig. 6A), the performance of homozygous Tg rats was signicantly poorer than that of wt, and on the last day of the training they spent a signicantly longer time to nd 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 measured with a probe test. Tg homozygous rats spent 12.9 4.7% of the time in the target quadrant (Fig. 6B), the percentage being signicantly lower compared to the wt group (40.6 3.3%; p = 0.0056, one-way ANOVA). The performance of Tg rats which spent 24.8 6.5% of the time in the target quadrant was intermediate between the other two groups. Also, wt animals crossed the target platform site 3.4 0.5 times, compared 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), signicantly higher levels were detected in homozygous animals compared to hemizygous animals (4.1 0.3 fold; p < 0.0001, unpaired t-test). After plotting these values against the latency of the animals recorded on the last day of training, a trend of positive correlation was found, even if statistical signicance was not reached, probably due to the small number of animals used in the study (Fig. 6D). The behavioral display of 13 month-old rats was also assessed in the MWM. Homozygous Tg animals showed an even more severe impairment in learning the task, as for each day of training, they took more time than wt controls to locate the hidden platform (Fig. 7). Hemizygous animals also showed a cognitive impairment, since they learned the task with a signicant delay compared to wt rats. As a direct consequence of the cognitive decline, memory recall was signicantly impaired in 13 month-old homozygous rats (p < 0.05, one-way ANOVA), since they spent less time in the target quadrant (24.1 2.8 compared to 38.3 3.4) and crossed the trained platform position fewer times (2 0 compared to 4.7 0.6) than wt animals. Soluble A trimer levels measured in cortical samples of Tg animals were found to be 3.5 0.5 (mean SEM) fold higher in homozygous rats than in hemizygous littermates, similar to what was observed in younger animals. Interestingly, a signicant positive correlation (p = 0.0064, linear regression) was found between the amount of A trimers and the latency measured on the last day of training, suggesting an effect of soluble A oligomers on the behavioral display of McGill-RThy1-APP Tg rats. As no difference was observed in the amyloid pathology developed by male and female Tg rats,both genders were equally represented in the behavioral tests and, as expected, their performance was always comparable (Fig. S3).

DISCUSSION Although several transgenic rats overproducing A PP were generated in the past, none showed the full amyloid pathology. We reasoned that low expression of the transgene in neurons is behind the failure to replicate the AD-like pathology in the rat model. Here we used a Thy1.2 driven expression construct of doubly mutated A PP that was highly expressed in neurons. Indeed, McGill-R-Thy1-APP rats show a direct effect of gene dosage on the amyloid pathology produced. In

fact, the complete amyloid phenotype,with dense brillar plaque deposition, is replicated only in homozygous animals, while in hemizygous littermates the intraneuronal accumulation of A remains the only hallmark of the amyloid pathology throughout their entire life, as in our previous Tg rat model coded UKUR25 [12]. Lack of mature amyloid plaque deposition was also observed in other previous Tg rat models [36,37] until the more recent publication from Flood and colleagues [18], in which the co-expression of two different FAD isoforms of the hA PP (A PP swe,ind and A PPswe ), alone or in combination with a human FAD variant of presenilin1 (PS1 f inn ) [38] led to the deposition of amyloid plaques in animals aged 9 to 18 months. The cognitive performance of triple Tg rats has been also investigated [19], demonstrating a signicant cognitive impairment as early as at 7 months of age, two months prior to plaque deposition, as well as a corresponding decit in LTP formation in the hippocampus of Tg animals. In the present model, a single transgene is expressed encoding for a variant of hA PP containing both the Swedish and Indiana mutations under the control of the murine Thy1.2 promoter [21]. The expression of the transgene is, therefore, highly restricted to neurons, and the levels of human A PP were undetectable in cerebellum, heart, kidney, liver, and thymus. hA -IR was detected inside the neurons as early as at one week after birth, and this phenotype was wellestablished in 23 month-old Tg rats. Soluble A was found to accumulate intraneuronally,at least partially in an oligomeric form, in most areas of the forebrain, and more prominently in entorhinal cortex and hippocampus, resembling what has been reported for early stages of AD [3941] and of Down syndrome [42]. A similar pre-plaque phase of iA accumulation has also been reported in a large number of mouse and rat transgenic models [43]. In the McGill-R-Thy1-APP rats, this phenotype was present in both hemi- and homozygous animals, with the former showing a much lighter pathology. From the age of 6 months, extracellular amyloid deposits were detected in homozygous Tg rats, starting invariably with a few dense-core plaques in the subiculum. In addition, smaller deposits were found at about this age in the entorhinal cortex, seemingly originating from A -loaded neurons (Fig. 2), an observation which is in agreement with what was reported in human specimens from AD cases [41,44] and reinforces the argument that at least some plaques originate from cellular debris leaking from A -burdened neurons. Plaque pathology increases with time, spreading by 13 months

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of age to the rest of the hippocampal formation and eventually to the neocortex. Extracellular A deposits were found to be both of dense (brillar, thioavin Spositive) and diffused nature, with the initial plaques in the subiculum being usually of the rst type. Interestingly, hemizygous animals failed to produce any detectable extracellular deposit of amyloid protein. This would indicate that in these animals the amount of A peptide generated is not sufcient for the progression from intra- to extracellular pathology. This supports our initial hypothesis that failure to develop full ADlike pathology in previously published rat Tg models, including our UKUR25 model, is caused by insufcient expression of A to progress to extracellular pathology. After the plaque deposition is initiated, other signs of degeneration were detected in McGill-R-Thy1-APP rats, such as formation of dystrophic neurites in the peri-plaque areas. Fibrillar, dense plaques were also found to be surrounded by activated (MHCII-positive) microglia, while no reactivity was detected in the proximity of diffused amyloid deposits, as already reported in specimens from AD patients (for a review see [45, 46]). Western blot analysis revealed A trimers as the main species detected in soluble homogenates from cortical samples of Tg rats, and their level was found to be higher in homozygous Tg rats than in hemizygous littermates. Naturally produced low-molecular weight (low-n) A oligomers have been demonstrated to have neurotoxic effects, even at low, nanomolar concentrations, provoking disruption of both LTP formation [47] and cognitive functions [48]. Such ndings are consistent with our behavioral/neurochemical observation in the Tg rats. In conclusion, McGill-R-Thy1-APP homozygous animals show a clear cognitive decline by 3 months of age when compared to age-matched wt and hemizygous littermates, at a stage of the amyloid pathology where A oligomer accumulation is well-established. In particular, homozygous Tg rats spent more time to locate the hidden platform during the training phase of the MWM, and their performance during the probe test showed a signicant impairment of spatial memory retrieval. Since the trimer levels were found to be around 4-fold higher in homozygous compared to hemizygous Tg rats, the intraneuronal accumulation of low-n A oligomers (trimers) may be suggested as being responsible for the behavioral decit observed. Such a view is supported by the trend for a positive correlation between the levels of A trimers and the escape latency in 3 month-old rats and the signicant correlation of

these two parameters in the 13 month-old rats, when trimers are still the main form of soluble A detected in homogenates from cortical samples of Tg rats. The effect of soluble oligomers of A on synaptic plasticity and behavior in animal models of AD has been recently reviewed [49]. Cell-derived A dimers, trimers, and tetramers were conrmed to interfere with the formation of LTP [47,50]. Both intracellular and extracellular accumulation of A peptide is also responsible for the dysregulation of the ERK/CREB/CRE signaling pathway [17,5154], a key mechanism for synaptic plasticity underpinning LTP formation, the disruption of which could explain the cognitive decits observed in vivo. The nding that McGill-R-Thy1-APP hemizygous rats displayed less behavioral impairment than homozygous littermates strongly suggests a dose-dependent interference of oligomeric A on CNS synaptic functions in this Tg model. This feature is of particular interest for the study of early phases of AD. In fact, it is becoming widely accepted that early (even premorbid) therapeutic intervention before or at the onset of the cognitive decline would have a better chance for a disease-modifying therapy [55]. McGill-R-Thy1-APP homo- and hemizygous lines, therefore, offer an interesting opportunity to study A dose-response effects on cognition, in vivo, at the earliest stages of the A amyloid pathology, taking advantage of the more predictable behavioral display of rats when compared to mice.

CONCLUSIONS In the McGill-R-Thy1-APP transgenic rats, the expression of a single transgene (hA PP swe,ind ) is sufcient to trigger the full A amyloid pathology. Tg rats developing an AD-like amyloid pathology would offer a suitable alternative model to study putative new therapies, given the richer behavioral display of this species and the close correlation between A oligomer load and the cognitive status observed.

ACKNOWLEDGMENTS The authors thank the skillful technical assistance of Dr. Anne Uimari, Ms. Riitta Sinervirta. and Ms. Anu Heikkinen in the production of the transgenic rat line. This project has been supported by CIHR grant MOP6717 to ACC. ACC is the holder of the Charles E.

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Frosst Merck Chair in Pharmacology. FC is the holder of the Charles E. Frosst Merck post-doctoral fellowship. WCL is the holder of a fellowship from University of the Andes, Merida, Venezuela. ACC would like to thanks Dr H. Van der Putten (Novartis) for providing the murine Thy1.2 promoter and Dr Alan Frosst and the Frosst family for their interest and support in these investigations. Authors disclosures available online (http://www.jalz.com/disclosures/view.php?id=222).

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