Proteolytical processing of mutated human amyloid precursor protein in transgenic mice

Molecular Brain Research 47 Ž1997. 108–116

Research report

Proteolytical processing of mutated human amyloid precursor protein in transgenic mice Christian Czech a , Pia Delaere ` a, Anne Franc¸oise Macq b, Michel Reibaud a, Sylvie Dreisler a, Nathalie Touchet a , Brigitte Schombert a , Martine Mazadier a , Luc Mercken a , Manfred Theisen c , Laurent Pradier a , Jean-Noel ¨ Octave b, Konrad Beyreuther d, a,) Gunter Tremp ¨ a

Rhone-Poulenc Rorer, Centre de Recherche de Vitry-AlfortÕille, 13 quai Jules Guesde, BP 14, 94403 Vitry-sur-Seine Cedex, France ˆ ´ b UniÕersite´ Catholique de LouÕain, Clos Chapelle aux Champs 30.31, 1200 Brussels, Belgium c Transgene S.A., 11 rue de Molsheim, 67082 Strasbourg, France d Zentrum fur ¨ Molekulare Biologie, UniÕersitat ¨ Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Accepted 3 December 1996

Abstract The evidence that bA4 is central to the pathology of Alzheimer’s disease ŽAD. came from the identification of several missense mutations in the amyloid precursor protein ŽAPP. gene co-segregating with familial AD ŽFAD.. In an attempt to study the proteolytical processing of mutated human APP in vivo, we have created transgenic mice expressing the human APP695 isoform with four FAD-linked mutations. Expression of the transgene was controlled by the promoter of the HMG-CR gene. Human APP is expressed in the brain of transgenic mice as shown by Western blot and immunohistology. The proteolytic processing of human APP in the transgenic mice leads to the generation of C-terminal APP fragments as well as to the release of bA4. Despite substantial amounts of bA4 detected in the brain of the transgenic mice, neither signs of Alzheimer’s disease-related pathology nor related behavioural deficits could be demonstrated. Keywords: Alzheimer’s disease; Amyloid precursor protein; FAD mutation; bA4; Transgenic mouse; HMG-CR promoter

1. Introduction Alzheimer’s disease ŽAD. is the most prevalent neurodegenerative disease. In AD, dementia is associated with massive accumulation of fibrillary aggregates in various cortical and subcortical region of the brain. These aggregates appear intracellularly as neurofibrillary tangles, extracellularly as amyloid plaques and as perivascular amyloid in cerebral blood vessels Žreviewed in w16x. The major proteinaceous component of the AD amyloid fibrils is a peptide of maximally 39–43 amino-acid residues termed the bA4 peptide w10,25,31x. The bA4 peptide is derived by proteolytic processing from the amyloid precursor protein ŽAPP. w23x. APP is cleaved by at least three different yet unknown, proteolytic activities. The a-secretase which cleaves within the bA4 region, thus, preventing bA4 formation w35x, cleavage at the N-terminus of bA4 by

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Corresponding author. Fax: q33 Ž1. 5571-3471.

the b-secretase and at the C-terminus by the g-secretase, releasing bA4 w15x. Evidence that APP is causally involved in the pathogenesis of AD came from the identification of several missense mutations in the APP gene, co-segregating with early-onset familial AD ŽFAD. Žreviewed by w29x.. These mutations are located either within or close to the bA4 sequence. Mutations replacing the amino-acid valine in position 717 Žaccording to APP770 numbering. by either isoleucine, glycine or phenylalanine are associated with early-onset FAD w11,2,32x. These mutations lead to an increase in the production of the more amyloidogenic form 1-42 of the bA4 peptide in transfected cells w36x which could trigger the pathological mechanism. However, recent reports suggests that missense mutations at this position could also lead to apoptosis in transfected COS cells w43x as well as activation of intracellular signaling mediated by a G0-dependent mechanism w22x. A double mutation at position 670r671 exchanging the amino acids lysine to asparagine and methionine to leucine segregates with AD

0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 0 3 9 - 9

C. Czech et al.r Molecular Brain Research 47 (1997) 108–116

in a large Swedish kindred w30x. Transfected cells expressing human APP with this mutation secrete up to 6-fold more of the bA4 peptide w3x. Two mutations at distinct additional locations within the bA4 sequence have been described. The exchange of the amino acid glutamate to glutamine at position 693 of APP results in a phenotype termed hereditary cerebral hemorrhage with angiopathy Dutch-type ŽHCHWA-D., resulting in massive deposition of bA4 in blood vessels leading to cerebral hemorrhage and subsequent death from stroke. It has been proposed that this mutation causes a structural change of bA4, thereby accelerating the rate of fibril formation w24,42,4x. Also within the bA4 region, a mutation replacing alanine in position 692 by glycine leads to a disease with the characteristic of both Alzheimer’s dementia and cerebral hemorrhage w17x. This mutation probably affects the a-secretase activity resulting in an increase of bA4 secretion w13x. These data strongly implicate that APP processing and bA4 release play a key role in the etiology of AD. However, the mechanisms responsible for the aggregation of bA4 into amyloid plaques and for neuronal degeneration are still poorly understood. An experimental animal model of AD would facilitate analysis of the mechanisms of in vivo bA4 release and the identification of risk factors leading to bA4 aggregation and amyloid plaque formation. Knowledge of the underlying mechanisms would also have an important impact on the development of therapeutic strategies. Here, we report the generation of transgenic mice expressing the APP695 isoform harboring four different mutations. We combined FAD mutations at the N-terminus K 670 N, M 671 L, the C-terminus V 717 I and within the bA4 region E 693 Q in one transgene. We intended to study the influence of this mutations on APP processing and bA4 release in vivo. A possible additive effect of four different mutations in one transgenic expression construct could accelerate the pathological process and provide a new model for studying the mechanisms leading to neuronal degeneration.

2. Materials and methods 2.1. Construction of the transgene The SmaI–ClaI fragment of human APP695 cDNA was subcloned in a Bluescript II KS cloning vector ŽStratagen.. Four different FAD mutations w11,24,30x have been introduced in the same human APP695 sequence. The mutations of codons 670 and 671 were created by the insertion of two hybridized oligonucleotides encoding the desired mutations in the BglII–EcoRI site of APP cDNA. A PCR-based approach was used to modify codons 693 and 717, respectively. In separate amplification reactions, codon 693 primers Žsense strand: 5X-CGG GAT CCG GTG TTC

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TTG CCC AAG ATG TG-3X ; antisense strand: 5X-CCG GAT CCC CCA CAT CTT GGG CAA AGA ACA CC-3X . or codon 717 primers Žsense strand: 5X-CGG GAT CCC GAC AGT GAT CAT CAT CAC C-3X ; antisense strand: 5X-CGG GAT CCG GTG ATG ATG ATC ACT GTC G-3X ., together with APP wild-type primers Žsense strand: 5X-CCG TGG AGC TCC TTC CCG-3X Žunique SacI site.; antisense strand: 5X-CCC ATC GAT TCT TAA AGC-3X Žunique ClaI site., were used for the amplification of specific fragment in a plasmid carrying human APP695 cDNA. The mutations of codons 693 and 717 were combined by using codon 717 mutated sequence as a template for the PCR reaction. Sequence analysis of all PCR products verified the presence of the different mutations and showed no additional mutations due to errors of the Taqpolymerase. The PCR products of interest were digested with the corresponding restriction enzymes and cloned by a three partner ligation into the APP 695-Bluescript vector. The pHMG vector w9x was linearized by digestion with BamHI, rendered blunt by Klenow fragment of DNA polymerase and cleaved by SalI. Finally, the SmaI–SalI fragment of mutated APP695 cDNA was cloned in the pHMG plasmid. 2.2. Generation and screening of transgenic mice The structure of the hybrid gene is shown in Fig. 1. The transgene was excised by digestion with NotI, leaving behind all the vector sequences and purified on an agarose gel according to standard procedures w19x. The linearized DNA, diluted to a final concentration of 2 ngrm l in 10 mM Tris–HCl ŽpH 7.4. 0.1 mM EDTA, was injected in one of the two pronuclei of fertilized mouse embryos ŽC57 black6rJ= SJL-F2 hybrids, IFFA CREDO.. The surviving embryos were subsequently transplanted into the oviduct of pseudopregnant foster mothers. Mice harboring the transgene were detected by Southern blot analysis of DNA prepared from tail samples. A 1200-bp Pst I–SalI fragment from the intron 1 of the pHMG plasmid, randomly labelled with w a- 32 PxdCTP served as probe for the detection of the transgene and of the endogenous mouse HMG gene as internal control. The Southern blot analysis showed no major re-arrangement or deletion of the transgene in none of the 15 independent lines obtained. Animals were handled according to the French guidelines for animal care. 2.3. Protein analysis Brain tissue from control or transgenic mice was homogenized on ice in 0.32 M sucrose solution containing 10 m grml of pepstatin, 10 m grml of leupeptin and 1 mM PMSF. Cellular debris were removed by centrifugation at 48C for 5 min with 1500 = g. The protein concentration in the supernatant was measured using the BCA protein assay ŽPierce.. SPA4-CT-transfected and -non-transfected SY5Y cells were lysed as described w6x. For immunoprecipitation,

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cell extract corresponding to 5 = 10 5 cells was used. For precipitation of APP and C-terminal fragments thereof, brain extracts were diluted to a final concentration of 1 mg in 50 m l of 10 = solubilization buffer Ž0.25 M Tris–HCl pH 7.5, 5% Žvrv. Triton X-100, 5% Žvrv. NP-40. and filled up to 500 m l with PBS Žphosphate-buffered saline. containing 10 m grml of pepstatin, 10 m grml of leupeptin and 1 mM PMSF. For precipitation of bA4, 2 mg of brain extracts were diluted in 100 m l of 1 = solubilization buffer complemented with 2% Žwrv. SDS, left on ice for 20 min, incubated at 1008 for 10 min and diluted to a final volume of 1 ml with 1 = solubilization buffer. Thirty m l of protein A Sepharose together with 10 m l of rabbit normal serum were added. The resulting solution was gently mixed for 1 h at room temperature. After centrifugation Ž13 000 = g, 1 min., the supernatant was recovered and immunoprecipitated for 2 h at room temperature with either 5 m l of anti-FdAPP polyclonal antiserum w41x, 5 m l of anti-APP-CT antiserum w27x or overnight at 48C with 15 m l of polyclonal anti-bA4 antiserum raised against a synthetic peptide which corresponds to bA4 1-40 together with 20 m l of protein A Sepharose. After centrifugation, the pellet was washed 3 = with 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.2% Žvrv. NP-40, 2 mM EDTA, twice with Tris– HCl pH 7.5, 500 mM NaCl, 0.2% Žvrv. NP-40, 2 mM EDTA, once with 10 mM Tris–HCl pH 7.5 and then denatured at 1008C for 10 min in 30 m l of Laemmli loading buffer. Proteins were separated under denaturing conditions on 7.5% Tris–glycine for APP, 12.5% Tris–tricine polyacrylamide gels for APP C-terminal fragments, and 10–20% gradient gels ŽNovex. for bA4, and transferred to a nitrocellulose membrane. The filter was blocked with 5% Žwrv. non-fat dry milk in TBST Ž50 mM Tris– HCl pH 8.1, 150 mM NaCl, 0.5% Žvrv. Tween-20. and incubated overnight 48C with the primary antibody at concentration of 500 ngrml. In case of bA4 detection, the filter was boiled for 5 min in PBS to enhance the signal. Binding of the primary antibody was detected with horseradish peroxidase-conjugated anti-IgG secondary an-

tibody ŽAmersham. followed by ECL detection system ŽAmersham. according to the manufacturer’s instruction or alkaline phosphatase-conjugated secondary anti-IgG antibody ŽPromega. and developed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate ŽGibcoBRL.. For re-probing, the filters were stripped of the bound antibodies by incubation in 100 mM b-mercaptoethanol, 2% Žwrv. SDS, 65.5 mM Tris–HCl pH 6.4 for 30 min at 558C. 2.4. Culture and metabolic labelling of mouse embryonic neurons Mouse embryo’s forebrains were taken at 14 days of gestation and dissected under a binocular microscope w26x. Cells were mechanically dissociated with a pipette in HAM-F12 culture medium ŽGibco. supplemented with 10% Žvrv. fetal calf serum, 0.6% Žwrv. D-glucose, 100 IU penicillin and 100 m grml streptomycin. Dissociated cells were then plated at a density of 3 = 10 6 cells in 35 mm plastic tissue culture dishes coated with polylysine Ž10 m grml. and grown at 378C in a humidified atmosphere at 10% Žvrv. CO 2 . Cytosine arabinofuranoside was added after 3 days of culture to prevent glial expansion. After 5 days of culture, cells were rinsed for 1 h with a culture medium without methionine and metabolic labelling was performed during an overnight incubation in the presence of 100 m Cirml of w 35 Sxmethionine. The extracellular radiolabelled proteins were immunoprecipitated with the SGY2134 anti-bA4 serum as described w34x. The immunoprecipitated proteins were separated on a 10–16% Tris–tricine SDS-polyacrylamide gel. After immersion in 1 M salicilate pH 5.0 for 20 min, the gel was dried under vacuum and exposed to an X-ray film. 2.5. Histological analysis Animals Ž7–13 months. were anaesthesized and transcardially perfused with 4% Žwrv. paraformaldehyde prepared in PBS. Tissues dissected from perfused mice were

Fig. 1. Schematic structure of the transgene HMG-APP695 SDL. Expression is controlled by the promoter of the HMG-CR gene, followed by genomic sequences of the first non-translated exon and the first 3.5-kb intron. Mutated human APP695 cDNA was cloned into the non-translated region of the second exon of the HMG-CR gene. The SV40 large T antigen polyadenylation sequence terminates transcription.

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post-fixed for 2 h in paraformaldehyde and embedded in paraffin. Coronal sections of 6 m m thickness were obtained at various hippocampal levels. They were stained with Hematoxylin eosin and Nissl stain for routine histology. Amyloid deposition was assessed histochemically by Congo red staining. Servier-Munger silver impregnation was used to detect bA4 deposits and neurofibrillary tangles. 2.6. Immunohistochemical staining Sections were pre-incubated in 80% Žvrv. formic acid during 5 min for bA4 immunostaining or in a citrate buffer using microwave oven heating Ž3 = 5 min, 750 W. for human APP immunostaining. They were incubated overnight with primary antibodies: anti-bA4 monoclonal antibody ŽDAKO. diluted 1r50; rabbit anti-bA4 sera diluted 1r1000, rabbit anti-GFAP ŽDAKO. sera diluted 1r600, antihuman APP monoclonal antibody 8E5 ŽAthena Neuroscience. diluted 1r3000. All immunostained sections were developed with diaminobenzidine ŽDAB. substrate using the streptavidin-biotin peroxidase system ŽAmersham. or the peroxidase anti-peroxidase method ŽDAKO., sections were counterstained with hematoxylin. Brain sections from transgenic animals and control littermates were always processed in parallel. Brain sections from Alzheimer’s patients were used as positive controls for all the techniques and antibodies. 2.7. BehaÕioural testing f 17-week-old male transgenic mice of line 20 SDL backcrossed 3 = into C57 black6rJ strain, were employed in the behavioural experiments. All mice were trained in the water-maze during 14 days. The first 10 days, the mice were trained for 4 trials per day with the escape platform positioned at a constant location in the centre of the SW quadrant. On each trial, the mouse was placed in the water at one of four start positions ŽN, W, E and S.. The mouse was allowed to swim until it found and climbed onto the hidden platform or until a maximum of 60 s had elapsed. If the mouse failed to locate the platform within this time, it was placed on the platform by the experimenter for 30 s. The swimming path of each mouse, the latency to escape and the distance covered were recorded for each trial ŽVideotrack 512 system, Viewpoint, Lyon, France.. A single probe trial was conducted on day 11. The platform was removed from the maze and the swimming path on each mouse was recorded while it searched for the missing platform over 60 s. The time spent searching the quadrant of the maze that formerly contained the platform was measured and compared with the time spent searching the others quadrants. On days 12–14, training continued for 4 trials per day with the escape platform returned to the same location that had been used on days 1–10.

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3. Results 3.1. Generation of transgenic mice To control the expression of the mutated human APP695 cDNA ŽhAPP695 SDL., we used a genomic construct with the promoter of the murine 3-hydroxy-3-methyl-glutaryl CoA reductase gene ŽHMG-CR.. The HMG-CR promoter is a housekeeping-type promoter that shows a strong and ubiquitous expression pattern with high expression in the brain w9,37x. The construct contained further, the 5X-flanking region, the first non-coding exon of the HMG-CR gene and the SV40 polyadenylation site. Fig. 1 outlines the map of the expression vector used for the generation of transgenic mice. The offspring was screened by Southern blot for the presence of the transgene. We obtained 15 independent transgenic lines with stable integration of the transgene. Most of the lines transmitted the transgene in a mendelian fashion. 3.2. Expression analysis of transgenic mice Brain extracts of heterozygous transgenic mice were analysed by immunoprecipitation with a polyclonal antiAPP antiserum w41x and subsequent Western blotting using the monoclonal 8E5 antibody for detection. Under the conditions used, 8E5 detected specifically human APP, no cross-reactivity with murine APP was observed. Four transgenic mouse lines expressed detectable amounts of human APP SDL in the brain, with line 20 SDL showing the highest level ŽFig. 2A.. In order to determine whether the total amount of APP in transgenic mice has been changed significantly, the membrane was stripped of the 8E5 antibody and stained for total APP with monoclonal antibody 22C11. Since the epitope of 22C11 is conserved between human and mouse w18x, it should, therefore, mark total APP levels. The results indicate that there is only moderate overexpression of APP in some of our transgenic lines compared to the control mice ŽFig. 2B.. Semi-quantitative estimation of total brain APP measured with antibody 22C11 and compared with anti-tubulin immunoreactivity, used as internal standard, revealed an f 30% increase of total APP in the highest expressing line 20 SDL Ždata not shown.. However, 22C11 cross-reacts with APP-like proteins ŽAPLPs. w39,40x which could lead to an underestimation of the expression level of the transgenic APP relative to the mouse APP. 3.3. Proteolytic processing of APP The double mutation at position 670r671 results in transfected cells in a several-fold increase of bA4 production w3x. This effect is supposed to be due to an increase of b-secretase activity w14,38x. In order to determine if the human APP is similarly processed in the transgenic mouse

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brain, we analysed the generation of C-terminal APP fragments. Line 20 SDL was used for this studies due to its strong expression of the transgene. Brain extracts of 9month-old heterozygote transgenic mice and non-transgenic littermates were immunoprecipitated with a polyclonal antiserum raised against the C-terminus of APP w27x. The detection of the proteins after Western blotting was performed with the monoclonal anti-bA4 antibody WO-2 w21x. The major recognition site of this antibody is mapped to bA4 residues 5–8 and, therefore, APP C-terminal fragments containing the bA4 sequence should be revealed. As a control, we used SY5Y cells stable transfected with a construct containing the last 100 residues of APP with the APP signal peptide termed SPA4-CT w6x. The signal peptide of SPA4-CT is cleaved off after translation and the resulting fragment is similar in size to bcleaved APP. The WO-2 antibody detected a band of f 12 kDa in the transgenic mouse brain similar to that obtained with the SPA4-CT construct ŽFig. 3A, lane 4.. We concluded that the 12-kDa product represents b-cleaved APP. No signal is detectable in the control mouse Žlane 3.. The slight difference in apparent molecular weight in lanes 1 and 4 results from the presence of two additional amino acids after the signal peptide in the SPA4CT construct w6x. Re-probing the Western blot with an affinity-purified polyclonal antibody directed against the C-terminus of APP w27x shows in addition the a-cleaved APP fragments ŽFig.

3B.. In the control mouse, the endogenous murine APP is cleaved at the a-cleavage site resulting in a fragment of f 10 kDa length. The 10-kDa a-secretase fragment in the transgenic mouse Žlane 4. is apparently stronger as compared to the control mouse Žlane 3., indicating that the transgenic APP is also cleaved by the a-secretase. The b-cleaved C-terminal fragment of APP has been shown w6,7x to be the precursor of bA4. To analyse whether bA4 is produced in the brain of the transgenic mice, brain extracts of 5-month-old, heterozygote transgenic mice of line 20 and age-matched controls were subjected to immunoprecipitation with a polyclonal antiserum raised against synthetic bA4. After Western blot, the membrane was stained with WO-2. A strong immunoreactive signal at 4 kDa, corresponding to bA4 could be detected in the brain of transgenic mice, whereas only a faint band is visible in non-transgenic mouse brain ŽFig. 4.. These results clearly demonstrate that human APP SDL expressed in transgenic mice leads to the production of bA4. 3.4. Production of b A4 peptide by transgenic neurons To address the question how APP is processed during embryonic development, cultured neurons were isolated from embryonic day 14 mouse embryos. The embryos were obtained by breeding heterozygous transgenic males of line 20 SDL with non-transgenic females of the same

Fig. 2. Western blot analysis of APP expression in transgenic mouse brain. Brain extracts from transgenic and control mice were immunoprecipitated with polyclonal anti-APP antiserum separated by SDS-PAGE and transferred to nitrocellulose membrane as described in Materials and methods. Each lane represents individual mice. A: transgenic human APP was detected with monoclonal 8E5 antibody staining. 8E5 does not react with mouse APP as shown by the absence of a specific signal in the lanes of the non-transgenic mice Žlanes 18 and 19. B: total APP was detected by stripping and restaining the membrane with monoclonal anti-APP antibody 22C11.

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Fig. 3. Western blot analysis of APP C-terminal fragments. Extracts from transgenic Žline 20 SDL. and control mouse brain, SPA4-CT-transfected SY5Y cells and -non-transfected SY5Y cells were immunoprecipitated with polyclonal anti-APP-CT antiserum separated by SDS-PAGE and transferred to nitrocellulose membrane as described in Materials and methods. A: APP C-terminal fragments resembling b-cleaved APP were detected in transgenic mouse brain and SPA4-CT-transfected SY5Y cells with monoclonal antibody WO-2. B: the a-secretase- and b-secretasecleaved APP fragments are revealed by stripping and re-probing the membrane with affinity-purified polyclonal anti-Jonas antiserum directed against the C-terminus of APP Žsee arrows..

genetic background. DNA was isolated from embryonic tissue and analysed by Southern blot for the presence of the transgene. After 5 days of culture, the cells were metabolically labelled and bA4 presence was analysed by immunoprecipitation of the conditioned medium with bA4 antiserum. A strong signal at 4 kDa corresponding to bA4 could be detected in the culture medium of the transgenic neurons while the signal was much less abundant in the culture medium of the control neurons ŽFig. 5.. The production of bA4 in the embryonic neurons demonstrated that APP is processed similarly in the developing mouse brain and in the adult brain.

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Fig. 4. Western blot detection of bA4 in transgenic mouse brain. Brain extracts from transgenic and control mouse brain were immunoprecipitated with polyclonal anti-bA4 antiserum, separated by SDS-PAGE and transferred to nitrocellulose membrane as described in Materials and methods. Staining with monoclonal antibody WO-2 detects bA4-immunoreactive signal at 4 kDa in lanes 1–3, representing individual transgenic mice of line 20 SDL. Only a faint band is detected in non-transgenic control mice Žlanes 4 and 5..

neuronal cell bodies and proximal dendrites. No immunostaining with bA4-specific antibodies could be detected in transgenic brain tissues. The absence of amyloid deposits and neurofibrillary tangles were confirmed with Congo red staining and Servier-Munger silver impregnation. Moreover, there was no astrocytosis as shown with GFAP immunostaining. Transgenic mice showed the same neuroanatomical and histological features as the non-transgenic littermates. 3.6. BehaÕioural testing We performed behavioural tests to analyse if the expression of the transgene had any functional consequences

3.5. Immunohistochemistry By using the human APP-specific antibody 8E5, intense APP immunoreactivity was detected in the neurons of numerous brain areas in transgenic mice of line 20 SDL as compared to brain sections of non-transgenic mice. Human APP immunoreactivity could be detected in pyramidal cells in cerebral cortex, in neurons of Ammon’s horn, enthorinal and piriform cortex, thalamic nuclei and basal ganglia ŽFig. 6.. The reaction product showed a granular appearance and was scattered within the cytoplasm of

Fig. 5. Detection of bA4 in conditioned medium of embryonic neurons. Cells were isolated from embryonic day 14 embryos and tested for the presence of the transgene as described in Materials and methods. The bA4 peptide was immunoprecipitated from the conditioned medium after metabolic labeling of embryonic neurons from non-transgenic littermates Žlane 1. and embryonic neurons from transgenic mice of line 20 SDL Žlane 2..

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Fig. 6. Coronal section through the piriform cortex of a 10-month-old transgenic mouse of line 20. Immunostaining with 8E5 revealed strong human APP immunoreactivity mostly in the cytoplasm of neuronal cells bodies and the proximal dendrites Ž=300..

particularly concerning cognitive changes similar to human dementia. It turned out that the genetic background of C57 black6rJ= SJL-F2 hybrids showed strong deficits in learning and could, therefore, not be employed in the Morris water-maze task for memory tests. However, after three backcrosses into C57 black6rJ genetic background, mice displayed learning capabilities as measured by a decrease in acquisition latencies in the Morris water-maze task. The animals could be analysed for cognitive changes resulting from the expression of the transgene. At the age of 4 months, transgenic mice of line 20 SDL and non-transgenic littermates controls were tested for deficits in spatial memory in the Morris water-maze task w28,33x. The tests were performed daily over a period of 2 weeks. Both groups learned within successive days of training to locate the submerged platform to escape from the water pool. After an initial learning phase of 6 days, both groups did not show any further improvement. No significant differences between transgenic and control mice could be demonstrated. A single probe trail was conducted on day 11. The submerged platform was removed and the swimming path of each mouse was recorded while it was searching the platform. Both groups showed the same preference for the quadrant formerly containing the now missing platform. Analysis of swimming distances revealed no differences in both tests Ždata not shown..

4. Discussion Several attempts have been made to model AD neuropathology in transgenic rodents Žfor review, see w12x. but

so far only two recent studies reported extracellular accumulation of bA4 and dystrophic neurites in transgenic mice expressing w8,20x. In one report, expression of APP with the FAD mutation V717F was driven by the PDGF-b promoter inducing human APP expression at levels ) 10fold higher than endogenous mouse APP levels w8x. In the other study, a hamster prion protein ŽPrP. cosmid vector was used in which the open reading frame of PrP was replaced by human APP containing the double mutations K670N, M671L w20x. In both cases, transgenic expression levels of ) 5 = the endogenous APP were reported. The observed phenotype seems to be related to the very high expression of human mutated APP. Evidence of extracellular accumulation of bA4 in rodents was also shown by a study utilizing retrovirus-transfected brain transplants, expressing human APP in the rat brain w1x. For the present study, we have generated transgenic mice expressing human APP695 with four different FAD mutations under the control of the HMG-CR promoter. The expression of the transgenic protein in the mouse brain could be detected by Western blot as well as by immunohistochemistry. Although the expression level of the human APP was only moderate compared to the endogenous mouse APP, we could demonstrate that the transgenic APP is processed into C-terminal fragments and bA4 as described for human APP in vitro and in vivo w15,30x. Transgenic human APP is secreted as shown by the appearance of a-secretase-cleaved fragments and substantial amounts are cleaved at the b-secretase site resulting in amyloidogenic C-terminal APP fragments. These fragments are further processed into soluble forms of bA4, which could be detected in embryonic neurons as well as in adult brains of transgenic mice.

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Although mice express high amounts of APP, we show here that endogenous mouse bA4 is only present in small quantities in the brain or in embryonic neurons. This argues that murine APP is not a good substrate for the protease activity leading to the release of bA4. However, human APP expressed in the transgenic mouse brain is processed as in the human brain. This make the transgenic mouse a valuable model to study in vivo the pathological effect of the FAD mutations and the cellular mechanism responsible for the processing of APP. It remains to be shown if there is also an increase of the more amyloidogenic bA4 species 1-42 resulting from the mutation valine to isoleucine at position 717 in the transgene. Despite the fact that we can show neuronal expression of human APP and substantial amounts of bA4 being produced thereof, we could not detect pathological changes resembling human AD in the brain sections of the transgenic mice. Also, an effect of the mutation within the bA4 region, which leads in humans to a phenotype characterized by deposition of amyloid in the blood vessels, could not be demonstrated. The absence of pathology might be explained by the lack of aggregation of bA4 in the transgenic mice. Indeed, we have not been able to identify ether immunohistochemicaly or biochemicaly aggregated forms of bA4. This might be due to insufficient levels of bA4, not reaching the critical threshold concentration for aggregation into amyloid plaques or the lack of additional factors promoting aggregation. Even in the absence of histopathological changes, functional impairments may result from the exogenous expression of human APP. As a simple test of cognitive deficits, the Morris water-maze test w28x has been widely used in aged mice and rats and to evaluate a variety of models of dementia w5x. For analysing the transgenic animals, the test has been adapted to assess for impairments in spatial memory tasks w33x. Transgenic animals and controls showed no difference in learning to locate the escape platform and both groups were able to adopt an effective search focused on the appropriate location when the platform was removed. These results indicate that the expression of the transgene does not lead to a substantial impairment in spatial memory. Moderate overexpression of human APP, even with FADlinked mutations, is not sufficient to induce detectable pathological changes in transgenic mice. Although we can detect widespread neuronal expression and proteolytical processing of transgenic human APP into bA4, both in cell culture and in vivo, this does not seem to be per se a pathological situation. A critical threshold level of bA4 has to be archived or additional factors need to be present to induce the pathology. However, the transgenic mice presented in this study might provide a valuable model to study the mechanism of additional genetic or environmental risk factors accountable for the aggregation of bA4 into amyloid plaques and neuronal degeneration, characteristic for Alzheimer’s disease.

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Acknowledgements We thank D. Schenk ŽAthena Neuroscience. for the antibody 8E5, U. Monning for polyclonal APP antisera, G. ¨ Multhaup for polyclonal bA4 antisera, N. Ida for b yA4 antibody W0-2, S. Younkin for antibody SGY2134 and T. Dyrks for the SPA4-CT-transfected SY5Y cell line. We also thank P. Barneoud for valuable discussion. We appre´ ciate excellent technical assistance by G. Ret and N. Clavel. This work was in part supported by the Queen Elisabeth Medical Foundation ŽBelgium. and the Bioavenir programme with the participation of the French Ministry of Research and the French Ministry of Industry. J.N.O. is Research Associate of the National Fund for Scientific Research ŽBelgium..

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