Characterisation of cytoskeletal abnormalities in mice transgenic for wild-type human tau and familial Alzheimer's disease mutants of APP and presenilin-1

Characterisation of cytoskeletal abnormalities in mice transgenic for wild-type human tau and familial Alzheimer's disease mutants of APP and presenilin-1

www.elsevier.com/locate/ynbdi Neurobiology of Disease 15 (2004) 47 – 60 Characterisation of cytoskeletal abnormalities in mice transgenic for wild-ty...

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www.elsevier.com/locate/ynbdi Neurobiology of Disease 15 (2004) 47 – 60

Characterisation of cytoskeletal abnormalities in mice transgenic for wild-type human tau and familial Alzheimer’s disease mutants of APP and presenilin-1 Allal Boutajangout, a Miche`le Authelet, a Ve´ronique Blanchard, b N. Touchet, c Gunter Tremp, b,c Laurent Pradier, b and Jean-Pierre Brion a,* a

Laboratory of Histology and Neuropathology, Universite´ Libre de Bruxelles, Campus Erasme, 1070 Brussels, Belgium Neurodegenerative Disease Group, Aventis Pharma, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France c Functional Genomics Department, Aventis Pharma, Centre de Recherche de Vitry-Alfortville, 94403 Vitry-sur-Seine, France b

Received 2 January 2003; revised 8 July 2003; accepted 12 September 2003

To study the role of AB amyloid deposits in the generation of cytoskeletal lesions, we have generated a transgenic mouse line coexpressing in the same neurons a wild-type human tau isoform (0N3R), a mutant form of APP (751SL) and a mutant form of PS1 (M146L). These mice developed early cerebral extracellular deposits of AB, starting at 2.5 months. A somatodendritic neuronal accumulation of transgenic tau protein was observed in tau only and in tau/PS1/APP transgenic mice, including in neurons adjacent to AB deposits. The phosphorylation status of this somatodendritic tau was similar in the two transgenic lines. The AB deposits were surrounded by a neuritic reaction composed of axonal dystrophic processes, immunoreactive for many phosphotau epitopes and for the human tau transgenic protein. Ultrastructural observation showed in these dystrophic neurites a disorganisation of the microtubule and the neurofilament network but animals that were observed up to 18 months of age did not develop neurofibrillary tangles. These results indicate that overexpression of mutant PS1, mutant APP and of wildtype human tau were not sufficient per se to drive the formation of neurofibrillary tangles in a transgenic model. The AB deposits, however, were associated to marked changes in cytoskeletal organisation and in tau phosphorylation in adjacent dystrophic neurites. D 2003 Elsevier Inc. All rights reserved. Keywords: Transgenic; Tau; Presenilin 1; APP; Mutation; Alzheimer

Introduction The two neuropathological hallmark lesions observed in the brain of patients with Alzheimer’s disease (AD) are the senile plaques and the neurofibrillary tangles. The main component of

* Corresponding author. Laboratory of Histology and Neuropathology, School of Medicine, Universite´ Libre de Bruxelles, 808 route de Lennik, Bldg. C-10, 1070 Brussels, Belgium. Fax: +32-2-5554121. E-mail address: [email protected] (J.-P. Brion). Available online on ScienceDirect (www.sciencedirect.com.) 0969-9961/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2003.09.007

senile plaques is an extracellular amyloid deposit made of a 39 to 43 amino acids peptide (the Ah peptide), generated by proteolysis from the amyloid precursor protein (APP). Neurofibrillary tangles are constituted of bundles of abnormal filaments called paired helical filaments (PHF), found in neurons and in dystrophic neurites associated to Ah deposits. These PHF are composed of highly phosphorylated forms of the microtubuleassociated protein tau. It is believed that the high degree of phosphorylation of tau is a critical event linked to microtubule disorganisation, generation of neurofibrillary lesions, and neuronal cell death and dysfunction (Goedert et al., 1997). In addition to sporadic cases, a small percentage of familial Alzheimer’s disease (FAD) cases are caused by autosomal dominant mutations of the APP gene or, more frequently, of the presenilin-1 (PS1) and PS2 genes (for a review, see Czech et al., 2000). Mutations in APP and PS1 have been shown to increase the production of Ah and of the longest forms of Ah (e.g. Ah1 – 42) that aggregate more readily into amyloid fibres. In patients affected with pathogenic PS1 or APP mutations, numerous neurofibrillary tangles are also observed in addition to abundant Ah deposits, suggesting that these mutations might also sensitise neurons to the formation of neurofibrillary tangles. The Ah deposits might also be directly responsible for the generation of neurofibrillary tangles (Hardy, 1999). The molecular relationship between the formation of senile plaques and NFT might be experimentally investigated in an in vivo model of AD that would be expected to develop both senile plaques and neurofibrillary tangles. The development of Ah amyloid deposits in in vivo models has been successfully achieved in transgenic models overexpressing mutated forms of APP (Games et al., 1995; Hsiao et al., 1996; Sturchler-Pierrat et al., 1997) and is greatly accelerated in transgenic models overexpressing both mutated forms of APP and PS1 (Borchelt et al., 1997; Holcomb et al., 1998). Neurofibrillary tangles were, however, not observed in these transgenic lines. However, transgenic mouse lines expressing a mutated form of tau identified in familial forms of fronto-temporal dementia were found to develop neurofibrillary tangles in the absence of Ah deposits

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(Allen et al., 2002; Go¨tz et al., 2001a; Lewis et al., 2000; Tanemura et al., 2002; Tatebayashi et al., 2002). Interestingly, in a tau mutant transgenic line injected intracerebrally with Ah42 fibrils (Go¨tz et al., 2001b) and in a double transgenic line expressing both a mutant of tau and a mutant of APP (leading to the formation of Ah deposits) (Lewis et al., 2001), an enhancement of the neurofibrillary pathology was reported, suggesting that Ah deposits can accelerate NFT formation. However, since tau mutations have not been found in Alzheimer’s disease, it is also important to investigate if amyloid deposits can induce the formation of a neurofibrillary pathology in animals expressing wild-type human tau proteins. In this study, we have generated and studied a mouse transgenic line expressing simultaneously a wild-type human tau isoform, and FAD mutants forms of both PS1 and APP leading to the formation of early and abundant Ah deposits.

Materials and methods Generation of transgenic mice Different transgenic lines were used in this study. The tau transgenic mice expressing the three-repeat human tau isoform, without N-terminal insert, under the control of a modified promoter of the 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase gene has been previously described (Brion et al., 1999). These animals were further screened for the presence of the tau transgene in genomic DNA by PCR amplification using a sense primer localised in exon 1 (5V-ATG GCT GAG CCC CGC CAG GAG3V) and an antisense primer localised in exons 7 and 9 (5VTGGAGGTTCACCAGAGCTGGG-3V). The cDNA encoding the human FAD mutant PS1 (M146L) was introduced under the control of the same modified promoter of the

Fig. 1. (A) PCR amplification on tail genomic DNA of a specific 392-bp tau fragment (lanes 1 – 4) of a specific 425-bp PS1 fragment (lanes 5 – 8), and of a specific 548-bp APP fragment (lanes 9 – 12) in nontransgenic (lanes 1, 5 and 9), PS1/APP transgenic (lanes 2, 6, 10), tau/PS1 transgenic (lanes 3, 7, 11) and tau/ PS1/APP transgenic mice (lanes 4, 8, 12). Bars and numbers on the left of lane 1 indicate the position and molecular weight of DNA fragments of the following sizes: 770, 612, 495, 392, 345 and 341 bp. (B – E) Immunoblotting analysis of the S1 (B, D, E) and S2 (C) fractions of transgenic and nontransgenic mice. The blots were probed with the human-specific antibodies to tau (B), to PS1 (C), to APP (D) and with the anti-a-tubulin antibody (E). The blots were performed on 4-month-old animals, corresponding to nontransgenic (lanes 1), tau/PS1 transgenic (lanes 2), PS1/APP transgenic (lanes 3) and tau/PS1/APP transgenic mice (lanes 4). Bars and numbers on the left of lanes 1 indicate the position and molecular weight (in kDa) of proteins used as markers: phosphorylase b (97.4 kDa), catalase (58.1 kDa), alcohol dehydrogenase (39.8 kDa), carbonic anhydrase (29.0 kDa).

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HMG-CoA reductase gene and PS1 transgenic mice were generated as previously reported (Boutajangout et al., 2002; Pradier et al., 1998). Transgenic mice were identified by PCR using oligomers for human PS1 (forward primer 5V-TAA TTG GTC CAT AAA AGG C-3V; reverse primer 5V-GCA CAG AAA GGG AGT CAC AAG-3V). Doubly transgenic mice for human tau and human mutated PS1M146L were generated by crossbreeding of single transgenic mice and have been previously described (Boutajangout et al., 2002). Regarding APP expression, a transgenic line expressing APP751 SL mutant bearing the Swedish (K670N, M671L) and London (V717I) FAD mutations under the control of the Thy promoter was used. This transgenic line (Thy-1 APP751 SL, line 28) has been previously characterised (Blanchard et al., in press; Wirths et al., 2002). Transgenic mice were genotyped by PCR using oligonucleotides for human APP (forward primer: 5V-GTA GCA GAG GAA GAA GTG-3V and reverse primer 5V-CAT GAC CTG GGA CAT TCT C-3V). Mice homozygote for human mutant PS1M146L were crossed with heterozygote Thy1-APP751SL mice to generate PS1 M146L/ APP751SL doubly transgenic mice (Blanchard et al., in press) and these PS1 M146L/APP751SL mice were crossed with HMG-tau mice to generate tau/PS1 M146L/APP751SL triple transgenic mice. The PCR amplification of the tau, PS1 and APP transgenes in these animals allows the amplification of 392, 425 and 548 bp fragments, respectively. All studies on animals were performed in compliance and following approval of Aventis Animal Care and Use Committee and in accordance with standards of the guide for the care and use of laboratory animals (National Research Council ILAR) and with respect to French and European Community rules.

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Source kindly provided the phosphospecific rabbit polyclonal tau antibodies to pThr212, pSer214, pSer262, pThr403, pSer404, pSer409 and pSer413. The rabbit polyclonal antibody to presenilin 1 (a kind gift from Prof. C. Van Broeckhoven, Antwerpen) is specific for the Nterminus of PS1 (Hendriks et al., 1998). A rat monoclonal antibody to PS1 raised to a fusion protein containing the N-terminus of human PS1 was obtained from Chemicon, USA (MAB1563). The mouse monoclonal anti-APP antibodies 3H5 (Philippe et al., 1993) (kindly provided by Dr. J.N. Octave) and 22C11 (purchased from Boehringer) recognise a N-terminal epitope on APP. For detection of Ah, we used a polyclonal antibody raised to amino acids 12 – 28 of the Ah peptide (Brion et al., 1991a,b), a polyclonal antibody specific for Ah42 (purchased from BioSource), and a mouse monoclonal antibody raised to amino acids 8 – 17 of the Ah peptide (purchased from Dako, clone 6F/3D). The following additional antibodies were also used: rabbit polyclonal antibodies to MAP2

Antibodies The B19 antibody is a rabbit polyclonal antibody raised to adult bovine tau proteins. This antibody reacts with all known adult and foetal tau isoforms in bovine, rat, mouse and human nervous tissue in a phosphorylation-independent manner (Brion et al., 1991a,b). The TP20 rabbit polyclonal antibody was raised against a synthetic peptide of human tau, and reacts with human tau and not with mouse tau (Brion et al., 1999). The AT8, AT180, AT270 and AT10 mouse monoclonal antibodies (purchased from Innogenetics) are specific for tau phosphorylated at Ser202 and Thr205 (AT8) (Goedert et al., 1995), at Thr231 (AT180), at Thr181 (AT270) (Goedert et al., 1994) and a specific PHF-tau epitope involving phosphorylation at Thr212 and Ser214 (AT10) (Hoffmann et al., 1997). The mouse monoclonal antibodies PHF-1 (kindly provided by Drs. P. Davies and S. Greenberg, New York) and AP422 (kindly provided by Drs M. Hasegawa and M. Goedert, Cambridge, UK) are specific for tau phosphorylated at Ser396/404 and Ser422, respectively (Hasegawa et al., 1996; Otvos et al., 1994). The tau-1 monoclonal antibody (Boehringer) recognises an epitope that needs unphosphorylation of Ser199/202 (Biernat et al., 1992). The tau monoclonal antibody TG-3 recognises a conformationand phosphorylation-dependent epitope requiring phosphorylation of Thr231 (Jicha et al., 1997b) and the tau monoclonal antibody Alz50 recognises a conformational epitope requiring both an Nterminal fragment and a C-terminal fragment (Jicha et al., 1997a) (TG-3 and Alz50 were kindly provided by Dr. P. Davies). Bio-

Fig. 2. Immunocytochemical detection of the transgenic proteins in tau/ PS1/APP transgenic mice. Triple immunolabelling of a tissue section of the brainstem with (A) the human tau-specific antibody, (B) the human APPspecific antibody and (C) the human PS1 antibody. The arrow points on a neuron expressing simultaneously the three transgenic proteins. The transgenic tau protein is also detected in cell processes in the neuropil (arrowhead in A). Scale bar: 25 Am.

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Fig. 3. Immunocytochemical detection of Ah deposits in tau/PS1/APP transgenic mice. These low magnifications show the extent of Ah deposits at 2.5 months (A), 4 months (B), 9 months (C) and 18 months (D). Scale bar: 1 mm.

(Brion et al., 1988), to a-synuclein (Affiniti) and mouse monoclonal antibodies to ubiquitin (clone MAB1510, Chemicon), to atubulin (Sigma, clone DM1-A), to MAP1 (Sigma), to synaptophysin (Clonab) to GAP43 (Boehringer) and the mouse monoclonal antibody RT97 recognising the multiphosphorylation repeats in high-molecular weight neurofilament protein (Coleman and Anderton, 1990). Preparation of brain homogenates The brains from transgenic mice were dissected and homogenised in four volumes of buffer A containing 0.1 mM 2-(Nmorpholino)ethansulfonic acid, 0.5 mM MgSO4, 1 mM EGTA, 2 mM DTT (dithiotreitol) pH 6.8, 0.75 mM NaCl, 2 mM PMSF (phenylmethyl sulfonyl fluoride), leupeptin (1 Ag/ml), pepstatin (1 Ag/ml), soybean trypsin inhibitor (1 Ag/ml), aprotinin (1 Ag/ml), 20 mM NaF and 0.5 mM sodium orthovanadate. The homogenates were centrifuged (20,000  g for 30 min at 4jC) and the supernatant used as a soluble fraction. The pellet was resuspended in buffer A added with 1% (v/v) Triton X-100 and 1% (w/v) SDS, incubated for 20 min and centrifuged at 20,000  g for 30 min; the supernatant was kept and used as the insoluble fraction.

Western blot analysis The amounts of proteins in each fraction were estimated with the Bradford method (BioRad reagent). Tissue samples (100 Ag of protein/lane) were run in 12% (w/v) polyacrylamide gels (SDSPAGE) and proteins were electrophoretically transferred to nitrocellulose membrane using a semidry electroblotter. For immunoblotting, the nitrocellulose sheets were blocked in semifat dry milk (10%, w/v, in Tris-buffered saline) for 1 h at room temperature and they were incubated with primary antibodies overnight followed by antirabbit or antimouse immunoglobulins conjugated to alkaline phosphatase (Sigma). Finally, the membranes were incubated in developing buffer (Tris 0.1 M, NaCl 0.1 M, MgCl2 0.05 M, pH 9.5) containing nitroblue tetrazolium at a concentration of 0.33 mg/ ml and 5-bromo-4-chloro-3-indolyl phosphate at a concentration of 0.175 mg/ml. The development was stopped by dipping the membranes in 10 mM Tris, 1 mM EDTA, pH 8. The levels of expression of transgenic proteins and change in tau phosphorylation were estimated by densitometric analysis using the NIH Image J program, and adjusted for protein loading based on immunoblots performed with the anti-a-tubulin antibody and the B19 tau antibody. Statistical comparisons were performed using ANOVA and Student’s t tests.

Fig. 4. (A – H) Histochemical and immunocytochemical detection of neuritic dystrophy associated to Ah deposits in tau/PS1/APP transgenic mice. (A, B) Gallyas and Congo red staining in a 9-month-old transgenic mouse. The Ah deposit in the transgenic mice is Congo red positive (A) and birefringent when observed between crossed polarization filters (B), but is not surrounded by Gallyas-positive neurites. In Alzheimer’s disease (inset in A), this Ah deposit is surrounded by Gallyas-positive neurites. A Gallyas-positive NFT is also visible in this inset. (C, D) APP immunolabelling and Congo red staining. Two amyloid deposits (birefringence shown in D) are surrounded by large APP-positive dystrophic neurites. A cluster of APPpositive neurites not associated to an amyloid deposit is also observed (arrow). Several neurons expressing the transgenic APP protein are present adjacent to the Ah plaques. (E, F) Double immunolabelling with the anti-APP antibody (E) and the antiubiquitin antibody (F) in a 12-month-old tau/ PS1/APP transgenic mouse. Most large dystrophic neurites around the Ah deposit are labelled by both antibodies. (G) Immunolabelling with the phosphotau antibody AT8. The amyloid deposit is surrounded by tau-positive thin dystrophic and beaded processes. (H) Double immunolabelling with the anti-APP antibody (green) and the p262 phosphotau antibody (red). The tau positive neurites are shorter than the APP-positive neurites and appear sometimes granular. (I – L) Association between neurons expressing the human tau protein and Ah deposits in tau/PS1/APP transgenic mice. (I, J) Immunolabelling with the human tau-specific antibody in the subiculum of a 9-month-old mouse. The amyloid deposits (birefringence shown in J after staining with Congo red) are often surrounded by dystrophic neurites containing the transgenic tau protein. (K) Immunolabelling with the human tauspecific antibody in the thalamus of a 12-month-old mouse. Several neurons expressing the human tau protein in the somatodendritic domain and numerous neurites are in close proximity of amyloid deposits (*, Congo red staining). (L) Double immunolabelling with the human specific tau antibody (green) and the anti-Ah antibody (red) in a 12-month-old mouse, showing two neurons with a pericaryal human tau immunoreactivity adjacent to an Ah deposit. Scale bar: 25 Am.

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Immunocytochemical analysis Brains were dissected from transgenic mice from 8 weeks up to 18 months and tissue blocks fixed by immersion with 10% (v/v) formalin or 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Tissue sample were embedded in paraffin and cut on a sliding microtome at a thickness of 10 Am. The immunohistochemical labelling was performed using the ABC method. Briefly,

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tissue sections were treated with H2O2 to inhibit endogenous peroxidase and incubated with the blocking solution (10%, v/v) normal horse serum in TBS (0.01 M Tris, 0.15 M NaCl, pH 7.4). After an overnight incubation with the diluted primary antibody, the sections were sequentially incubated with horse antimouse antibodies conjugated to biotin (Vector) followed by the ABC complex (Vector). The peroxidase activity was revealed using diaminobenzidine as chromogen. For immunolabelling with the

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Ah antibodies, rehydrated tissue sections were pretreated with 100% formic acid for 10 min before incubation with the blocking solution. Double immunolabelling was performed using fluorescent markers. The first antibody was detected using an antirabbit or an antimouse antibody conjugated to FITC (Jackson) and the second antibody detected using an antirabbit or an antimouse antibody conjugated to biotin, followed by streptavidin conjugated to Alexa 594 (Molecular Probes). For triple immunolabelling, selected areas in tissue sections were photographed after double fluorescent immunolabelling and then immunolabelled with the third antibody using the peroxidase – antiperoxidase method and diamininobenzidine as chromogen. Formalin-fixed sections were also stained with the Gallyas silver-staining method; additional sections were stained with Congo red and examined under crossed polarisation filters, or stained with haematoxylin – eosin or with the Nissl method. Tissue sections were examined with a Zeiss Axioplan microscope and digital images acquired using an Axiocam HRc camera. Ultrastructural analysis and immunolabelling in electron microscopy Control and transgenic animals were anesthetised with chloral hydrate and perfused intracardially with a solution of 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. Tissue blocks were quickly dissected and further fixed by immersion in the same fixative or with 4% (w/v) glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 90 min. For immunolabelling, 50-Am-thick tissue sections were cut on a vibratome, cryoprotected by incubation in 30% (w/v) sucrose and frozen in isopentane cooled at 80jC. After thawing, the tissue sections were treated with 1% (w/v) sodium borohydride for 30 min and processed for immunolabelling with the ABC method, as previously reported (Brion et al., 1999). After washing in Millonig’s buffer with 0.5% (w/v) sucrose for 24 h, the tissue sections were postfixed in 2% (w/v) OsO4 for 30 min, dehydrated and embedded in Epon. Semithin sections were stained with toluidine blue. Ultrathin sections were counterstained with uranyl acetate and lead citrate and observed with a Zeiss EM 809 at 80 kV. Measurements of the diameter of filaments were performed on digitalised images using the public domain NIH Image J program.

Results Generation of transgenic lines The full characterisation of the tau, PS1 M146L, APP 751SL single and double transgenic line have been previously reported (Blanchard et al., in press; Boutajangout et al., 2002; Brion et al., 1999; Leutner et al., 2000). Although a detailed behavioural analysis of triple transgenic animals was not performed in the present study, the animals examined up to 18 months did not show alterations in their motor activity or an obvious behavioural phenotype. We present here the biochemical and histopathological characterisation of these triple transgenic animals and their comparison with single and double transgenic animals. The PCR amplification of the tau, PS1 and APP transgenes in these animals allows the amplification of specific fragments absent from non-

transgenic animals, the three fragments being detected simultaneously only in the triple transgenic animals (Fig. 1A). Expression of the tau, PS1 and APP transgenic proteins: immunoblotting analysis By immunoblotting, the TP20 antibody specific for human tau detected several closely spaced tau bands between 39 and 55 kDa in the tau, tau/PS1 and tau/PS1/APP transgenic mice, but not in the control mice (Fig. 1B). The antibody to the N-terminus of human PS1 labelled a 29-kDa N-terminal PS1 fragment in the S2 fraction of tau/PS1, PS1/APP and tau/PS1/APP transgenic animals (Fig. 1C), as previously reported in tau/PS1 animals (Boutajangout et al., 2002). The antibody to human APP labelled a 100-kDa band only in PS1/APP and tau/PS1/APP animals (Fig. 1D). The relative mean expression of transgenic tau and PS1 proteins were compared between double (n = 6) and triple transgenic animals (n = 5) aged from 2 to 18 months, after normalisation using tubulin loading (Fig. 1E), and were not observed to be significantly different (for tau expression: P = 0.11, and for PS1 expression: P = 0.10 for comparison between tau/PS1 and tau/PS1/APP mice).

Table 1 Tau immunoreactivity in somatodendritic compartment and in dystrophic neurites around Ah deposits in transgenic mice Antibody Epitope to tau

B19 TP20 AT270 AT8 AT180 AT10 PHF-1 Tau-1 AP422 PThr212 PSer214 PSer262 PThr403 PSer404 PSer409 Pser413 Alz50 TG3

Bovine tau (h, m)* 32 – 41 (h)* Thr181 (P) Ser202/ Thr205 (P) Thr231 (P) Thr212/ Ser214 (P) Ser396/404 (P) Ser199/202 (NP) Ser422 (P) Thr212 (P) Ser214 (P) Ser262 (P) Thr403 (P) Ser404 (P) Ser409 (P) Ser413 (P) 7 – 9 and 312 – 342** Thr231***

Tau IR in somatodendritic compartment

Tau-positive neurites in amyloid plaques

TG tau

TG tau/PS1/APP

TG PS1/APP

TG tau/PS1/APP

+

+

+

+

+ +

+ +

+ +

+ + +

+

+

+ +

+ +

+

+

+

+ + + + + + + + +

+

+

+

+

+ +

+ +

+ + + + + +

+

+

+ +

The immunoreactivity was studied with antibodies requiring a phosphorylated epitope (P), an unphosphorylated epitope (NP), with phosphorylationindependent antibodies (*), with antibodies requiring a conformational epitope (**) or a conformational- and phosphorylated epitope (***). The numbers refer to the position of amino acids in the longest human tau isoform. The immunoreactivity was estimated on a qualitative scale: , absent; +, present. h, human; m, mouse.

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Expression of the tau, PS1 and APP transgenic proteins: immunocytochemical analysis The distribution of the transgenic human tau protein in tau/PS1/ APP mice was similar as that observed in tau- and tau/PS1 animals, as previously reported (Boutajangout et al., 2002; Brion et al., 1999). Neurons exhibiting a somatodendritic accumulation of human tau were found in many areas, for example, in the hippocampus, the cortex, the basal ganglia, the cerebellum, the brainstem (Fig. 2A) and in the spinal cord. The transgenic tau proteins were also detected in axonal tracts. The transgenic human APP was detected in most neurons in tau/PS1/APP mice (Fig. 2B), as in PS1/APP mice; the APP immunoreactivity was the strongest in cell bodies and was also detected in many axonal tracts. The transgenic PS1 protein was detected in neurons as previously reported (Fig. 2C). Triple immunohistochemical labelling demonstrated that transgenic tau, APP and PS1 proteins were coexpressed in many neurons (Fig. 2). Formation of A deposits in PS1/APP and tau/PS1/APP transgenic mice First, conspicuous extracellular Ah deposits were observed at 2.5 months in tau/PS1/APP transgenic mice and were limited to the subiculum (Fig. 3A), as reported previously in PS1/APP transgenic mice (Blanchard et al., in press). A similar progression of Ah deposits in other brain areas was observed during aging of tau/PS1/ APP mice. At 18 months of age (Fig. 3D), Ah deposits were observed in many cortical areas, in basal ganglia’s, in the cerebellum, brainstem and (less abundantly) in the gray matter of the spinal cord. These Ah deposits were immunolabelled with the

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different Ah antibodies and most Ah deposits were stained by Congo red. Ah deposits in the walls of small and middle-size blood vessels were also observed (not shown). A punctuate Ah immunoreactivity, localised in or adjacent to cell bodies, was also observed in neurons in PS1/APP and tau/PS1/APP mice. This Ah immunoreactivity appeared early and might correspond to intracellular accumulation of Ah (not shown). Analysis of the phosphorylation status of tau-immunoreactive cells As previously reported in tau transgenic mice (Brion et al., 1999), an accumulation of the transgenic tau protein in the somatodendritic compartment of a population of neurons was observed in the tau/PS1/APP transgenic mice. These neurons were more frequent in the hippocampus and the cortex, but were ubiquitously distributed and also found in basal ganglia such as the thalamus (Fig. 4K). This somatodendritic tau accumulation was detected with the human specific tau antibody TP20 and was never found in control animals. A similar number of cells with somatodendritic tau accumulation were present in tau and in tau/SP1/APP animals. The phosphorylation status of this somatodendritic tau was studied with a panel of phosphorylation-dependent tau antibodies (Table 1) and observed to be positive with the antibodies AT180, AT270, tau-1, pSer214, pThr403 and pSer404, both in tau and in tau/PS1/APP transgenic mice. Analysis of the neuritic changes associated to A deposits To assess for the presence of dystrophic neurites associated to the Ah deposit in PS1/APP and in tau/PS1/APP transgenic mice,

Fig. 5. Tau immunoreactivity associated to Ah deposits. (A – F) Immunolabelling of dystrophic neurites around Ah deposits in 9-month-old (A – E) and 18month-old (F) tau/PS1/APP transgenic mice, with the B19 tau antibody (A), the Alz50 conformation-dependent tau antibody (B), and the phosphotau antibodies AT10 (C), AT270 (D), pThr262 (E) and PHF-1 (F). Scale bar: 25 Am.

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we used both silver staining methods and immunocytochemistry. In contrast to what is observed in AD (inset in Fig. 4A), the Gallyas silver staining methods did not identify grossly dilated and dystrophic neurites (Figs. 4A,B) in PS1/APP or in tau/PS1/APP transgenic mice. On the contrary, clusters of dystrophic neurites associated to an amyloid core could be identified using several antibodies. A first group of dystrophic neurites were made up of large and rounded processes, strongly immunolabelled with antihuman APP (Fig. 4C) and antiubiquitin antibodies (these two immunoreactivities were generally colocalised; Figs. 4E,F). Clusters of dystrophic neurites in the absence of Ah deposits could also be occasionally observed (Figs. 4C,D). The second group of dystrophic neurites was characterised by assessing the phosphorylation status of tau in neurites associated to the Ah deposit, using a panel of phosphorylation-dependent tau antibodies (Table 1). The tau antibodies B19 (Fig. 5A), AT8 (Fig. 4G), AT270 (Fig. 5D), pThr212, pSer262 (Fig. 5E), PHF-1 (Fig. 5F) strongly labelled short, segmental or granular profiles associated to the Ah deposit, clearly different from the APP positive globular processes (see Fig. 4H). This immunoreactivity could not be observed in the absence of Ah deposit and was rarely seen before 4 months. The AP422 antibody and the conformationdependent Alz50 (Fig. 5B) and AT10 (Fig. 5C) tau antibodies also labelled these short profiles, but not the conformation-dependent tau antibodies TG-3. A similar pattern of phosphotau immunore-

activity was observed in PS1/APP and tau/PS1/APP transgenic mice (Table 1). Dystrophic neurites could also be demonstrated with antibodies to the human tau transgenic protein in tau/PS1/APP but not in PS1/ APP transgenic mice. The human tau protein accumulated in some dystrophic neurites around Ah deposits (Figs. 4I,J) and neurons exhibiting a somatodendritic accumulation of this human tau protein and adjacent to Ah deposits were frequently observed (Figs. 4K,L). Dystrophic processes around Ah deposits were also identified with antiphosphorylated neurofilament antibodies (Fig. 6A). Globular axonal dilatations at distance from Ah deposits were identified with the Bodian staining (Fig. 6B) and the neurofilament antibodies (Fig. 6C). The MAP2 (Fig. 6D) and MAP1 antibodies did not label any of these dystrophic neurites, but they were also labelled with the anti-a-synuclein antibody (Fig. 6E), and the GAP-43 and synaptophysin antibodies (not shown). In addition to these neuritic changes, many amyloid deposits in tau/PS1/APP mice were associated to hypertrophied GFAP-positive astrocytes and to microglial cells, as observed in PS1/APP mice (Blanchard et al., in press). The Gallyas, the Bodian silver-staining methods and the Congo red staining did not detect neurofibrillary lesions in the forebrain or in the spinal cord of the tau/PS1/APP mice examined up to 18 months of age, including in neurons with a somatodendritic accumulation of human tau and localised nearby Ah deposits.

Fig. 6. Cytoskeletal immunoreactivity of dystrophic neurites in tau/PS1/APP transgenic mice. (A) Immunolabelling with the RT97 neurofilament antibody, showing neurofilament positive dystrophic neurites around Ah deposit. Bundles of normal adjacent axons are also labeled. (B, C) Axonal dilatations identified by Bodian staining (B) in a 9-month-old mouse, and by antineurofilament immunolabelling (C) in a 12-month-old mouse. (D) Immunolabelling with the antiMAP2 antibody. Dendritic processes around the amyloid deposit are immunolabelled but the dystrophic processes are unlabelled. (E) Immunolabelling with the anti-a-synuclein antibody. The dystrophic process around the amyloid deposit are labeled. Scale bar: 25 Am.

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Fig. 7. Ultrastructural aspect of Ah deposit and dystrophic neurites in tau/PS1/APP transgenic mice. (A) Low magnification showing the amyloid core surrounded by dystrophic neurites of various sizes. (B) Higher magnification of dystrophic neurites adjacent to an amyloid deposit. They contain accumulations of dense bodies, mitochondria and microtubular elements. (C) High magnification of the periphery of the amyloid deposit, showing that some amyloid fibres (Am) are in close contact with membraneous profiles (arrows). (D) Immunolabelling with the human APP antibody. A dilated processes adjacent to an amyloid deposit (Am) is strongly positive. The APP immunoreactivity is associated with the dense bodies. (E) Immunolabelling with the phosphotau AT8 antibody. Two thin processes (arrows) adjacent to an amyloid deposit (Am) are strongly AT8 positive. The AT8 immunoreactivity is associated to microtubules or to adjacent amorphous material. Scale bars: (A) 10 Am; (B) 0.5 Am; (C) 0.25 Am; (D, E) 1 Am.

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Fig. 8. Ultrastructural aspect of the fibrillar pathology in tau/PS1/APP transgenic mice. (A) A dystrophic neurite contains a large crystal-like structure. The higher magnification in B shows that it is composed of an arrangement of straight filaments alternating with a ‘‘bead-on-a string’’ aspect. (C) A degenerating neurite exhibits large dense bodies and numerous fibrillar elements. In the higher magnification in D, they appear as 10 nm-wide filaments showing a swirling arrangement. (E) Higher magnification of an axonal process with an atrophied myelin sheath, showing an accumulation of clear vesicular organelles, and numerous tubular structures. In the higher magnification in F, the latter appear as straight 25-nm-wide microtubular elements, often organised in small bundles running in various directions. Scale bars: (A, C) 1 Am; (E) 0.5 Am; (B, D, F) 200 nm.

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Ultrastructural analysis of the amyloid plaques and dystrophic neurites

Ah deposits was mainly associated to microtubules with some diffuse labelling adjacent to these microtubules (Fig. 7E).

Both the amyloid deposit and the cellular components of the amyloid plaques were analysed at the ultrastructural level in tau/ PS1/APP and in PS1/APP mice. The most frequent plaques were constituted of one or more core of amyloid composed of extracellular amyloid filaments (Fig. 7A). At the periphery of the amyloid core, the amyloid fibres were, however, often in contact with membranous profiles, possibly derived from the Golgi or the reticulum (Fig. 7C). Several types of cell processes surrounded the amyloid core. Astroglial processes were identified by their high content in intermediate type (10-nm wide) glial filaments. The amyloid core was also surrounded by globular dystrophic neurites (myelinated and nonmyelinated) containing multilaminar dense bodies, multivesicular bodies, mitochondria and profiles of smooth endoplasmic reticulum (Fig. 7B). Regularly, these dystrophic neurites contained numerous tubular elements (with a clear centre) running in various directions (Fig. 8E,D); these tubules had a regular diameter of 26.2 F 2.9 nm (mean F SD). This diameter was similar to that of microtubules clearly identified in adjacent dendrites and axons (28.5 F 2.1 nm, mean F SD). Sometimes, clusters of dystrophic neurites, without amyloid core, were also observed. A few dystrophic neurites contained bundles of straight tubular structures with a clear centre. These tubular elements had a regular diameter of 18.4 F 2.0 nm (mean F SD) and were assembled with a regular spacing between them. These tubular elements were sometimes assembled in crystal-like structure by admixing of bundles arranged in various angulations (Figs. 8A,B). The latter inclusions were structurally reminiscent of Hirano bodies. Other dystrophic neurites contained swirling filaments with a diameter of 11.8 F 1.3 nm (mean F SD), similar to neurofilaments (Figs. 8C,D). The immunolabelling in electron microscopy with the anti-APP antibody showed that the APP immunoreactivity was mainly associated with the laminar dense bodies in globular dystrophic neurites (Fig. 7D). The immunolabelling with the AT8 antibody indicated that this immunoreactivity in small neurites adjacent to

Analysis of phosphorylation of tau protein by Western blotting The phosphorylation of tau in the soluble and insoluble brain fractions of control, tau and tau/PS1/APP mice was investigated by immunoblotting using the AT8, AT180, pS214 and PHF1 phosphorylation-dependent tau antibodies (Fig. 9). Tau proteins in the soluble and insoluble fractions exhibited an immunoreactivity with all these phosphorylation-dependent tau antibodies in control and transgenic lines, although less intensely with the pS214 antibody. Although the pattern of tau labelling differed between antibodies, the phosphotau antibodies labelled more intensely than the B19 tau antibody tau species with slower electrophoretic mobilities in the soluble and insoluble fractions, as shown in representative immunoblots in Fig. 9. The densitometric analysis for tau phosphorylation was performed taking into account the total tau levels (as assessed by using the B19 tau antibody) for normalisation purposes. Statistical analysis (n = 3 animals for controls and each transgenic line), however, did not reveal significant differences of tau phosphorylation between control, tau and tau/PS1/APP animals in the soluble and insoluble fractions.

Discussion Numerous transgenic models have been generated with the aim to modelise the formation of brain lesions observed in AD (for a review, see Go¨tz, 2001; Janus et al., 2000; Van Leuven, 2000). Transgenic mice engineered to express FAD mutants of APP develop amyloid deposits, but these mice do not develop NFT. However, these transgenic models might lack some critical elements necessary for the formation of NFT, such as the expression of a proper human tau protein, expression of a tau isoform normally not expressed in the adult mouse and a sufficient length of exposure to Ah deposits of the neurons expressing this human

Fig. 9. Immunoblotting analysis of the soluble (A – E) and insoluble (F – J) fractions from brain homogenates of 18-month-old nontransgenic mice (lanes 1), tau transgenic mice (lanes 2) and tau/PS1/APP transgenic mice (lanes 3). The blots were probed with the B19 antitau antibody (A, F) or the phosphorylationdependent tau antibodies PHF1 (B, G), AT180 (C, H), AT8 (D, I) and pSer214 (E, J). Bars and numbers on the left of lanes 1 indicate the position and molecular weight (in kDa) of proteins used as markers: catalase (58.1 kDa) and alcohol dehydrogenase (39.8 kDa).

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tau protein in the brain of these transgenic mice. The triple transgenic model that we have characterised in the present study is a model that seems to alleviate at least partially these factors. These mice express a wild-type human tau protein that colocalises with transgenic APP and PS1 proteins in neurons, and they express a tau isoform (0N3R) normally not expressed in adult mouse that express only 4R tau isoforms. Due to the combination of the expression of mutants of APP and PS1 proteins, they developed an aggressive phenotype in terms of extracellular Ah deposits observed as early as 2.5 months and up to 18 months. In previous reports of transgenic mice developing Ah deposits, the age of onset of amyloid deposition generally varied from 6 months up to 25 months (for a review, see Janus et al., 2000), leading to a shorter exposure to Ah deposits. Despite this long exposure to extracellular Ah deposits and humanisation of these triple transgenic mice by expression of a human tau isoform, they did not develop authentic NFT, as evidenced by silver staining, Congo red staining, immunolabelling and ultrastructural observation. These results argue against a direct role of extracellular Ah as a strong inducer of NFT formation in the absence of a preexisting NFT pathology. Indeed, the enhancement of the neurofibrillary pathology reported in transgenic lines expressing mutant tau proteins and injected intracerebrally with Ah42 fibrils (Go¨tz et al., 2001b) or crossed with a transgenic line developing Ah deposits (Lewis et al., 2001) might reflect the ability of Ah to potentialise this preexisting NFT pathology. Ah might have a similar role in Alzheimer’s disease (Delacourte et al., 1999). The development of a neuronal tau fibrillary pathology independently of an Ah pathology has been successfully obtained in transgenic models overexpressing mutant tau proteins (Allen et al., 2002; Go¨tz et al., 2001a; Lewis et al., 2000; Tanemura et al., 2002; Tatebayashi et al., 2002). On the contrary, transgenic models overexpressing wild-type tau proteins did not develop neurofibrillary tangles (Boutajangout et al., 2002; Brion et al., 1999; Go¨tz et al., 1995; Ishihara et al., 1999; Probst et al., 2000; Spittaels et al., 1999) although in some transgenic models a moderate (Ishihara et al., 2001) or a glial (Higuchi et al., 2002) fibrillary pathology has been reported. This greater ability of mutant tau proteins, by contrast to wildtype tau proteins, to generate a neurofibrillary pathology, might be related to their ability to accelerate the formation of tau filaments in vitro (e.g. Nacharaju et al., 1999). Since tau mutations have not been described in Alzheimer’s disease, a relevant animal model of neurofibrillary lesions would also be expected to be generated in the absence of these mutations. Higher levels of expression of human tau, as obtained in other tau transgenic models, might be useful to further investigate the effects of Ah deposits. However, this high tau expression induce striking neuronal lesions independently of Ah deposits (Brion et al., 1999; Ishihara et al., 1999; Probst et al., 2000; Spittaels et al., 1999) and can make difficult the interpretations of the respective roles of high tau expression and Ah deposits. Although the use of different tau isoforms than the 0N3R used in our model or a combination of them might prove to be important in tau transgenic models of neurofibrillary lesions induced by amyloid deposits, it should be pointed that all six isoforms have been identified in PHF-tau proteins (Goedert et al., 1989). The 0N3R isoform is not a minor species in PHF (Greenberg et al., 1992; McLaughlin et al., 1997) and transgenic mice expressing a mutant 0N4R (Allen et al., 2002) develop neurofibrillary lesions, indicating that the presence of inserts 1 and 2 are not a prerequisite for the development of NFT.

A somatodendritic accumulation of phosphorylated tau was observed in tau/PS1/APP mice; the phosphorylation status of this somatodendritic tau was, however, limited to a subset of phosphorylation sites identified in PHF-tau proteins and was similar as the one observed in tau only transgenic mice, as previously observed (Brion et al., 1999), suggesting that the expression of the mutants PS1 and APP transgenic proteins did not affect tau phosphorylation in these neurons. Despite this absence of authentic neurofibrillary pathology, a neuritic dystrophy was, however, present around the Ah deposits in PS1/APP and in tau/PS1/APP mice and at least some of these dystrophic processes belonged to neurons expressing the human tau protein in tau/PS1/APP mice. This neuritic dystrophy was similar in PS1/APP and in tau/PS1/APP mice and consisted in large, dilated processes or in short segmental or granular processes. The large dystrophic neurites exhibited an abundance of membranous bodies, similar to those observed in Alzheimer’s disease (Terry et al., 1964) and in models of APP (Masliah et al., 1996) and PS1/APP (Kurt et al., 2001) transgenic mice, and that can be interpreted as alterations observed in reactive and degenerating axons (Lampert, 1967). The dystrophic processes were not identified with the dendritic markers MAP2 and MAP1 but were labelled with axonal markers (phosphorylated high-molecular weight neurofilaments, GAP43, a-synuclein, synaptophysin), indicating that they mainly arise from axonal processes, a finding in agreement with a previous study (Phinney et al., 1999). The large dystrophic processes were also identified by an overlapping APP and ubiquitin immunoreactivity, and APP was ultrastructurally colocalised with the dense bodies. This suggest that ubiquitinated proteins, including APP, accumulates in these membranous bodies in dystrophic neurites, possibly as a result of disturbances of axoplasmic transport. The smaller processes were identified by their strong tau immunoreactivity, especially phosphorylated tau immunoreactivity. An immunoreactivity for some phosphorylated tau epitopes around amyloid deposits has been identified in previous studies in more aged animals (e.g. generally with the AT8 antibody; Masliah et al., 2001; Sturchler-Pierrat et al., 1997) but we performed in the present study a complete analysis of the pattern of tau phosphorylation in these dystrophic neurites, by using a panel of tau antibodies to phosphorylated epitopes and to conformation-dependent epitopes. We observed a net increase of phosphorylated tau immunoreactivity for several amino acids known to be phosphorylated in PHF-tau proteins (Thr181, Ser202/thr205, Thr231, Ser396/Ser404, Thr212, Ser214, Ser262, Thr403, Ser404, Ser413) and interestingly with phosphorylated and/or conformation sites that seems more specific for PHF-tau proteins (AT10 epitope, Alz50 epitope, AP422 epitope). Since these tau immunoreactivities were topographically concentrated around amyloid deposits, this suggests that Ah deposits might locally activate tau protein kinases, a result compatible with some in vitro observations (Alvarez et al., 1999; Busciglio et al., 1995; Takashima et al., 1998). The tau immunoreactivity of these neurites was similar in PS1/APP and in tau/PS1/APP mice, suggesting that this local increased tau phosphorylation affected at least in part the endogenous tau protein. The immunoblotting analysis did not, however, reveal significant changes of tau phosphorylation between the tau only and the tau/PS1/APP mice, suggesting that this local increased tau phosphorylation around amyloid deposits was relatively moderate and not detectable in brain homogenates.

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Other indications of a cytoskeletal pathology in neuronal dystrophic processes was the presence of straight tubular elements (with a diameter compatible with microtubules) disposed in various directions or grouped in small bundles and neurofilament-positive dystrophic processes and dilatations, possibly related to disturbances of axoplasmic transports (Masliah et al., 2001). The Hirano bodies detected in dystrophic neurites have been previously observed in AD and other conditions (Hirano, 1994) and might reflect alterations of the actin network. In conclusion, we have characterised a triple transgenic line expressing a human tau wild-type isoform, a mutated form of human PS1 and a mutated form of APP. These mice developed early extracellular Ah deposits. These Ah deposits were associated with neuritic and cytoskeletal alterations, and induced an increased phosphorylated tau immunoreactivity. These results confirm in vivo that Ah deposits affect tau phosphorylation, but suggest also that a long exposure to Ah in vivo was insufficient per se to drive the formation of an authentic neurofibrillary pathology, in a transgenic model expressing a wild-type human tau and without preexisting neurofibrillary pathology. Since neuronal lesions due alone to the high expression of tau or a mutant of tau are not found in this model (with a relatively lower level expression of human tau), it represents also a more pathophysiological situation reminiscent of the human disease and it will be adequate to test additional stress factors (e.g. oxidative stress, age-related metabolic modifications) that might be necessary to obtain the full neuropathological picture of AD.

Acknowledgments This study was supported by grants from the Belgian FRSM, Alzheimer Belgique, the International Alzheimer Research Foundation and the Foundations Hoguet and Jean Brachet.

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