Enhanced accumulation of tau in doubly transgenic mice expressing mutant βAPP and presenilin-1

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Enhanced accumulation of tau in doubly transgenic mice expressing mutant βAPP and presenilin-1 Eriko Samura a , Mikio Shoji a,⁎, Takeshi Kawarabayashi a , Atsushi Sasaki b , Etsuro Matsubara c , Tetsuro Murakami a , Xu Wuhua a , Shuta Tamura a , Masaki Ikeda d , Koich Ishiguro e , Takaomi C. Saido f,g , David Westaway h , Peter St. George Hyslop h , Yasuo Harigaya i , Koji Abe a a Department of Neurology, Division of Neuroscience, Biophysical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama, 700-8558 Japan b Department of Pathology Gunma University Graduate School of Medicine, Maebashi, Japan c Department of Alzheimer's Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Japan d Department of Neurology, Gunma University Graduate School of Medicine, Maebashi, Japan e Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Japan f Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Japan g Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada h Departments of Medicine (Neurology) and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada i Department of Neurology, Maebashi Red Cross Hospital, Maebashi, Japan

A R T I C LE I N FO

AB S T R A C T

Article history:

Aβ amyloidosis and tauopathy are characteristic changes in the brain of Alzheimer's disease.

Accepted 27 December 2005

Although much evidence suggests that Aβ deposit is a critical initiation factor, the

Available online 19 May 2006

pathological pathway between Aβ amyloidosis and tau accumulation remains unclear. Tau accumulation was examined in the doubly transgenic mouse (APP-PS) expressing βAPPKM670/

Keywords:

671NL

Alzheimer's disease

deposits were detected from 8 weeks. Tau accumulation appeared at 4.5 months and markedly

Doubly transgenic mouse

increased in dystrophic neurites around Aβ amyloid. Accumulated tau was phosphorylated,

Presenilin-1

conformationally altered, and argyrophilic. Expression of tau and accumulation of sarkosyl-

Tau

insoluble phosphorylated tau were increased in APP-PS brains compared with those of Tg2576

Aβ

mice. Straight or twisted tubules mimicking paired helical filament were revealed at electron

Tg2576

microscopic level in 16-month-old APP-PS. These findings suggest that mutant presenilin-1

(Tg2576) and presenilin-1 L286V (PS-1 L286Vtg). Accelerated and enhanced Aβ amyloid

accelerated Aβ-induced tauopathy and further promoted fibril formation of tau. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Alzheimer's disease (AD), one of the most devastating brain diseases, is a medical, sociological, and economic problem due

to the escalating increase in the elderly population in modern society. About 5% of the population over 65 years old suffer from dementia. These serious problems immediately demand social care systems and development of treatment for

⁎ Corresponding author. Fax: +81 86 235 7368. E-mail address: [email protected] (M. Shoji). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.134

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dementia. The majority of patients with dementia are AD. AD brains are invariably characterized by two pathological features: initial Aβ amyloidosis by extracellular deposition of Aβ42(43) and Aβ40, and subsequent tauopathy characterized by intracellular accumulation of neurofibrillary tangles (NFT) comprised of abnormal aggregates of phosphorylated tau. Because familial AD-linked mutations of amyloid β protein precursor (βAPP), presenilin-1 (PS-1), and presenilin-2 increase the extracellular concentration of Aβ42(43) or protofibril Aβ, these peptides are likely to be an initiating factor in the evolution of all types of AD (Scheuner et al., 1996). This Aβ cascade from Aβ deposits to the final appearance of tauopathy and neuronal cell losses is the major hypothesis to explain all steps of the pathogenesis of AD. Recent transgenic mouse studies have confirmed this hypothesis in part. Overproduction of mutant βAPP in the mouse brain causes substantial Aβ amyloid deposits (Games et al., 1995; Hsiao et al., 1996; Kawarabayashi et al., 2001; Sturchler-Pierrat et al., 1997). Memory disturbance and subsequent pathological events of AD were reproduced in these mouse models. Doubly

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transgenic mice expressing mutant βAPP and mutant PS-1 showed robust acceleration of Aβ amyloid deposits (Holcomb et al., 1998; McGowan et al., 1999; Wengenack et al., 2000). However, no NFT was recognized in these mice. Although multiple tau gene mutations cosegregated with familial frontotemporal lobe dementia, the brain pathology is tau accumulations without Aβ amyloid. Appearance of NFT was enhanced by Aβ amyloid in Tg2576 × tau P301L doubly transgenic mice (Lewis et al., 2001). Injection of Aβ amyloid into the brain also induced NFT in tau P301L transgenic mice (Gotz et al., 2001). These findings suggest that Aβ amyloidosis may induce tauopathy secondarily, and that these pathological relationships play critically important roles in the development of all types of Alzheimer's disease. Here, we generated doubly transgenic mice, APP-PS, proband mice from a cross between Tg2576 mouse expressing mutant βAPP695K670N/M671L (Hsiao et al., 1996) and a transgenic mouse expressing presenilin-1 L286V (PS-1 L286Vtg, line 198) (Citron et al., 1997). APP-PS showed substantial acceleration of Aβ amyloid deposits and the early appearance of core plaques. Associated dystrophic neurites showed numerous tau

Fig. 1 – Early appearance of Aβ amyloidosis in brains of APP-PS. APP-PS mice (A–C, G–I) and Tg2576 mice (D–F). (A–F) Hemibrain section stained with Ab9204; (A, D) 2 months old (8 weeks); (B, E) 9 months old; (C, F) 16 months old. Development of plaque was accelerated in APP-PS brains compared with that of Tg2576. In APP-PS, core plaques appeared from 8 weeks of age (A, arrow). In Tg2576, core plaques were detected from 9 months (E, arrowhead). (G–I) Staining of 16-month-old APP-PS (G, I: BA-27; H: BC-05). Core plaques were stained with both BA-27 (G) and BC-05 (H). They were labeled by BA-27 more predominantly than by BC-05. (I) Amyloid angiopathy and subpial amyloid deposits were strongly stained with BA-27. Scale bars = 2 mm in panels A–F and 20 μm in panels G–I.

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accumulations. These tau accumulations were phosphorylated, conformationally changed, and argyrophilic. Sixteenmonth-old mice showed accumulation of straight or twisted tubules in these dystrophic neurites. The accumulation level of tau was increased and further phosphorylated in APP-PS. These findings suggested that extensive deposition of Aβ amyloid accelerated secondary tauopathy in dystrophic neurites of senile plaques of the mouse brain.

2.

Results

In APP-PS, core plaques appeared from 8 weeks (2 months) of age (Fig. 1A, arrow). The number of core plaques stained by Ab9204 increased according to age, and these plaques spread

widely into the cerebral cortices. At 9 months of age, small and round-shaped core plaques were observed in the hippocampus, thalamus, basal ganglia, brain stem, and cerebellar cortex in addition to many cortical core plaques (Fig. 1B). They were labeled by BA-27 more predominantly (Fig. 1G) than by BC-05 (Fig. 1H). Amyloid angiopathy was also detected by BA-27 (Fig. 1I). After 9 months, these small round core plaques developed into large core plaques. Many large core plaques and diffuse plaques were observed throughout the brain at 16 months of age (Fig. 1C). Aβ42-positive diffuse plaques appeared at 14 months and their number increased with age. No plaques were observed in Tg2576 brains at 2 months (Fig. 1D). Core plaques were detected in the cerebral cortex of Tg2576 from 8 months, and a few plaques were detected at 9 months (Fig. 1E, arrowhead). At 16 months of age, some

Fig. 2 – Progress and distribution of tau accumulation in brains of APP-PS. APP-PS (A–C, G–J) and Tg2576 mice (D–F, K–N). (A–F) Staining of the cerebral cortex with AT8; (A, D) 4.5 months old; (B, E) 9 months old; (C, F) 16 months old. Phosphorylated tau appeared earlier and more abundantly in dystrophic neurites around core plaques in APP-PS mice than in Tg2576. AT8-positive dystrophic neurites appeared at 4.5 months of age in the APP-PS brain (A) and at 14 months of age in Tg2576 (F, arrow). (G–N) Staining of 16-month-old APP-PS (G–J) and Tg2576 (K–N); (G, K) AT8; (H, L) PHF-1; (I, M) Alz50; (J, N) Gallyas–Braak silver stain. Accumulation of phosphorylated, conformationally changed and argyrophilic tau was enhanced in APP-PS. Scale bars = 130 μm in panels A–F and 50 μm in panels G–N.

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cortical plaques and only a few diffuse plaques were detected in the cortex of Tg2576 brains (Fig. 1F). Thus, development of core plaque was accelerated 4-fold in APP-PS brains (8 months to 8 weeks) compared with Tg2576. A small number of AT8-positive dystrophic neurites appeared in the APP-PS brain at 4.5 months of age (Fig. 2A). At 9 months of age, many dystrophic neurites around core plaques were stained by AT8. Plaque cores were also labeled weakly by AT8 (Fig. 2B). The number and size of AT8-positive dystrophic neurites increased with age. Almost 90% of core plaques showed AT8-positive dystrophic neurites in the cortex of APP-PS at 16 months of age (Figs. 2C and G). In Tg2576 brains, no AT8-positive dystrophic neurites were detected at 4.5 and 9 months of age (Figs. 2D and E). Small numbers of dystrophic neurites were labeled by AT8 in Tg2576 at 14 months of age, and they were obvious at 16 months (Fig.

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2F, arrow, and Fig. 2K). The number and size of AT8-positive neurites in Tg2576 were much less than those of APP-PS. The time lag between the appearance of amyloid deposition and that of phosphorylated tau was 2.5 months in APP-PS (2– 4.5 months) and 6 months in Tg2576 (8–14 months). Thus, phosphorylated tau appeared earlier and more abundantly in dystrophic neurites around core plaques in APP-PS mice. In APP-PS at 16 months old, the accumulated tau in dystrophic neurites around core plaques was labeled by PHF1 (Fig. 2H). About 70% of dystrophic neurites were labeled by Alz-50 (Fig. 2I). Gallyas–Braak silver staining showed extensive labeling of core plaques and surrounding large dystrophic neurites in APP-PS (Fig. 2J). The Gallyas-positive dystrophic neurites were observed in about 20% of core plaques in the whole brain and 50% of those in the cerebral cortex. Gallyaspositive dystrophic neurites were large and round shaped but

Fig. 3 – Electron microscopic examination of the 16-month-old APP-PS. (A) A core plaque surrounded by many dystrophic neurites filled with dense laminar bodies. (B) In the peripheral area of the core plaque (A), some unmyelinated neurites were filled with filaments. (C) Large magnification of the neurite (B) showed that filaments were tubular structures of 14 nm ˜ diameter. (D) Another type of dystrophic neurite filled with different types of filaments. (E) The filaments in panel D were tubular structures of 20-nm diameter. A helical structure was suggested in these tubules although it was not regularly observed ˜ (arrow). (F) IEM showed that dystrophic neurites around the core plaque were labeled with AT8. Asterisk shows core plaque. (G) In dystrophic neurites, straight or tubular filaments and nonfilamentous amorphous substances were labeled by AT8. Scale bars = 22 μm in panel A, 3 μm in panel B, 410 nm in panel C, 2.1 μm in panel D, 750 nm in panel E, 5.4 μm in panel F, and 530 nm in panel G.

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different from the dotlike small round dense stain detected by AT8 or PHF-1. In contrast, core and dystrophic neurites were labeled weakly by AT8, PHF-1, and Alz50 staining in Tg2576 mice at 16 months of age (Figs. 2K–M). Gallyas-positive dystrophic neurites were small and few in Tg2576 (Fig. 2N). Thus, accumulation of phosphorylated, conformationally changed and argyrophilic tau was enhanced in APP-PS. Electron microscopic examination was performed using the cerebral cortex of APP-PS at 16 months old. Typical amyloid star was surrounded by many dystrophic neurites containing dense laminated bodies (Fig. 3A). Some dystrophic neurites were filled with filaments of ∼14 nm in diameter corresponding to straight tubules in NFT (Fig. 3B). No regular helical structure was observed in these tubules (Fig. 3C). Another type of dystrophic neurites was filled with ∼20-nmdiameter filaments (Fig. 3D). These filaments were almost longitudinally parallel and had a twisted tubules appearance although this was not regularly periodical (Fig. 3E). IEM showed that these tubules and nonfilamentous amorphous substances in dystrophic neurites were labeled with AT8 (Figs. 3F and G). The presence of straight and twisted tubules mimicking paired helical filaments (PHF) suggested that extensive accumulation of phosphorylated and conformationally altered tau closely associated with tubule formation in dystrophic neurites of APP-PS. Western blot analysis of TS-soluble fractions from control non-Tg mouse, Tg2576, and APP-PS at 3 months of age showed 66-kDa bands and lower 55-kDa band labeled by tau-C. The 66kDa tau level was the highest in the APP-PS brain, followed by those in the Tg2576 brain and lowest in the control non-Tg mouse (Fig. 4A, arrow). Larger bands were also detected by tauC as described (Planel et al., 2001). The signal intensity increased 1.3-fold in the Tg2576 brains and 1.6-fold in the APP-PS brains compared to the non-Tg mouse. Marked accumulation of phosphorylated tau was observed by PHF-1 (Fig. 4B) and PS-199 (Fig. 4C) in sarkosyl-insoluble fractions of Tg2576 and APP-PS brains at 12 months of age. The level of

phosphorylated tau in the APP-PS brains was 1.05-fold by PHF1 and 2.8-fold by PS-199 compared to those in the Tg2576 brains (Figs. 4B and C). These findings suggested that overexpression of mutant APP or Aβ-induced tau expression, and that mutant PS-1 further facilitated tau expression leading to tau phosphorylation in the sarkosyl-insoluble fraction of APPPS brain.

3.

Discussion

As expected, Aβ amyloidosis was accelerated in our APP-PS brains. Doubly transgenic mice (PSAPP) from a cross between Tg2576 and PS-1 M146L transgenic mice showed that Aβ42 deposits started at 10 weeks of age and extensively accumulated (Holcomb et al., 1998; McGowan et al., 1999; Wengenack et al., 2000). The amyloid burden increased an average of 179fold in PSAPP mice from 12 to 54 weeks of age (Wengenack et al., 2000). Because Aβ deposits were observed from 8 weeks in the present APP-PS, the appearance of Aβ amyloidosis is earlier than other findings of doubly transgenic mice with APP and mutant PS-1 and the rate of acceleration was estimated as 4-fold. Small core plaques constituting Aβ40 and Aβ42 in the early phase developed large core plaques with dystrophic neurites that were distributed widely in addition to the cerebral cortex. These distributions and large plaque formation throughout the brain were characteristic changes in APPPS. Highly distributed diffuse plaques consisting of Aβ42 in the late phase were also observed in addition to those in Tg2576. The appearance and distribution of amyloid angiopathy labeled by Aβ40 were also accelerated in APP-PS. These findings suggested that APP-PS is one of most useful AD mouse models representing earlier and more severe Aβ amyloidosis in the brain. Except for the marked acceleration of Aβ amyloidosis, the early appearance and enhanced accumulation of tau were characteristic changes in APP-PS. The period from amyloid

Fig. 4 – Western blot analysis of non-Tg, Tg2576 and APP-PS brains. (A) TS-soluble fractions of 3-month-old mice stained with Tau-C. Tau-C detected 66 kDa tau band (arrow) and 55 kDa lower band. The level of tau was the highest in the APP-PS brain, followed by those in the Tg2576 brain, and the lowest in the control non-Tg brain. The signal intensity increased 1.3-fold in the Tg2576 brains and 1.6-fold in the APP-PS brains compared to the non-Tg mouse. (B, C) Sarkosyl-insoluble fractions of 12-month-old mice stained with PHF-1 (B) and PS-199 (C). Both antibodies detected a 66-kDa tau band. The level of phosphorylated tau was highest in the APP-PS brain, followed by those in the Tg2576 brain, and lowest in the control non-Tg brain. The level of phosphorylated tau in the APP-PS brains was increased 1.05-fold by PHF-1 and 2.8-fold by PS-199 compared to the Tg2576 brains.

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deposits until tau accumulation was shorter at 2.5 months in APP-PS than the 6 months in Tg2576. The number and intensity of phosphorylated tau, conformationally changed tau (Wolozin et al., 1986), and argyrophilic tau were prominently enhanced in APP-PS. Tau accumulation in dystrophic neurites associated with senile plaque Aβ amyloid was first described in APP23 mice expressing βAPPKM670/671NL (Sturchler-Pierrat et al., 1997) and Tg2576 single mice (Tomidokoro et al., 2001a,b). In APP-PS mice, accumulation of tau was more prominent than those in APP23 mice or single Tg2576 mice. These findings suggest the possibility that mutated PS-1 may facilitate Aβ-induced tau accumulation and special modifications, such as phosphorylation, conformationally alteration, argyrophilicity, and sarkosyl-insoluble aggregation lead to NFT formation. In in vitro studies, Aβ42 promoted both tau aggregation and hyperphosphorylation (Rank et al., 2002). Aggregated Aβ increased the phosphorylated tau in rat primary cultured septal cholinergic neurons (Zheng et al., 2002). Aggregated Aβ peptides induced tau phosphorylation associated with increased APP expression in injured neurons (Le et al., 1997). Spherical aggregates of Aβ amyloid activate GSK-3β (Hoshi et al., 2003). In cultured cells treated with Aβ, PS-1 mutations significantly increased neuritic dystrophy and AD-like changes in tau such as hyperphosphorylation and increased tau protein levels (Pigino et al., 2001). Amyloid Aβ deposits have a marked effect on the dendritic microarchitecture in the cortex, even in the relative absence of phosphorylated alterations (Le et al., 2001a). Recent studies reported that double mutant progeny between Tg2576 and JNPL3, a tau P301L doubly transgenic mouse, developed Aβ deposits at the same age and enhanced NFT pathology in the limbic and olfactory cortex (Lewis et al., 2001). Injection of Aβ42 amyloid into the brain also induced tau accumulation in P301L mutant tau transgenic mice and caused 5-fold increases in the numbers of Gallyas silver stain-positive and hyperphosphorylated NFTs in neurons in the amygdala (Gotz et al., 2001). We have already shown that phosphorylated tau accumulates in lipid rafts followed by accumulation of Aβ dimers in lipid rafts of Tg2576 brain (Kawarabayashi et al., 2004). All these findings support the possibility that tauopathy is induced by Aβ amyloidosis. In contrast, doubly transgenic mouse overexpressing PS-1 M146L and human tau increased tau phosphorylation, but it was not sufficient to induce the formation of NFT (Boutajangout et al., 2002). Typical cotton wool plaques in familial Alzheimer's disease patients with PS-1 without exon 9 were accompanied by very little PHF-tau (Le et al., 2001b; Verkkoniemi et al., 2001). These findings suggested that the acceleration effect in tauopathy by mutation of PS-1 is weak compared with that in Aβ amyloidosis. However, a novel PS-1 mutation (Gly183Val) associated with Pick's disease without β-amyloid plaques has been recently reported (Dermaut et al., 2004). Thus, a direct association between mutant PS-1 and tauopathy may be considered. Western blot analysis of Tg2576 and APP-PS brains supported these findings. At 3 months of age of each mouse, marked enhanced expression of tau was observed in Tg2576 brains. Enhanced expression of tau was further induced in APP-PS. Because no amyloid deposition was

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observed in Tg2576 at 3 months of age, overexpression of soluble Aβ or mutant βAPP was suggested to be the cause of enhanced tau expression in Tg2576. Overexpression of mutant PS-1 or the early appearance of Aβ amyloid was also considered to be the cause of further tau induction in APP-PS. These findings suggested the possibility that tau expression is reactively regulated by the amount of βAPP, Aβ, or mutant PS-1 in Tg2576 and APP-PS brains. Other reactive changes of tau, such as phosphorylation and insolubility, were also facilitated in Tg2576 and APP-PS brains. Marked accumulation of phosphorylated tau at serine 396/serine 404 was recognized in sarkosyl-insoluble fractions of Tg2576 and APP-PS brains. Accumulation of phosphorylated tau at Ser199 was prominent in APP-PS mice, suggesting that mutant PS-1 facilitated phosphorylation of insoluble tau further in APP-PS mice. All these findings indicated that Aβ amyloidosis or mutant PS-1 causes reactive overexpression of tau and special modification of tau leading to NFT formation. To clarify this hypothesis, the presence of NFT was examined by EM study. EM showed massive straight or twisted tubules in some dystrophic neurites around amyloid cores. Some of the tubules were stained with AT8 by IEM. We previously reported that Tg2576 developed many dystrophic neurites around an amyloid core and that these neurites contained many dense multilaminar bodies. However, no straight or twisted tubules were detected even in 29-monthold Tg2576 (Sasaki et al., 2002). In PDAPP mice, phosphorylated tau immunoreactivity was observed as clusters distributed along filamentous structures accumulating in the dystrophic neurites and around neurotubules in the axons. However, no paired helical filaments were observed (Masliah et al., 2001). Kurt et al. (2003) examined APP/PS-1 (M146L) double transgenic mice and found straight filaments (10–12 nm wide) in dystrophic neurites. They also found PHF-like structures in a dark, atrophic neuron in one double Tg. EM findings of APP-PS mice suggested that additional expression of mutant PS-1 or the early appearance of Aβ amyloid induced by mutant PS-1 further facilitates modification of accumulated tau leading to NFT formation in dystrophic neurites around an amyloid core although the tubules did not show periodicity. These findings corresponded to the Aβ cascade hypothesis that Aβ amyloidosis facilitates secondary tauopathy. Because curative therapy, such as Aβ vaccine, is now developing, this clarification of the special relationship between Aβ amyloidosis and tauopathy may contribute to the next generation of therapy for tauopathy.

4.

Experimental procedures

4.1.

Subjects

Generation of the transgenic lines overexpressing FAD Presenilin-1 L286V (PS-1 L286Vtg: 198) is described elsewhere (Citron et al., 1997). PS-1 L286Vtg, line 198, were crossed with Tg2576 expressing βAPP KM670/671NL to generate doubly transgenic mouse APP-PS. We analyzed here 18 APP-PS and 19 PS-1 L286Vtg, line 198, at 1–16 months, 30 age-matched Tg2576 mice, and 28 non-Tg littermates as control.

198 4.2.

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Immunostaining

After Tg and non-Tg littermates were sacrificed under ether anesthesia, brains were removed and cut sagittally in the midline. One hemisphere was fixed in 4% paraformaldehyde with 0.1 M phosphate buffer (pH 7.6) for 8 h and embedded in paraffin. Five-micrometer-thick sections were prepared for immunostaining and Gallyas–Braak silver stain. For Aβ and tau immunostaining, sections were immersed in 0.5% periodic acid for blocking intrinsic peroxidase and treated with 99% formic acid for 3 min. After blocking with 5% normal goat or horse serum in 50 mM phosphate-buffered saline (pH 7.4) containing 0.05% Tween 20 and 4% Block Ace (Snow brand, Sapporo, Japan), sections were incubated overnight with primary antibodies. The specific labeling was visualized by Vectastain Elite ABC kit (Vector, Burlingame, CA). Tissue sections were counterstained with hematoxylin.

4.3.

Antibodies

The following antibodies were used in this study: tau-C against C-terminal human and mouse tau (1:200, 422–438 a.a. of human tau 441) (Ishiguro et al., 1995, 1999), anti-PS-199 antibody to phosphorylated tau at serine 199, 1:500) (Ishiguro et al., 1995, 1999), PHF-1 against phosphorylated tau at serine 396/serine 404 (1:400) (Greenberg and Davies, 1990), AT8 to phosphorylated tau at serine202/threonine205 (1:400) (Innogenetics, Gent, Belgium), and anti-PHF-tau antibody Alz-50 (1:100) (Wolozin et al., 1986). For Aβ immunostaining, Ab9204 against normal L-aspartate at position 1 (0.1 μg/ml) (Saido et al., 1995), BA-27 to carboxyl-terminus of Aβ40 (0.5 μg/ml) (Suzuki et al., 1994), and BC-05 to Aβ42 (0.1 μg/ml) (Suzuki et al., 1994) were used.

4.4.

Electron microscopy (EM)

The brain tissue of 16-month-old APP-PS mice was immersed in the fixative (2.5% glutaraldehyde, 0.1 M phosphate buffer (PB), pH7.4, for 4 h and washed several times in 0.1 M PB containing 7% sucrose. Blocks were then postfixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in Quetol 812 (Nisshin EM, Tokyo, Japan). Ultrathin sections were stained with uranyl acetate and lead acetate prior to observation with an electron microscope. Cerebral cortical tissues were fixed in PLP for 6 h at 4 °C for immunoelectron microscopy (IEM). After washing in PBS containing a graded series of sucrose, the tissues were embedded in OCT compound and rapidly frozen in liquid nitrogen. Frozen sections (6–8 μm) of the tissues were incubated with AT8 (1:100) followed by the addition of biotinylated anti-mouse Ig and streptavidin/biotin–peroxidase complex (Nichirei, Tokyo, Japan). After immunostaining, the sections were embedded in Quetol 812, and ultrathin sections were cut. Some sections were stained with uranyl acetate.

4.5.

Western blot

Half of the mouse brains were weighed and homogenized using a motor-driven Teflon glass homogenizer for 20 strokes in 9 volumes of Tris–saline buffer (TS) with protease inhibitors

(TS inhibitors: 50 mM Tris–HCl and 150 mM NaCl, pH 7.6, 0.5 mM DIFP, 0.5 mM PMSF, 1 μg/ml TLCK, 1 μg/ml antipain, 1 μg/ml leupeptin, 0.1 μg/ml pepstatin, 1 mM EGTA). The homogenate was centrifuged at 55,000 rpm for 60 min at 4 °C and the supernatant was analyzed as the TS-soluble fraction. After washing with 10 volumes of TS inhibitors, the pellet was homogenized again in 4 volumes of 1% sarkosyl in TS inhibitors, incubated on ice for 30 min, and centrifuged at 55,000 rpm for 60 min at 4 °C. The supernatant was analyzed as the sarkosyl-soluble fraction. The pellet was washed twice with 10 volumes of 1% sarkosyl in TS inhibitors, and the remaining pellet was analyzed as the sarkosyl-insoluble fraction. Each sample was boiled at 70°C in SDS sample buffer, separated on 4–12% NuPAGE Bis–Tris Gel (Invitrogen, Carlsbad, CA), and electrotransferred to Immobilon P (Millipore, Bedford, MA) at 100 V for 1.5 h. The blots were labeled by tau-C, PHF-1, and anti-PS-199. The signal intensity of labeled protein using Supersignal (Pierce, Rockford, IL) was quantified by the luminoimage analyzer (LAS 1000-mini, Fuji film, Tokyo).

Acknowledgments This work was supported by Grants-in Aid for Primary Amyloidosis Research Committee (S. Ikeda and T. Ishihara); surveys and research on special disease from the Ministry of Health, Labor and Welfare of Japan and by Grants-in Aid for Scientific Research (B) (16390251, 15390273), Scientific Research (C) (16590829, 15590879 and 16500213), and Scientific Research on Priority Areas (C) – Advanced Brain Science Project – from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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