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Keywords:

  • Alzheimer's disease;
  • amyloid β peptide;
  • β-secretase inhibitor;
  • beta-site APP cleaving enzyme 1;
  • hydroxymethylcarbonyl isostere;
  • KMI inhibitor

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid β peptide (Aβ), is generated from amyloid precursor protein (APP) by β- and γ-secretase-mediated cleavage. Because β-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Aβ and grow normally, a β-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel β-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits β-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Aβ production in vivo in the soluble fraction compared with vehicle, but the level of Aβ in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Aβ production in both the soluble and insoluble fractions. Our results indicate that the β-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.

Abbreviations used:

amyloid β peptide

AD

Alzheimer's disease

APP

amyloid precursor protein

BACE

beta-site APP cleaving enzyme

BBB

blood–brain barrier

CTF

C-terminal fragment

DMSO

dimethyl sulfoxide

FBS

fetal bovine serum

FL

full-length

GuHCl

guanidium chloride

HEK

human embryonic kidney

HMC

hydroxymethylcarbonyl

PBS

phosphate-buffered saline

sAPPα

soluble extracellular fragment of APP generated by α-secretase

sAPPβ

soluble extracellular fragment of APP generated by β-secretase

TBS

Tris-buffered saline

Z-VLL-CHO

N-benzyloxycarbonyl-valine-leucine-leucinal

Alzheimer's disease (AD) is a progressive neurodegenerative disorder whose primary pathogenic event is the extracellular accumulation of amyloid β peptide (Aβ), followed by oxidative damage to neurons that ultimately results in dementia (Selkoe 2001; Hardy and Selkoe 2002; Citron 2004a). The generation of Aβ by limited proteolysis of amyloid precursor protein (APP) is a key event in the pathogenesis of AD. The proteases known to be involved in the production of Aβ are β-secretase and γ-secretase, which were recently identified as beta-site APP cleaving enzyme 1 (BACE1) (Vassar et al. 1999; Hussain et al. 1999; Sinha et al. 1999; Yan et al. 1999; Lin et al. 2000) and presenilin complex respectively (Yu et al. 2000; Francis et al. 2002; Takasugi et al. 2003). β-Secretase mediates the initial step of Aβ production by β-cleavage of APP, releasing a large soluble fragment, soluble extracellular fragment of APP generated by β-secretase (sAPPβ). The C-terminal fragment (CTF) of APP (APP-CTF, C99) is then cleaved by γ-secretase at several positions, leading to the formation of the primary pathogenic species, Aβ42, and the secondary pathogenic species, Aβ40.

Because Aβ42 generation is the rate-limiting step in the Alzheimer's amyloid cascade hypothesis (Selkoe 2001; Hardy and Selkoe 2002; Citron 2004a), the reduction of Aβ production by inhibition of the initial key enzyme, BACE1, is highly desirable (Citron 2004b). In addition, BACE1 knockout mice lack Aβ and are phenotypically normal, suggesting that a therapeutic BACE1 inhibitor may be free of mechanism-based side-effects (Luo et al. 2001, 2003; Roberds et al. 2001; Ohno et al. 2004).

To design the KMI compound, a BACE1 inhibitor, we focused on the substrate sequence of the APP β-cleavage site P4-P4′ site; EVKM*DAEF. We synthesized KMI-008, a novel transition-state mimic BACE1 inhibitor, containing the unnatural amino acid phenylnorstatine [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] and a hydroxymethylcarbonyl (HMC) isostere, as a lead compound (Shuto et al. 2003). The HMC isostere is a highly effective, transition-state mimic analogue, in which the replacement of isostere renders the scissile bond uncleavable. The HMC isostere has been used to develop inhibitors for disease-related aspartic proteases, such as the human immunodeficiency virus protease (Abdel-Rahman et al. 2002). We recently synthesized smaller, effective BACE1 inhibitors, KMI-358 and KMI-370, by modifying their chemical structures at the P1′ site (Kimura et al. 2004) and successfully developed the more potent inhibitor, KMI-429, by further modifying both the P1′ and P4 sites (Kimura et al. 2005).

In the present study, we injected KMI-429 into the hippocampus of APP transgenic or wild-type mice to evaluate its in vivo inhibitory effects on β-secretase activity. We found that KMI-429 effectively inhibited Aβ production as well as the release of sAPPβ. These results indicate that the novel BACE1 inhibitor KMI-429 effectively reduces Aβ production and may have therapeutic potential in AD.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

BACE1 inhibitors

KMI inhibitors (KMI-358 and KMI-429) were synthesized as described previously (Kimura et al. 2004, 2005). N-benzyloxycarbonyl-valine-leucine-leucinal (Z-VLL-CHO) was purchased from Calbiochem (San Diego, CA, USA) (Abbenante et al. 2000). All inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted with sodium phosphate-buffered saline (PBS) before use. The vehicle solution contained DMSO at the same concentration as that in inhibitor solutions.

Cell culture

Human embryonic kidney (HEK)293 cells that stably express human BACE1 (BACE1-HEK293 cells) were maintained in Dulbecco's Modified Eagle's medium (Sigma, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1 mg/mL geneticin/G418 (Sigma). The cells were grown on 60-mm tissue culture dishes in a 5% CO2 incubator at 37°C, until they became 80–100% confluent. The cells were then washed twice with PBS, and FBS-free medium with or without KMI-429 was added, followed by incubation for 6 h. To quantify the amount of wild-type sAPPβ in the conditioned medium, the medium was collected, concentrated with trichloroacetic acid, and subjected to western blot analysis using anti-sAPPβ(wt) polyclonal antibody, which recognizes the C-terminus of sAPPβ (Shuto et al. 2003; Kimura et al. 2004). Soluble extracellular fragment of wild-type APP generated by α-secretase (sAPPα) in the conditioned medium, full-length APP (FL-APP), BACE1 and β-actin in the cell lysate were quantified by western blot analysis using 6E10 (Signet Laboratory, Dedham, MA, USA), anti-APP (Sigma), anti-BACE1 (MoBiTec, Göttingen, Germany) and anti-β-actin antibodies (Sigma) respectively. The amount of Aβx−40in the conditioned medium was determined by sandwich ELISA using the monoclonal antibodies BNT77 and BA27 (Iwata et al. 2004).

Animal experiments

All animal experiments were performed in compliance with the RIKEN institutional guidelines. Three to five-month-old male Tg2576 (Hsiao et al. 1996) and male C57BL/6J mice (Japan SLC, Shizuoka, Japan) were used for the experiments. Mice were anesthetized with sodium pentobarbital and placed in a stereotaxic apparatus before bilateral injection of KMI inhibitor or Z-VLL-CHO, at doses indicated in figure legends. Inhibitors were injected in 1 µL PBS into the hippocampus (stereotaxic coordinates: anteroposterior, 2.6 mm; mediolateral, 3.1 mm; dorsoventral, 2.4 mm) using a 26S-gauge needle equipped with a 0.5-mL motorized Hamilton syringe (KD Scientific, Boston, MA, USA). Sixty seconds after insertion of the needle, an inhibitor solution was injected at a constant flow rate of 0.1 µL/min. The injection needle was kept in place for an additional 5 min to prevent reflux of fluid.

Extraction of Aβ and ELISA

Extraction of Aβ from the hippocampus and fractionation were carried out as described previously (Iwata et al. 2004). The amounts of Aβx−40 and Aβx−42 in the Tris-buffered saline (TBS)-extractable fraction (soluble fraction) and guanidinium chloride (GuHCl)-extractable fraction (insoluble fraction) were determined by sandwich ELISA using the monoclonal antibodies BNT77/BA27 and BNT77/BC05 respectively (Iwata et al. 2004). In the case of APP transgenic mice, Aβ levels were expressed as a ratio of FL-APP to correct for individual differences in expression levels of APP. Statistical analysis was done by student's t-test.

Quantitative western blot analysis

The hippocampal homogenate was separated into a precipitate and supernatant by centrifuging at 200 000 g for 20 min at 4°C. The levels of FL-APP and APP-CTF in the precipitate, and sAPPβ in the supernatant were quantified by western blot analysis using anti-APP (corresponding to the C-terminal of human APP695, amino acid 676-695, Sigma) and anti-sAPPβNL polyclonal antibody, which was produced in rabbits by injecting synthetic peptides, is specific to the soluble N-terminal fragment of APP with Swedish mutation respectively. 6E10 (Signet Laboratory) recognizes human-type APP and was used to detect FL-APP. Immunoreactive bands on the membrane were visualized using an ECLplus kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and band intensity was determined with a densitometer (LAS-3000; Fuji Photo Film, Tokyo, Japan), using Science Laboratory 2001 Image Gauge software (Fuji Photo Film). The amount of immunoreactive FL-APP, APP fragments and BACE1 in each sample were calculated in the linear range, based on a standard curve constructed from one of the samples.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

KMI-429 dose-dependently inhibited β-secretase activity in BACE1-HEK293 cells

We first evaluated the inhibitory effect of KMI-429 on cellular β-secretase activity (Fig. 1a). KMI-429 dose-dependently inhibited secretion of sAPPβ in the conditioned medium from BACE1-HEK293 cells. Its IC50 for cellular BACE1 activity was 42.8 nm (Fig. 1b). The IC50 value of KMI-429 was similar to that obtained in a test-tube experiment using recombinant BACE1 and an artificial substrate (3.9 nm) (Kimura et al. 2005). KMI-429 also inhibited the secretion of soluble Aβ into the conditioned medium (Fig. 2). The cell-permeable inhibitor Z-VLL-CHO strongly inhibited Aβ production in a culture system.

image

Figure 1. Dose-dependent inhibition of β-secretase activity in cultured cells by KMI-429. (a) Chemical structures of the KMI inhibitors. (b) Inhibition of β-secretase activity in a cell culture system. Amounts of sAPPβ released into the conditioned medium from BACE1-HEK293 cells treated with or without inhibitors were measured by quantitative western blot analysis. Values are mean ± SD of three independent experiments. *p < 0.05, **p < 0.005, ***p < 0.0005 versus vehicle-treated group. Sample western blots are shown for sAPPβ, sAPPα, FL-APP, BACE1 and β-actin.

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image

Figure 2. Inhibition of Aβ production in cultured cells by β-secretase inhibitors. Amount of Aβ released into the conditioned medium from BACE1-HEK293 cells was measured by sandwich ELISA. Values are mean ± SD of 3–5 independent experiments (vehicle, n = 4; 10−8− 10−5 m KMI-429, n = 3; 10−4 m KMI-429, n = 5; 10−4 m KMI-358, n = 4; 10−5 m Z-VLL-CHO, n = 4). *p < 0.005 versus vehicle-treated group.

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KMI-429 inhibited sAPPβ secretion in the hippocampus of APP transgenic (Tg2576) mouse

We next examined the in vivo inhibitory effects on β-secretase activity using APP transgenic (Tg2576) mice. KMI-358, KMI-429 or vehicle was injected into the hippocampus, where BACE1 is strongly expressed (Irizarry et al. 2003), and levels of sAPPβ and FL-APP were measured 1.5 or 3 h later by quantitative western blotting (Fig. 3a). After a 3-h incubation, mice that had received 2.5 nmol KMI-429 showed a significant decrease of 20% in sAPPβ level in the hippocampus compared with mice injected with vehicle alone; the injection had no significant effect on β-secretase activity after incubation for 1.5 h. KMI-429 consistently decreased the hippocampal APP-CTFβ level at 3 h after incubation (Fig. 3b). However, mice injected with 2.5 nmol KMI-358 showed no significant decrease in β-secretase activity after incubation for 3 h. Injection of either 2.5 nmol KMI-358 or KMI-429 had no effect on BACE1 protein expression (data not shown).

image

Figure 3. Inhibition of β-secretase activity in APP transgenic (Tg2576) mice by intrahippocampal injection of KMI-429 and KMI-358. KMI-429 (2.5 nmol), KMI-358 (2.5 nmol) or vehicle was injected into the hippocampus of Tg2576 mice; 1.5 and 3 h later, sAPPβ levels and APP-CTFβ were quantified by quantitative western blot analysis. Levels of APP fragments were expressed as sAPPβ/FL-APP (a) and APP-CTFβ/FL-APP (b) respectively to correct for individual differences in the expression levels of APP. Values are mean ± SEM for four mice. *p < 0.005 versus vehicle-injected group.

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KMI-429 inhibited Aβ production in the hippocampus of APP transgenic (Tg2576) and wild-type mice

We next investigated changes in Aβ levels in the hippocampus of Tg2576 mice 3 h after injection of inhibitors (Fig. 4). Z-VLL-CHO, a potent cell-permeable and reversible inhibitor of β-secretase (Abbenante et al. 2000), was used as a control. KMI-358 or KMI-429 at 10 mm in DMSO was injected into the hippocampus. Because it is insoluble in the same amount of DMSO, Z-VLL-CHO was diluted four-fold to a final concentration of 2.5 mm. KMI-429 treatment effectively reduced levels of Aβ40 (60.7 ± 9.2%, p < 0.002) and Aβ42 (65.4 ± 10.3%, p < 0.001) in the soluble fraction (TBS-extractable fraction) and led to a slight reduction in levels of both Aβ species in the insoluble fraction (GuHCl-extractable fraction). Significant changes in Aβ levels in either fraction were not observed in the KMI-358 or Z-VLL-CHO groups.

image

Figure 4. Reduction of hippocampal Aβ levels in Tg2576 mice by KMI inhibitor treatment. Levels of Aβ40 (a) and Aβ42 (b) in the soluble fraction and Aβ40 (c) and Aβ42 (d) in the insoluble fraction of the hemilateral hippocampal formation were measured by sandwich ELISA 3 h after bilateral injection of KMI-358 (10 nmol), KMI-429 (10 nmol), Z-VLL-CHO (2.5 nmol) or vehicle into the hippocampal formation of Tg2576 mice. Aβ levels were divided by FL-APP values to correct for individual differences. Values are mean ± SEM from four mice. *p < 0.005 versus vehicle-injected group.

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Finally, we examined the inhibitory effects of KMI-429 on Aβ production in the hippocampus of wild-type mice (C57BL/6J) (Fig. 5). A high dose (10 nmol) of KMI-429 produced a significant decrease in Aβ levels, compared with those in the vehicle-treated group in both the soluble (Aβ40, 42.9%; Aβ42, 39.8% reduction) and insoluble (Aβ40, 34.6%; Aβ42, 31.0% reduction) fractions. A low dose (2.5 nmol) of KMI-429 also decreased Aβ levels in both the soluble (Aβ40, 38.1%; Aβ42, 31.5% reduction) and insoluble fractions (Aβ40, 32.0%; Aβ42, 38.0% reduction). Thus, the inhibitory effects of KMI-429 were more effective in wild-type mice than in the Tg2576 mice.

image

Figure 5. Reduction of hippocampal Aβ levels in wild-type mice by KMI-429. Aβ40 (a) and Aβ42 (b) levels in the soluble fraction and Aβ40 (c) and Aβ42 (d) levels in the insoluble fraction were measured by sandwich ELISA 3 h after the bilateral injection of 2.5 nmol or 10 nmol KMI-429, or vehicle into the hippocampal formation of C57BL/6J mice. Values are mean ± SEM from six mice. *p < 0.05, **p < 0.005, ***p < 0.0005 versus vehicle-injected group.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

β-Secretase is a key enzyme in Alzheimer's disease (AD) therapy (Citron 2004b). BACE1 is the major β-secretase responsible for the generation of Aβ in the brain (Cai et al. 2001; Luo et al. 2001). Because the inhibition of BACE1 protease activity leads to a reduction in the level of Aβ, the development of a BACE1 inhibitor is of interest to numerous research groups (Sinha et al. 1999; Abbenante et al. 2000; Hong et al. 2000, 2002; Hom et al. 2003, 2004; Tamamura et al. 2003; Coburn et al. 2004). For example, the Elan Pharmaceuticals team has reported the development of a BACE1 inhibitor, H-KTEEISEVN(Stat)VAEF-OH, that replaces the Swedish APP Leu (P1) and Asp (P1′) residues with uncleavable statine (Stat) and Val residues respectively (IC50 0.03 µm) (Sinha et al. 1999). Recently they designed smaller BACE1 inhibitors by either truncating its N- and C-termini (IC50 0.12 µm) or modifying its central core to contain hydroxyethylene (IC50 0.03 µm) (Hom et al. 2003, 2004). The development of the hydroxyethylene transition-state isostere inhibitor OM99-2 was based on the X-ray crystallographic structure of the complex with a recombinant BACE1 protease domain (IC50 0.002 µm) (Hong et al. 2000). This group further developed the hydroxyethylene BACE1 inhibitor GT-1017 (IC50 0.002 µm), using an X-ray structure-based modification of OM99-2 (Hong et al. 2002). Tamamura et al. (2003) designed a hydroxyethylamine dipeptide isostere inhibitor, TK-19 (IC50 0.047 µm). In addition, the non-peptide inhibitor compound 1, which was identified as aminopentyl oxyacetamide, reproducibly inhibited β-secretase in five different enzymatic assays with an IC50 of 25 µm (Coburn et al. 2004).

However, only some of these compounds resulted in valid inhibitory effects in cell culture systems and evidence that verified inhibitory effects in vivo was much less direct. Recently, in vivo experiments were reported by Chang et al. (2004). Although intraperitoneal injection of carrier peptide-conjugated BACE1 inhibitor in APP transgenic (Tg2576) mice resulted in a decrease in Aβ levels in both plasma and brain, levels of brain Aβ42, the primary pathogenic amyloid form, were not measured. In the present study, we examined the in vivo inhibitory effects of a novel HMC isostere BACE1 inhibitor, KMI-429, (Kimura et al. 2005) that has high APP β-cleavage inhibitory activity. Finally, we successfully demonstrated that KMI-429 inhibited β-secretase activity, and reduced both Aβ40 and Aβ42 levels in the hippocampi of APP transgenic (Tg2576) and wild-type mice.

KMI-429, as well as KMI-358, inhibited β-secretase activity in a dose-dependent manner in BACE1-expressing HEK293 cells in culture (Kimura et al. 2004). The IC50 value for KMI-429 was nearly the same as that in the in vitro experiment, whereas the IC50 value for KMI-358 in culture was higher than that in the in vitro experiment (650 vs. 16 nm). These data suggest that membrane permeability of KMI-429 is higher than that of KMI-358. For KMI-429, an oxalyl moiety was replaced with tetrazole carbonyl derivatives. This substitution in the structure of KMI-429 may have affected membrane permeability and, furthermore, the in vivo inhibitory effect on Aβ production and sAPPβ secretion. Z-VLL-CHO, a potent cell-permeable and reversible β-secretase inhibitor, inhibited Aβtotal (IC50 0.7 µm) and Aβ42 (IC50 2.5 µm) in Chinese hamster ovary cells stably transfected with wild-type APP751 (Abbenante et al. 2000), and in our BACE1-HEK293 cells (Fig. 2), but did not reduce the level of Aβ in our in vivo experimental paradigm, possibly owing to low membrane permeability, similar to KMI-358.

The fact that a greater effect on the reduction of both sAPPβ and Aβ levels was noted at 3 h than at 1.5 h after KMI-429 injection into the hippocampus suggests that KMI-429 is barely metabolized in the body. On the other hand, BACE1 protein levels in the hippocampus did not change significantly after KMI treatment, indicating that KMI-429 directly affects BACE1 protease activity without altering its level of expression in vivo.

The two doses of KMI-429 decreased hippocampal Aβ levels in the wild-type mice equipotently (30–40% reduction), indicating that the maximal inhibitory effect was obtained with the lowest dose tested. Intrahippocampal amounts of Aβ, APP and APP fragments were estimated as the sum of the amounts released from neuronal presynapses that projected from the extrahippoampus, such as the entorhinal cortex, to the hippocampus and from the intrahippocampal neuronal circuits. In our experimental paradigm, a 30–40% reduction in the Aβ level was regarded as the maximal inhibitory effect of KMI-429 on Aβ production in the intrahippocampal neuronal circuits.

KMI-429 treatment reduced levels of both soluble and insoluble Aβ in the hippocampi of wild-type mice. However, in APP transgenic mice its effects were more potent in the soluble than the insoluble fraction. These results may reflect a difference in the half-life of Aβ between the wild-type and APP transgenic mice. Cirrito et al. (2003) observed that the half-life of Aβ in brain interstitial fluid of young APP transgenic mice (PDAPP mice) was approximately 2 h. On the other hand, Iwata et al. (2000) previously reported that the half-life of multiple radiolabeled Aβ1−42, injected into rat hippocampi, was 17.5 min. These data suggest that the rate of Aβ clearance cannot reach a steady-state level in APP transgenic mice, which have substantially higher levels of Aβ than wild-type mice, although the experimental paradigms of these two studies are different. Aβ levels are likely to be higher in the APP transgenic mice. Aβ may translocate from the soluble to the insoluble fraction immediately after secretion. Consequently, Aβ levels in the soluble fraction of transgenic mice are more likely to be effectively influenced by KMI-429 treatment than Aβ levels in the insoluble fraction. Recent studies suggest that soluble forms of Aβ, which are composed of monomeric, oligomeric and protofibrillar forms, induce synaptic dysfunction and progressive neurotoxicity (Selkoe 2002). Therefore, a reduction in soluble Aβ levels by KMI-429 is desirable to decelerate Aβ deposition and prevent neurotoxicity, even after the initial formation of amyloid plaques.

β-Secretase inhibitors must penetrate the blood–brain barrier (BBB) to reach the brain parenchyma; such penetration is particularly crucial for inhibitors used to treat AD. Compounds that freely pass through the BBB are generally smaller than 500 Da (Hong et al. 2002). However, it has been shown that the human immunodeficiency virus protease inhibitor Indinavir (molecular mass 613 Da) traverses the BBB and successfully elicits its inhibitory effects (Martin et al. 1999). We designed KMI-429 with the aim of retaining its molecular mass in this range. As the molecular mass of KMI-429 (753 Da) is much smaller than that of other inhibitors (e.g. 917 Da) (Turner et al. 2001; Chang et al. 2004; Citron 2004b), KMI-429 may potentially permeate the BBB.

Our KMI-429 BACE1 inhibitor might be an effective therapeutic reagent for AD as it is the first inhibitor shown to reduce the level of soluble and insoluble Aβ40, and soluble and insoluble Aβ42, in both APP transgenic and wild-type mice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported in part by a Grant-in-Aid for the Scientific Research on Priority Areas, the Advanced Brain Science Project, the Frontier Research Program and the 21st century Center of Excellence program from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by research grants from the Ministry of Health, Labor, and Welfare, Japan. We thank Yukio Matsuba, Kaori Watanabe and Misaki Sekiguchi for their technical assistance, and Tomoya Kotake and Takashi Hamano for their valuable advice. We also thank Takeda Chemical Industries, Ltd. for kindly providing us with anti-Aβ monoclonal antibodies for sandwich ELISA and Dr Karen Hsiao-Ashe (Department of Neurology, University of Minnesota) for kindly providing Tg 2576 mice.

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  3. Materials and methods
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  5. Discussion
  6. Acknowledgements
  7. References
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