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Oxidative stress accelerates amyloid deposition and memory impairment in a double-transgenic mouse model of Alzheimer's disease
Neuroscience Letters 587 (2015) 126–131
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Research article
Oxidative stress accelerates amyloid deposition and memory impairment in a double-transgenic mouse model of Alzheimer’s disease Takuya Kanamaru a , Naomi Kamimura a,∗ , Takashi Yokota a , Katsuya Iuchi a , Kiyomi Nishimaki a , Shinya Takami c , Hiroki Akashiba c , Yoshitsugu Shitaka c , Ken-ichiro Katsura b , Kazumi Kimura b , Shigeo Ohta a a
Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki-city, Kanagawa 211-8533, Japan b Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan c Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
h i g h l i g h t s • We have established a novel mouse model of Alzheimer’s disease (the APP/DAL mouse). • Pathological changes and memory impairment are accelerated in the APP/DAL mouse. • The APP/DAL mouse is useful for examining the role of oxidative stress in the progression and pathogenesis of Alzheimer’s disease.
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Article history: Received 28 September 2014 Received in revised form 11 November 2014 Accepted 16 December 2014 Available online 18 December 2014 Keywords: Alzheimer’s disease Amyloid  APP transgenic mice DAL mice Oxidative stress
a b s t r a c t Oxidative stress is known to play a prominent role in the onset and early stage progression of Alzheimer’s disease (AD). For example, protein oxidation and lipid peroxidation levels are increased in patients with mild cognitive impairment. Here, we created a double-transgenic mouse model of AD to explore the pathological and behavioral effects of oxidative stress. Double transgenic (APP/DAL) mice were constructed by crossing Tg2576 (APP) mice, which express a mutant form of human amyloid precursor protein (APP), with DAL mice expressing a dominant-negative mutant of mitochondrial aldehyde dehydrogenase 2 (ALDH2), in which oxidative stress is enhanced. Y-maze and object recognition tests were performed at 3 and 6 months of age to evaluate learning and memory. The accumulation of amyloid plaques, deposition of phosphorylated-tau protein, and number of astrocytes in the brain were assessed histopathologically at 3, 6, 9, and 12–15 months of age. The life span of APP/DAL mice was significantly shorter than that of APP or DAL mice. In addition, they showed accelerated amyloid deposition, tau phosphorylation, and gliosis. Furthermore, these mice showed impaired performance on Y-maze and object recognition tests at 3 months of age. These data suggest that oxidative stress accelerates cognitive dysfunction and pathological insults in the brain. APP/DAL mice could be a useful model for exploring new approaches to AD treatment. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases involving cognitive impairment. The discovery of AD-responsible mutations in the human amyloid precursor pro-
∗ Corresponding author. Tel.: +81 44 733 1859; fax: +81 44 733 9268. E-mail address: [email protected] (N. Kamimura). http://dx.doi.org/10.1016/j.neulet.2014.12.033 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
tein (APP) has enabled construction of transgenic animal models. PDAPP [1], Tg2576 [2], and APP23 [3] are mouse models of AD harboring these mutations and showing several features of AD. However, such models rarely entirely replicate human pathological features, such as neurofibrillary tangles (NFTs) or neuronal degeneration [1–3]. Aging is the most important risk factor for AD. Growing evidence suggests that age-dependent oxidative stress is a characteristic feature of AD brains. Several studies have reported elevated oxi-
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dation of DNA, RNA, proteins, and lipids in AD brains [4,5]. The plasma levels of antioxidants, such as vitamin C and E, are lower in patients with AD than in the healthy aged population, and the activity of antioxidant enzymes, such as catalase and glutathione peroxidase, is also impaired in AD brains [6,7]. Furthermore, subjects with mild cognitive impairment exhibit elevated oxidative stress, lower antioxidant levels, and impaired antioxidant enzyme activity [8,9]. Thus, oxidative stress plays important roles in the onset and progression of AD pathogenesis. We have constructed transgenic mice (DAL mice) expressing a dominant-negative mutant form of mitochondrial aldehyde dehydrogenase 2 (ALDH2), which detoxifies 4-hydroxy-2-nonenal (HNE), an end product of lipid peroxidation [10]. Lipid peroxidation is a major source of oxidative stress-mediated injury that directly damages neuronal membranes and contributes to oxidative damage in the pathogenesis of neurodegenerative disorders [4,5]. DAL mice exhibited a decreased ability to detoxify HNE in cortical neurons and an accelerated accumulation of HNE in the brain [10]. ALDH2 protein was localized in the mitochondria and we found increased levels of HNE adduct proteins in the mitochondrial fraction of DAL mice [11]. DAL mice show age-dependent neurodegeneration and cognitive decline and have a shortened lifespan. Additionally, ALDH2-deficient cells have a greater level of oxidative stress, as shown by oxidation–reduction sensitive green fluorescent protein (GFP) [11]. In order to investigate the role of oxidative stress in AD, we produced double-transgenic mice (APP/DAL mice) by crossbreeding DAL mice with APP mice. We investigated the pathological changes in this novel transgenic mouse model and assessed their performance on learning and memory tasks. 2. Materials and methods 2.1. Mice Tg2576 (APP) mice [2], expressing human APP containing the Swedish mutation (Lys670 Asn and Met671 Leu), were licensed from the Mayo Foundation for Medical Education and Research (Rochester, MN, USA). DAL101 (DAL) mice, expressing the dominant-negative form of mitochondrial aldehyde dehydrogenase 2 (ALDH2) [10], were purchased from Mitos Co., Ltd. (Kawasaki, Japan). We crossbred the APP with DAL mice to bear double transgenic (APP/DAL), single transgenic (APP and DAL), and wild-type mice. These mice were maintained by brother-sister mating to produce progeny with same genetic background. All experimental protocols were approved by both the Animal Care Committee of Nippon Medical School and Institutional Animal Care and Use Committee of Astellas Pharma Inc. Astellas Pharma Inc., Tsukuba Research Center was awarded Accreditation Status by the AAALAC International. 2.2. Immunohistochemistry Mouse brains were collected after anesthesia and perfusion. Brains were fixed with 4% paraformaldehyde and embedded in paraffin. For A staining, the sections were treated with 99% formic acid for 5 min at room temperature and incubated in Immunosaver (Nisshin EM Corp., Tokyo, Japan) according to the manufacturer’s instructions. We used anti-A40 (0.5 g/mL, AnaSpec Inc., Fremont, CA, USA) and anti-A42 (0.4 g/mL, AnaSpec Inc., Fremont, CA, USA) primary antibodies and visualized the staining with an ABC kit (Vectastain; Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). For paired helical filament (PHF)tau staining, sections were incubated in anti-PHF-tau (1 g/mL, Thermo Fisher Scientific Inc., Waltham, MA, USA) and counterstained with Congo red (Cosmo Bio Co., Ltd., Tokyo, Japan).
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Anti-glial fibrillary acidic protein (GFAP) antibody (0.1 g/mL, Dako, Glostrup, Denmark) was used to stain astrocytes. Semiquantification of A deposits was carried out by counting the number of plaques per coronal section per mouse (n = 5). Semiquantification of phosphorylated tau protein was measured as the mean area of PHF-tau protein on the periphery of each amyloid plaque per five visual fields (1.0 mm2 ; × 20) per section per mouse (n = 5; ImageJ, National Institutes of Health, Bethesda, MD, USA), while that of astrocytes was measured by counting the number of GFAP-positive cells in the CA1 region of the hippocampus and in the cerebral cortex adjacent to the CA1 per visual field (×20). 2.3. Y-maze spontaneous alternation test The Y-maze spontaneous alternation test, as previously described, examined recognition memory [12]. Briefly, mice were placed into the start arm and allowed to habituate to the maze environment for 10 min. The next day, the mice were placed at the end of the start arm and allowed to move freely through the maze for 8 min. The percentage of spontaneous alternations was calculated as the ratio of the number of alternations to the total number of arm entries. 2.4. Object recognition test The object recognition test was used as a second measure of recognition memory [13]. Mice were habituated in a test chamber (25 cm wide × 25 cm long × 40 cm high) for 24 h before training. During the training session, two identical objects (round filter units, diameter 33 mm, height 27 mm) were placed in the chamber and mice were allowed to explore for 10 min. The following day, one of the familiar objects was replaced with a novel object (plastic cone, diameter 25 mm, height 30 mm). Object recognition index was defined as the percentage of time spent sniffing or touching the novel object with the nose during a 5 min session. All training and testing trials were video recorded and analyzed using EthoVision XT8.5 (Noldus Information Technology, Wageningen, Netherlands). 2.5. Statistical analysis Data were analyzed using an unpaired two-tailed student’s ttest or analysis of variance (ANOVA) followed by Tukey’s test. The Kaplan–Meier method was used to calculate survival curves, and survival periods were compared with the log-rank test. A P < 0.05 was considered statistically significant. JMP9 software (SAS Institute Inc., Cary, NC, USA) was used for all statistical analysis. 3. Results 3.1. Life span and body weight of APP/DAL mice Kaplan–Meier survival curves show that mice of all genotypes began dying by 120 days of age, but after 240 days, the rate of death among APP/DAL mice was more rapid than that of the other genotypes (Supplementary Fig. 1A). APP/DAL mice had a significantly shortened life span compared with other mice (log-rank test, P < 0.0001). Body weights of all mice were recorded monthly (Supplementary Fig. 1B). APP/DAL mice weighed significantly lesser than WT or DAL mice (two-way repeated measures ANOVA, P < 0.0001). There were no significant differences in body weight between APP and APP/DAL mice (P = 0.12). 3.2. Onset of impairment in spatial learning and memory in APP/DAL mice We used Y-maze and object recognition tests to examine whether oxidative stress leads to an acceleration of spatial learning
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Fig. 1. Performance of mice on the Y-maze and object recognition test. (A and B) Alternation rate of 3- or 6-month-old mice in the spontaneous alternation Y-maze test (n = 8–10). (C and D) Exploratory preference of 3- or 6-month-old mice in the object recognition test (n = 8–10). Data are expressed as mean ± S.D. Unpaired t-test, *P < 0.05, **P < 0.01.
and memory impairments in APP mice with aging. The mice were examined at 3 and 6 months of age in both behavioral tests. At 3 months, the alternation rate of Y-maze in APP/DAL mice was significantly lower than the other three genotypes (53.6% compared with approximately 65%; P < 0.01; Fig. 1A). At 6 months, alternation rates in APP/DAL mice were also significantly lower than WT (P < 0.01), whereas APP mice showed a trend (not significant) towards lower alternation rates than WT and DAL mice (Fig. 1B). Similarly, in the object recognition test, exploratory preference for the novel object was significantly lower in APP/DAL mice than in the other three groups at 3 months of age (60.0% compared with approximately 71%; P < 0.05, Fig. 1C) and at 6 months (Fig. 1D). APP and DAL mice tended to show lower exploratory preference than WT mice at 6 months, with no significant differences among the groups. 3.3. Accumulation of amyloid ˇ deposition in the brains of APP/DAL mice To investigate whether oxidative stress accelerates accumulative amyloid  deposition in the brain, we counted amyloid plaques in APP and APP/DAL mouse brain sections immunostained for A40 or A42 (Fig. 2). No plaques were detected at 3 months of age in either genotype (data not shown). At 6 months, APP mouse brains remained free of plaques, whereas plaques were detected in the brain sections of APP/DAL mice. At 12–15 months, significantly more A40 (67.0 ± 8.9 plaques per section; P = 0.035) and A42 (83.2 ± 9.4; P = 0.012) plaques were detected in APP/DAL mice than in APP mice (41.4 ± 2.5 and 45.4 ± 5.0, respectively)(Fig. 2B–C). 3.4. Deposition of phosphorylated tau proteins in the brains of APP/DAL mice To investigate whether oxidative stress increases NFTs, composed of hyperphosphorylated tau proteins and formed by arrays of PHFs, we stained brain sections with a monoclonal antibody against PHF-tau and Congo Red (Fig. 3). Sections from APP/DAL mice showed PHF-tau-positive staining surrounding most Congo Redpositive deposits (Fig. 3A). The area stained with PHF-tau antibody
was significantly larger in APP/DAL mice at 9 months compared to APP mice (unpaired t-test, P = 0.0096; Fig. 3B). At 12–15 months, APP/DAL mice showed a trend towards larger PHF-tau areas in the brain, but the difference was not significant (P = 0.18). No PHF-tau was detected at 3 or 6 months of age in either genotype (data not shown).
3.5. Gliosis in the brains of APP/DAL mice Neuronal degeneration leads to an increase in astrocyte number, and gliosis is a pathological marker of many neurodegenerative diseases, including AD. We counted astrocytes in brain sections immunostained for anti-GFAP, a marker of astrocyte activation (Fig. 4). Sections from APP/DAL mice showed a greater number of astrocyte clusters than APP mice in the CA1 region of the hippocampus and cerebral cortex at 12 months of age (Fig. 4A). An age-dependent increase was observed in GFAP-positive cells in both the hippocampus and cerebral cortex of APP/DAL mice (nonparametric test; Spearman’s rank correlation coefficient = 0.74, 0.72, P = 0.0002, 0.0004, respectively; Fig. 4B), whereas no change was observed in the brains of APP mice. At 6, 9, and 12–15 months of age there were significantly more astrocytes in the brains of APP/DAL mice than in that of APP mice (P < 0.05).
4. Discussion In the present study, we have shown that oxidative stress accelerates AD-like pathology, such as memory impairment and amyloid plaques, in APP transgenic mice. APP/DAL mice developed early spatial learning and memory impairments in both the Y-maze and object recognition test. The onset of memory impairment in APP/DAL mice was considerably earlier than that in APP mice. The onset of impairment in other double transgenic AD mice models is later than 3 months of age (e.g., 15–16 months in PS1Tg2576 [14], 8 months in PS2APP [15], 4–5 months in PS2Tg2576 [16], and 4 months in PS1/APP [17]). These results suggest APP/DAL mice could be a useful model of AD for behavioral tests.
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Fig. 2. Accumulation of amyloid  deposits in the brains of APP/DAL mice. (A) Coronal brain sections of APP and APP/DAL mice. Amyloid plaques were immunostained with a monoclonal antibody against A42. Scale bar: 500 m. (B) Semiquantification of A40 and A42 plaques (n = 5 per age). Data are mean ± S.E.M. Unpaired t-test, *P < 0.05.
According to a previous report, amyloid plaques begin being deposited in the brains of APP mice (Tg2576) at 7–8 months of age [2,18]. However, we show that APP/DAL double transgenic mice exhibit brain A deposition by 6 months of age. In addition, we
observed accelerated hyperphosphorylated tau protein accumulation and age-dependent increases in astrocytes. Increased levels of the glutathione-HNE adducts and accumulation of HNE-adducted proteins, such as proteasome and neprilysin,
Fig. 3. PHF-tau protein deposits on the periphery of an amyloid plaque in APP/DAL mouse brain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Coronal cortical sections of brains from APP and APP/DAL mice (cortex) at 9 months of age. Tau proteins were immunostained with a monoclonal antibody against PHF-tau protein (blue). Amyloid plaques were stained with Congo Red (red). Scale bar: 100 m. Insets show plaques at higher magnification. (B) Semi-quantitative analysis of PHF-tau protein in the brain. Data are expressed as mean ± S.E.M. (n = 5).
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Fig. 4. Astrocyte proliferation in the APP/DAL mouse brain. (A) Coronal hippocampal (CA1) and cortical sections of APP and APP/DAL mice at 12 months of age, immunostained for GFAP. Scale bar: 100 m. (B) Semi-quantitative analysis of GFAP-positive cell in the brain. Data are mean ± S.E.M. (n = 5). Unpaired t-test, *P < 0.05, **P < 0.01.
a major protease for A, have been observed in brains of patients with AD [19–21]. Toxicity associated with A in turn leads to the formation of HNE, resulting in a vicious cycle of HNE and A accumulation. Proteomics studies report a large number of HNE-adducted proteins in AD brains, including ATP synthase, ␣enolase, aconitase, aldolase, and manganese superoxide dismutase [22]. Moreover, brain tissue from subjects with mild cognitive impairment showed greater HNE and protein-bound HNE levels, suggesting that HNE could contribute to the early stages of AD [8,23]. We previously demonstrated the effect of HNE on AD pathogenesis and progression in ALDH2 deficient mice [10]. Importantly, ALDH2 deficiency enhances oxidative stress, as judged by oxidation–reduction sensitive GFP [11]. The ALDH2*2 polymorphism of the ALDH2 gene, responsible for a deficiency of ALDH2 activity, is specific to North–East Asians and associated with ethanol sensitivity [24]. We previously reported that ALDH2 deficiency is a risk factor for late-onset AD in the Japanese population [25], which was replicated in a Chinese study [26]. Several studies have reported the presence of A in the mitochondrial membrane of neurons in postmortem brain specimens from patients with AD and in brains of AD animal models [27–29]. Hence, A may initiate mitochondrial membrane lipid peroxidation, which is then detoxified by ALDH2.
5. Conclusion We have established a novel mouse model of AD having accelerated AD-like pathological changes and memory impairment. Early onset of the behavioral phenotype in APP/DAL mice would be useful for research. Furthermore, the APP/DAL mouse is a valuable model to examine the specific role of HNE in AD progression and pathogenesis. It also provides a tool for exploring new approaches, such as lipid accessible antioxidant molecules used to treat mild cognitive impairment and AD.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Oxidative stress accelerates amyloid deposition and memory impairment in a double-transgenic mouse model of Alzheimer’s disease Takuya Kanamaru a , Naomi Kamimura a,∗ , Takashi Yokota a , Katsuya Iuchi a , Kiyomi Nishimaki a , Shinya Takami c , Hiroki Akashiba c , Yoshitsugu Shitaka c , Ken-ichiro Katsura b , Kazumi Kimura b , Shigeo Ohta a a
Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki-city, Kanagawa 211-8533, Japan b Department of Neurological Science, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan c Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
h i g h l i g h t s • We have established a novel mouse model of Alzheimer’s disease (the APP/DAL mouse). • Pathological changes and memory impairment are accelerated in the APP/DAL mouse. • The APP/DAL mouse is useful for examining the role of oxidative stress in the progression and pathogenesis of Alzheimer’s disease.
a r t i c l e
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Article history: Received 28 September 2014 Received in revised form 11 November 2014 Accepted 16 December 2014 Available online 18 December 2014 Keywords: Alzheimer’s disease Amyloid  APP transgenic mice DAL mice Oxidative stress
a b s t r a c t Oxidative stress is known to play a prominent role in the onset and early stage progression of Alzheimer’s disease (AD). For example, protein oxidation and lipid peroxidation levels are increased in patients with mild cognitive impairment. Here, we created a double-transgenic mouse model of AD to explore the pathological and behavioral effects of oxidative stress. Double transgenic (APP/DAL) mice were constructed by crossing Tg2576 (APP) mice, which express a mutant form of human amyloid precursor protein (APP), with DAL mice expressing a dominant-negative mutant of mitochondrial aldehyde dehydrogenase 2 (ALDH2), in which oxidative stress is enhanced. Y-maze and object recognition tests were performed at 3 and 6 months of age to evaluate learning and memory. The accumulation of amyloid plaques, deposition of phosphorylated-tau protein, and number of astrocytes in the brain were assessed histopathologically at 3, 6, 9, and 12–15 months of age. The life span of APP/DAL mice was significantly shorter than that of APP or DAL mice. In addition, they showed accelerated amyloid deposition, tau phosphorylation, and gliosis. Furthermore, these mice showed impaired performance on Y-maze and object recognition tests at 3 months of age. These data suggest that oxidative stress accelerates cognitive dysfunction and pathological insults in the brain. APP/DAL mice could be a useful model for exploring new approaches to AD treatment. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases involving cognitive impairment. The discovery of AD-responsible mutations in the human amyloid precursor pro-
∗ Corresponding author. Tel.: +81 44 733 1859; fax: +81 44 733 9268. E-mail address: [email protected] (N. Kamimura). http://dx.doi.org/10.1016/j.neulet.2014.12.033 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
tein (APP) has enabled construction of transgenic animal models. PDAPP [1], Tg2576 [2], and APP23 [3] are mouse models of AD harboring these mutations and showing several features of AD. However, such models rarely entirely replicate human pathological features, such as neurofibrillary tangles (NFTs) or neuronal degeneration [1–3]. Aging is the most important risk factor for AD. Growing evidence suggests that age-dependent oxidative stress is a characteristic feature of AD brains. Several studies have reported elevated oxi-
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dation of DNA, RNA, proteins, and lipids in AD brains [4,5]. The plasma levels of antioxidants, such as vitamin C and E, are lower in patients with AD than in the healthy aged population, and the activity of antioxidant enzymes, such as catalase and glutathione peroxidase, is also impaired in AD brains [6,7]. Furthermore, subjects with mild cognitive impairment exhibit elevated oxidative stress, lower antioxidant levels, and impaired antioxidant enzyme activity [8,9]. Thus, oxidative stress plays important roles in the onset and progression of AD pathogenesis. We have constructed transgenic mice (DAL mice) expressing a dominant-negative mutant form of mitochondrial aldehyde dehydrogenase 2 (ALDH2), which detoxifies 4-hydroxy-2-nonenal (HNE), an end product of lipid peroxidation [10]. Lipid peroxidation is a major source of oxidative stress-mediated injury that directly damages neuronal membranes and contributes to oxidative damage in the pathogenesis of neurodegenerative disorders [4,5]. DAL mice exhibited a decreased ability to detoxify HNE in cortical neurons and an accelerated accumulation of HNE in the brain [10]. ALDH2 protein was localized in the mitochondria and we found increased levels of HNE adduct proteins in the mitochondrial fraction of DAL mice [11]. DAL mice show age-dependent neurodegeneration and cognitive decline and have a shortened lifespan. Additionally, ALDH2-deficient cells have a greater level of oxidative stress, as shown by oxidation–reduction sensitive green fluorescent protein (GFP) [11]. In order to investigate the role of oxidative stress in AD, we produced double-transgenic mice (APP/DAL mice) by crossbreeding DAL mice with APP mice. We investigated the pathological changes in this novel transgenic mouse model and assessed their performance on learning and memory tasks. 2. Materials and methods 2.1. Mice Tg2576 (APP) mice [2], expressing human APP containing the Swedish mutation (Lys670 Asn and Met671 Leu), were licensed from the Mayo Foundation for Medical Education and Research (Rochester, MN, USA). DAL101 (DAL) mice, expressing the dominant-negative form of mitochondrial aldehyde dehydrogenase 2 (ALDH2) [10], were purchased from Mitos Co., Ltd. (Kawasaki, Japan). We crossbred the APP with DAL mice to bear double transgenic (APP/DAL), single transgenic (APP and DAL), and wild-type mice. These mice were maintained by brother-sister mating to produce progeny with same genetic background. All experimental protocols were approved by both the Animal Care Committee of Nippon Medical School and Institutional Animal Care and Use Committee of Astellas Pharma Inc. Astellas Pharma Inc., Tsukuba Research Center was awarded Accreditation Status by the AAALAC International. 2.2. Immunohistochemistry Mouse brains were collected after anesthesia and perfusion. Brains were fixed with 4% paraformaldehyde and embedded in paraffin. For A staining, the sections were treated with 99% formic acid for 5 min at room temperature and incubated in Immunosaver (Nisshin EM Corp., Tokyo, Japan) according to the manufacturer’s instructions. We used anti-A40 (0.5 g/mL, AnaSpec Inc., Fremont, CA, USA) and anti-A42 (0.4 g/mL, AnaSpec Inc., Fremont, CA, USA) primary antibodies and visualized the staining with an ABC kit (Vectastain; Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). For paired helical filament (PHF)tau staining, sections were incubated in anti-PHF-tau (1 g/mL, Thermo Fisher Scientific Inc., Waltham, MA, USA) and counterstained with Congo red (Cosmo Bio Co., Ltd., Tokyo, Japan).
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Anti-glial fibrillary acidic protein (GFAP) antibody (0.1 g/mL, Dako, Glostrup, Denmark) was used to stain astrocytes. Semiquantification of A deposits was carried out by counting the number of plaques per coronal section per mouse (n = 5). Semiquantification of phosphorylated tau protein was measured as the mean area of PHF-tau protein on the periphery of each amyloid plaque per five visual fields (1.0 mm2 ; × 20) per section per mouse (n = 5; ImageJ, National Institutes of Health, Bethesda, MD, USA), while that of astrocytes was measured by counting the number of GFAP-positive cells in the CA1 region of the hippocampus and in the cerebral cortex adjacent to the CA1 per visual field (×20). 2.3. Y-maze spontaneous alternation test The Y-maze spontaneous alternation test, as previously described, examined recognition memory [12]. Briefly, mice were placed into the start arm and allowed to habituate to the maze environment for 10 min. The next day, the mice were placed at the end of the start arm and allowed to move freely through the maze for 8 min. The percentage of spontaneous alternations was calculated as the ratio of the number of alternations to the total number of arm entries. 2.4. Object recognition test The object recognition test was used as a second measure of recognition memory [13]. Mice were habituated in a test chamber (25 cm wide × 25 cm long × 40 cm high) for 24 h before training. During the training session, two identical objects (round filter units, diameter 33 mm, height 27 mm) were placed in the chamber and mice were allowed to explore for 10 min. The following day, one of the familiar objects was replaced with a novel object (plastic cone, diameter 25 mm, height 30 mm). Object recognition index was defined as the percentage of time spent sniffing or touching the novel object with the nose during a 5 min session. All training and testing trials were video recorded and analyzed using EthoVision XT8.5 (Noldus Information Technology, Wageningen, Netherlands). 2.5. Statistical analysis Data were analyzed using an unpaired two-tailed student’s ttest or analysis of variance (ANOVA) followed by Tukey’s test. The Kaplan–Meier method was used to calculate survival curves, and survival periods were compared with the log-rank test. A P < 0.05 was considered statistically significant. JMP9 software (SAS Institute Inc., Cary, NC, USA) was used for all statistical analysis. 3. Results 3.1. Life span and body weight of APP/DAL mice Kaplan–Meier survival curves show that mice of all genotypes began dying by 120 days of age, but after 240 days, the rate of death among APP/DAL mice was more rapid than that of the other genotypes (Supplementary Fig. 1A). APP/DAL mice had a significantly shortened life span compared with other mice (log-rank test, P < 0.0001). Body weights of all mice were recorded monthly (Supplementary Fig. 1B). APP/DAL mice weighed significantly lesser than WT or DAL mice (two-way repeated measures ANOVA, P < 0.0001). There were no significant differences in body weight between APP and APP/DAL mice (P = 0.12). 3.2. Onset of impairment in spatial learning and memory in APP/DAL mice We used Y-maze and object recognition tests to examine whether oxidative stress leads to an acceleration of spatial learning
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Fig. 1. Performance of mice on the Y-maze and object recognition test. (A and B) Alternation rate of 3- or 6-month-old mice in the spontaneous alternation Y-maze test (n = 8–10). (C and D) Exploratory preference of 3- or 6-month-old mice in the object recognition test (n = 8–10). Data are expressed as mean ± S.D. Unpaired t-test, *P < 0.05, **P < 0.01.
and memory impairments in APP mice with aging. The mice were examined at 3 and 6 months of age in both behavioral tests. At 3 months, the alternation rate of Y-maze in APP/DAL mice was significantly lower than the other three genotypes (53.6% compared with approximately 65%; P < 0.01; Fig. 1A). At 6 months, alternation rates in APP/DAL mice were also significantly lower than WT (P < 0.01), whereas APP mice showed a trend (not significant) towards lower alternation rates than WT and DAL mice (Fig. 1B). Similarly, in the object recognition test, exploratory preference for the novel object was significantly lower in APP/DAL mice than in the other three groups at 3 months of age (60.0% compared with approximately 71%; P < 0.05, Fig. 1C) and at 6 months (Fig. 1D). APP and DAL mice tended to show lower exploratory preference than WT mice at 6 months, with no significant differences among the groups. 3.3. Accumulation of amyloid ˇ deposition in the brains of APP/DAL mice To investigate whether oxidative stress accelerates accumulative amyloid  deposition in the brain, we counted amyloid plaques in APP and APP/DAL mouse brain sections immunostained for A40 or A42 (Fig. 2). No plaques were detected at 3 months of age in either genotype (data not shown). At 6 months, APP mouse brains remained free of plaques, whereas plaques were detected in the brain sections of APP/DAL mice. At 12–15 months, significantly more A40 (67.0 ± 8.9 plaques per section; P = 0.035) and A42 (83.2 ± 9.4; P = 0.012) plaques were detected in APP/DAL mice than in APP mice (41.4 ± 2.5 and 45.4 ± 5.0, respectively)(Fig. 2B–C). 3.4. Deposition of phosphorylated tau proteins in the brains of APP/DAL mice To investigate whether oxidative stress increases NFTs, composed of hyperphosphorylated tau proteins and formed by arrays of PHFs, we stained brain sections with a monoclonal antibody against PHF-tau and Congo Red (Fig. 3). Sections from APP/DAL mice showed PHF-tau-positive staining surrounding most Congo Redpositive deposits (Fig. 3A). The area stained with PHF-tau antibody
was significantly larger in APP/DAL mice at 9 months compared to APP mice (unpaired t-test, P = 0.0096; Fig. 3B). At 12–15 months, APP/DAL mice showed a trend towards larger PHF-tau areas in the brain, but the difference was not significant (P = 0.18). No PHF-tau was detected at 3 or 6 months of age in either genotype (data not shown).
3.5. Gliosis in the brains of APP/DAL mice Neuronal degeneration leads to an increase in astrocyte number, and gliosis is a pathological marker of many neurodegenerative diseases, including AD. We counted astrocytes in brain sections immunostained for anti-GFAP, a marker of astrocyte activation (Fig. 4). Sections from APP/DAL mice showed a greater number of astrocyte clusters than APP mice in the CA1 region of the hippocampus and cerebral cortex at 12 months of age (Fig. 4A). An age-dependent increase was observed in GFAP-positive cells in both the hippocampus and cerebral cortex of APP/DAL mice (nonparametric test; Spearman’s rank correlation coefficient = 0.74, 0.72, P = 0.0002, 0.0004, respectively; Fig. 4B), whereas no change was observed in the brains of APP mice. At 6, 9, and 12–15 months of age there were significantly more astrocytes in the brains of APP/DAL mice than in that of APP mice (P < 0.05).
4. Discussion In the present study, we have shown that oxidative stress accelerates AD-like pathology, such as memory impairment and amyloid plaques, in APP transgenic mice. APP/DAL mice developed early spatial learning and memory impairments in both the Y-maze and object recognition test. The onset of memory impairment in APP/DAL mice was considerably earlier than that in APP mice. The onset of impairment in other double transgenic AD mice models is later than 3 months of age (e.g., 15–16 months in PS1Tg2576 [14], 8 months in PS2APP [15], 4–5 months in PS2Tg2576 [16], and 4 months in PS1/APP [17]). These results suggest APP/DAL mice could be a useful model of AD for behavioral tests.
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Fig. 2. Accumulation of amyloid  deposits in the brains of APP/DAL mice. (A) Coronal brain sections of APP and APP/DAL mice. Amyloid plaques were immunostained with a monoclonal antibody against A42. Scale bar: 500 m. (B) Semiquantification of A40 and A42 plaques (n = 5 per age). Data are mean ± S.E.M. Unpaired t-test, *P < 0.05.
According to a previous report, amyloid plaques begin being deposited in the brains of APP mice (Tg2576) at 7–8 months of age [2,18]. However, we show that APP/DAL double transgenic mice exhibit brain A deposition by 6 months of age. In addition, we
observed accelerated hyperphosphorylated tau protein accumulation and age-dependent increases in astrocytes. Increased levels of the glutathione-HNE adducts and accumulation of HNE-adducted proteins, such as proteasome and neprilysin,
Fig. 3. PHF-tau protein deposits on the periphery of an amyloid plaque in APP/DAL mouse brain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Coronal cortical sections of brains from APP and APP/DAL mice (cortex) at 9 months of age. Tau proteins were immunostained with a monoclonal antibody against PHF-tau protein (blue). Amyloid plaques were stained with Congo Red (red). Scale bar: 100 m. Insets show plaques at higher magnification. (B) Semi-quantitative analysis of PHF-tau protein in the brain. Data are expressed as mean ± S.E.M. (n = 5).
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Fig. 4. Astrocyte proliferation in the APP/DAL mouse brain. (A) Coronal hippocampal (CA1) and cortical sections of APP and APP/DAL mice at 12 months of age, immunostained for GFAP. Scale bar: 100 m. (B) Semi-quantitative analysis of GFAP-positive cell in the brain. Data are mean ± S.E.M. (n = 5). Unpaired t-test, *P < 0.05, **P < 0.01.
a major protease for A, have been observed in brains of patients with AD [19–21]. Toxicity associated with A in turn leads to the formation of HNE, resulting in a vicious cycle of HNE and A accumulation. Proteomics studies report a large number of HNE-adducted proteins in AD brains, including ATP synthase, ␣enolase, aconitase, aldolase, and manganese superoxide dismutase [22]. Moreover, brain tissue from subjects with mild cognitive impairment showed greater HNE and protein-bound HNE levels, suggesting that HNE could contribute to the early stages of AD [8,23]. We previously demonstrated the effect of HNE on AD pathogenesis and progression in ALDH2 deficient mice [10]. Importantly, ALDH2 deficiency enhances oxidative stress, as judged by oxidation–reduction sensitive GFP [11]. The ALDH2*2 polymorphism of the ALDH2 gene, responsible for a deficiency of ALDH2 activity, is specific to North–East Asians and associated with ethanol sensitivity [24]. We previously reported that ALDH2 deficiency is a risk factor for late-onset AD in the Japanese population [25], which was replicated in a Chinese study [26]. Several studies have reported the presence of A in the mitochondrial membrane of neurons in postmortem brain specimens from patients with AD and in brains of AD animal models [27–29]. Hence, A may initiate mitochondrial membrane lipid peroxidation, which is then detoxified by ALDH2.
5. Conclusion We have established a novel mouse model of AD having accelerated AD-like pathological changes and memory impairment. Early onset of the behavioral phenotype in APP/DAL mice would be useful for research. Furthermore, the APP/DAL mouse is a valuable model to examine the specific role of HNE in AD progression and pathogenesis. It also provides a tool for exploring new approaches, such as lipid accessible antioxidant molecules used to treat mild cognitive impairment and AD.
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