アルツハイマー病、他の認知症、老化 Alzheimer's Disease, Other Dementia, Aging
P3-1-197 海馬長期増強におけるアミリン受容体を介したベータアミロイド抑制 The Amylin Receptor Regulates Beta-amyloid Inhibition of Hippocampal Long-Term Potentiation ○木村良一1,2 ○Ryoichi Kimura1,2, David MacTavish2, Jing Yang4, David Westaway2,3,4, Jack H. Jhamandas2 兵庫医大・医・生体情報1, アルバータ大学・医・神経学2, アルバータ大学・生化学3, アルバータ大学・プリオン及びタンパク質フォールディング病センター4 Dept Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo1, Dept Medicine and Centre for Neurosci, Univ of Alberta, Edmonton, Canada2, Dept Biochemistry, Univ of Alberta, Edmonton, Canada3, CPPFD, Univ of Alberta, Edmonton, Canada4 Alzheimer's disease (AD) and diabetes are two important human conditions that are strongly linked epidemiologically. Human amylin, an amyloidogenic peptide first isolated from pancreatic islets of diabetic patients, is the endogenous ligand for the amylin receptor and shares similarities in its biophysical and neurotoxic properties with β-amyloid peptide (Aβ), which accumulates at abnormally high levels in the brain areas of AD patients subserving the memory and cognition. The amylin receptor is a potential target receptor for expression of the deleterious actions of soluble oligomeric Aβ species. In this study, we investigated whether the amylin receptor antagonist, AC253, neutralizes the depressant effects of Aβ1-42 and human amylin on hippocampal long-term potentiation (LTP). Furthermore, we examined whether depressed levels of LTP observed in transgenic mice, which overexpress amyloid precursor protein (TgCRND8), could be restored with AC253. In mouse hippocampal brain slices, field excitatory postsynaptic potentials were recorded from the stratum radiatum layer of the CA1 area in response to electrical stimulation of Schaeffer collateral afferents. LTP was induced by 3-theta burst stimulation protocols. Aβ1-42 (50 nM) and human amylin (50 nM), but not Aβ42-1 (50 nM), depressed LTP evoked using both stimulation protocols. Preapplication of AC253 (250 nM) blocked Aβ- and human amylin-induced reduction of LTP without affecting baseline transmission or LTP on its own. In contrast to wild-type controls, where robust LTP is observed, 6- to 12-month-old TgCRND8 mice show blunted LTP that is significantly enhanced by application of AC253. Our data demonstrate that the effects of Aβ1-42 and human amylin on LTP are expressed via the amylin receptor, and moreover, blockade of this receptor increases LTP in transgenic mice that show increased brain amyloid burden. Amylin receptor antagonists could serve as potentially useful therapeutic agents in AD.	P3-1-198 アルツハイマー病とシヌクレイノパチーにおけるストレス防御システム関連タンパク質の検討 Involvement of oxidative stress-sensor protein, Keap1, in brains with Alzheimer's disease and synucleinopathy ○丹治邦和1, 丸山敦史2, 小田桐紗織3, 森文秋1, 伊東健2, 柿田明美4, 高橋均5, 若林孝一1 ○Kunikazu Tanji1, Atsushi Maruyama2, Saori Odagiri3, Fumiaki Mori1, Ken Itoh2, Akiyoshi Kakita4, Hitoshi Takahashi5, Koichi Wakabayashi1 弘前大学医学研究科 脳神経病理学講座1, 弘前大学医学研究科 分子生体防御学講座2, 弘前大学医学研究科 神経解剖・細胞組織学講座3, 新潟大学脳研究所 脳疾患標本資源解析学分野4, 新潟大学脳研究所 病理学分野5 Dept. of Neuropathol., Hirosaki Univ. Sch. of Med., Hirosaki, JAPAN1, Dept. of Stress Response Science, Hirosaki Univ. Sch. of Med., Hirosaki, JAPAN2, Dept. of Neuroanatomy, Cell Biology and Histology, Hirosaki Univ. Sch. of Med., Hirosaki, JAPAN3, Dept. of Pathol. Neurosci., Center for Bioresource-based Res., Univ. of Niigata, Niigata, JAPAN4, Dept. of Pathol., Brain Res. Inst., Univ. of Niigata, Niigata, JAPAN5 Oxidative stress has been proposed as a potential mechanism for neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis. In response to oxidative stress, the levels of numerous cytoprotective products are increased via alteration of the Kelch-like ECH-associated protein 1 (Keap1) and NF-E2 related factor 2 (Nrf2) system. p62, one of the Nrf2 targets, has been known to be incorporated into a wide spectrum of cytoplasmic inclusions in neurodegenerative diseases, and interacts with Keap1. However, it remains unclear whether Keap1 is associated with the pathogenesis of neurodegenerative diseases. In this study, we investigated the relationship between p62 and Keap1 in the brains of patients with AD, PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We found that Keap1 co-immunoprecipitated with p62 in diseased and control brains. Further pathological analyses showed that Keap1 was clearly co-localized with p62 in cytoplasmic inclusions observed in AD, PD, DLB and MSA brains. Quantitative reverse transcription-polymerase chain reaction analysis revealed that several Nrf2 target genes were significantly increased in AD brains compared to controls. Because oxidative stress has been shown in a number of studies to precede the cardinal neuropathological manifestations in neurodegeneration, the sequestration of Keap1 in cytoplasmic inclusions may be a consequential response against stressors. Our results imply that inclusion formation exerts a cytoprotective role within neurons and glial cells, in which Nrf2 target genes are potentially activated.
P3-1-199 アルツハイマー病におけるα4β2ニコチン性コリン神経系へのアミロイド蓄積の影響 Effect of amyloid deposition on α4β2 nicotinic cholinergic system in Alzheimer's disease ○尾内康臣1, 寺田達弘1, 大星有美1, 吉川悦次2, 二ッ橋昌実2, 植木孝俊3, 小川美香子4, 間賀田泰寛4 ○Yasuomi Ouchi1, Tatsuhiro Terada1, Yumi Oboshi1, Etsuji Yoshikawa2, Masami Futatsubashi2, Takatoshi Ueki3, Mikako Ogawa4, Yasuhiro Magata4 浜松医大・MPRC・生体機能イメージ1, 浜松ホトニクス・中研2, 浜松医大・医・神経解剖3, 浜松医大・MPRC・分子病態イメージ4 Dept Biofunct Imaging, Hamamatsu Univ Sch Med, Hamamatsu1, Hamamatsu Photonics KK, Hamamatsu2, Dept Neuroanat, Hamamatsu Univ Sch Med, Hamamatsu3, Dept Molecul Imaging, Hamamatsu Univ Sch Med, Hamamatsu4 An α4β2 nicotinic acetylcholine receptor (nAchR) is considered to engage in regulations of various brain functions such as emotion, attention and memory. Here, we report the levels of nAchRs binding that are associated with frontal cognitive function in AD patients and the effect of amyloid burden on the α4β2 nAchRs using positron emission tomography (PET) with [18F]2FA, an agonistic tracer for α4β2 nAchRs, and [11C]PIB. [Methods] Twenty-five cognitively normal subjects and 20 AD patients participated in this study. All subjects were evaluated with multiple neuropsychological tests. They underwent MRI and PET with two tracers. The parametric images of [18F]2FA uptake and [11C]PIB accumulation in the whole brain was generated in reference to the corpus callosum ([18F]2FA BPr) and cerebellum ([11C]PIB SUVR), respectively. We used regions-of-interest and parametric mapping analysis methods for various statistics. [Results] Frontal lobe function (FAB) scores correlated with levels of [18F]2FA BPr in the prefrontal and supraparietal cortices of healthy subjects. There was significantly greater reduction in [18F]2FA BPr in the broad brain regions including the thalamus, basal forebrain, temporal and frontal cortices in AD patients. In addition, the basal forebrain [18F]2FA BPr was positively associated with the FAB scores, and the [18F]2FA BPr level in those regions was negatively correlated with [11C]PIB SUVR in the precuneus. [Discussion] A positive correlation of [18F]2FA binding with only FAB scores indicates that the α4β2 nAchRs system may be involved in specific cognitive processes such as attention and execution. Impairment of this system in the basal forebrain may be specifically important in AD. β-amyloid-related α4β2 nicotinic cholinergic dysfunction plays a pivotal role in higher cognition impairment in humans.	P3-1-200 神経細胞における小胞体ストレスはAβ分泌の抑制とSyntaxin5を含むER-Golgi SNAREの発現量の上昇を引き起こす ER stress suppress Aβ secretion and induces the expression of ER-Golgi SNARE including Syntaxin5 proteins in neuronal cells ○須賀圭1, 齋藤綾子1, 三嶋竜弥1, 赤川公朗1 ○Kei Suga1, Ayako Saito1, Tatsuya Mishima1, Kimio Akagawa1 杏林大・医・細胞生理1 Dept. Cell Physiol., Kyorin Univ. Sch. of Med., Tokyo, Japan1 Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER, which occurs under a variety of conditions, and has been implicated in many neurodegenerative diseases such as Alzheimer's disease (AD). During ER stress, how such stress signal propagates throughout the cell and how it affects the processing of AD-related membrane protein such as βAPP are poorly understood. Syntaxins (Syx) are involved in trafficking of various membrane proteins between specific intracellular compartments. Among them, Syntaxin5 (Syx5) is thought to function in transporting vesicles between the ER and the Golgi apparatus as SNAREs. We have previously shown that, among Syxs, Syx5 isoforms specifically interacted with presenilin (PS) holoproteins and Syx5 overexpression resulted in the accumulation of β-amyloid precursor protein (βAPP) in the ER to cis-Golgi compartment, an attenuation of the amount of the C-terminal fragment (APP-CTF), and a reduction in the secretion of β-amyloid peptide (Aβ peptide). We also showed that overexpression of Syx5 mimicked the effect of ER stress inducer (Brefeldin A) that inhibits the transport of vesicles from the ER to the Golgi. In order to understand the role of Syx5 in the processing and trafficking of βAPP under ER stress, we examined the mechanisms of ER stress-induced upregulation of the Syx5 containing ER-Golgi SNAREs and their effect on the processing of the βAPP in neuronal cells. We found that ER stress induces the de novo synthesis of mRNA and increase in the certain ER-Golgi SNAREs including Syx5 in the vesicular compartment. While ER stress suppressed the secretion of endogenous Aβ peptide, mild knock-down of Syx5 proteins resulted in the increase of Aβ secretion in neuronal cells. We would like to discuss the relationship between the ER stress response and the βAPP metabolism pathways by focusing on the ER-Golgi SNAREs.
P3-1-201 アストログリア細胞におけるエンドサイトーシス障害の検索 Endocytic dysfunction in astrocytes of aged cynomolgus monkey brains ○木村展之1,2, 岡林佐知2,3, 小野文子2,3 ○Nobuyuki Kimura1,2, Sachi Okabayashi2,3, Fumiko Ono2,3 国立長寿医療研究センター　認知症先進医療開発センター　アルツハイマー病研究部1, 基盤研　霊長類センター2, 予防衛生協会3 Dept AD Res, CAMD, NCGG, Aichi, Japan1, TPRC, NIBIO, Ibaraki, Japan2, Corp. Prod and Res of Lab Primates, Ibaraki, Japan3 We previously reported that age-related endocytic dysfunction causes intracellular Aβ accumulation and neuritic swelling in neurons, but it remains unclear whether endocytic dysfunction affects glial cells. In the present study, we investigated various aged monkey brains histopathologically, and we found that enlarged endosomes accumulated in astrocytes, indicating that aging also causes endocytic dysfunction in astrocytes. We also carried out in vitro studies by using primary cultured rat and monkey astrocytes, and investigated whether endocytic dysfunction affects astroglial functions such as Aβ phagoctosis. In rat and monkey astrocytes, siRNA-induced downregulation of dynein heavy chain (DHC) reproduced endocytic dysfunction as previously reported. In DHC-knockdown astrocytes, we did not observe significant changes in Aβ uptake. However, in DHC-knockdown astrocytes, Aβ accumulated in enlarged early endosomes even at 2 hours after Aβ treatment, suggesting that the transport of incorporated Aβ from early endosomes to lysosomes would be disturbed. Moreover, such Aβ accumulation in enlarged early endosomes of astrocytes was observed in aged monkey brains. From these findings, age-related endocytic dysfunction would be associated with Alzheimer's disease pathology by disrupting Aβ clearance in astrocytes as well as neuronal dysfunctions previously reported.	P3-1-202 アミロイドβによる記憶障害を改善するペプチドの同定 Identification of a peptide that improves amyloidβ-induced cognitive dysfunction ○宮野貴士1, 真鍋裕子1, 佐藤翔太1, 勝孝1, 井上剛1 ○Takashi Miyano1, Yuko Manabe1, Shota Sato1, Takashi Katsu1, Tsuyoshi Inoue1 岡山大院・医歯薬・生体分子解析学1 Dept Biophys Chem, Okayama Univ, Okayama, Japan1 Because there are no established medical treatments for Alzheimer's disease (AD), it is very important to find effective therapeutic strategy. Low aggregated form of amyloid β (Aβ oligomer) is proposed as a trigger of the AD. Previous studies showed that acute intracerebroventricular (i.c.v.) injection of Aβ oligomer in mice leads to AD-like symptoms including cognitive dysfunction. However, medicinal treatments to recover this cognitive dysfunction are largely unknown. In this study, using these AD-like model mice, we explored peptides that improve memory disorder induced by Aβ oligomer. To assess the cognitive dysfunction, we used a novel object recognition task that is often used as a behavior test for learning and memory. As a control, we first injected artificial cerebrospinal fluid (ACSF) into the lateral ventricles bilaterally (3.5 μl in each side), and the i.c.v. injection of ACSF did not affect the memory performance. However, an i.c.v. injection of Aβ oligomer (1 μM) impaired the memory performance. Next, we examined effects of a peptide sequence on the Aβ oligomer-mediated memory impairment. An i.c.v. injection of mixture of this peptide (10 μM) and Aβ oligomer (1 μM) fully recovered the memory impairment. Additionally, an i.c.v. administration of this peptide (10 μM) alone did not affect the memory retention. Finally, we also investigated effects of the peptide on different models of cognitive deficits that are induced by MK-801, an NMDA receptor antagonist. In contrast to the Aβ oligomer-mediated cognitive deficits, an i.c.v. injection of the peptide (10 μM) did not improve the NMDA receptor-mediated cognitive deficits. These results clearly show that this peptide improves Aβ oligomer-mediated cognitive dysfunction in mice, and also suggest that the memory improvement by this peptide is Aβ specific.
P3-1-203 反応性アストロサイトによるAβ産生：アルツハイマー病のアミロイド代謝異常に関する新知見 Reactive astrocytes but not neurons are responsible for Aβ production: A new aspect for the pathogenesis of Alzheimer`s disease ○滝川修1, 奥野海良人1, 吉見立也1, 岡田健1, 三河隆太1, 高柳亜希子1 ○Osamu Takikawa1, Alato Okuno1, Tatsuya Yoshimi1, Ken Okada1, Ryuta Mikawa1, Akiko Takayanagi1 国立長寿医療研究センター　認知症先進医療開発センター治療薬探索研究部　リード分子探索研究室　ラジオアイソトープ管理室（併任）1 Dept Drug Discovery, CAMD, Nat Center for Geriat Geront1 Purpose: In Alzheimer's disease (AD), amyloid β (Aβ) which results in deposit of amyloid plaques is implicated in the neurodegeneration. The mechanisms underlying the Aβ generation, however, remain still poorly understood. Quinolinic acid (QA) is an endogenous neurotoxin. A high production of QA has been demonstrated in the hippocampus of AD brains, and the injection of QA into the mouse hippocampus has been shown to kill most neurons with a marked increase of Aβ. We attempt to identify the cells responsible for the QA-induced increase of Aβ. Methods: One μl of QA saline solution (40 nmol) was injected into the hippocampus of mice. The hippocampal Aβ40/42 levels were measured by ELISA. To identify the cells producing Aβ, the expression of APP was examined for GFAP+ astrocytes and Iba1+ microglia, by immunohistochemistry. The QA-induced changes in the APP, GFAP, and Iba1 levels were also analyzed by Western blotting. Results: Hippocampal Aβ increased by QA injection in a time-dependent manner and this increase was associated with the infiltration of APP+ cells, GFAP+ astrocytes, and IbaI+ microglia. Immunostaining revealed the APP expression to be localized in GFAP+ reactive astrocytes, but not Iba1+ microglia. A western blot analysis indicated the QA-dependent increase of APP to correlate closely with that of GFAP, but not IbaI. Conclusion: GFAP+ reactive astrocytes are therefore the cells responsible for the elevation of Aβ in the QA-treated hippocampus. In fact, a high production of Aβ from reactive astrocytes was confirmed by an in vitro study with primary culture of astrocytes. Discussion: Aβ is believed to originate from neurons, but concrete evidence is lacking. Based on the above findings, we propose that reactive astrocytes are another source of Aβ in AD brains. Our proposal is consistent with the fact that amyloid plaques are always surrounded by GFAP+ reactive astrocytes, which may be generating Aβ for the formation of amyloid plaques.	P3-1-204 アルツハイマー病画像診断を目的とした19F MRIによる脳内アミロイド斑の検出（その2） Development of a novel 19F MRI probe for detecting amyloid deposition in Alzheimer's disease, part 2 ○柳沢大治郎1, 森川茂廣2, 田口弘康1, 椎野顯彦3, 犬伏俊郎3遠山育夫1 ○Daijiro Yanagisawa1, Shigehiro Moirikawa2, Hiroyasu Taguchi1, Akihiko Shiino3, Toshiro Inubushi3, Nor Faeizah Ibrahim1, Ikuo Tooyama1 滋賀医大・分子神経科学研究センター1, 滋賀医科大学 医学部 看護学科2, 滋賀医大・MR医学総合研究センター3 MNRC, Shiga Univ Med Sci, Otsu, Japan1, Dept Fundamental Nursing, Shiga Univ Med Sci, Otsu, Japan2, Biomed MR Sci Res Ctr, Shiga Univ Med Sci, Otsu, Japan3 Fluorine-19 (19F) magnetic resonance imaging (19F MRI) would be a promising approach to image amyloid deposition in the brain. However, the preferable features of 19F MRI probe for amyloid detection remains unclear. MR signal of the probe dramatically reduces when the probe binds to amyloid plaques because of inhibition of molecular mobility. In the present study we therefore investigated a preferable probe to keep high MR signal under binding to amyloid plaques. We have synthesized a series of compounds as potential 19F MRI probes. They are fluorescent chemicals, and each has different length of polyethylene glycol (PEG) chain between core structure and 19F atom. The compounds were injected APP/PS1 mice, an animal model of Alzheimer's disease. Then, MR signals were measured using a 7.0 T horizontal-bore MR scanner. The APP/PS1 mice injected with a compound in which the number of ethylene glycol group in the PEG chain is seven, named as XP7, showed marked 19F MR signals in the area corresponding to the brain, in comparison to wild-type mice. When a compound in which the number of ethylene glycol group in the PEG chain is five, named as XP5, was injected, 19F MR signals in APP/PS1 mice became lower. On the other hand, no 19F MR signals were detected in both APP/PS1 mice and wild-type mice injected with compounds in which the number of ethylene glycol group in the PEG chain were eleven and thirteen, named as XP11 and XP13, respectively. These results suggest the potential benefit of XP7 as a 19F MRI probe for amyloid detection. Furthermore, a proper length of PEG chain is useful to avoid the reduction in MR signal under binding to amyloid plaques.
P3-1-205 アルツハイマー病モデルマウス脳におけるDNAの酸化ダメージの定量的検出 Quantitative detection of oxidative DNA damage in brains of the triple transgenic Alzheimer's disease mouse model ○秋本頼子1, 岡素雅子1,2, 中別府雄作1,2 ○Yoriko Akimoto1, Sugako Oka1,2, Yusaku Nakabeppu1,2 九州大・生医研・脳機能制御学1, ヌクレオチドプール研究センター2 Div. Neurofunc. Genomics, Med. Inst. Bioreg. Kyushu Univ, Japan1, Res. Ctr. Nucleotide pool, Kyushu Univ, Japan2 Modified nucleotides are known to cause various biological effects (e.g. cancer, neurodegenerative diseases). 8-oxo-guanine and 8-oxo-adenine are major oxidization products of guanine and adenine, respectively. These modified nucleobases can lead to mutations, altered protein synthesis, or cell death. 8-oxo-guanine is known to pair with adenine and cytosine, thus causing GC to TA or AT to CG transversion mutations. 8-oxo-adenine can form a base pair with cytosine or guanine, thereby it may cause AT to GC transition and AT to CG transversion mutation, respectively. It has been reported that these major oxidized lesions are highly accumulated in the postmortem brain tissues of Alzheimer's disease patients. In the present study, we focused on the accumulation of 8-oxo-deoxyadenosine and 8-oxo-deoxyguanosine in brains of the triple-transgenic Alzheimer's disease mouse model (3xTg-AD). Prior to the quantification, we prepared and purified 13C, 15N-labeled 8-oxo-deoxyadenosine as an internal control. We previously found that 8-oxo-deoxyadenosine was effectively generated by X-ray irradiation. 13C, 15N-labeled deoxyadenosine was exposed to X-rays and then 13C, 15N-labeled 8-oxo-deoxyadenosine was separated and purified by HPLC. DNA samples were prepared from hippocampi, cerebral cortices, cerebelli, brain stems and striata of non-transgenic control mice and 3xTg-AD homozygous (3xTg-AD-H) mice harboring PS1M146V, APP(Swe), and tauP301L transgenes,.The absolute levels of 8-oxo-deoxyadenosine and 8-oxo-deoxyguanosine in each brain region were determined by LC-MS/MS analysis using the stable isotope-labeled internal standards.	P3-1-206 神経細胞由来エクソソームがもつアミロイドβペプチド除去能力に関する研究 Coordinated function of neurons and microglia to clear Alzheimer's amyloid-β protein by usage of exosomes ○湯山耕平1, 孫慧1, 五十嵐靖之1 ○Kohei Yuyama1, Hui Sun1, Yasuyuki Igarashi1 北海道大学大学院　先端生命科学研究院1 Faculty of Advanced Life Science, Hokkaido University, Sapporo1 Exosomes are extracellular vesicles, which are released from a variety of cells including neurons. Several proteins related to neurodegenerative disorders are reported to be associated with neuronal exosomes, such as amyloid β-protein (Aβ) in Alzheimer's disease, α-synnuclein in Parkinson's disease, and prion protein in Creutzfeldt-Jakob disease. In this study, we examined the potency of the exosomes against Aβ fibrilization and clearance. First, we collected exosomes from culture supernatants of neuro2a (N2a) cells or mouse primary cortical neurons by sequential ultracentrifugation. Then, we performed thioflavin assay to examine the ability of the exosomes on Aβ assembly. The incubation mixture of monomeric Aβ with the exosomes contained higher levels of amyloid fibrils than that without the exosomes, indicated the exosomes significantly enhanced the amyloid formation of Aβ. Based on the recent report that microglia can engulf exosomes, we exposed the incubation mixtures of Aβ with or without exosomes to microglia and then measured Aβ uptaken into microglia. The exosomes markedly increased Aβ incorporation into microglial cells. Furthermore, we found that secretion of neuronal exosomes was modulated by sphingolipid metabolism. Their release were largely decreased by treatment with a N-sphingomyelinase (SMase) inhibitor and N-SMase siRNA, whereas significantly increased by treatment with a sphingomyelin synthase (SMS) inhibitor and SMS siRNA. And interestingly, SMS knockdown also decreased extracellular Aβ levels in transwell culture system of N2a and microglial cells. These results suggest that neuronal exosomes accelerate Aβ assembly on its surface and transports Aβ into microglia to degrade. The potential of neuronal exosomes against Aβ clearance might be useful for the therapy of Alzheimer's disease.

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