一般演題（ポスター）

一般演題（ポスター） Alzheimer’s Disease

3P-27 QUANTITATIVE ANALYSIS OF GSK3β ACTIVITY IN CELLS AND BRAINS KRISHNANKUTTY AMBIKA，Kimura Taeko，Yonezawa Ryo，Saito Taro，Asada Akiko，Hisanaga Shin-ichi MOLECULAR NEUROSCIENCE, DIVISION OF BIOLOGY, TOKYO METROPOLITAN UNIVERSITY Tauopathy neurodegeneration including Alzhimers disease（AD）is characterized by the intracellular accumulation of hyperphosphorylated tau. More than 40 phosphorylation sites are reported in AD tau. A characteristic feature is Ser-Pro or Thr-Pro Phosphorylation, which are catalysed mainly by GSK3β and CDK5. Together with their co-localization with tau aggregates in brains of the patients, CDK5 and GSK3β have been considered as prime candidates for AD pathogenesis. Both GSK3β and CDK5 are Proline directed protein kinases, but their phosphorylation site preference is somewhat different. Phosphorylation of tau by GSK3β is accelerated by prime phosphorylation by CDK5. However, it is not known well how these protein kinases cooperate in generation of AD abnormal phosphorylation epitopes. GSK3β activity is regulated by phosphorylation at Ser9, and its activity is usually estimated by Ser9 phosphorylation using phospho-specific antibody. The use of phospho-Ser9 antibody enabled us to measure relative changes in the GSK3β activity but did not provide the absolute kinase activity. Considering GSK3β as a primary pathological kinase, it is important to understand the activity of GSK3β in pathological brains. In this study, we measured the absolute activation of GSK3β in various cultured cells, neurons and mouse brains using Phos-tag SDS-PAGE. GSK3β has two major phosphorylation sites；Ser9 and Tyr216. Phosphorylation at Ser9 inactivates GSK3β whereas that at Y216 is proposed to activate the kinase. We first analysed GSK3β phosphorylation in CHO-K1 cells using Phos-tag SDS-PAGE, in which phosphorylated GSK3β was retarded extraordinarily. GSK3β was separated into three bands；non-phosphorylated, Tyr216 phosphorylated, and Tyr216 and Ser9 double phosphorylated GSK3β. GSK3β expressed in CHO-K1 cells were mostly active with Tyr216 phosphorylation. Insulin treatment increased Ser9 inhibitory phosphorylation but most part of GSK3β still remained in an active phosphorylation state. We would like to report GSK3β activation in different types of cells and different regions of brain.		3P-28 An autophagy-inducing herbal extract alleviates the pathology of Alzheimer’s disease Liao Yung-Feng¹，Chen Rita P.-Y.²，Huang Chang-Jen² ¹Institute of Cellular and Organismic Biology, Academia Sinica，²Institute of Biological Chemistry, Academia Sinica Alzheimer’s disease（AD）is a progressive neurodegenerative disease and is the most common form of dementia in the elderly. AD patients suffer from memory impairment and cognitive deficits. The lack of disease-modifying therapeutics for AD has imposed a huge social burden. It thus becomes a pressing issue to develop effective therapeutic and prevention strategies against AD. The pathological hallmarks of AD include formation of amyloid-β（Aβ）plague in extracellular space and aggregation of Tau protein within neurons. It is widely believed that accumulation of Aβ and Tau is a causative event in the AD pathogenesis. Both Aβ- and Tau-elicited neurotoxicity could significantly contribute to the onset and progression of AD. We have now identified a herbal extract（HE238）that exhibit potent biological efficacy in suppressing the neurotoxicity elicited by amyloidopathy and tauopathy. Our data show that treatments with HE238 can effectively induce autophagy and neprilysin to promote the clearance of Aβ and Tau in cultured cells. Oral administration of HE238 for 2 month also significantly improves the cognitive function in an Aβ42-injection mouse model. Together, the dual modalities existing in the active ingredients of HE238 obviously present an enormous resource for AD-alleviating agents.

3P-29 The abundance of nonphosphorylated tau among heterogeneously phosphorylated tau species in vivo in mouse and human tauopathy brains Kimura Taeko¹，Hatsuta Hiroyuki²，Masuda-Suzukake Masami³，Hosokawa Masato³，Ishiguro Koichi⁴，Akiyama Haruhiko³，Murayama Shigeo²，Hasegawa Masato³，Hisanaga Shin-ichi¹ ¹Dept. of Biol. Sci., Tokyo Metro. Univ.，²Tokyo Metropolitan Institute of Gerontology，³Department of Neuropathology and Cell Biology, Tokyo Metropolitan Institute of Medical Science，⁴Juntendo University Tauopathy is neurodegenerative diseases characterized by aggregates of hyperphosphorylated tau. Previous intensive studies have identified many disease-related phosphorylation sites on tau. However, it is not understood how tau is hyperphosphorylated and how hyperphosphorylated tau forms aggregates. It is neither clear yet what extent these sites are phosphorylated in disease brains but also normal brains. Most previous studies have used phospho-specific antibodies in analysis of tau phosphorylation. They were useful but did not provide information of nonphosphorylated tau. Here, we applied the method of Phos-tag SDS-PAGE, in which phosphorylated proteins are retarded extraordinary, making it possible to analyze in vivo phosphorylated, as well as nonphosphorylated, tau more quantitatively. Tau in adult mouse brains was heterogeneously phosphorylated with nonphosphorylated 0N4R isoform strongest. Perinatal tau and tau in cold water-stressed tau showed the similar extent of high phosphorylation. Tau in normal aged human brain was separated into more than 8 discrete bands. Among them, nonphosphorylated 0N3R and 0N4R tau were strongest. A slightly higher phosphorylation of tau, which may represent the initial step of hyperphosphorylation, was detected in Alzheimer’s disease（AD）patients at Braak stage V. This phosphorylation state of tau was pelleted by centrifugation and Sarkosyl-soluble tau in either AD or corticobasal degeneration（CBD）brains showed a similar phosphorylation profiles to tau in normal human brains, suggesting that hyperphosphorylation occurs in aggregated tau. These results indicate that tau is present in multiple phosphorylation states in mouse and human brains and nonphosphorylated forms are highly expressed among them.		3P-30 Sustained rise in body temperature exacerbates the pathologies of Alzheimer disease in mice. Enomoto Hiroyuki¹，Kato Reiko²，Nunome Mari¹，Abudullah Mohammad¹，Gong Jien-Sheng¹，Jung Cha-Gyun¹，Yamashita Hitoshi²，Michikawa Makoto¹ ¹Dept of Biochem, Nagoya City Univ Grad Sch Med Sci，²Chubu Univ. Climate change is affecting our health and economy in diverse ways and global warming is one of the most serious public health threats facing people in the world. Although in industrialized countries, many people live in a comfortable thermal conditions, environmental temperature keeps increasing annually. Effect of temperature on mammals have not been understood well. To explore the effect of high ambient temperature on memory functions in the elderly, we evaluated pathophysiology of Alzheimer disease（AD）under the different thermal conditions using APP-Tg mice. Surprisingly, compared to the room temperature at 23℃, the body temperature was significantly higher（about 0.5~0.7℃）in the Tg mice reared at 30℃. The APP-Tg mice reared at 30℃ showed impaired spatial memory function compared with those reared at 23℃. The levels of Aβ deposits in the cerebral cortex and hippocampus of APP-Tg mice（17-month-old）reared at 30℃ were greater than those of mice at 23℃. In the hippocampus, Aβ peptides level was 1.85-fold higher in the mice reared at 30℃ than those at 23℃. The levels of all the HSPs（HSP90, 70, 60 and 27）examined were increased in the mice reared at 30℃ compared with those at 23℃. In addition, the levels of hyper-phosphorylated tau was significantly increased in the brains of mice reared at 30℃. The phosphorylation levels of JNK, ERK and p38MAPK were also increased in the cortex of mice reared at 30℃ compared with those at 23℃. When the cultured neuronal cells were incubated at 35, 37, and 39℃, the Aβ levels synthesized increased in a temperature-dependent manner, and γ-secretase activity also increased in a temperature-dependent manner. Although, effect of temperature on the phosphorylation state of tau in vitro remains to be addressed, these lines of evidence suggest that even in mammals, thermal conditions affects body temperature and higher body temperature may enhance AD pathophysiology including Aβ synthesis/deposition and tau phosphorylation.

3P-31 Intracellular Aβ Oligomers Cause Tau-Independent Spine Alteration and Defect of Axonal and Dendritic Transport Umeda Tomohiro¹，Silverman Michael²，Mori Hiroshi¹，Tomiyama Takami¹ ¹Department of Neuroscience, Osaka City University Graduate School of Medicine，²Department of Biological Sciences and Department of Molecular Biology and Biochemistry, Simon Fraser University Objectives： Mounting evidence indicates that extracellular amyloid β（Aβ）oligomers cause synaptic dysfunction and that this toxicity requires tau in the dendrites. Meanwhile, it has been suggested that intraneuronal accumulation of Aβ proceeds extracellular Aβ, and is an early event in Alzheimer’s disease. It remains unclear whether intraneuronal Aβ also contributes to synaptic alteration, and if so, whether the toxicity requires tau. Methods： To address these questions, mouse/rat primary neurons were transfected with human APP with or without the Osaka（E693Δ）mutation which induces intracellular accumulation of Aβ oligomers. The morphology of dendritic spines, and axonal or dendritic transport of BDNF, mitochondria, and transferrin receptor（a marker of dendritic recycling endosomes）were evaluated. For comparison, the effect of extracelluar Aβ on dendritic spines was examined by adding Aβ into untransfected neurons at concentrations comparable to those in culture media of wild-type APP-transfectants. To study the necessity of tau, primary neurons from tau-deficient mice were also analyzed following to APP transfection. Results： Neurons expressing APP Osaka, but not wild-type APP, accumulated Aβ oligomers within cells. APP Osaka-transfectants showed reduced numbers of total and mushroom-type spines, but wild-type APP-transfectants and Aβ-added untransfectants did not. The flux values of BDNF, mitochondria, and the transferrin receptor transport in axons and dendrites were reduced only in APP Osaka-transfectants. Intracellular Aβ-induced aberrant spine morphology was observed even in tau-deficient neurons. Conclusions： Intraneuronal Aβ oligomers disrupted synaptic integrity independently of tau, and this toxicity was accompanied by an impairment of axonal and dendritic trafficking.		3P-32 Differential effects of angiotensin II receptor blockers on Aβ generation Liu Junjun，Liu Shuyu，Murakami Saki，Sugakawa Yusuke，Kami Ayako，Tanabe Chiaki，Maeda Tomoji，Zou Kun，Komano Hiroto Department of Neuroscience School of Pharmacy Iwate Medical University Angiotensin II receptor blockers（ARBs）are widely prescribed for the medication of systemic hypertension and congestive heart failure. It has been reported that ARBs can reduce the risk for the onset of Alzheimer’s disease（AD）and have beneficial effects on dementia. Neurotoxic amyloid β-protein（Aβ）is believed to play a causative role in the development of AD. However, whether ARBs regulate Aβ generation remains largely unknown. Here, we studied the effect of ARBs on Aβ generation and found that telmisartan significantly increased Aβ40 and Aβ42 generation, but decreased the Aβ42/Aβ40 ratio. However, losartan, valsartan and candesartan did not increase Aβ generation, while olmesartan significantly increased Aβ42 generation. We also found that telmisartan increased the Aβ generation through angiotensin type 1a receptor（AT1a）and the receptor-related phosphotidylinositide 3-kinases（PI3K）pathway. Our findings revealed the different effects of ARBs on Aβ generation and provide new evidence for the relationship between antihypertensive treatment and AD pathogenesis.

3P-33 The role of Rap1A in Cas/HEF1 assocated signal transducer-induced neuronal death Gomi Fujiya，Uchida Yoko Molecular Neurobiology, Tokyo Metropolitan Institute of Gerontology Aβ plays an important role in the early pathogenesis of AD. However, the molecular mechanisms of neuronal death by Aβ remain to be fully elucidated. We analyzed the gene expression profile of neurons exposed to Aβ for 15 hours comprehensively by microarray. One of induced genes was Cas/HEF1 assocated signal transducer（Chat, also reported as NSP3 or SHEP1）. Chat binds to Eph receptor in N-terminus, and binds to Cas in C-terminus. Binding to Cas leads to activation of Rap1. Chat palys a role in cell movement, cell attachment, and axonal guidance. Northern blot analysis showed Chat expression of Aβ treated neurons was 2.8 times higher than those of control neurons, and the expression of Chat in the cortices of AD model mouse, Tg2576 was significantly higher than that in wild mouse. Overexpression of Chat in rat cultured cortical neurons accelerated cell death. C-terminal region of Chat was reported to interact with Cas family proteins, but the co-expression of Chat and p130Cas or NEDD9 interfered Chat-induced neuronal death.The Chat C-terminal region has a guanine nucleotide exchange factor（GEF）like region and is reported to interact with Rap2 and R-Ras and Rap1A, but its GEF activity is obscure. To exclude Chat’s Cas-dependent small G protein activation, Chat Y635E which does not interact with Cas was used. As small G proteins, R-Ras, Rap1A, their constitutively active, and dominant negative mutants were used. Chat Y635E induced accelerated neuronal death was reduced when dominant negative form of Rap1A was co-transfected. Reversely, constitutively active Rap1A induced neuronal death acceleration even without Chat transfection. This result indicates that Rap1A activation was working downstream of Chat in Chat induced accelerate neuronal death pathway.		3P-34 Leptin inhibits expression of neprilysin in cultured astrocytes Yamamoto Naoki^1,2，Tanida Mamoru³，Ono Yoko²，Kasahara Rika²，Suzuki Kenji²，Sobue Kazuya⁴ ¹Fac. Pharm., Sci., Hokuriku Univ.，²Dept. Pharmacy, Ritsumeikan Univ.，³Dept. Physiology II, Kanazawa Med. Univ.，⁴Dept. Anesthesiol., Nagoya City Univ. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of Alzheimer’s disease（AD）. Pathogenesis of AD is characterized by accumulation of extracellular deposits of amyloid β-protein（Aβ）in the brain. The balance between production and degradation of Ab proteins is critical to amyloid accumulation and resulting disease. The major Aβ-degrading enzymes in the brain are neprilysin（NEP）and insulin-degrading enzyme（IDE）, which may promote Aβ deposition in patients with sporadic late-onset AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin induces Aβ degradation by inducing NEP and IDE expression of astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase（ERK）in primary cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in astrocyte-cultured medium. These results suggest that leptin decreases Aβ degradation by NEP through activation of ERK.

3P-35 Coffee reduces BACE1 expression in human neuroblastoma SH-SY5Y cells. Fukuyama Kazuya¹，Kakio Shota¹，Enoki Soichiro²，Kobata Kenji²，Suzuki Toshiharu³，Funakoshi-Tago Megumi¹，Tamura Hiroomi¹ ¹Facalty of Pharmacy, Keio University，²Graduate School of Pharmaceutical Sciences, Josai University，³Faculty of Pharmacy, Hokkaido University Objectives Coffee is one of the most world-widely consumed beverage on a daily bases. Recent epidemiological studies have reported that 3-5 cups of coffee per day can reduce the risk of Alzheimer’s disease（AD）by 65%. However, the precise molecular mechanisms of the effects of coffee are yet uncertain. Beta secretase（BACE1）is the enzyme that produces amyloid beta by cleaving amyloid precursor protein（APP）at N-terminal and is a potential therapeutic target for AD. Therefore, we investigated the effects of coffee on BACE1 expression in human neuroblastoma SH-SY5Y cells.Methods SH-SY5Y cells were cultured in Ham’s F-12/DMEM（1：1）medium supplemented with 15% FBS. The cells were exposed to coffee, decaffeinated coffee, or coffee extracts up to 2.0%（v/v）. After 15 hours, the whole cell lysates were isolated and subjected to immunoblotting for BACE1. The amount of Aβ40 and Aβ42 in the culture medium was measured with ELISA. Results Coffee reduced BACE1 expression in a dose-dependent manner. Mixture of decaffeinated coffee and caffeine（100 μM）showed significant suppression in BACE1 expression, whereas decaffeinated coffee or caffeine（100 μM）itself showed little effect. The active constituents of coffee were produced by roasting process of coffee beans. Discussion Coffee reduced BACE1 expression and, Aβ40 and Aβ42 production in the human neuroblastoma SH-SY5Y cells. Our data suggest that coffee might reduce BACE1 expression by interaction between caffeine and constituents of roasted coffee. This activity may contribute to the preventive effects of coffee on AD. Further studies to identify active components and to elucidate the mechanism of the effects are needed to clarify the molecular basis of prevention of the disease associated with daily coffee consumption.		3P-36 Effect of Aβ on exosome release from astrocytes in culture Abdullah Mohammad，Enomoto hiroyuki，Nunome Mari，Gong JianSheng，Michikawa Makoto Dept of Biochem, Nagoya City Univ, School of Med Sci In the central nervous system, exosomes and apoE-HDL are known to be secreted form glia cells and play important roles in the clearance of amyloid β-proiten（Aβ）. Exosomes are small extracellular vesicles（30-100 nm）derived from the endosomal system and secreted by variety of cell types such as neurons, astrocytes and oligodendrocytes. Exosomes are suggested to play important roles in Aβ deposition and clearance. Aβ is well known to induce neuronal cell death, whereas little is known about its effect on astrocytes. The limited information of the effect of Aβ on astrocytes led us to perform experiments to study the effect of Aβ on release of exosome from astrocytes. We characterized and analyzed release of exosomes and apoE, both of which are known to remove/clear Aβ from the brain, in the culture medium of rat astrocytes in culture. Exosome release was determined by western blot analysis using exosome specific marker proteins, flotillin and HSP90. We found that exosome and apoE-HDL were successfully separated by density gradient ultracentrifugation. Their release was confirmed by distribution of their specific markers and lipids, and electron microscopic analysis. Exosome release was significantly reduced by Aβ1-42 treatment in cultured astrocytes accompanied by an increased JNK phosphorylation. Whereas, apoE-HDL release remained unchanged. A JNK inhibitor recovered the decreased levels of exosome induced by Aβ treatment to levels similar to those of control, suggesting that Aβ1-42 inhibits exosome release via stimulation of JNK signal pathway. Because, exosome is shown to remove Aβ in the brain, our findings suggest that increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway.

3P-37 Diosgenin decreases the expression of HSC70 and improves memory function in Alzheimer’s disease model mice. Yang Ximeng，Kuboyama Tomoharu，Tohda Chihiro Division of Neuromedical Science, Department of Bioscience, Institute of Natural Medicine, University of Toyama We previously found that diosgenin, a constituent of Dioscorea Rhizoma, restored axonal degeneration and improved memory function in Alzheimer’s disease model mice（5XFAD）. In this study, we aimed to investigate diosgenin-elicited expression change of intracellular molecules, to gain insight about diosgenin mechanism leading to axonal regrowth and memory improvement. Vehicle solution or diosgenin（0.1 μmol/kg/day, p.o.）was treated to wild-type or 5XFAD（male, 24-27 weeks old）for 15 days. Object recognition memory of diosgenin-treated 5XFAD was significantly improved. After the behavioral test, cortical lysates were compared on 2D-PAGE. We focused several proteins that showed drastic changes in the expression level and analyzed those by MALDI-TOF/MS. Heat shock cognate 70（HSC70）was identified as the protein decreased by diosgenin treatment in 5XFAD. Next, diosgenin（0.1, 1 μM）or the inhibitor of HSC70, VER-155008（50, 500 and 5000 nM）was treated for 4 days to cultured cortical neuron（ddY, E14）. Diosgenin as well as VER-155008 decreased the expression level of HSC70 and increased axonal density. VER-155008 also decreased in HSC70 expression and increased in axonal density although VER-155008 should be just an activity inhibitor of HSC70. Diosgenin（0.1 μmol/kg/day, p.o.）or VER-155008（10 μmol/kg/day, i.p.）was treated to ddY（male and female, 6-9 weeks old）for 4 days, or wild-type and 5XFAD（female, 32-38 weeks old）for 17 days. Diosgenin and VER-155008 treatment enhanced object recognition memories in normal mice and improved the memories in 5XFAD. These results suggest that a decrease in HSC70 may relate to memory improvement. We are now investigating specific functions of HSC70 in the 5XFAD brain.		3P-38 Cytosolic Asparatate Aminotransferase Relates to Axonal Growth Control under Aβ Treatment Kobayashi Ryo¹，Watari Hidetoshi^1,2，Shimada Yutaka²，Tohda Chihiro¹ ¹Division of Neuromedical Science, Institute of Natural Medicine, University of Toyama，²Department of Japanese Oriental Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama Alzheimer’s disease（AD）is the most common cause of dementia. We are investigating anti-AD drugs from traditional Japanese Kampo medicine, especially kamikihito（KKT）. In this study, we aimed to identify the direct binding proteins of KKT. The drug affinity responsive target stability（DARTS）method was used to identify the direct binding proteins of KKT, and cytosolic aspartate aminotransferase（cAST）was identified. Primary culture cortical neurons were treated with amyloid beta（Aβ）（25-35）, and the cAST expression and activity were evaluated by Western blotting and an AST activity assay, respectively. To investigate the effect of the inhibition and knockdown of cAST on the KKT activity, cortical neurons were treated with O-（carboxymethyl）hydroxylamine hemihydrochloride（OCHH；an AST inhibitor）or transfected with siRNA for cAST, and the degree of axonal atrophy was evaluated under those conditions. DARTS analysis showed that a 42 kDa protein was protected by proteolysis via KKT coexistence. MALDI-TOF/TOF analysis indicated that the protein was cAST. cAST in Aβ（25-35）-treated neurons showed no change in the expression level but low activity. In contrast, treatment with KKT reversed the cAST activity to control level. Treatment of cortical neurons with Aβ（25-35）significantly decreased the axonal density. KKT treatment restored the axonal density, whereas the KKT-induced increase in axonal density was diminished by OCHH treatment or knockdown of cAST. In normal condition, the down regulation of cAST was not related to axonal damage. Aβ（25-35）-triggered cAST inactivation may relate to axonal atrophy. KKT up-regulates the cAST activity probably via direct binding to cAST, resulting in axonal growth. Functional roles of cAST in AD pathology are under investigation.

3P-39 Tau phosphorylation via microtubule-affinity-regulating-kinase（MARK）/PAR-1 as an initial step in the pathological cascade leading to neurodegeneration Ando Kanae¹，Hayashishita Motoki¹，Oka Mikiko¹，Maruko-Otake Akiko²，Ohtake Yosuke³，Iijima Koichi M.^4,5 ¹Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University，²Department of Neuroscience, Thomas Jefferson University，³Shriners Hospitals Pediatric Research Center, Temple University School of Medicine，⁴Department of Alzheimers Disease Research, Laboratory of Genetics and Pathobiology, National Center for Geriatrics and Gerontology，⁵Department of Experimental Gerontology, Graduate School of Pharmaceutical Sciences, Nagoya City University Tau is a microtubule-binding protein localized to the neuronal axons, where it regulates microtubule stability. However, tau is hyper-phosphorylated and accumulated in the cytosol in the brain neurons under pathological conditions including Alzheimer’s disease（AD）. Tau phosphorylation at Ser262 has been suggested to occur in the early stages in AD pathogenesis and to play critical roles in tau toxicity. It is not clear how elevated levels of tau phosphorylated at Ser262 leads to neurodegeneration. By using Drosophila as a model system, we found that the events causally related to disease pathogenesis such as expression of β-amyloid peptide or depletion mitochondria from the presynaptic terminals increases the levels of tau phosphorylated at Ser262 via microtubule-affinity-regulating-kinase（MARK）/PAR-1. Tau phosphorylation at Ser262 and Ser356 causes accumulation of tau with a prominent effect on tau species that are not phosphorylated at proline-directed kinase-target sites（SP/TP sites）. Tau phosphorylated at Ser262 and Ser356 were subjected to further phosphorylation at disease-associated SP/TP sites. These results suggest that tau phosphorylation at Ser262 and Ser356 via MARK/PAR-1 is a critical step initiating a cascade that leads to accumulation of toxic tau species, and targeting such tau species may be an effective strategy to block the cascade of events leading to neuron loss in diseased brains.