Poster

Poster 19 Neurological Diseases 3 ポスター 19 神経疾患3

P19-1 Atomic force microscopy observation of the ganglioside-induced amyloid β aggregation in the presence of metal ions 金属イオン存在下でガングリオシドに誘起されるアミロイドβ凝集体形成のAFM観察 Ito Koichiro（伊藤広一郎），松原輝彦，佐藤智典 Faculty of Sci. and Tech., Keio University The Alzheimer’s disease (AD) is the most common and serious neurodegenerative disease. In AD brains, amyloid β protein (Aβ) deposition and higher concentrations of metal ions such as Zn²⁺, Cu²⁺ and Fe³⁺ than healthy brains have been found. The Aβ binds to the monosialoganglioside GM1 in neuronal cell membranes and form toxic aggregations and fibrils. It has also been reported that metal ions form some complexes with Aβ and promote and/or inhibit Aβ aggregation. However, few studies have been reported for the effects of metal ions on the ganglioside-induced Aβ fibril formation. Therefore, we investigated the aggregations of Aβ₄₂ on lipid bilayers in the presence of metal ions. A GM1-containing lipid bilayer by accumulation of GM1/sphingomyelin/cholesterol (10:45:45) monolayer onto phospholipid-coated mica. Metal ions such as Cu²⁺, which was reported that can make some complexes with Aβ₄₂, were interacted with Aβ₄₂ on the lipid bilayer. After that, the morphology of Aβ₄₂ aggregations was observed by atomic force microscopy (AFM). It was found that morphology of Aβ₄₂ aggregations were different depending on the concentration of Cu²⁺. Particularly, the number and total amount of Aβ₄₂ fibrils was increased at high concentration of Cu²⁺ close to senile plaque in AD brains, suggesting that the fibril formation was promoted by Cu²⁺ at high concentration. These results indicate that copper ion influences the ganglioside-induced Aβ fibril formation.		P19-2 Tau Phosphorylation at AT8 Pathological Site during Brain Development Tau Phosphorylation at AT8 Pathological Site during Brain Development Dilina Tuerde^1,2，Kotaro Furusawa²，Toshiyuki Takasugi²，Taeko Kimura²，Shinsuke Ishigaki¹，Kanae Ando²，Gen Sobue¹，Shin-ichi Hisanaga² ¹Graduate School of Medicine, Nagoya University ²Department of Biological Sciences, Tokyo Metropolitan University Tau is a microtubule (MT) associated protein (MAP), stabilizing MTs in axon of neurons and then supporting neuronal network in healthy brains. In contrast, in Alzheimer`s disease (AD) brains, tau is abnormally phosphorylated more than 40 sites, and aggregated into neurofibrillary tangles (NFTs) in neurons undergoing degeneration. Among many pathological phosphorylation sites, phosphorylation at the AT8 site has been most frequently used for diagnosis of AD. The AT8 site comprises two phosphorylation sites at Ser202 and Thr205, although some literatures demonstrate it as a consequence of triple phosphorylation at Ser199, Ser202 and Thr205. However, it is not completely understood yet how the AT8 reactivity is generated in AD brain and how it contributes to AD development. Tau hyperphosphorylation at specific residues occurs not only in AD brains but also in fetal and early postnatal brains. We found that AT8 is one of highly phosphorylated sites in fetal and neonatal stages of mouse, and it suddenly disappear during 2 to 3 weeks after birth when neuronal circuit is established. Moreover, hypothyroidism delayed tau dephosphorylation at the AT8 site specifically about 3 days. These results indicate direct relationship between neuronal development and AT8 site phosphorylation. We think it important to understand the role of AT8 phosphorylation in neuronal maturation at a molecular level. Here, we examined the effect of overexpression of wild-type human tau or AT8 site mutants, both unphosphorylatable Ala and phosphomimetic Asp in developing neurons. Our study would shed light on a physiological role of the AT8 phosphorylation in tau and also provide more efficient approaches to tackle AD.

P19-3 Tubulin degeneration causes tau abnormalities チューブリン変性を起点としたタウ異常性の獲得の可能性 Fujiwara Hitomi（藤原ひとみ）¹，渡辺祥司²，岩田実里²，上田翔洋¹，延原美香¹，和田聡子¹，御園生裕明²，宮坂知宏¹ ¹Dept of Neuropathology, Faculty of Life and Medical Sciences, Doshisha Univ, Kyoto, Japan ²Lab for Cognition, Memory and Aging, Graduate School of Brain Science, Doshisha Univ, Kyoto, Japan Tauopathy is a type of neurodegenerative disorder including Alzheimer’s disease defined by formation of tau filamentous inclusion in affected neurons. Tau is localized in axon and assumed to promote microtubule stabilization in healthy neurons, whereas hyperphosphorylated tau is accumulated in somatodendrite of tauopathy neurons. Intriguingly, microtubules (tubulin) loss are inevitably found in the neurons. Although it is believed that excessive phosphorylation of tau is involved in neurodegeneration, the causal relationship between tau abnormalities and microtubule loss remains unclear. To address whether tubulin degeneration induces tau pathology, we developed a miRNA-mediated knockdown system of tubulin-specific chaperon E (Tbce), a factor needed for de novo synthesis of tubulin, in mouse primary hippocampal neurons. The knockdown of Tbce led to enhance localization of alpha-tubulin in cell body at DIV (days in vitro) 7, while that of acetylated-alpha-tubulin in the compartment was reduced. In these conditions, total tau and phospho-Ser202/Thr205 tau protein were increased in cell body without alteration of tau mRNA expression level. These indicate that the Tbce knockdown induces reduction of active tubulin, which has the capability to polymerize microtubules, as well as accumulation of phosphorylated tau in somata. These results suggest that disruption of mechanism to maintain tubulin and/or microtubule properties promotes tau pathology.		P19-4 Elevated mRNA expression and low methylation of SNCA in Japanese Alzheimer's disease subjects 日本人アルツハイマー病患者ではSNCA遺伝子 mRNA発現が上昇し、メチル化率が低下する Funahashi Yu（舟橋裕），吉野祐太，森崇明，吉田卓，山崎聖広，尾崎優樹，佐尾知子，伊賀淳一，上野修一 Department of Neuropsychiatry, Molecules and Function, Ehime University Graduate School of Medicine, Shitsukawa, Ehime, Japan Despite the continuing debate about the amyloid hypothesis in Alzheimer’s disease (AD), the precise pathogenesis is still unclear. Mixed pathology is common and multiple different protein aggregates are seen in human postmortem brains. Aggregates consisting of the alpha-synuclein protein encoded by the Synuclein Alpha gene (SNCA) are common in both dementia with Lewy bodies and AD. We examined SNCA mRNA expression and methylation rates of the CpG island at intron 1 of SNCA in peripheral leukocytes in 50 AD and age- and sex-matched control subjects to verify whether alpha-synuclein pathology affects the AD pathogenesis. SNCA mRNA expression in AD subjects was significantly higher than that in control subjects (1.62±0.73 versus 0.98±0.50, p < 0.001). We found significant differences between AD and control subjects at seven CpG sites (average rate; 8.8±2.7 versus 9.5±2.5, respectively: p = 0.027). The methylation rates tended to be lower in AD subjects at all CpG sites. We conclude that mRNA expression and methylation of SNCA intron 1 are altered in AD, which may be caused by Lewy body pathology in AD.

P19-5 Synphilin-1 has neuroprotective effect on Parkinson's disease-model cells by inhibiting apoptosis Synphilin-1はパーキンソン病モデル細胞においてアポトーシス抑制による神経保護作用を有する Shishido Takeo（宍戸丈郎）¹，永野義人¹，荒木睦子¹，高橋哲也¹，松本昌泰²，丸山博文¹ ¹Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical and Health Sciences ²Sakai City Medical Center, Sakai City Hospital Organization [Background]Synphilin-1 represents a cytoplasmic protein that interacts with α-synuclein in neuron and labels the central core of Lewy bodies which is the pathological hallmark of Parkinson’s disease (PD). The interaction of synphilin-1 with α-synuclein indicates that synphilin-1 may also play a central role in PD pathogenesis. However, the biological functions of synphilin-1 are not fully understood.[Objective]In this study, we investigated whether synphilin-1 would have neuroprotective effects on in vitro PD-models using 1-methyl-4-phenylpyridinium (MPP⁺) which preferentially cause dopaminergic cell death.[Materials and Methods]We established SH-SY5Y cells stably overexpressing synphilin-1. The cells treated with MPP⁺ were evaluated to check the cell viability, apoptosis and reactive oxygen species (ROS) production using immunoblotting and immunostaining methods.[Results]Trypan blue exclusion assay showed that synphilin-1 significantly inhibited neuronal cell death induced by MPP⁺. Confocal study showed that synphilin-1 partially restored nuclear condensation and fragmentation caused by MPP⁺. Furthermore, immunoblotting studies indicated that the protein expression levels of cleaved- poly-ADP-ribose polymerase (PARP) and cleaved-caspase3 were decreased in cells expressing synphilin-1 compared to those expressing empty vector. Production of ROS induced by MPP⁺ was also significantly reduced in synphilin-1 expressing cells compared to control cells.[Conclusion]These data suggested that synphilin-1 could have a neuroprotective function by inhibiting apoptosis and ROS production in the molecular mechanism of PD.		P19-6 Neuroprotective effect of molecular hydrogen due to stomach-brain interaction in Parkinson's disease model mice パーキンソン病モデルマウスにおける胃・脳連関を介した分子状水素の神経保護作用 Uemura Yuya（上村優哉），新山哲史，吉井佑典，井上太海，野田百美 Lab. Pathophysiol. Grad. Sch. Pharm., Kyushu Univ. Molecular hydrogen (H₂), as a new medical gas, has protective effects in neurological disorders including Parkinson’s disease (PD). We have shown that drinking water with saturated H₂ (H₂ water) significantly reduced the loss of dopaminergic neurons in PD model mice using both acute and chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In addition, the neuroprotective effect of drinking H₂ water in PD mice might be due to stomach-brain interaction via release of gastric hormone, ghrelin. To confirm the contribution of ghrelin in H₂ water-drinking PD model mice, ghrelin-knock out (KO) mice were used. Despite the speculation, the effect of H₂ water was still observed in ghrelin-KO PD model mice. To　further check the involvement of ghrelin, possible contribution of ghrelin-induced vagal afferent effect was tested by performing subdiaphragmatic vagotomy before treating with H₂ water and administration of MPTP. The protective effect of H₂ water was still observed in the vagotomized mice in substantia nigra, suggesting that stimulation of vagal afferent nerves is not involved in H₂-induced neuroprotection. Other neuroprotective substitutes in ghrelin-KO mice were speculated because H₂-induced neuroprotection was not cancelled by ghrelin receptor antagonist, D-Lys³ GHRP-6, in ghrelin-KO PD model mice, unlike in wildtype PD model mice. Our results indicate that ghrelin and ghrelin-like factors are important for H₂-induced neuroprotection. The mechanism how H₂ induced ghrelin release is now under investigation.

P19-7 Investigation of Parkinsonian neurotoxin-susceptible lysosomal proteins パーキンソン病関連神経毒感受性リソソームタンパク質の探索 Miyara Masatsugu（宮良政嗣）^1,2,3，三浦ゆり⁴，徳永航³，中西剛¹，太田茂^3,5，古武弥一郎³ ¹Gifu Pharmaceutical University, Gifu, Japan ²Research Fellow of Japan Society for the Promotion of Science, Tokyo, Japan ³Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan ⁴Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan ⁵Wakayama Medical University, Wakayama, Japan Parkinson's disease (PD), a progressive and chronic neurodegenerative disorder, is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Although its etiology is unclear, recent evidence suggests that lysosomes play vital roles in its pathogenesis. Based on our previously established cellular model of PD with progressive cell death using low concentrations of the Parkinsonian neurotoxin MPP⁺, we reported impaired autophagic degradation concomitant with reduced lysosomal hydrolase cathepsin D activity. In the present study, we explored MPP⁺-susceptible lysosomal proteins with the aim of identifying the key proteins involved in impaired autophagic degradation caused by low-level exposure to this Parkinsonian neurotoxin. Human neuroblastoma SH-SY5Y cells were exposed to low concentrations of MPP⁺ for 48 h, and post-nuclear supernatant (PNS) was prepared using a commercial kit. The PNS was then subjected to OptiPrep^TM density gradient ultracentrifugation, and any organelles present in the resulting seven fractions were evaluated by Western blotting. The third lightest fraction contained organelle marker proteins and the highest concentrations of LAMP1 and cathepsin D, lysosomal proteins. Currently, we are in the process of completing proteomic analysis of this fraction to identify the potential target proteins. In addition, the lysosomal biogenesis enhancer trehalose partially suppressed the impairment of autophagic degradation and mild MPP⁺ exposure-caused cell death, although these had limited effects. In conclusion, Parkinsonian neurotoxin-susceptible lysosomal proteins have the potential to be specific and potent targets in the development of PD therapy via autophagic degradation enhancement.		P19-8 HDAC3 inhibition ameliorates memory function via regulating microglial phenotype in Alzheimer's disease model mice アルツハイマー病モデルマウスにおいてHDAC3阻害はマイクログリアの表現型制御を介して記憶を改善させる Kuboyama Tomoharu（久保山友晴），東田千尋 Div of Neuromedical Science, Instit of Natural Med, Univ of Toyama Many studies suggest that Alzheimer’s disease is an inflammatory disease. Brain inflammation is related to activation of microglia, especially M1 subtype. On the other hand, another type of microglia in the brain, M2 microglia plays a role of anti-inflammatory and neurotrophic effects. We previously clarified that HDAC3 inhibition induced predominance of M2 microglia and recovered locomotor function in spinal cord injured mice. Therefore, this study is aimed to clarify HDAC3 inhibition increases predominance of M2 microglia and restored memory function in Alzheimer’s disease model mice. An HDAC3 inhibitor, RGFP966 was intraperitoneally administered to 5XFAD mice, a transgenic model of Alzheimer’s disease. RGFP966 improved novel object recognition memory in 5XFAD mice. When microglia in the brain of 5XFAD mice were eliminated by intracerebroventricular administration of clophosome, the effect of RGFP966 was diminished. In cultured microglia, amyloid β treatment skewed toward M1 microglia, whereas RGFP966 treatment skewed toward M2 microglia when treated 24 h after amyloid β addition. These results indicate that HDAC3 inhibition increased predominance of M2 microglia and ameliorated memory in 5XFAD mice. We propose RGFP966 is a novel anti-Alzheimer’s disease drug candidate which regulates microglial phenotype.

P19-9 Effect of educational attainment on Alzheimer's disease-related neuroimaging biomarkers in healthy controls, and participants with mild cognitive impairment and Alzheimer's disease アルツハイマー病における教育期間が神経画像バイオマーカーに与える影響 Wada Masataka（和田真孝）¹，野田賀大¹，品川俊一郎²，澤田恭助¹，尾久守侑¹，垂水良介¹，津川幸子¹，山縣文¹，宮崎貴浩¹，三村將¹，中島振一郎¹ ¹Department of Neuropsychiatry, Keio University School of Medicine ²Department of Psychiatry, Jikei University School of Medicine Background: Cognitive reserve is the acquired capacity reflecting a functional brain adaptability/flexibility in the context of aging. Educational attainment is thought to be one of the most important contributors to cognitive reserve. Objective: The goal of this research is to examine the relationships among duration of education and Alzheimer's Disease (AD) related neuroimaging biomarkers such as amyloid-β deposition, glucose metabolism, and brain volumes in each stage of AD. Methods: We reanalyzed a part of the datasets of the Alzheimer's Disease Neuroimaging Initiative. Participants aged from 55 to 90 (inclusive) years old and diagnosed with mild cognitive impairment (MCI) or AD, as well as healthy controls (HC). Multiple regression analyses were conducted to investigate the relationships among duration of education and amyloid-β deposition (n=825), brain metabolism (n=1304), and brain volumes (n=1606) among three groups using data for 18F-Florbetapir (AV-45) imaging, Fludeoxyglucose (FDG) Positron Emission Tomography, and T1-weighted magnetic resonance imaging. Results: Duration of education had no correlations with amyloid-β deposition or brain metabolism in any groups. However, duration of education was positively associated with the total brain volume only in participants with MCI. Conclusions: Our findings suggest that education may exert a protective effect on total brain volume in the MCI stage but not in HC or AD. Thus, education may play an important role in preventing the onset of dementia through brain reserve in MCI.		P19-10 Genetic silencing of the mitochondrial protein p13 protects against experimental parkinsonism ミトコンドリア蛋白p13の遺伝子サイレンシングは実験的パーキンソン病モデルに保護的に働く Shintani Norihito（新谷紀人）¹，小椋紗恵¹，井上直紀¹，師田洋平¹，植野寛貴¹，橋本均^1,2,3 ¹Lab. of Mol. Neuropharmacol., Grad. Sch. of Pharmaceut. Sci., Osaka Univ. ²Mol. Res. Cent. for Children's Mental Dev., United Grad. Sch. Child Dev., Osaka Univ. ³Instit. for Datability Sci., Osaka Univ. We recently identified a novel 13-kDa protein (p13) that may be involved in mitochondrial function, however, its pathophysiological roles have been remained poorly understood. Here, we investigated the mitochondrial function of p13 and its involvement in the pathogenesis of Parkinson's disease (PD) using mitochondrial toxin-induced PD models. Our data show that p13 overexpression induces mitochondrial dysfunction and apoptosis and its knockdown attenuates toxin-induced mitochondrial dysfunction and apoptosis in dopaminergic SH-SY5Y cells. Importantly, heterozygous p13-deficient mice obtained by using the CRISPR/Cas9 system exhibit tolerances against toxin-induced motor deficits and the loss of dopaminergic neurons in the substantia nigra. Accordingly, these results firstly revealed the neuronal p13 function, and suggest that manipulating p13 expression might be a promising avenue for therapeutic intervention in PD.