一般口演（若手道場）
若手道場 アルツハイマー病と認知症２
Wakate Dojo: Alzheimer's Disease and Dementia 2
座長：今泉 和則（広島大学分子細胞情報学）・永井 裕崇（神戸大学） |
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| 2022年6月30日 15:00～15:15 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06a2-01
アルツハイマー病モデルマウスの認知機能低下を防ぐ血中因子の探索
Blood factors preventing cognitive decline in Alzheimer’s disease
*井城 綸沙(1)、東田 千尋(1)
1. 富山大学和漢医薬学総合研究所
*Tsukasa Iki(1), Chihiro Tohda(1)
1. Univ of Toyama, Toyama, Japan
Keyword: Alzheimer's disease, 5XFAD mouse, environmental enrichment, brain-peripheral interaction
In recent years, "healthy aging" is focused as desirable situation in the rapid progress of aging society. It was reported that some people could maintain the cognitive function even with Aβ plaque deposition, which is a pathological cause of Alzheimer’s disease. The phenomenon is considered due to high cognitive reserve that may prevent cognitive decline even in neurogenerative disease. Cognitive reserve is recognized as a capacity of the brain to sustain activity, which is related to educational level, leisure activities, social interaction, and exercise. Therefore, the cognitive reserve is attracting the most attention as a key concept for prevention of dementia. However, its true nature is not clear, and little is known about the molecular mechanism. This study aimed to identify responsible molecules facilitating cognitive reserve. In this study, environmental enrichment (EE) was defined as a socially, mentally, and physically stimulating environment for mice. In 5XFAD mice (Alzheimer’s disease model), Aβ accumulation starts at about 6 weeks after birth, and cognitive impairment occurs approximately 16 weeks after the birth. In this study, 12-week-old 5XFAD mice were housed in a standard environment or EE for 8 weeks. Cognitive impairment evaluated by an object recognition memory test occurred in mice under standard environment at 20 weeks of age. On the other hand, the object recognition memory was not declined in EE group. From the viewpoint of the brain-peripheral organs interaction, we focused on some factors in blood, which functionally related to the cognitive upkeep. After the object recognition test, blood was collected from the inferior vena cava to collect plasma. Plasma from standard environmental mice and plasma from EE mice were comprehensively analyzed by mass analysis and were quantitatively compared. Upregulated and downregulated proteins in plasma of EE mice are now investigated concerning function, brain-targeting property, and source organs. Previous other studies of EE-related cognitive enhancement focused on some brain-specific events such as neurogenesis. However, we suppose that brain-peripheral organs interaction is involved in the cognitive upkeep by EE. Responsible molecules which are derived from peripheral organs and affect brain function are our focus as key factors of cognitive reserve. | | 2022年6月30日 15:15～15:30 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06a2-02
前頭側頭型認知症ミクログリアにおける脂質代謝異常機構解明と神経変性に及ぼす影響
Blockade of endo-lysosomal pathway promotes progranulin-deficient microglial toxicity via proinflammatory lipids
*橋本 恵(1,2)、Eric J Huang(2)
1. お茶の水女子大学アカデミック・プロダクション、2. カリフォルニア大学サンフランシスコ校病理部
*Kei Hashimoto(1,2), Eric J Huang(2)
1. Academic Production, Ochanomizu University, Tokyo, Japan, 2. Department of Pathology, University of California, San Francisco, CA, USA
Keyword: microglial toxicity, frontotemporal dementia, lipid metabolism, neurodegeneration
While microglia are key cells to maintain brain functions by preventing damage, it is becoming clear that aging changes microglial functions toward to having neuronal aggressive character. However, it remains unclear the details how aged microglia influences neurodegeneration.Dominant mutations in the human Progranulin (GRN) gene dramatically reduce Progranulin levels in the brain and are causally connected to frontotemporal degeneration. To investigate how Progranulin deficiency promotes neurodegeneration, we have shown that Grn knockout (Grn-/-) mice develop hyperactivation of microglia, which affects cytoplasmic TDP-43 aggregation and neuronal loss via excessive complement production (Zhang J*, Velmeshev D*, Hashimoto K*, Huang Y*, et al., Nature, 2020). Another phenotype in Grn-/- mice is the abnormal lipidosis in aged brain (Evers BM, et al., Cell Rep, 2017; Marschallinger J, et al., Nat Neurosci, 2020; Logan T, et al., Cell, 2021), however, the mechanisms leading to these lipid phenotypes and how the lipid metabolic defect impact neurodegeneration in Grn-/-mouse brain are not revealed.Here, we combined sucrose density gradient-based subcellular fractionation of Grn+/+ and Grn-/- mouse brains with proteomic and lipidomic analyses to show that Progranulin deficiency caused proteomic changes that affected the endo-lysosomal pathway and altered lipidomic profiles in Grn-/- brain early endosomes and lysosomes. Interestingly, confocal microscopic analyses showed that drastic accumulation of lipids was occurred in Grn-/- microglia and neurons, not in astrocytes and oligodendrocytes. To determine the detail mechanisms how Grn-/- microglia show lipid metabolic defect, we performed lipidomic profiling in primary microglia and microglia conditional media from Grn+/+ and Grn-/- mice at postnatal day 3, and analyzed subcellular localization of lipids in Grn+/+ and Grn-/-primary microglia. These data indicated that lipidomic alteration in Grn-/- microglia were caused by the failure to engage lipids to lysosome-mediated degradation, leading to increase in lipid storage and secretion. Using microglia-neuron co-culture system, we showed that the lipid-enriched Grn-/- microglia promote dendritic degeneration and cell death in Grn-/- neurons, which can be neutralized by monoclonal antibody E06, a functional blocker of oxidized phospholipids. These results propose novel insights how endo-lysosomal block in Grn-/- microglia promote disrupt microglia-neuron homeostasis, thereby promoting neurodegeneration by the accumulation and secretion of proinflammatory lipids. Hereafter, further studies should clarify how endo-lysosomal lipid trafficking is inhibited by progranulin deficiency. | | 2022年6月30日 15:30～15:45 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06a2-03
タウの軸索局在および微小管重合能を決定づけるリン酸化部位の同定
Identification of tau phosphorylation sites which affect its functions on microtubules and axonal localization in neurons
*姚 芷薇(1)、辰本 彩香(1)、鳥居 知宏(2)、御園生 裕明(2)、宮坂 知宏(1)
1. 同志社大学大学院生命医科学研究科、2. 同志社大学大学院脳科学研究科
*Zhiwei Yao(1), Ayaka Tatsumoto(1), Tomohiro Torii(2), Hiroaki Misono(2), Tomohiro Miyasaka(1)
1. Faculty of Life and Medical Sciences, Doshisha University, 2. Graduate School of Brain Science, Doshisha University
Keyword: Tau, phosphorylation, Alzheimer's disease, microtubule-associated protein
Tau is a microtubule-associated protein (MAP) that is localized to the axon, and is also known as a component of neurofibrillary tangles in Alzheimer's disease (AD). The pathologically deposited tau is not only lost its physiological functions to bind and stabilize microtubules (MT), but also mis-localized into the soma and dendrites. Therefore, dysregulation of the functions and intracellular localization of tau is considered to be a key step to cause neurodegeneration in AD brains. Among seventies of putative candidates, critical phosphorylation site(s) of tau that affects its functions or intracellular localization in neuronal cells had remain obscure. Our previous report showed that the substitution of eight selected Ser/Thr residues in proline-rich region 2 to Glu (8E-tau) disrupts the MT binding and axonal localization of tau. Here we further tried narrow down such Ser/Thr sites that reproduce tau abnormalities. We subclassed these phosphorylation sites into three groups and created phospho-mimetic mutants: S198-T205 (4E-tau), T212/S214 (2E1-tau), and T231/S235 (2E2-tau). GFP-tagged wild-type (WT-tau) and the mutant tau were transiently expressed in rat primary cultured neurons. The molecular dynamics of WT-tau and mutants were analyzed using the fluorescence recovery after photobleaching (FRAP) assay, and their intracellular localization were analyzed using the immunocytochemistry. Recombinant tau proteins were purified and subjected to the MT-binding or -assembly assay in vitro. We found that the 2E1-tau exhibited higher mobility than WT-tau and was comparable to 8E-tau in FRAP analyses, indicating reduced binding of 2E1-tau to MTs. However, the Glu mutations in T212, S214 did not significantly affects MT-binding ability of tau in vitro. Considering the dissociation between these data, we speculate that phosphorylation at Thr212 and Ser214 regulates MT-binding in conjunction with intrinsic phosphorylation at other sites in living cells. Interestingly, 4E-tau exhibited apparent reduction only in MT assembly without affecting on its MT binding. Furthermore, we confirmed that 2E1-tau and 2E2-tau showed a significantly smaller axon/dendrite ratio than WT-tau. Thus, dephosphorylations at T212, S214, T231, and S235 are critical for the axonal localization of tau. Because the double phosphorylation in T212/S214 sites has been specifically identified in pathologically deposited tau, they may be essential for the abnormal behaviors of tau in the pathogenesis of AD. | | 2022年6月30日 15:45～16:00 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06a2-04
ヒトiPS細胞を用いた、アルツハイマー病モデルの作製及び17β-エストラジオール治療効果の確認
Human-Derived Induced Pluripotent Stem Cells-Based Disease Models of the Alzheimer’s Disease (AD) Patients Expressed AD Phenotypes and Responded to 17β-Estradiol Treatment
*Supakul Sopak(1)、前田 純宏(1)、岡野 栄之(1)
1. 慶應義塾大学医学部生理学教室
*Sopak Supakul(1), Sumihiro Maeda(1), Hideyuki Okano(1)
1. Department of Physiology, Keio University School of Medicine, Tokyo, Japan
Keyword: ALZHEIMER'S DISEASE, INDUCED PLURIPOTENT STEM CELL, 17β-ESTRADIOL, HORMONE THERAPY
アルツハイマー病（Alzheimer’s Disease: AD）は最も有病率が高い認知症であり、有効な治療法の開発が困難な疾患の一つである。特にADは男女で発症機序が異なることも知られ、女性が男性より有病率が高く、男性は女性より進行が早い。また、エストロゲン等の性ホルモンの低下がADの発症に関わると考えられており、閉経後の女性AD患者に対してホルモン補充療法を試みる臨床研究が多く行われてきたが、有効/部分的有効/無効と一定の結論は得られていない。近年の臨床研究では、エストロゲンの投与方法、投与タイミング、及び患者の遺伝型（ApoEなど）を層別化し、エストロゲンのADに対する効果が検証されてきたがその細胞生理学的な根拠は薄い。そこで我々は、様々なAD患者由来iPS細胞を神経細胞に分化誘導・表現型解析を行い、それぞれの患者疾患モデルにおけるエストロゲンへの反応性の違いに基づき、ホルモン療法のヒト神経細胞に対する効果の検証、患者ごとの効果予測を行っている。これまで、健常者由来、家族性アルツハイマー病患者由来2例（APP KM670/671NL, APP V717L）、孤発性アルツハイマー病患者2例（ApoE ε3/ε3, ApoE ε3/ε4）などの既存iPS細胞から、新規分化誘導法により神経細胞モデルを作製した。このモデルを用いて、過去に報告されたADの表現型（ELISAによるAβ分泌量の上昇・Ca imagingによる神経細胞の活性化の上昇など）の確認にも成功している。さらに、iPS細胞から分化誘導を行なった神経細胞では、エストロゲン受容体（ERα、ERβ）が発現しており、エストラジオール投与後の効果として、短期的には神経細胞興奮性の上昇を認め、長期的効果としては神経突起分岐促進などが確認できた。これらは、ヒトiPS細胞由来神経が、性ホルモンへの反応性を検証する為に適した系であることを示していた。今後は、性ホルモンの効果が、細胞株間、男女間、疾患群と健常者群において異なるかどうかなどを検証する。これらは、性ホルモンの効果におけるAD患者の層別化に役立つ可能性がある。 | |
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