ポスター発表 (Poster Sessions)

情報伝達とその調節 Signal Transduction and Modulation
P2-1-37 マウス末梢神経系有髄神経線維におけるSrc-MPP6-4.1G-CADM4 蛋白複合体 Src-MPP6-4.1G-CADM4 protein complex in mouse peripheral nervous system myelinated nerve fibers ○寺田信生¹, 齊藤百合花², 大野伸彦², 山内淳司³, 大野伸一² ○Nobuo Terada¹, Yurika Saitoh², Nobuhiko Ohno², Jyunji Yamauchi³, Shinichi Ohno² 信州大学・医学部・保健学科・基礎作業療法学講座¹, 山梨大学大学院・医学工学総合研究部・解剖分子組織学教室², 国立成育医療研究センター研究所³ Dept Occupational Therapy, School Health Science, Shinshu Univ School Medicine, Nagano¹, Dept Anatomy Molecular Histol, Int Grad School Medicine Engineer, Univ Yamanashi, Yamanashi², Natl Res Institute Child Health Development, Tokyo³ Schmidt-Lanterman incisures (SLIs) are a specific feature of myelinated nerve fibers in the peripheral nervous system (PNS). Recently, we reported a membrane skeleton-related molecular complex in SLI, membrane protein palmitoylated 6 (MPP6)-protein 4.1G-cell adhesion molecule 4 (CADM4). Additionally, we will report localization of a signal transduction protein, Src, in the SLIs of mouse sciatic nerves, and its phosphorylation states in Y527 and Y418 (P527 and P418, respectively) under normal conditions or deletion of a membrane skeletal protein, 4.1G. In adult mouse sciatic nerves, Src was immunolocalized in SLIs as a cone-shape, as well as in paranodes and some areas of structures reminiscent of Cajal bands. By immunostaining in normal nerves, P527-Src was strongly detected in SLIs, whereas P418-Src was much weaker. Developmentally, P418-Src was detected in SLIs of early postnatal mouse sciatic nerves. The staining patterns for P527 and P418 in normal adult nerve fibers were opposite those in primary culture Schwann cells and a Schwannoma cell line, RT4-D6P2T. In 4.1G-deficient nerve fibers, which had neither 4.1G nor the MPP6 in SLIs, the P418-Src immunoreactivity in SLIs was clearly detected at a stronger level than that in the wild type. An immunoprecipitation study revealed Src interaction with MPP6. These findings indicate the Src-MPP6-4.1G-CADM4 protein complex in SLIs have a role in signal transduction in PNS.	P2-1-38 視床下部神経細胞でのGnRHによるERKの活性化へのPYK2の関与 Involvement of PYK2 in GnRH-induced ERK activation in cultured hypothalamic neurons ○山本秀幸¹, 仲嶺三代美¹, 前田紀子¹, 徳誠吉¹ ○Hideyuki Yamamoto¹, Sayomi Higa-Nakamine¹, Noriko Maeda¹, Seikichi Toku¹ 琉球大院・医・生化学¹ Dept Biochem, Univ of Ryukyus, Nishihara, Japan¹ Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). GnRH neurons have a GnRH receptor belonging to the G-protein-coupled receptors (GPCRs). In previous work, we found that ErbB4 was involved in the activation by GnRH of extracellular signal-regulated protein kinase (ERK) in immortalized GnRH neurons (GT1-7 cells)¹⁾. In addition, we found that ErbB4 was cleaved after GnRH treatment. In the present study, we further examined the intracellular signaling of ERK activation and ErbB4 cleavage. The down-regulation of protein kinase C by PMA treatment strongly inhibited ERK activation and ErbB4 cleavage. The inhibitor and siRNA for protein kinase C delta strongly inhibited ERK activation, but not ErbB4 cleavage. Src existed in the activated form without GnRH, while GnRH activated protein tyrosine kinase PYK2. The inhibitor for Src completely inhibited the activation of Src, PYK2, and ERK. Furthermore, we found that siRNA for PYK2 significantly inhibited ERK activation. These results indicated that protein kinase C delta, Src, and PYK2 were involved in the GnRH-induced activation of ERK in GnRH-neurons.1) S. Higa-Nakamine et al., J. Cell. Physiol., 2012, 2492-2501.
P2-1-39 周産期の低酸素状態における呼吸性ニューロンの解析 Analysis of modulation on respiratory neurons in the hypoxia during perinatal period ○志賀真理¹, 西山紋惠¹, 下村英毅², 谷澤隆邦², 荒田晶子¹ ○Mari Shiga¹, Ayae Nishiyama¹, Hideki Shimomura², Takakuni Tanizawa², Akiko Arata¹ 兵庫医科大学　生理学生体機能部門¹, 兵庫医科大学　小児科学² Dept Physiol, Hyogo College of Med, Hyogo, Japan¹, Dept.Pediatrics, Hyogo College of Med, Hyogo, Japan² The respiratory hypoxia detection shows increase in respiratory rhythm caused by mainly the influence on peripheral chemoreceptor. It is well known that the central chemoreception detects bicarbonate. However, the central effects of hypoxia on brainstem and spinal cord during developmental stage had not been investigated. Our previous studies using isolated brainstem spinal cord preparations showed that respiratory rhythm was facilitated in hypoxic condition at embryonic day 18 (E18) and postnatal day 1 (P1) rats; in contrast, respiratory rhythm decreased in P2-4. In this study, we examined which neuron type is major effecter on respiratory rhythm in developmental stage. We investigated extracellular recordings of respiratory neurons recorded from the rostral ventrolateral medulla (RVLM) during development. In hypoxic condition in P2-P4, the intraburst frequency of inspiratory neuron did not change but that of expiratory neurons was significant decrease, and tonic neurons in RVLM showed a marked depression in hypoxic condition. In contrast, the intraburst frequency of expiratory neurons was significant increase but that of inspiratory neuron did not change, and also tonic neurons in RVLM showed a marked facilitation in hypoxic condition in E18. These results showed that tonic and expiratory neurons in RVLM might play a crucial role of the modulation of central chemoreceptor on respiratory network in hypoxic condition in the perinatal period.	P2-1-40 AddicsinによるTomoregulin-1細胞膜発現量の制御 Addicsin regulates the cell surface expression level of Tomoregulin-1 ○荒野拓^1,2, 藤崎真吾², 池本光志^1,2 ○Taku Arano^1,2, Shingo Fujisaki², Mitsushi J Ikemoto^1,2 独立行政法人産業技術総合研究所バイオメディカル研究部門分子複合医薬研究グループ¹, 東邦大学大学院理学研究科生物分子科学専攻² Biomedical Research Institute, AIST, Ibaraki, Japan¹, Graduate School of Science, Toho University, Chiba, Japan² Addicsin is a negative modulator of Na⁺-dependent neural glutamate transporter excitatory amino acid carrier 1. Addicsin is mainly localized at endoplasmic reticulum (ER) and has various physiological functions by forming hetero-complexes with many factors. As previously reported, we have identified Tomoregulin-1 (TR1) as a novel addicsin-associating factor by performing a yeast two-hybrid screen. TR1 is a type-I transmembrane protein containing two follistatin modules and an epidermal growth factor domain, and is involved in the Nodal and BMP signaling. TR1 bound directly to addicsin in vitro and in vivo and drastically changed its intracellular localization to the ER by overexpression of addicsin. However, physiological functions of addicsin remain largely unknown. Here, we show that addicsin regulates the cell surface expression level of TR1 and may participate in its post-translational modification. In COS7 cells, the biotinylation analysis demonstrated that the cell surface expression level of TR1 was significantly reduced to 40% by co-overexpression of addicsin with TR1. Furthermore, the molecular weight of TR1 at the plasma membrane slightly increased compared with that of TR1 at cytoplasm. Although the cell surface expression level of a TR1 deletion mutant lacking the region at amino acids of 228-266 containing an addicsin binding region (dTR1) significantly decreased to 10% compared with that of TR1, no increase in the molecular weight of dTR1 at plasma membrane was observed. As TR1 has several post-translational modification motifs, addicsin may regulate the post-translational modification of TR. At present, we have been investigating the post-translational modifications of TR1 at plasma membrane. We will further discuss the molecular mechanism of addicsin-mediated TR1 cell surface expression.
P2-1-41 マウスドーパミン作動性神経の初代培養系における大型有芯小胞からのドーパミン分泌とCAPS2の関係 Involvement of CAPS2 in dopamine release from large dense-core vesicles in mouse primary dopaminergic neurons ○井口大壽¹, 小林翔太¹, 定方哲史^2,3, 篠田陽^1,2, 古市貞一^1,2 ○Hirotoshi Iguchi¹, Shota Kobayashi¹, Tetsushi Sadakata^2,3, Yo Shinoda^1,2, Teiichi Furuichi^1,2 東京理科大・理工・応用生物科学¹, 科学技術振興機構/CREST², 群馬大学・先端科学研究指導者育成ユニット³ Dept. of Appl. Biol. Sci., Fac. of Sci. and Technol., Tokyo Univ. of Sci., Chiba, Japan¹, JST/CREST, Saitama, Japan², Adv. Sci. Res. Leaders Develop. Unit, Gumma Univ. Gumma, Japan³ One of the important proteins to control the release of neuropeptides and biogenic amines from large dense-core vesicle (LDCV) in neuronal/neuroendocrine cells is calcium-dependent activator protein for secretion (CAPS) family proteins. CAPS2, one of the two CAPS family proteins, has been shown to promote BDNF release and its knockout mice have impaired social interaction and increased anxiety. It is also known that CAPS2 is highly expressed in dopaminergic neurons of the midbrain substantia nigra (SN) and ventral tegmental area (VTA) playing an important role in brain function (such as voluntary movement, attention, cognition and reward predict). However, it's yet unclear how DA release from LDCVs, but not small synaptic vesicles, is regulated by CAPS2 proteins in the SN and the VTA neurons. To address this issue, we first established a method to prepare primary cultures of DA neurons in mouse system as previously reported in rat system with modification. Tyrosine hydroxylase (TH)-positive DA neurons were confirmed to endogenously express CAPS2 in the primary culture. We analyzed dynamics of DA release from cultured DA neurons prepared from wild-type and CAPS2 KO mice by utilizing the TH promoter-driven vesicular monoamine transporter 2 (VMAT2)-pHluorin fusion protein or Fluorescent False Neurotransmitter FFN511. In this presentation, we will discuss a role of CAPS2 in the kinetics of DA release from LDCVs.	P2-1-42 C1ドメインとC末の塩基性アミノ酸クラスターはジアシルグリセロールキナーゼβの突起伸長能及びbranchingに重要である Both C1 domain and a basic amino acid cluster at C-terminus are important for branching and neurite induction of diacylglycerol kinase β ○白井康仁¹, 加野拓也¹, 上月健², 上田修司¹, 山之上稔¹, 坂根郁夫³, 齋藤尚亮² ○Yasuhito Shirai¹, Takuya Kano¹, Takeshi Kouzuki², Shuji Ueda¹, Minoru Yamanoue¹, Fumio Sakane³, Naoaki Saito² 神戸大院・農¹, 神戸大学・バイオシグナル研², 千葉大院・理³ Grad. Sch. of Agricul. Sci., Kobe Univ.¹, Biosignal Res. Ctr., Kobe Univ.², Grad. sch. of Sci., Chiba Univ.³ Diacylglycerol kinase (DGK) is a lipid kinase which converts diacylglycerol (DG) to phosphatidic acid (PA). In other words, DGK plays an important role in signal transduction by regulating the balance between two signaling lipids, DG and PA. Among ten mammalian subtypes, DGKβ is enriched in neurons. We recently reported that DGKβ induces neurite branching, contributing to formation of neural network and higher brain function including memories. However, detail molecular mechanism of the neurite outgrowth is still unknown. Therefore, we constructed various mutants of DGKβ, and compared their enzyme activity, intracelullar localization, and ability to induce neurite in SHSY-5Y cells.Even when RVH domain and EF-hand motifs were deleted, the mutant, showed similar enzyme activity, plasma membrane localization, and induction of neurite outgrowth to those of wild type. In contrast, further deletion of C1 domain lost to plasma membrane localization and ability to induce neurites although it possessed equal enzyme activity to wild type. When 30 amino acids were deleted from the C-terminus, the mutants lost all of the enzyme activity, the plasma membrane localization and the ability to induce neurite outgrowth. These results indicate that it is necessary for the neurite induction of DGKβ to localize on the plasma membrane through having C1 domain and C-terminus. In addition, we also found that C-terminus region of DGKβ is indispensable for enzyme activity. Furthermore, C1A and C1B mutants. which have a mutation of cysteine residue in C1 A and C1 B domain respectively, and RK/E mutant, which has substitutions of arginin and lysine to glutamic acid in a cluster of basic amino acids at C-terminus, lost the plasma membrane localization and neurite extension ability, indicating that both entire structure of C1 domain and a C-terminal basic amino acids cluster were important for neurite extension of DGKβ.
P2-1-43 モータータンパク質ミオシンVIによる胚性腫瘍細胞株P19細胞の増殖抑制 Predominant inhibition of proliferation activity for self-replication by the motor protein myosin VI in pluripotent P19 cells ○高巳奇¹, 宝田剛志¹, 福森良¹, 米田幸雄¹ ○Miki Kou¹, Takeshi Takarada¹, Ryo Fukumori¹, Yukio Yoneda¹ 金沢大学大学院　医薬保健学総合研究科　創薬科学¹ Dept Pharmacal, Univ of Kanazawa, Ishikawa, Japan¹ In our pervious study, mRNA for the motor protein Myosin VI (Myo6) was selectively up-regulated in the hippocampus within 24 h after the extremely traumatic experience, water-immersion restraint stress, which occurred prior to a drastic but transient decrease in proliferation of neural progenitors in the dentate gyrus of adult mouse brains. In this study, we further investigated the role of Myo6 in both proliferation and differentiation in mouse embryonal carcinoma P19 cells endowed to proliferate for self-replication and differentiate into neurons and astroglia. Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type Hes5 amongst different bHLH proneural genes and cell cycle regulators examined without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the aggregate size and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. These results suggest that Myo6 may play a pivotal role in the mechanism underlying the suppression of adult neurogenesis relevant to hippocampal atrophy seen in patients with posttraumatic stress disorder.	P2-1-44 培養中枢神経細胞における神経活動依存的なNurr1 (Nr4a2) 核内受容体の発現調節 Activity-dependent regulation of the expression of Nr4a nuclear receptor in cultured central neurons ○徳岡宏文¹, 畑中貴之¹, 一瀬宏¹ ○Hirofumi Tokuoka¹, Takayuki Hatanaka¹, Hiroshi Ichinose¹ 東京工業大学大学院　生命理工学研究科分子生命科学専攻¹ Tokyo Institute of Technology, Tokyo¹ Nr4a family (nuclear receptor subfamily 4 group A), a subfamily of orphan nuclear receptors, is composed of NGFI-B (Nr4a1), Nurr1 (Nr4a2), and Nor1 (Nr4a3). Nurr1 is implicated to be involved in psychiatric disease, as Nurr1^+/- mice show behaviors potentially relevant to schizophrenia, and gene mutations in the Nurr1 allele were found in schizophrenia patients. We previously demonstrated that Nurr1 is required to maintain the expression of genes important for dopaminergic neurons in the adult mouse midbrain. However, those nuclear receptors are expressed in many other types of neurons. Notably, Nr4a genes are supposed to be an immediate early gene, raising a possibility that Nr4a genes may play a role in neural activity-dependent modulation of neural functions. In this study, we investigated if the expression of Nurr1 is regulated by neural activity using cultured neurons of mouse hippocampus, or subiculum and entorhinal cortex. At resting condition, Nurr1 is expressed in only minority of neurons, with low expression level. Forskolin induced robust expression of Nurr1 in a few hours as examined by immunocytochemistry. Raising neural network activity by bicuculline and high K⁺ increased both the level of Nurr1 expression and the number of Nurr1-positive neurons. This enhancement of Nurr1 expression was inhibited by cadmium chloride, a blocker of voltage-dependent calcium channels. Meanwhile, blockade of neural activity by tetrodotoxin reduced the Nurr1 expression level. These results suggest that Nurr1 expression is regulated by neural activity, through voltage-dependent calcium channels.
P2-1-45 プルキンエ細胞における活動依存的な小胞体内腔Ca²⁺動態の可視化解析 Imaging of activity-dependent Ca²⁺ dynamics in the endoplasmic reticulum of Purkinje cells ○大久保洋平¹, 鈴木純二¹, 金丸和典¹, 飯野正光¹ ○Yohei Okubo¹, Junji Suzuki¹, Kazunori Kanemaru¹, Masamitsu Iino¹ 東京大学大学院医学系研究科細胞分子薬理学¹ Dept Pharmacol, Univ of Tokyo, Tokyo¹ The endoplasmic reticulum (ER) is the major intracellular Ca²⁺ store. Although Ca²⁺ mobilization from the ER is considered to be involved in various physiological and pathophysiological functions in neurons, direct measurements of luminal Ca²⁺ dynamics in intact neurons have not been carried out. Recently, we developed a series of genetically-encoded Ca²⁺ indicators that are optimized for the detection of luminal Ca²⁺ dynamics in the intracellular organelles (calcium-targeted entrapped protein indicators; CEPIAs). Here we used one of these newly developed indicators to image spatiotemporal ER Ca²⁺ dynamics in response to synaptic inputs to Purkinje cells in cerebellar slice preparations. To our surprize, parallel fiber (PF) inputs induced an immediate increase in the ER Ca²⁺ concentration, which was followed by a large-amplitude decrease. The PF-induced ER Ca²⁺ dynamics were observed locally in dendritic shafts and spines in an input-specific manner. The PF-induced increase and decrease in the ER Ca²⁺ content were dependent on AMPA receptors and metabotropic glutamate receptors, respectively. Climbing fiber (CF) inputs induced a global increase in the ER Ca²⁺ concentration that was also dependent on AMPA receptors. ER Ca²⁺ concentrations showed relatively slow recovery after PF-induced Ca²⁺ release. This ER Ca²⁺ refilling would be mediated by both the lateral diffusion of luminal Ca²⁺ and the activity of sarco(endo)plasmic reticulum Ca²⁺ ATPase. The refilling rate was enhanced by CF-induced Ca²⁺ influx. Thus, we succeeded in imaging physiologically relevant ER Ca²⁺ dynamics in Purkinje cells, and our results indicate that the neuronal ER functions not only as a source of Ca²⁺ but as a sink for the depolarization-induced Ca²⁺ influx via the plasma membrane.	P2-1-46 GPCRに対するGタンパク質の挙動のSPRによる観察 G protein behavior to GPCR observed using SPR sensor ○河西奈保子¹, , 井上鈴代², 瀬山倫子², 岩崎弦², 堀内勉², 林勝義², 為近恵美², 鳥光慶一³, 住友弘二¹ ○Nahoko Kasai¹, Sannna Rauhamaki¹, Suzuyo Inoue², Michiko Seyama², Yuzuru Iwasaki², Tsutomu Horiuchi², Katsuyoshi Hayashi², Emi Tamechika², Keiichi Torimitsu³, Koji Sumitomo¹ NTT物性基礎研¹, NTTマイクロシステム研², 東北大学大学院工³ NTT Basic Research Labs, NTT Corp., Atsugi¹, NTT Microsystem Integration Labs², Tohoku Univ., Sendai³ G protein-coupled receptors (GPCRs) are members of a large membrane receptor family that locate on a cell surface membrane and transduce signals from outside the cell to activate internal signal pathways. GPCRs are involved in many diseases and are the target of 1/3 of the recent medical drugs. Moreover, the 2012 Nobel Prize in chemistry was awarded for understanding the GPCR function. GPCRs' conformations can be changed as a result of ligand binding to activate the associated G-protein by exchanging its α -subunit bound GDP for GTP. The GTP bound G-protein α can be released from β - and γ -subunits and affect intracellular pathways depending on the G-protein types. There have been many studies attempting to understand GPCR functions, however, signaling pathways for most GPCR families have yet to be defined, and a real-time examination of the several target proteins to GPCR is required. In this study, we examined the adsorption/desorption of molecules related to GPCR reactions using a surface plasmon resonance sensor system (SPR) in real time and in a label-free condition. A μ-type opioid receptor, whose signal transduction pathways are widely known, was used as a target GPCR. The μ-opioid receptor and G protein were isolated from rat cortex and prepared for SPR measurement. SPR was combined with an antibody-immobilized sensor array chip and microfluidics. The chip contained both sample and reference spots, which made it possible to realize a precise and highly sensitive measurement almost simutaneously. The SPR measurement enabled us to observe the different adsorption/desorption behavior of the G-protein α -subunit against μ-opioid receptors treated with either an agonist or an antagonist. Furthermore, we could observe G protein α release caused by excess GTP probably due to the exchange reaction of GDP to GTP. These results suggest that our SPR system is a powerful tool for examining the GPCR reaction, and we can expect it to be applied to other GPCR proteins.
P2-1-47 メタアンフェタミン感受性亢進マウスの海馬における転写調節の変化 A change in transcriptional regulation in the hippocampus of mice sensitized with methamphetamine ○車地曉生¹, 板坂典郎¹, 石渡小百合¹, 西川徹¹ ○Akeo Kurumaji¹, Michio Itasaka¹, Sayuri Ishiwata¹, Toru Nishikawa¹ 東京医科歯科大学大学院　精神行動医科学分野¹ Tokyo Med&Dent Univ Graduate School, Sect Psychiatry & Behav Sci¹ The repeated administration of methamphetamine (MAP) elicits a long-term augmentation in the behavioral response to a subsequent challenge of the drug. This sensitization has been considered to be a useful model of stimulant-induced psychosis and/or schizophrenia.Accumulating evidence suggests an important role of hippocampus in the pathophysiology. The aim of the present study was to investigate whether genes responsive to stress, e.g., Btg2, Fos and Dusp1, may contribute to the behavioral sensitization in the brain area of mice. Adult male mice (C57BL) were subcutaneously injected with 1.0 mg/kg of MAP or saline once daily for 10 days, and then the animals were treated with a challenge dose (0.24 mg/kg, s.c) of the drug or saline on day 24 or 25 of withdrawal following the repeated administration. To evaluate the behavioral effects of the challenge, the spontaneous vertical and horizontal movements including locomotion, rearing, and head movements were quantified using a Supermex instrument (Muromachi-kikai, Co., Ltd., Tokyo, Japan). The gene expression of the hippocampus was examined by a previously reported quantitative RT-PCR method (Kurumaji et al., 2011). An enhanced motor activity examined for 60min after the challenged drug was observed in mice that received repeated treatment with MAP compared to those treated with saline. In addition, the MAP challenge induced a higher level in the mRNA of Dusp1 in the hippocampus of mice sensitized with MAP than in those of the saline-treated mice, while the challenge similarly produced a significant increase in the level of mRNAs of other genes, i.e., Fos and Btg2, in both of the repeatedly treated groups. Hence, the present study suggests the differential transcriptional regulation by the challenged MAP of hippocampal Dusp1 in the behaviorally sensitized animals. Dusp1 signaling in the hippocampus may be involved in a regulating network underlying the activated MAP sensitivity.	P2-1-48 スパインにおけるArf6活性化制御因子EFA6Aとsorting nexin-1との相互作用 Ultrastructural localization of EFA6A , a gunanine nucleotide exchange factor for Arf6, and its interaction with sorting nexin-1 at the dendritic spine ○阪上洋行¹, 福島大輔¹, 深谷昌弘¹ ○Hiroyuki Sakagami¹, Daisuke Fukushima¹, Masahiro Fukaya¹ 北里大学医学部解剖学¹ Department of Anatomy, Kitasato University School of Medicine, Kanagawa, Japan¹ EFA6A is a specific guanine nucleotide exchange factors (GEF) for Arf6, a small GTPase that regulates endocytic membrane trafficking and remodeling of the actin cytoskeleton. Recent evidence indicates the functional importance of the EFA6A-Arf6 pathway in the formation and maintenance of dendrites and dendritic spines. To understand the molecular network of the EFA6A-Arf6 pathway in the neuron, we first examined the ultrastructural localization of EFA6A by immunoelectron microscopic analyses. In the hippocampus, immunoreactive signals for EFA6A were distributed in dendritic fields as numerous puncta, which partially overlapped with phalloidin and PSD-95. Pre-embedding immunoelectron microscopy showed the predominant localization of EFA6A in dendritic spines of hippocampal CA1 neurons. In addition, more than 20% of all silver particles for EFA6A were associated with intracellular membranous structures. In dendritic spines, post-embedding immunoelectron microscopy showed the accumulation of gold particles for EFA6A just beneath the excitatory postsynaptic density (PSD) in addition to inside the PSD. Using a yeast two-hybrid screening, we identified sorting nexin 1 (SNX1), a member of the retromer complex involved in protein sorting within the endocytic pathway, as a novel EFA6A-binding protein. Immunohistochemical analysis revealed the partial colocalization of EFA6A with SNX1 in dendritic spines. The present findings suggest that EFA6A may regulate endosomal trafficking regulates through the activation of Arf6 in the proximity of SNX1.

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