Poster

Poster 17 Axons, Trafficking ポスター 17 軸索・細胞内輸送

P17-1 A microtubule mediated model for the differential branch regulation of axonal arbor 軸索枝間の競合をもたらす微小管制御モデル Imanaka Chiaki（今中千秋）¹，葛西真里菜¹，島田聡史⁴，小西慶幸^2,3 ¹Department of Applied Chemistry and Biotechnology,Facl.Eng.,Univ.Fukui ²Life Science Innovation Center,University of Fukui ³Facl.Eng.,Univ.Fukui ⁴Department of Human and Artificial Intelligent Systems,Facl.Eng.,Univ.Fukui In the reconstruction of branched axonal pattern, competition between adjacent branches occurs via unknown mechanisms. We reported that retraction of branches was inhibited selectively in longer branches compared with adjacent shorter branches because of higher efficiency of axonal transport (Seno et al., 2016). Although the difference in microtubule stability between branches is supposed to contribute to branch-dependent axonal transport, it is unknown what kind of mechanism causes this difference. We carried out the simulation using a model in which the stability (lifetime) of the microtubule simply depends on the protrusion length, and found that the lifetime difference of the microtubules depending on the branch length occurred even in the vicinity of the branch point as observed in the actual axon. Furthermore, it was predicted that stabilization of the whole microtubule increases the difference of microtubule lifetime between branches. Indeed, difference of tyrosinated/de-tyrosinated tubulin signal ratio, indicator of the stability of microtubules, between branches was increased by stabilizing microtubules with taxol.Collectively our study establishes a novel model for axonal branch-dependent microtubule regulation and revealed the mechanism of positive feedback exists in the long branch. It is also suggested that the competition between branches may depend on the stability of microtubules throughout the axons. It would be possible to examine by analyzing the relationship between axonal transport and morphological changes of axonal branches.		P17-2 Distribution regularity of mitochondria in axons 軸索内におけるミトコンドリアの分布規則性 Hori Ikuma（堀生実）¹，松本望¹，三宅誠司³，小西慶幸^1,2 ¹Facl.Eng.,Univ.Fukui ²Life Science Innovation Center,University of Fukui ³Department of Ophthalmology, Faculty of Medical Sciences, University of Fukui Mitochondria, synthesizing ATP consumed as energy in the body, is present in most cells including neurons. In neurons, mitochondria have important functions for branching and elongation of axons as well as other neural functions. In axons, mitochondria are transported by axonal transport via microtubules and motor protein. On the other hand, stationary mitochondria stopping transport are also widely distributed and are considered to be involved in axon branching and elongation. The systems regulating and distribution of stationary mitochondria remain to be elucidated.In this study, we investigated the distribution patterns of stationary mitochondria. First, based on the knowledge that two thirds of the mitochondria in the axon are stationary mitochondria, the number of mitochondria contained in each compartment of axons was measured by using cerebellar granule nervous. We made two evaluations based on this. Firstly, we compared the probability distribution of mitochondria with the Poisson distribution. The probability distribution did not follow the Poisson distribution, suggesting that it is not a random distribution. Secondly, by calculating the distribution concentration index called I_δ index, it was suggested that there is a tendency of equal distribution. In addition, we performed a similar analysis using retinal ganglion cells. The results showed a tendency of equal distribution similarly to cerebellar granule cells.In summary, we found a tendency for the stationary mitochondria to be evenly distributed within the axons of two different types of neurons, suggesting the possibility that there is a universal system controlling the distribution of stationary mitochondria.

P17-3 Mechanism of axon retraction with 3-nitro tyrosine 3-ニトロチロシンによる軸索退縮のメカニズム Hirai Masahiro（平井真大）¹，葛西祐介¹，坪田雅英¹，小西慶幸^1,2 ¹Facl.Eng.,Univ.Fukui ²Life Science Innovation Center,Univ.Fukui Reactive oxygen is a major cause of age-dependent disease. It can reacts with nitric oxide (NO) to generate peroxynitrite, and produces tyrosine 3-nitrotyrosine (3-NT). Nitration of proteins is considered to be associated with various neurodegenerative diseases as a target of oxidative stress. 3-NT has been shown to cause apoptosis in dopamine producing cells depending on the dopamine synthesis system. Furthermore, even in non-dopamine producing neurons, 3-NT is taken in α-tubulin, but it is not well understood how this affects neuronal functions. Therefore, we investigated the effect of 3-NT on cerebellar granule cells which are non-dopamine producing cells. We found that the length of the axon is shortened depending on the concentration of 3-NT. Administrating of 3-NT locally to axons by using microfluidic device caused axonal shorting, suggested that 3-NT directly act, on the axons. By western blotting it was confirmed that 3-NT was specifically incorporated into α-tubulin. We hypothesized three possible mechanisms for the 3-NT dependent axonal shortening; destabilization of microtubules, activation of apoptotic pathways, and disruption of axonal transport. To validate these hypotheses, we first analyzed acetylation and Δ tyrosylation of tubulins, indicators of microtubule stability. We found no significant difference in neurons administered with 3-NT compared with control neurons. Similarly, no significant difference was observed in the amount of cleaved Caspase 3, which is induced by the apoptotic pathways. Thus, it was suggested that 3-NT shortens axons without destabilizing microtubules nor activating the apoptotic pathways.		P17-4 The Molecular Mechanism Regulating Axonal Localization of the Secretion-Related Protein CAPS2 分泌関連タンパク質CAPS2の軸索局在を制御する分子機構 Shimizu Kazuki（清水一貴）¹，川本一夫¹，定方哲史²，佐野良威¹，古市貞一¹ ¹Dept. of Appl. Biol. Sci., Fac. of Sci. and Tech., Tokyo Univ. of Sci. ²Adv. Scie. Res. Leaders Dev. Unit., Gunma Univ. Graduate School of Med. CAPS2 (Ca2+-dependent activator protein for secretion 2) regulates exocytosis of secretory vesicles containing the brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) both of which are critical for neuronal development, function and survival. There are several alternative splicing subtypes in CAPS2. We previously reported an aberrant increase in expression of a rare CAPS2 subtype lacking exon 3 (deletion of exon3, "dex3") in some autistic patients. Unlike the major type harboring exon 3 ("CAPS2"), dex3 subtype expressed in cultured neurons does not localize to axons, resulting in a deficit in axonal release of BDNF. Genetically-modified mice expressing only dex3 exhibit impairments in synapse morphology and function, and autistic-like social behavior. In the present study, to clarify the molecular mechanism underlying axonal localization of CAPS2, we carried out the imaging analysis of fluorescent protein tagged CAPS2 and dex3 expressed in primary-cultured neurons. We prepared various CAPS2 deletion mutants fused to the red fluorescent protein tdTomato and the Flag tag. Our imaging data confirmed that dex3 lacks the ability of axonal distribution, suggesting the presence of a critical region for axonal transport in which at least the amino-acid residues encoded in exon3 are involved. We also performed a search for molecules acting on the axonal localization of CAPS2. Protein extracts prepared from cerebella of WT and dex3 mice were analyzed by coimmunoprecipitation assay with anti-CAPS2 antibody followed by SDS-PAGE and silver staining. We have found difference in protein banding pattern between WT and dex3 mice up to now, although we have to confirm it by further analysis.

P17-5 Effect of Chemical chaperone on ER-Golgi SNARE expression and Aβ peptide production in neuronal cells 神経細胞におけるER-Golgi SNAREの発現とAβペプチド合成に対するケミカルシャペロンの効果 Suga Kei（須賀圭）^1,2，山本幸子¹，西野将史^1,2，寺尾安生²，赤川公朗²，丑丸真¹ ¹Dept. Chem. Kyorin Univ. Sch. of Med. ²Dept. Cell Phys. Kyorin Univ. Sch. of Med. Endoplasmic reticulum (ER) stress and caspase3-dependent apoptosis have been implicated in neurodegenerative diseases such as Alzheimer’s disease. We have been focusing on the neuronal function of ER-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs). We previously showed that ER stress upregulates de novo synthesis of ER-Golgi SNAREs Syntaxin5 (Syx5) in Neuroblastoma-Glioma hybrid cell line NG108-15 (Exp. Cell Res. , 2015) and in rat hippocampal neurons (Neurosci. Lett., 2015). While ER stress caused the reduction of β-amyloid peptide (Aβ peptide), knockdown of Syx5 protein enhanced the secretion of Aβ. The reduction of Aβ secretion by ER stress was significantly suppressed by Syx5 knockdown. In addition, sustained ER stress promoted caspase3-dependent apoptosis followed by degradation of Syx5 proteins by activated caspase3 (Data in Brief, 2015, 2016). Recently, we investigated the effects of chemical compounds which modulate different sites in ER stress signaling on the expression of ER-Golgi SNAREs and the processing of βAPP. Furthermore, we reported that a chemical chaperone 4-phenylbutyrate (PBA) showed alleviation of caspase3-dependent apoptosis induced by ER stress in NG108-15 cells. However, we did not know whether the observed effects of PBA in NG108-15 cells are also applicable to primary cultured neurons. In order to know the protective mechanism of the ER stress modulator on the processing of βAPP, the amount of secreted Aβ, intracellular Aβ, and the ratio of Aβ peptides, were examined in neuronal cells. We will present the data for the effect of PBA on the expression of ER-Golgi SNAREs and the production of Aβ peptides under ER stress condition in neuronal cells and would like to discuss the results.		P17-6 SKF-10047, a prototype Sigma-1 receptor agonist, facilitated the membrane trafficking and uptake activity of serotonin transporter and its mutant by the mechanism independent of Sigma-1receptor シグマ1受容体アゴニストであるSKF-10047はシグマ1受容体を介さない機序でセロトニントランスポーターとその変異体の膜輸送と活性を促進する Sakai Norio（酒井規雄），浅野昌也，横田智香，臼杵直人，山本光，秀和泉，田中茂 Dept of Pharmacol Neurosci, Grad Sch of Biomed&Health Sci The function of serotonin transporter (SERT) is regulated via its membrane trafficking. Our previous studies have revealed that the SERT C-terminal deletion mutant (SERTΔCT) showed the robust decrease in its membrane trafficking and was retained at endoplasmic reticulum (ER), suggesting that SERTΔCT would be an unfolded protein. The accumulation of unfolded membrane protein in ER could be the cause of ER stress. It has been reported that the Sigma-1receptor (SigR1) attenuates the ER stress via its chaperone activity. In order to find the drugs that accelerate the membrane trafficking and relieve the ER stress, we investigates the effects of SKF-10047, a prototype SigR1 agonist, on the membrane trafficking and uptake activity of SERT and SERTΔCT expressed in COS-7 cells. The 24hr-treatment of SKF-10047 (> 200 μM) accelerated the SERT membrane trafficking, and robustly upregulated the activity of SERTΔCT. Interestingly, these effects of SKF-10047 on SERT functions also remained in the cells in which the SigR1 expression was knocked-down by shRNA, suggesting that SKF-10047 exerted these effects on SERT via a mechanism independent of SigR1. The cDNA array study showed several candidate genes, which is involved in the mechanism of SKF-10047 actions. Among them, syntaxin 3, a cellular receptor for transport vesicles which participate in exocytosis, was significantly upregulated by the treatment of SKF-10047 (> 200 μM) for 48 hours. These results suggest that SKF-10047 would be a candidate drug for the relief of ER stress, which caused by the accumulation of unfolded membrane proteins.