若手道場口演（Wakate Dojo）

Wakate Dojo Neural Excitability, Synapse and Glia 2 若手道場口演神経興奮性・シナプス・グリア2

7月26日（金）8:30～8:50　第10会場（万代島ビル　6F　会議室） 2WD10am1-1 ジスキネジア関連分子PRRT2のシナプス調節における役割 Daisuke Hatta（八田大典）¹，Daiki Nagai（永井大己）²，Kentaro Nagata（永田健太郎）¹，Yuma Hori（堀祐真）¹，Yuka Jiuchi（地内友香）¹，Kaori Watanabe（渡辺かおり）¹，Akira Kinoshita（木下晃）³，Koh-ichiro Yoshiura（吉浦孝一郎）³，Naohiro Kurotaki（黒滝直弘）⁴，Keiro Shirotani（城谷圭朗）^1,2，Nobuhisa Iwata（岩田修永）^1,2 ¹長崎大院医歯薬・ゲノム創薬学 ²長崎大・薬 ³長崎大・原研遺伝 ⁴香川大・医・臨床心理学 Mutations of PRRT2 (proline-rich transmembrane protein 2) cause various neurological disorders represented by PKD (paroxysmal kinesigenic dyskinesia), which is characterized by short and frequent attacks of hyperkinetic movements precipitated in a voluntary movement-dependent manner. Since most of PRRT2 mutations found in PKD patients lead to a premature stop codon, PKD is believed to be caused by loss-of-function of PRRT2; however, we do not exclude gain-of-abnormal-function. Although there are a few reports showing involvement of PRRT2 in regulation of synaptic vesicles, its definitive pathophysiological functions are still unclear. Here, we generated Prrt2 knock-in (KI) mice which have premature stop codon at the position corresponding to the most frequent PKD-associated PRRT2 mutation (c.649dupC), and investigated expression of Prrt2 mutant mRNA and protein. We found that the mutant mRNA is reduced by approximately 80% in the brain of Prrt2 KI mice compared to wild-type mice, which decrease the mutant protein level below detection threshold, indicating that PKD might be caused by loss-of-function of PRRT2. To explore molecules associated functionally with PRRT2, we examined Prrt2 KI-induced changes in expression levels of representative 50 synaptic proteins in various brain regions (cerebellum, neocortex, basal ganglia and hippocampus). We found that levels of 11 proteins are decreased selectively in the basal ganglia and the neocortices, suggesting that the cortico-basal ganglia motor loop might be the most susceptible neural circuit to loss of PRRT2, in which the neuronal hyperactivity cause PKD seizure. Among the proteins decreased by Prrt2 mutation, synaptogyrin 1 and synaptogyrin 3 (Syngr1/3) were reduced drastically, which are reported as synaptic vesicle-associated proteins involved in inhibitory modulation of synaptic vesicle release and activation of dopamine transporter. Thus, PRRT2 might regulate neurotransmitter release and dopamine reuptake by interacting with the protein. We are currently investigating a detailed relationship between PRRT2 and the proteins.		7月26日（金）8:50～9:10　第10会場（万代島ビル　6F　会議室） 2WD10am1-2 アミロイドβ毒性に対する海馬神経ネットワークの恒常性維持機構 Hiroki Ishikawa（石川裕貴），Tetsuya Hori（堀哲也），Naoto Saitoh（齋藤直人）同志社大院生命医科学 Alzheimer's disease (AD) is characterized by the accumulation of the amyloid-β (Aβ) peptide within the extracellular space of the brain. Aβ may exert its neurotoxic effects via multiple mechanisms and in particular through degradation of excitatory synaptic transmission associated with impaired synaptic plasticity. However, homeostatic plasticity might assure neuronal network stability against Aβ accumulation because AD has long precritical period in spite of the progressive accumulation. We focus on the presymptomatic mechanism which must be the sources of the ideas for the prevention medicine of AD. In this study, we applied 10 nM Aβ42 to cultured hippocampal neurons at 20 day in vitro (DIV) and examined chronic toxicity of Aβ42 on the neural network activity from 20 DIV to 28 DIV. First, we analyzed the pyramidal cells firing rate by calcium imaging using CaMKIIα-GCaMP6f. The Firing rate was maintained at 36°C in spite of the presence of Aβ42, whereas it was transiently 24 DIV reduced by Aβ42 at 28°C. These results might indicate that Aβ42 has toxic effect to the firing rate, but homeostatic plasticity recovers from the damage under the cultured condition. Second, we focused on the number of synapses and expression level of voltage-gated Na channel 1.6 (Nav1.6). The number of excitatory synapses increased by 50% at 24 DIV. The result might suggest that neural network attempted to cancel the Aβ42 toxicity by the amount of the excitatory synapse. On the other hand, the expression level of Nav1.6 at axon initial segments didn't change. It might suggest that Aβ42 has no significant effect on action potential generation. Considering that the firing rate was not changed at 36°C, it was expected that Aβ42 caused serious damage to synaptic transmission in the initial phase. Third, we purified total RNA from cultured hippocampal neurons at 24 DIV, and performed microarray analysis. As a result, we identified the upregulation of oxidative phosphorylation-related genes. Their genes might try to recover Aβ42-induced mitochondrial dysfunction. In summary, our results indicate that neural network resists Aβ toxicity by increasing the number of excitatory synapse and upregulation of mitochondrial function to keep the network activity.

7月26日（金）9:10～9:30　第10会場（万代島ビル　6F　会議室） 2WD10am1-3 ペンチレンテトラゾール誘発性キンドリングモデルにおけるてんかん発生にともなうAMPA受容体のin vivo PETイメージング Yusuke Shibata（柴田裕介），Takuya Takahashi（高橋琢哉），Tomoyuki Miyazaki（宮崎智之），Chinatsu Ikeda（池田千夏），Waki Nakajima（中島和希），Mai Hatano（波多野真衣）横浜市大院医生理 The AMPA receptors(AMPARs) is a glutamate receptor responsible for excitatory neurotransmission of the central nervous system. Recent studies have reported abnormal quantitative changes in AMPARs in epilepsy. For the reason, epileptic drugs by antagonizing the AMPARs have been developed. However, since diversity exists in the quantitative variation of AMPARs in such epilepsy pathology, In our laboratory, we developed AMPARs PET probe and follow AMPARs localization over time in whole brain. Therefore, in this study, we investigated whether quantitative fluctuation of AMPARs associated with epileptogenesis can be detected by this PET medicine, and found that AMPARs therapy confirmed whether drugs are effective in identifying epilepsy. Pentylenetetrazole (PTZ) model as a drug induced epilepsy model in which increase of AMPARs was reported, and re-administered 5 weeks after drug withdrawal from occurrence of epileptogenesis to detect latency until seizure occurred. PET imaging was taken of the before occurrence of epileptogenesis, 3 weeks and 5 weeks. Furthermore, Talampanel, an antagonist of AMPARs was administered at the time of re-administration, and the effect was examined. Compared to the control, the latency to seizure was significantly shorter(p <0.05) in the PTZ. From PET imaging, PTZ significantly increased the amount of AMPARs in the striatum and thalamus compared with the control(P<0.05). Furthermore, Talampanel significantly suppressed the seizure latency in the PTZ group until the same time as the control group (p <0.05). in this results, it was shown that this PET drug is useful for discriminating patients effectively for therapeutic drugs targeting AMPA receptor in epilepsy.