神経化学教育口演セッション1　Neurochemistry Educational Oral Session1

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神経化学教育口演セッション1 Neurochemistry Educational Oral Session1

EO1-1 ミエリン-軸索間相互作用に依存して発現変化するニューロン遺伝子の同定國澤和生^1,2，清水健史^1,2，長内康幸^1,2，小林憲太³，Bhat Manzoor A.⁴，池中一裕^1,2 総合研究大学院大学・生命科学・生理科学¹，生理学研究所・分子神経生理²，生理学研究所・ウイルスベクター開発室³，テキサス健康科学センター大学・生理学⁴ Myelinated axons are composed of four distinct regions：the node of Ranvier, paranode, juxtaparanode and internode, characterized by the presence of specific component proteins. The paranodal junction consists of neurofascin155, Caspr and Contactin. Although myelin sheaths are thought to have crucial effects on cognition and motor function, the significance of paranodal junction in exerting higher brain function and in neurological diseases remains unclear. In the previous study, PLPCre-ERT recombinase-inducible ablation of neurofascin155 in oligodendrocytes led to disorganization of paranodal junction and reduction in nerve conduction velocity. Firstly, we analyzed the myelinated optic nerve of the tamoxifen-injected mice by immunofluorescence for alterations in neurofascin155 and Caspr localization. We found that the number and length of these positive signals at the paranode were significantly decreased at 60 days after tamoxifen injection. To examine whether disruption of paranodal junction affects the neuronal gene expression, we prepared total RNA from the retina of oligodendrocyte specific conditional neurofascin155 knockout mice and proceeded to microarray analysis. Interestingly, we found that expression level of neuronal genes dramatically changed in response to the ablation of the paranodal junction, and some of the identified gene expression can be primarily observed in neurons by in situ hybridization. These results suggest that opening of paranodal region affect gene expression profile in their myelinating neurons．		EO1-2 細胞外vimentinはIGF1Rを介して軸索伸展を促進する執行美智子，久保山友晴，東田千尋富山大学、和漢医薬学総合研究所、神経機能学分野　Vimentin, an intermediate filament protein, is generally known as an intracellular protein related to cell adhesion and cell migration. Recently, we reported that vimentin secreted from astrocytes promoted axonal growth in cultured mouse cortical neurons. The effect of extracellular vimentin in neurons was a new finding, but its signal pathway was unrevealed. In this study, we aimed to clarify the signaling mechanism of extracellular vimentin that facilitates axonal growth. Primary cultured rat cortical neurons（SD, E17）were treated with human recombinant vimentin（1.75 μM）for 10 min, and then cell lysates from vehicle treated or vimentin-treated neurons were analysed with phosphoprotein arrays. As a result, IGF1R was identified as a highly phosphorylated molecule by vimentin stimulation. In primary cultured mouse cortical neurons（ddY, E14）, vimentin（1.75 μM）as well as IGF1（1.3 μM）facilitated axonal growth at 6 days after the treatment. Vimentin-elicited axonal growth was completely inhibited by pretreatment with an IGF1R inhibitor（IGF1-analog）and with a neutralizing antibody of IGF1R. Phosphorylation level of IGF1R by vimentin was peaked at 30 min after the stimulation. The level was higher than that by IGF1 treatment at a similar dose. ELISA experiment confirmed that IGF1 was not released from neurons into culture medium within 30 min after the vimentin stimulation. These results suggest that vimentin stimulates IGF1R, leading to axonal growth. Our results show the new signaling of axonal growth via IGF1R by extracellular vimentin. The finding may provide a novel strategy to induce axonal growth in neurodegenerative diseases.

EO1-3 扁桃体-中脳水道周囲灰白質回路は恐怖学習を制御し記憶の強さを決定する小澤貴明¹，Ycu Edgar A.¹，Ahmed Touqeer²，Kumar Ashwani¹，Koivumaa Jenny¹，Johansen Joshua P.¹ 理化学研究所脳科学総合研究センター記憶神経回路研究チーム¹，National University of Sciences and Technology, Atta-ur-Rahman School of Applied Biosciences, Islamabad, Pakistan² During auditory fear conditioning, animals learn that an auditory tone predicts an aversive outcome（electric shock）. Learning finally reaches a steady state at a certain memory strength（termed the learning asymptote）. Beyond this further training is ineffective at producing learning unless the strength of the aversive outcome is increased. We previously found a learning dependent reduction in shock-evoked responses in lateral nucleus of amygdala（LA）neurons. A similar reduction also occur in the periaqueductal gray（PAG）which relays aversive shock signals to LA. PAG receives inhibitory input from the central nucleus of amygdala（CeA）which is activated by tones after fear learning. We hypothesized that a CeA-PAG pathway provides negative feedback on PAG to negatively regulate shock-evoked responses in LA neurons and that this reduction sets fear learning asymptotes. We used a 4-day fear conditioning paradigm（training day 1, 3；testing day 2, 4）in which rats reached learning asymptote after initial training（day 1）as learning was not enhanced by overtraining（day 3）. We found that：（1）optogenetic inhibition of CeA-PAG afferents at learning asymptote disinhibited predicted shock-evoked responses in LA neurons and increased behavioral learning levels through the activation of LA. （2）optogenetic additive activation of shock-evoked responses in LA pyramidal cells during overtraining increased learning asymptotes. （3）optogenetic inhibition of shock-evoked LA neuronal activity during overtraining abolished the increase in fear learning induced by a higher shock intensity. These results demonstrate a circuit mechanism for setting adaptive levels of fear memory strength and shows that disrupting this circuit produces exaggerated fear learning．		EO1-4 大型動物の広範な中枢神経領域に遺伝子導入可能なアデノ随伴ウィルスベクターの開発飯田麻子¹，滝野直美¹，宮内ひとみ¹，柴田宏昭²，小野文子³，村松慎一¹ 自治医科大学大学院医学研究科臨床神経学¹，独立行政法人医薬基盤研究所　霊長類医科学研究センター²，一般社団法人予防衛生協会³ Viral vectors, in particular vectors derived from adeno-associated virus（AAV）, have been shown to be suitable for the transduction of neurons. Infusion of recombinant AAV vectors into target brain regions via stereotaxic surgery results in continuous, long-term transgene expression. However, for diseases that affect large areas of the central nervous system（CNS）, local injection of AAV vectors yields less-than-optimal results. Here, we generated pseudotype AAV9/3 vectors that express green fluorescent protein（GFP）under control of neuron-specific synapsin I promoter, and demonstrated efficient and selective neuronal transduction in broad areas of primate CNS. Six adult cynomolgus monkeys were administered the AAV9/3 vector（6.2×10¹² vector genome）via intrathecal injection. Four weeks after the injection, robust GFP expression was detected in the spinal cord of monkeys. The vectors efficiently transduced bilateral ventral horn motor neurons. Moreover, widespread and selective neuronal transduction without significant inflammation was observed in the cerebellum and cerebrum. Although the exact mechanism of how the vectors penetrate the pia or cross the cerebrospinal fluid-blood barrier and enter the parenchyma remains to be elucidated, the observed pattern of transduction in cerebellar granule cells around mossy fibers suggests that the vectors spread along the fibers. These results suggest that AAV9/3 vectors are effective vehicles for therapeutic gene delivery into the CNS for treating neurological diseases including Alzheimer disease and amyotrophic lateral sclerosis.