神経化学教育口演セッション2　Neurochemistry Educational Oral Session2

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神経化学教育口演セッション2 Neurochemistry Educational Oral Session2

EO2-1 ラット傷害顔面神経のグリコーゲン合成竹澤洋亮¹，馬場麻人³，高坂新一²，中嶋一行^1,2 創価大学工学部生命情報工学科¹，国立精神・神経医療センター神経研究所²，奥羽大学歯学部口腔機能分子生物学講座生物学分野³ Transection of adult rat facial nerve causes transient functional down-regulation of motoneurons accompanying glial activation/proliferation. Although much energy is required in these processes, it is poorly understood how energy-supplying system is regulated in an axotomized facial nucleus. In this study, we investigated glycogen metabolism and its-related molecules in the ipsilateral nucleus. The amounts of glycogen in the injured nucleus significantly increased during days 5-14 after axotomy, and then returned to those as in control nucleus at 3 weeks post-transection. Immunohistochemical analysis using anti-glycogen antibody showed that the amounts of glycogen in the injured side at days 7 post-injury increased compared to those in the control side. The glycogen increased in the ipsilateral side was co-localized with injured motoneurons. Furthermore, glycogen metabolism-related proteins were analyzed. There was no significant difference in glycogen phosphorylase levels between contralateral and ipsilateral nucleus for 5 weeks after injury. In contrast, the ratio of phosphorylated glycogen synthase to total glycogen synthase was found to be markedly elevated at 7 days after axotomy, and gradually decreased to the control levels, suggesting that glycogen synthesis is temporally suppressed. Therefore, these results substantiated that the injured facial motoneurons regulate the glycogen synthesis through the activities of glycogen synthase for their survival and/or regeneration.		EO2-2 P2X7受容体依存型虚血耐性のメカニズム解析平山友里^1,2，松尾由理³，小泉修一² 山梨大学医学工学総合研究部リエゾンアカデミー¹，山梨大学医学工学総合研究部薬理学講座²，北里大学薬学部薬理学教室³ A brief ischemic episode（preconditioning；PC）induces resistance to a subsequent much severe ischemic injury. This phenomenon, known as ischemic tolerance, is an endogenous process that provides robust neuroprotection. We previously showed that astrocytes rather than neurons, by upregulating P2X7 receptors upon stimulation with PC, have an essential role for the induction of ischemic tolerance. However, the downstream signals after activation of P2X7 receptor remain unknown. Here we show hypoxia inducible factor-1α（HIF-1α）, a well-known master molecule that controls oxygen homeostasis, is important for astrocyte-mediated ischemic tolerance. Using a middle cerebral artery occlusion（MCAO）model in mice, we investigated the spatiotemporal pattern of HIF-1α protein expression by immunohistochemical staining. PC（15-min MCAO）evoked upregulation of HIF-1α in ipsilateral striatum, where astrocytic activation and ischemic tolerance were induced. The HIF-1α expression was mainly observed in neurons and astrocytes, but interestingly, neuronal HIF-1α showed an early and transient increase（from 1 day to 3 days after PC）, whereas astrocytic one revealed slow and long-lasting increase（from 3 days～）. Furthermore, HIF-1α in neurons was independent of P2X7 receptors but that in astrocytes was totally dependent on the receptors, which was demonstrated by P2X7 receptor knockout（P2X7-KO）mice. BzATP, a P2X7 receptor agonist, directly induced upregulation of HIF-1α in primary cultured astrocytes obtained from control but not in P2X7-KO mice. Taken together, these findings suggest that HIF-1α should have an indispensable role as a downstream signal of astrocyte-mediated ischemic tolerance, and be a master molecule for regulation of astrocytic phenotype．

EO2-3 気分制御における視床室傍核の関与加藤智朗¹，藤森典子¹，水上浩明²，小澤敬也²，加藤忠史¹ 理化学研究所脳科学総合研究センター精神疾患動態研究チーム¹，自治医科大学分子病態治療研究センター遺伝子治療研究部² The paraventricular thalamic nucleus（PVT）is classified as the epithalamus and receives many afferents containing important neurotransmitters and neuropeptides related to mood regulation such as 5-HT, noradrenaline, crh and orexin. In spite of the connections with mood and emotion related brain areas, the functional roles of this region in mood regulation remains unknown. To examine the importance of the PVT in mood regulation, we generated the mice in which synaptic transmission of PVT neurons were specifically impaired by tetanus toxin（Nakashiba et al. 2008）and observe wheel running activity for half a year to evaluate autonomous behavioral change. In the wheel running activity measurement, the female mice with impaired PVT neuronal transmission displayed spontaneous depression-like episodes with reduced activity lasting for about 2 weeks more frequently than control mice. These results indicate that the PVT might be included in the neural circuits to regulate mood and its proper function is necessary for mood stability.To investigate the characteristic features of the PVT, we performed gene expression analysis of the PVT together with other 3 brain regions implicated in mood regulation such as central amygdala nucleus（CeA）, prefrontal cortex（PFC）and paraventricular hypothalamic nucleus（PVN）. This analysis revealed that the genes related to mitochondrial function like oxidative phosphorylation and cholesterol biosynthesis were highly enriched in the PVT. These results provide insight into putative target of new mood stabilizers．		EO2-4 ニューロプシンによる抑制性神経ネットワークの特異的制御機構河田美穂，田村英紀，塩坂貞夫奈良先端科学技術大学院大学バイオサイエンス研究科神経機能科学研究室 Neuropsin is an important modulator of the nervous system. Recent our studies have reported that neuropsin proteolytically removes proteoglycan binding domain of neuregulin-1（NRG-1）, a risk factor of schizophrenia, allows binding its specific receptor, ErbB4, and leads to upregulation of GABAergic signaling. However, the physiological role of neuropsin-NRG-1 signaling in vivo remains unclear. To analyze this issue, we tried intraperitoneal injection of excite toxin, kainic acid to induce hyper active state of mouse brain and examined the expression of c-fos immunoreactivity in the hippocampus to compare neuropsin-deficient and wild type mice. Four hours after kainic acid injection, strong c-fos immunoreactivity was evident in ErbB4 positive GABAergic neurons in wild type mice. In contrast, only low levels of c-fos immunoreactivity was observed in ErbB4 positive GABAergic neurons of the neuropsin-defiecient mice. We also found that phosphorylation level of ErbB4 was significantly decreased in neuropsin-defecient mice than that in wild mice. We did not detect a significant difference in the expression of Nrg-1 mRNA between wild type and neuropsin-deficient mice. These results suggest that neuropsin has a critical role for excitation of ErbB4 positive neurons and consequently regulates the activity of GABAergic neurons.

EO2-5 脱髄時におけるカテプシンCとシスタチンFの遺伝子相互作用李佳益^1,2，Wisessmith Wilaiwan^1,2，清水崇弘²，田中謙二²，池中一裕² 総合研究大学院大学生命科学研究科生理科学専攻¹，生理学研究所　分子神経生理研究部門² Cystatin F, a papain-like lysosomal cysteine proteinase inhibitor, and its main substrate, Cathepsin C, have been demonstrated to be crucial factors in demyelinating diseases. It is found that the expression of Cathepsin C and Cystatin F are profoundly elevated and matched with ongoing demyelination/remyelination. However, their accurate functional role in demyelinating diseases is still unclear. To clarify their function in the pathological process of demyelination, we used a spontaneous chronic demyelination mouse model, named heterozygous PLP transgenic 4e（PLP4e/-）mice. Meanwhile, Flexible Accelerated-STOP-Tetracycline Operator Knockin（FAST）system was applied to up or down regulate Cathepsin C or Cystatin F gene expression. In situ hybridization revealed that in PLP4e/- mice, conditional knock down of Cystatin F gene in microglia lead to the down regulation of Cathepsin C mRNA levels. On the contrary, Cathepsin C gene expression is enhanced by up regulating Cystatin F in demyelinating condition. What is interesting is that, even not in demyelination background, Cystatin F knock down mice also show lower Cathepsin C expression level compared with wild type mice. It means that Cathepsin C gene and Cystatin F gene interact with each other through some unknown mechanism. It has important significance to clarify this mechanism for understanding their accurate roles in this disorder. Further study is needed to estimate the possible pathway and substrate of Cystatin F that influences Cathepsin C gene expression．		EO2-6 脱髄性疾患におけるカテプシンCとシスタチンFの役割 Wisessmith Wilaiwan¹，李佳益^1,2，清水崇弘¹，田中謙二³，池中一裕^1,2 生理学研究所　分子神経生理研究部門¹，総合研究大学院大学生命科学研究科生理科学専攻²，慶應義塾大学精神神経科学教室³ Multiple sclerosis（MS）is the most common demyelinating disease in the CNS. Studies on therapeutic attack on MS have been significantly increased in number in recent years. There are several animal models that can be used for the study for addressing and establishing MS treatment. We previously found CathepsinC（CatC）was also induced during chronic demyelinated lesions plp^4e/- mouse. Expression of its inhibitor Cystatin F（CysF）was induced during early phase of demyelination but ceased its expression in chronic demyelinated lesions. To study the role of CatC and CysF involvement in demyelinating disease, we generated mouse lines, in which CatC or CysF level can be manipulated by the use of FAST system. When remyelination is active in plp^4e/- mouse at 4 months, CysF Knockdown（CysFKD）mouse significantly worsened demyelination. Conversely, at the chronic demyelination phase（8 months）CatC knockdown（CatCKD）mouse diminished demyelination. CatC overexpression（CatCOE）mouse also gave a similar result with CysFKD mouse showing early demyelination. In MOG-EAE model, CatC expression was found in the chronic demyelinating region but not CysF. CatCKD showed delayed demyelination in EAE. Conversely, CatCOE in microglia significantly enhanced demyelination. This result is similar with that using chronic demyelination model, plp^4e- mouse. We have discovered CatC and CysF are strongly related with demyelinating diseases in both acute and chronic phase, with different cause and state of pathology. The conclusion of this study determined that balance of CatC and CysF expression plays an important role in chronic demyelinating diseases．