若手道場口演（Wakate Dojo）

Wakate Dojo Development and Regeneration 4 若手道場口演発生・発達・再生4

7月26日（金）16:10～16:30　第10会場（万代島ビル　6F　会議室） 2WD10aa2-1 ショウジョウバエ視神経系における段階的な層・カラム特異的投射の分子機構 Hiroki Takechi（武智広樹），Satoko Hakeda (Suzuki)（羽毛田（鈴木）聡子），Takashi Suzuki（鈴木崇之）東工大院生命理工 The Drosophila visual system is a suitable model to study the layered and columnar structure of functional organization of neuronal connections. Axonal targeting of photoreceptor neuron R8 occurs in three steps during development. In the first step, during third instar larvae, R8 axons recognize its correct targeting column. In the second step, during early pupal stage, R8 axons keep distances with each other and stay at the medulla neuropil surface. In the third step, in mid pupal stage, R8 axons extend filopodia and target to the final layer M3. In this study, we manipulated the transmembrane protein Golden Goal (Gogo) and the seven-pass transmembrane cadherin Flamingo (Fmi) in R8 specific manner and found that Fmi regulates Gogo localization at the filopodia and functions cooperatively to recognize columns in the first step, while Gogo interferes with Fmi in filopodia extension in the second step. This bimodal behaviour of Gogo was achieved by phospho-status of Gogo cytoplasmic domain. Analyses using phospho- and dephospho- mimic Gogo transgenes revealed that dephosphorylated Gogo is required for recognizing columns, while phosphorylated Gogo suppresses filopodia extension through downstream effector Adducin (hts), and thereby keeps distance between the axons. The temporal switch between dephospho-Gogo and phospho-Gogo appears to be the key mechanisms for R8s to determine its three dimensional position inside the brain. We have some data that supports the idea that the Gogo phosphorylation is regulated by secretion of insulin signal from glial cells that coincides with the onset of the second step. These results suggest that insulin signal from glial cells serves as a bimodal switch to change the Gogo function to enable the formation of the columnar-layered structure of the visual system.		7月26日（金）16:30～16:50　第10会場（万代島ビル　6F　会議室） 2WD10aa2-2 慢性期SCIマウスの運動機能および軸索伸長に対するdiosgeninの作用 Aoi Nakano（中野葵），Chihiro Tohda（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 Motor dysfunction in spinal cord injury (SCI) is caused by disruption of descending tracts, and axons of interneurons involving propriospinal neurons. Reconnecting an upper neuron to a lower target neuron, and a motor neuron to skeletal muscle are primarily required for regaining function. Although several treatable options have been proposed in experimental levels, practical applications are hardly established. Especially after shifting to the chronic phase, axonal growth is more inhibited by several disturbance factors, suggesting potent axonal repairing activity is required in chronic SCI. However, successful axonal growth strategies that improve motor function in chronic phase are very few. We have focused on diosgenin as a prominent compound that extends and repairs axons. Previous our studies found that diosgenin extended axons in cultured cortical neurons and mouse brain (Tohda et al.,2012; Tohda et al.,2013). Therefore, this study aimed to investigate effects of diosgenin on the motor function in chronic phase SCI by directly administering diosgenin to the injured area. SCI mice were prepared by contusion injury at T11 cord using ddY mice (8-weeks old, female). Diosgenin was continuously applied to the injured cord level by an intrathecal canula jointed with an osmotic minipump. The diosgenin administration was started 31 days after injury. During the diosgenin treatment for 56 days, motor functions were evaluated once in 2days. Motor function was evaluated by the Basso Mouse Scale for Locomotion (BMS) and Toyama Mouse Score (TMS, Shigyo et al.,2014). Diosgenin-injected group showed significant improvement of hindlimb movements in BMS and TMS. Cage climbing test also indicated a positive effect of diosgenin on hindlimb movement. Considering the effectiveness of locally administered diosgenin, axonal growth is expected in the spinal cord neurons. Histochemical analyses are now on going. Diosgenin treatment significantly extended axons in primary cultured spinal cord neurons seeded on CSPG-coated dish. Previously, we identified 1,25D₃-MARRS as a direct binding protein of diosgenin in the brain. (Tohda et al.,2012; Tohda et al.,2013) However, it couldn't be denied that diosgenin might stimulate other molecules in the spinal cord. The direct binding protein of diosgenin in the spinal cord neurons are now investigated. Since pharmacological intervention in chronic SCI is very expected, diosgenin is probably a promising drug candidate.

7月26日（金）16:50～17:10　第10会場（万代島ビル　6F　会議室） 2WD10aa2-3 成長円錐内の高温部位はTRPV2メカノセンサー活性を増強し、軸索伸長を促進する Mai Oda（織田麻衣）¹，Shouta Sugio（杉尾翔太）¹，Yuko Iwata（岩田裕子）²，Kohki Okabe（岡部弘基）³，Katsuhiko Ono（小野勝彦）⁴，Yasuki Ishizaki（石崎泰樹）¹，Koji Shibasaki（柴崎貢志）¹ ¹群馬大院医分子細胞生物 ²国立循環器病センター ³東京大薬合成化学 ⁴京都府立医大神経発生 We previously reported that TRPV2 activation promoted axonal outgrowth through its activation by growth cone movements in developing sensory neurons (J. Neurosci. 2010, JPS 2016, FASEB J. 2017). Further, we analyzed the intracellular temperature distribution in DRG neurons by an intracellular temperature-imaging method. We found that hot regions (2-3°C high) were localized in the growth cones. Since it expected that the hot regions might accelerate the sensitivity of TRPV2 for mechanical stimuli, we analyzed the mechanical stimuli-evoked TRPV2 currents at various temperatures by a whole cell patch-clamp recording. We found that over 39°C condition dramatically enhances TRPV2-mechanosensor activity. These results suggested that the hot regions in growth cones contribute to accelerate axonal outgrowth through the TRPV2 sensitization. In this study, we measured the intracellular temperature changes every 2 seconds as a real-time imaging. We found that the hot regions in growth cones were not always constant, and dynamically changed depending on the movements of growth cones. We next examined the changes of axonal outgrowth, when the hot regions were abolished. We applied high concentration of fluorescent polymer thermometer (FPT) to DRG cultures, and removed all of hot regions. The WT axons in 39°C culture condition were significantly longer than those in 37°C condition. Compared with WT results, the FPT-treated axons in both 37°C and 39°C conditions were shorter. In addition, the axons, prepared from motor/sensory neuron-specific TRPV2 CKO mice, were not affected by the FTP treatment. These results indicate that hot regions in growth cones enhance sensitivity of TRPV2 against the mechanical stimuli generated by the growth cone movements. We previously reported that mechanical stimuli-evoked TRPV2 activation enhanced growth cone motility and actin dynamics to promote axonal outgrowth. In this study, we examined the F-actin clustering at 37°C or 39°C conditions, and found that 2°C heating promoted the clustering through TRPV2 activation. Taken together, we conclude that hot regions in growth cones enhance actin rearrangement through sensitization of TRPV2-mechanosensor activity, and promote axonal outgrowth.