シンポジウム9　シナプス可塑性に関わる分子基盤への多角的アプローチ

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シンポジウム9 シナプス可塑性に関わる分子基盤への多角的アプローチ

S9-1 スパイン局在転写調節因子スパイカーはドレブリンと結合することによってスパインで安定化し機能する山崎博幸，白尾智明群馬大学大学院医学系研究科神経薬理学 Dendritic spines are small, actin-rich protrusions on dendrites. The actin cytoskeleton is a central player in dendritic spine morphogenesis. Drebrin is an actin-binding protein, which is thought to initiate the spine formation through forming unique drebrin-actin complex at postsynaptic sites. In this study, we have isolated a drebrin-binding protein, spikar. Spikar was localized mainly in nuclei and dendritic spines in cultured neurons. Using RNA interference（RNAi）approaches, we found that spikar knockdown resulted in decrease of dendritic spines and filopodia. In addition, electrophysiological study demonstrated that spikar knockdown resulted in a decrease of mEPSC frequency. These data suggested that spikar is involved in the spine formation. Rescue and overexpression experiment using mutated-NLS spikar（mNLS-spikar）showed that extranuclear spikar is involved in spine formation by modulating de novo spine formation and retraction of existing spines. We then examined whether the spine-formation activity of mNLS-spikar would require the presence of drebrin. We cotransfected mNLS-spikar and drebrin-shRNA into cultured neurons and measured the numbers of spines and filopodia. The drebrin knockdown abolished the mNLS-spikar-induced increase in spine and filopodium density. The inhibition of mNLS-spikar function by drebrin knockdown was rescued by the co-expression of an RNAi-resistant drebrin mutant. We further employed fluorescence recovery after photobleaching（FRAP）to evaluate the stability of spikar in dendritic spines. In drebrin-knockdown neurons, the stable fraction of mNLS-spikar was smaller than that of control neurons. These data indicate that extranuclear spikar facilitates spine and filopodium formation depending on drebrin.		S9-2 シナプス可塑性を支えるAMPA受容体の細胞内輸送機構松田信爾^1,2 慶応義塾大学医学部生理学教室¹，科学技術振興機構　さきがけ² Synaptic plasticity is thought to be one of the underlying mechanisms for learning and memory. Long-term depression（LTD）is one of the most well studied synaptic plasticity. It has been clarified that the endocytosis of AMPA type glutamate receptor（AMPA receptor）is the molecular basis for LTD induction. However, it is still unclear how the endocytosis of AMPA receptor is regulated. Recently, we clarified that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, the interaction between stargazin and AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Therefore, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors-transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes-through its sequential binding to AP-2 and AP-3A.

S9-3 活動依存的遺伝子Arcのシナプス調節と記憶制御機構奥野浩行¹，遠藤俊裕²，金亮²，石井雄一郎²，湊原圭一郎¹，荒木杏菜¹，崎村建司^3,4，尾藤晴彦^2,4 京都大院・医・メディカルイノベーションセンター¹，東京大・医・神経生化学²，新潟大・脳研・細胞神経生物学³，JST・クレスト⁴ Immediate-early genes（IEGs）, a class of genes that are rapidly and transiently up-regulated by extracellular stimuli, are dynamically regulated by synaptic activity in neurons. Arc（also known as Arg3.1）is one of such highly responsive, neuron-specific IEGs whose expression is tightly coupled with cognitive information processing in the brain. Arc plays critical roles in AMPA receptor（AMPA-R）trafficking, synaptic plasticity, experience-dependent cortical reorganization, and long-term memory formation. It remains, however, paradoxical why Arc, which is upregulated by strong synaptic activity that induces persistent forms of plasticity and learning, also critically contributes to weakening synapses by promoting AMPA-R endocytosis during various forms of synaptic and homeostatic plasticity. Here we show a preferred targeting of Arc protein to inactive synapses. We found that this synaptic targeting of Arc was regulated by dynamic interaction between Arc and the inactive form of the beta isoform of CaMKII（CaMKIIβ）. We also found that the degree of synaptic Arc accumulation was more sustained during a period of inactivity following strong induction, and correlated with removal of surface GluA1 from individual synapses. Furthermore, lack of the Arc gene in vivo caused cognitive deficits. Taken together, our findings suggest that the plasticity-related gene Arc plays a role in a novel"inverse"synaptic tagging process that facilitates the clearance of surface AMPA-R and contributes to regulate cognitive functions.		S9-4 連合性長期記憶への多角的アプローチ石川保幸前橋工科大学・システム生体工学科 Activity-dependent synaptic plasticity is widely accepted to provide a cellular basis for learning and memory. Synaptic associativity could be involved in activity-dependent synaptic plasticity, because it distinguishes between local mechanisms of synaptic tags and cell-wide mechanisms that are responsible for the synthesis of plasticity-related proteins. An attractive hypothesis for synapse specificity of long-term memory is synaptic tagging：synaptic activity generates a tag, which captures the PRPs（plasticity-related proteins）derived outside of synapses. Here we show that neuropsin, a plasticity-related extracellular protease, is engaged in synaptic tag setting for late associativity in vitro and behavioral tag setting for LTM in vivo. First, we investigated about neuropsin dependent late associativity using electrophysiological technique. Neuropsin was involved in synaptic tagging during LTP at basal and apical dendritic inputs. Moreover, neuropsin is involved in synaptic tagging and cross-tagging during LTP at apical dendritic inputs via integrin β1 and calcium/calmodulin-dependent protein kinase II signalling. Furthermore we next addressed whether a neuropsin was involved in behavioral tag setting. Behaviorally, weak training, which induces short-term memory（STM）but not LTM, can be consolidated into LTM by exposing animals to novel but not familiar environment 1 h before training. We found that neuropsin deficient mouse impaired such transformation short-term into long-term memory. These results suggest neuropsin as a tag setting for synaptic plasticity and memory.

S9-5 Phldb2はLTD誘導後のシナプスでのAMPA受容体のエンドサイトーシスを制御する謝敏かく^1,2,3，八木秀司⁴，猪口徳一⁵，岡雄一郎⁵，黒田一樹^1,3，柚崎通介⁶，松田信爾⁶，石川保幸⁷，佐藤真^1,2,5,8 福井大・医・組織細胞形態学・神経科学領域¹，福井大・子どものこころの発達研究センター²，福井大・生命科学複合研究教育センター³，兵庫医科大・医・解剖学・神経科学部門⁴，大阪大学大学院・医・解剖学講座⁵，慶應大・医・神経生理⁶，前橋工科大・システム生体工学⁷，大阪大学大学院・連合小児発達学研究科・こころの発達神経科学講座⁸ Long-term depression（LTD）is an essential mechanism underlying plasticity and has been intensively studied with hippocampal neurons because of their involvement in learning and memory formation. Phosphatidylinositol 3,4,5-triphosphate（PIP₃）plays important roles in a diverse range of neuronal functions. Although PIP3 is concentrated at the dendritic spines, and is a crucial for maintaining AMPAR clustering during long-term potentiation（LTP）, how it is regulated LTD is not completely elucidated. Here, we show pleckstrin homology-like domain family B member 2（Phldb2）, which pleckstrin homology（PH）domain is highly sensitive to PIP₃, was expressed in the hippocampus. The PI3K inhibitor induces a decrease in number of spines with Phldb2-positive head, hindering fast Phldb2 entry into spines. Moreover, the Phldb2 interacts with GluR2 and is required for the accumulation of GluR2 in the spine. Next, we asked whether or not Phldb2 is involved in synaptic plasticity. We observed that NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced long-term depression were blocked in hippocampal neurons of the Phldb2-knockout mice. Therefore, it is likely that Phldb2 plays an important role for the synaptic plasticity.