Poster

Poster 10 Synapses ポスター 10 シナプス

P10-1 Effect of drebrin knockout on synaptic plasticity ドレブリンノックアウトを用いたシナプス可塑性の解析 Shirao Tomoaki（白尾智明）¹，関野祐子²，安田浩樹²，児島伸彦⁴ ¹Dept. of Neurobiology and Behavior, Gunma Univ. Grad. Sch. of Med., Maebashi, Japan ²Lab. of Human-Cell based Drug Discovery, Grad. Sch. of Pharm. Sci., Univ. of Tokyo, Tokyo, Japan ³Dept. of Physiol., Saga Univ. Sch. of Med., Saga, Japan ⁴Dept. of Life Sci., Faculty of Life Sci,, Toyo University, Itakura, Japan Drebrin increases F-actin stability by elongating the helical crossover of F-actin. Drebrin is highly concentrated in dendritic spines and exit it in an activity-dependent manner. Interestingly, drebrin is classified into two major alternatively spliced isoforms, embryonic-type and adult-type (drebrin E and drebrin A, respectively) Drebrin E is expressed in the embryonic and early postnatal brain and is replaced by the drebrin A during development. We have shown that drebrin A has a larger stable fraction than drebrin E in dendritic spines, suggesting that the drebrin isoform conversion from drebrin E to drebrin A results in the accumulation of drebrin-bound stable F-actin in dendritic spines. In this study, we analyzed the synaptic plasticity of drebrin A knockout (DAKO) mice and drebrin E and A double knockout (DXKO) mice using brain slice electrophysiological techniques. In DAKO mice, in which drebrin E is expressed throughout development and adulthood, long-term potentiation (LTP) is impaired in adult. On the other hand, low frequency stimulation induced robust metabotropic glutamate receptor 5 (mGluR5)-dependent long-term depression (LTD) in both developing and adult brains of DAKO. In DXKO mice, while it induced NMDAR-dependent LTD in the developing hippocampus in wild-type mice, it did not induce LTD in developing DXKO. Therefore, while drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from drebrin E to drebrin A prevents robust mGluR5-dependent LTD. Taken together, we propose drebrin is involved in LTP and LTD mechanism via regulating the F-actin stability in dendritic spines.		P10-2 Drebrin interacts with the kinase domain of CaMKIIβ and stabilizes CaMKIIα/β hetero-oligomer in the inner region of dendritic spines ドレブリンはCaMKIIβのキナーゼドメインに結合しCaMKIIα/βヘテロオリゴマーを樹状突起スパインの内部領域に安定化させる Yamazaki Hiroyuki（山崎博幸），白尾智明 Department of Neurobiology and Behabior, Gunma University School of Medicine Drebrin is a major F-actin binding protein in neurons, and it is localized in the center of dendritic spines. We have isolated CaMKIIβ as a drebrin-binding protein and investigated drebrin-CaMKIIβ interaction. Domain analysis by using co-immunoprecipitation assay showed that drebrin interacts with the kinase domain of CaMKIIβ but not the association domain. This indicates that drebrin binding with CaMKIIβ does not interfere in CaMKIIα/β oligomerization. CaMKIIβ is localized in dendritic spines more than in dendritic shaft. However, drebrin knockdown (KD) caused diffuse localization of CaMKIIβ in dendrites, it suggests that drebrin holds CaMKIIβ in dendritic spines. Moreover, the spine localization of CaMKIIα was also weakened in the drebrin-KD neurons. These results suggest that drebrin stabilizes CaMKIIα/β hetero-oligomer in dendritic spines by interacting with CaMKIIβ. To analyze the drebrin-CaMKIIβ interaction in more detail, we used super-resolution microscopy (N-STORM) to elucidate the localization in a dendritic spine on the nanometer scale. Our data showed that CaMKIIβ is widespread in a dendritic spine and partially co-localize with drebrin in the inner region. Additionally, NMDA receptor activation disrupted the colocalization of CaMKIIβ and drebrin, suggesting that active form of CaMKIIβ is free from drebrin. Previous study shows that CaMKIIβ detaches from F-actin by autophosphorylation. Together, we conclude that drebrin stabilizes CaMKIIβ in the inner region of dendritic spines as drebrin/CaMKII/F-actin tripartite complex, and this interaction is regulated by neuronal activity.

P10-3 Molecular mechanisms of proBDNF-induced dendritic spine shrinkage proBDNFによって誘導されるスパイン退縮の分子メカニズム Mizui Toshiyuki（水井利幸），小島正巳 Molecular and Cellular Pathology Research Team, Biomedical Research Institute, AIST, Osaka, Japan Dendritic spines are small actin-rich protrusions from neuronal dendrites that from the postsynaptic part of most excitatory synapses, and their morphological changes are associated with synaptic plasticity and mental disorders. However, the underlying mechanism is not fully understood. Brain-derived neurotrophic factor (BDNF), through the activation of tropomyosin-related kinase B (TrkB), modulates synaptic transmission and modulates some forms of synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD). We recently showed that the precursor form of BDNF (proBDNF) affected dendritic spines through a pan-neurotrophin receptor p75NTR and decreased the amplitude of synaptic transmission. In the present study, we analyzed the effect of proBDNF on the morphology of dendritic spines in Banker-style low-density culture of hippocampal neurons. We found that proBDNF significantly decreased the sizes of spine heads. On the other hand, there were no significant differences on cell viability, total dendritic protrusion density and the density of synapsin I antibody-labeled pre-synaptic terminals. Notably, proBDNF significantly reduced the intensity of rhodamine-conjugated phalloidin in the dendritic spines. Stabilization of actin filaments by jasplakinolide blocked the proBDNF-induced spine shrinkage. Furthermore, we quantified cofilin phosphorylation changes induced by proBDNF stimulation. We found that cofilin phosphorylation levels were down-regulated apparently. These results suggest that proBDNF induced shrinkage of dendritic spines are dependent on reorganization of actin cytoskeleton via cofilin phosphorylation.		P10-4 Chemico-genetic discovery of molecules underlying tripartite-synaptic function in vivo Takano Tetsuya¹，Baldwin Katherine²，Croucher Jamie¹，Uezu Akiyoshi¹，Bradshaw Tyler¹，Bindu Dhanesh¹，Soderblom Erik²，Shimogori Tomomi³，Eroglu Cagla¹，Soderling Scott¹ ¹The Department of Cell Biology, Duke University Medical School ²Duke Proteomics and Metabolomics Shared Resource and Duke Center for Genomic and Computational Biology, Duke University Medical School ³RIKEN, Brain Science Institute Astrocytes are the most abundant glial cells in the brain. Interactions of astrocytes with synapses via thin perisynaptic astrocytic processes, called tripartite synapses, are critical for proper synaptic connectivity and function. The number of astrocytes and the extent of their interactions with synapses have increased throughout evolution, indicating a close link between astrocytes and cognition. In contrast to neuronal synaptic structures, however, the molecular composition and mechanisms of the astrocytic perisynaptic structures are largely unknown. Here, we developed a new approach Split-BioID2 to identify the molecules and determine their functions at the tripartite synapse. Split-BioID was split BirA2 into inactive N-terminal (N-BirA) and C-terminal (C-BirA) halves and expressing them on the surface of cells (using a GPI-anchor). We found that BirA2 activity of Split-BioID2 was reconstituted extracellularly at close contacts between astrocytes and neurons in mice brain. By Split-BioID2, we obtained more than 100 proteins that might be responsible for tripartite-synapse functions.

P10-5 Branched sialylated N-glycans are accumulated in brain synaptosomes and interact with Siglec-H 脳シナプトソームに集積している枝分かれしたシアル酸化N結合型糖鎖はシグレックHと相互作用する Yoshimura Takeshi（吉村武）^1,2,3，半田麻衣^2,3，小西博之⁴，深田優子⁵，真鍋良幸⁶，田中克典^6,7，Bao Guang-ming⁶，木山博資⁴，深瀬浩一⁶，池中一裕^2,3 ¹Dept. of Child Development and Mol. Brain Sci., United Grad. Sch. of Child Development, Osaka Univ. ²Div. of Neurobiology and Bioinformatics, NIPS, NINS ³Dept. of Physiol. Sci., Sch. of Life Sci., SOKENDAI ⁴Dept. of Funct. Anatomy and Neurosci., Nagoya Univ. Grad. Sch. of Med. ⁵Div. of Membrane Physiol., NIPS, NINS ⁶Dept. of Chem., Grad. Sch. of Sci., Osaka Univ. ⁷Biofunct. Synthetic Chem. Lab., RIKEN Cluster for Pioneering Res. Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G’2F (Galβ1-3GlcNAcβ1-2Manα1-6[Galβ1-3GlcNAcβ1-2Manα1-3]Manβ1-4GlcNAcβ1-4[Fucα1-6]GlcNAc-). A2G’2F has a branched sialic acid structural feature, and branched sialylated A2G’2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G’2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G’2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G’2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G’2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.