TOP一般演題(ポスター)
 
一般演題(ポスター)
Synapse II
2P-01
Subcellular localization of the SRF coactivators, MKL1 and MKL2, in the brain:possible involvement in dendritic spine morphology.
Satou Natsumi1,Ishibashi Yuta1,Ohtsuka Toshihisa2,Tobita Yamato2,Tsujii Junya1,Ishikawa Mitsuru1,Fukuchi Mamoru1,Tsuda Masaaki1,Tabuchi Akiko1
1Lab. of Mol. Neurobio., Grad. Sch. of Med. & Pharm. Sci., Univ. of Toyama,2Dep. of Biochem., Faculty of Med./Grad. Sch. of Med., Univ. of Yamanashi

Morphological changes of neurons and gene expression play important roles in the formation of proper neuronal circuit and plasticity. Megakaryoblastic leukemia(MKL)family members, MKL1 and MKL2, the serum response factor(SRF)coactivators, have actin-binding motifs suggesting that actin-mediated morphological change affects MKL-mediated gene expression.
Our previous studies have demonstrated that MKL1 and MKL2 are highly expressed in the brain and play an important role in activating SRF-mediated gene expression and regulating dendritic morphology in rat cortical neurons. Nuclear and cytoplasmic extraction from cultured cortical neurons and western blotting revealed that they are localized in both cytoplasm and nucleus. However, little is known about the subcellular localization of MKL.
In this study, we have analyzed the subcellular distribution of MKL1 and MKL2 in the rat brain and found that they are relatively concentrated in the postsynaptic density(PSD)fraction which was insoluble in 1% triton X-100. Conversely, they were not detectable in the crude synaptic vesicles(CSV). Furthermore, RNAi-mediated knock down of MKL1 and MKL2 was performed in long-cultured cortical neurons with synapses. As a result, the knock-down caused a decrease of spines with mushroom-shape, indicating that they play important roles in maturation of spines.
Brain-derived neurotrophic factor(BDNF)influences dendritic morphology. We have found that MKL1 and MKL2 are phosphorylated in neurons stimulated with BDNF. Therefore, it is thought that BDNF signaling pathway may mediate MKL phosphorylation and regulate dendritic spine morphology. To address this, we investigate the relationship between phosphorylation of MKL and alteration of dendritic spine morphology.
2P-02
Studies on possible PSD-core structure of type I excitatory synapse from rat forebrain
Suzuki Tatsuo,Guo Weiheng,Zhao LiYing
Dept. Neuroplasticity, Shinshu Univ. Grad. Sch. Med.

Postsynaptic density(PSD)is a specialized cytoskeleton, localizing immediately underneath the postsynaptic membrane and play important roles in signal processing upon receiving neurotransmitter and in generation of synaptic plasticity. Model for architecture of PSD of type I excitatory synapse comprises of several scaffolding proteins including shank, PSD-95, GKAP and homer, to which various molecules involved in postsynaptic signaling are associated. On the contrary, PSD-lattice structure has long been known to be an architectural base for type I PSD, of which major constituents are not clearly known. Lattice-like structure of type I PSD can be seen after solubilization of PSD with deoxycholate. We identified such lattice-like structures under other conditions, such as purification of PSDs from forebrains of immature rats and purification of PSDs from brains rapidly frozen after decapitation. The latter condition produces lean PSD(Suzuki et al., 1994, 63:1529-1537). To know the structural organization of synapses at molecular level, we investigated systematically purification process of PSD and postsynaptic membrane rafts(PSRs)from synaptic plasma membrane(SPM)of rat forebrain after treatment with three different detergents, Triton X-100, n-octyl β-D-glucoside and 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate(CHAPSO)at varied concentrations(Zhao et al., J. Neurochemistry, 2014, 131:147-162). We found clear difference in the separation of subsynaptic structures among these detergents. In particular, we identified several novel subsynaptic fractions, and one of them contained mainly mesh-like structures, which resembled previously reported PSD-lattice structure. This preparation may be of use to resolve the molecular architecture of type I excitatory PSDs of mammalian brain.
2P-03
SUMO1 Affects Synaptic Function, Spine Density and Memory
Matsuzaki Shinsuke1,2,Takamura Hironori1,3,Amano Genki1,Sato Hiroki1,Han Sarina1,Miyoshi Ko1,3,Fraser Paul4,Katayama Taiichi1,3
1Molecular Brain Science, United Graduate School of Child Development, Osaka University,2Anatomy and Neuroscience, Graduate School of Medicine, Osaka University,3Molecular Research Center for Children’s Mental Development, United Grad. Sch. of Child Development, Osaka Univ.,4Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto

Small ubiquitin-like modifier-1(SUMO1)plays a number of roles in cellular events and recentevidence has given momentum for its contributions to neuronal development and function. Here, wehave generated a SUMO1 transgenic mouse model with exclusive overexpression in neurons in aneffort to identify in vivo conjugation targets and the functional consequences of their SUMOylation.A high-expressing line was examined which displayed elevated levels of mono-SUMO1 and increasedhigh molecular weight conjugates in all brain regions. Immunoprecipitation of SUMOylated proteinsfrom total brain extract and proteomic analysis revealed ~95 candidate proteins from a variety offunctional classes, including a number of synaptic and cytoskeletal proteins. SUMO1 modification ofsynaptotagmin-1 was found to be elevated as compared to non-transgenic mice. This observationwas associated with an age-dependent reduction in basal synaptic transmission and impairedpresynaptic function as shown by altered paired pulse facilitation, as well as a decrease in spinedensity. The changes in neuronal function and morphology were also associated with a specificimpairment in learning and memory while other behavioral features remained unchanged. Thesefindings point to a significant contribution of SUMO1 modification on neuronal function which mayhave implications for mechanisms involved in mental retardation and neurodegeneration.
2P-04
Analysis of Arhgef2 phosphorylation at Ser885.
Oda Kaishu
NagoyaUniv.

In neuron, Rho family small GTPases regulate axon elongation or dendritic spine formation through F-actin cytoskeleton. The small G proteins function as molecular switches, cycling between inactive GDP-bound state and active GTP-bound state. Arhgef2(Lfc or GEF-H1)is a major Rho guanine exchange factor(RhoGEF), that activates RhoA by exchanging GDP for GTP. Depending on PKA, phosphorylation at Ser885 of Arhgef2 inactivates its GEF activity. In vitro investigation of the Ser885 phosphorylation has been conducted, however, the effect of such phosphorylation in vivo yet remains unknown. Assuming that phosphorylation of Arhgef2 at Ser885 alters neuronal morphology, we tried to produce Ala885 mutant Arhgef2 mouse with CRISPR/Cas9 system targeting Arhgef2 Ser885. We checked F0 mice and have detected knock-in allele in some mice. We will analyze RhoA activity, neuronal morphology and behavior in Arhgef2 mutant mice.
2P-05
5-HT2A receptor antagonist, Ketanserin induces change in the localization of an actin-binding protein drebrin in hippocampal neurons.
Oka Takero,Roppongi Reiko,Hanamura Kenji,Shirao Tomoaki
Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine

Serotonergic transmission plays important modulatory roles in higher brain function and is deeply involved in the pathogenesis of psychiatric disorders. Patients with depression have decreased level of serotonin in their brain. We have reported that an actin-binding protein, drebrin at postsynaptic sites plays a pivotal role in the effect of a serotonin-norepinephrine reuptake inhibitors imipramine(Kojima et al., 2010). Our previous study also demonstrated that activation of 5-HT2A receptor reduces clusters of drebrin in dendritic spines of cultured hippocampal neurons(Roppongi et al., 2013)through the activation of NMDA receptors. To examine the role of endogenous 5-HT2A receptor activity on the distribution of drebrin-binding actin cytoskeleton in neurons, we examined the effect of 5-HT2A receptor antagonist, Ketanserin on the localization of drebrin in cultured hippocampal neurons. Primary hippocampal neurons prepared from hippocampi of mouse embryo are treated with Ketanserin for 15 min at 21 days in vitro. Immunocytochemical analysis using anti-drebrin antibody showed that Ketanserin induces accumulation of drebrin in cell body with cluster-like distribution. These results suggest that endogenous 5-HT2A receptor activity regulates the distribution of drebrin in mature neurons.
2P-06
Allopregnanolone induces increase of excitatory but not inhibitory synapses via protein kinase A activation
Shimizu Hideo1,2,Ishizuka Yuta1,Yamazaki Hiroyuki1,Shirao Tomoaki1
1Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine,2Division of Pharmacology, National Institute of Health Sciences

Allopregnanolone(APα;5α-pregnan-3α-ol-20-one)is a steroid synthesized in both the periphery and central nervous system. Because APα was suggested to improve the symptoms of depression and Alzheimer´s disease, which involve synaptic dysfunction and loss, we hypothesized that APα increases excitatory synapses. Drebrin, an actin binding protein, facilitates the accumulation of other postsynaptic proteins in dendritic spines. Additionally, drebrin accumulation in dendritic spines is inhibited by soluble amyloid β oligomers, which are involved in the pathogenesis of cognitive decline. These suggest that the accumulation of drebrin within the dendritic spine is a good marker of mature synaptic function. In this study, we investigated whether APα increases mature excitatory synapse density by the analyses of dendritic spine morphology and drebrin accumulation. We prepared primary cultures of hippocampal neurons by Banker´s method. After the cells were incubated for 20 days in vitro, they were treated with various dosages of APα(0.1, 0.3 and 1 μM)for 24 hours, and then analyzed morphologically and immunocytochemically. 0.3 and 1 μM APα significantly increased dendritic spine density. The length and width of these dendritic spines were not altered by APα treatment. In addition, drebrin cluster density was increased by 0.3 and 1 μM APα treatment. Moreover, APα increased VGLUT1 cluster density but not VGAT. These data suggest that APα increases mature excitatory synapses. Interestingly, the protein kinase A inhibitor H-89 pretreatment inhibited the APα-induced increase in drebrin cluster density, while sole treatment of H-89 did not alter the density of drebrin clusters in control neurons. These results demonstrate that APα increases mature excitatory synapses via activation of protein kinase A.