AMPA受容体の輸送と機能
Molecular regulation of AMPA receptor traffic and function
O2-6-1-1
Arf6活性調節因子BRAG2/IQSEC1のスプライスバリアント依存的なシナプス局在
Splice variant-dependent synaptic localization of the Arf6 activator BRAG2/IQSEC1 in the adult mouse brain

○深谷昌弘1, 原芳信1, 阪上洋行1
○Masahiro Fukaya1, Yoshinobu Hara1, Hiroyuki Sakagami1
北里大・医・解剖1
Dept Anat, Kitasato Univ Sch Med, Sagamihara1

Brefeldin A-resistant Arf-GEF 2 (BRAG2)/IQSEC1 is a guanine nucleotide exchange factor that selectively catalyzes GDP-GTP exchange on ADP ribosylation factor 6 (Arf6). Arf6 is known as a small GTPase that regulates membrane trafficking between plasma membrane and endosomes. A recent report suggests that BRAG2 directly binds to Α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs), and is involved in the hippocampal long-term depression by regulating the endocytosis of AMPARs at excitatory synapses (Scholz et al., 2010). However, the anatomical evidence and molecular mechanisms of the postsynaptic localization of BRAG2 remains to be elucidated. In this study, we report that long C-terminal splice variants of BRAG2 are highly enriched in the postsynaptic density (PSD) fraction compared to short C-terminal splice variants, and selectively localized at the excitatory PSD accompanied by colocalization with AMPARs in the adult mouse brain. On the other hand, immunoreactive signals for BRAG2-pan antibody showed both postsynaptic and endosome-related distribution in the somato-dendritic region. Using yeast two-hybrid and immunoprecipitation assays, we show that long C-terminal splice variants of BRAG2 bind to PDZ domains of PSD-95. Taken together, these findings suggest that BRAG2 localizes and functions at excitatory postsynaptic sites and endosomes in a splice variant-dependent manner, serving a mechanistic basis for the internalization of AMPARs from postsynaptic sites to the intracellular membrane components through the BRAG2-Arf6 signaling.
O2-6-1-2
X染色体連鎖性精神遅滞原因遺伝子産物OPHN1のシナプス可塑性制御の解析
X-linked Mental Retardation Protein OPHN1 regulates synaptic plasticity by endocytic zone positioning via the interaction with Homer

○小林亜希子1,2, 萩原正敏1
○Akiko Kobayashi1,2, Nael Nadif Kasri3, Linda Van Aelst2, Masatoshi Hagiwara1
京都大学大学院 医学研究科 形態形成機構学1, コールド・スプリング・ハーバー研究所2
Dept Anatomy and Developmental Biology, Univ of Kyoto, Kyoto1, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA2, Donders Institute for Brain, Nijmegen, The Netherlands3

Synaptic strength is tightly regulated by the number of AMPA receptors expressed at the synapse. Here we show that the EZ positioning depends on the physical interaction between X-linked mental retardation protein Oligophrenin-1 (OPHN1) and post-synaptic scaffold protein Homer 1b/c. The disruption of this interaction caused the displacement of EZ from synapses, accompanied with a decrease in surface expressed AMPA receptors and reduced synaptic transmission. Furthermore, the disruption of OPHN1-Homer 1b/c interaction impaired long-term potentiation (LTP) by blocking the recycling of AMPA receptors. Thus OPHN1 contributes to maintain EZs adjacent to the PSD via physical interaction with Homer, and these findings provide new insight into how loss of function of OPHN1 leads to the abnormal glutamatergic development and synaptic plasticity in MR.
O2-6-1-3
海馬AMPA受容体リン酸化の定量解析
Stoichiometry of hippocampal AMPA receptor phosphorylation

○細川智永1, 美津島大2, 高橋琢哉2, 林康紀1
○Tomohisa Hosokawa1, Dai Mitsushima2, Takuya Takahashi2, Yasunori Hayashi1
独立行政法人理化学研究所脳科学総合研究センター1, 横浜市立大学大学院医学研究科生理2
RIKEN BSI, Saitama, Japan1, Dept. of Physiol., Grad Sch. Med., Yokohama City Univ., Yokohama, Japan2

It has been proposed that the phosphorylation of AMPA type glutamate receptors (AMPAR) regulates the receptor trafficking and channel activity, thereby playing an important role in synaptic plasticity such as long-term potentiation (LTP) and depression (LTD). However, the actual stoichiometry of phosphorylation, critical information to fully attest the model, is not known because of the lack of appropriate techniques for its measurement. Using phostag SDS-PAGE, we established a method to estimate the proportion of phosphorylated AMPAR from tissue. It turns out that the phosphorylated receptor at S831 and S845, so far implicated in AMPAR regulation, is almost negligible level in adult hippocampal tissue. We estimated that only one out of XXX and XXX synapses contains one molecule of phosphorylated receptor at S831 and S845, while majority of other synapse does not contain single phosphorylated AMPAR. Neuronal stimulation increased phosphorylation but the amount was still very low. There was no difference in the phosphorylation pattern between synaptic and non-synaptic receptors. Our results provide compelling evidence that argues against the prevailing model of synaptic plasticity and impels us to reconsider its mechanism.
O2-6-1-4
セマフォリン3Aが海馬学習依存的なAMPA受容体のシナプスへの移行を媒介する
Semaphorin3A mediates learning-induced AMPA receptor trafficking at hippocampal synapses

○高橋葵1, 山下直也1, 実木亨2, 五嶋良郎1, 高橋琢哉2
○Aoi Takahashi1, Naoya Yamashita1, Susumu Jitsuki2, Yoshio Goshima1, Takuya Takahashi2
横浜市立大学大学院医学研究科分子薬理神経生物学1, 横浜市立大学大学院医学研究科生理学2
Dept. mol Pharmacol & Neurobio., Yokohama city Univ. Grad. Sch. Med. Yokohama1, Dept. Physiol., Yokohama city Univ. Grad. Sch. Med. Yokohama2

Synaptic plasticity is important for the formation and stability of neuronal circuits and provides substrates for learning and memory. Several lines of evidence suggest that axon guidance molecules regulate synaptic connectivity and maintenance. We previously found that Semaphorin3A (Sema3A), a repulsive axon guidance molecule, regulates dendritic spine morphology. However, its role on synaptic plasticity is still unknown. Here, we show evidence that Sema3A signaling induces AMPA receptor (AMPAR) trafficking to regulate hippocampal learning. Over-expression of PlexinA (PlexA)-CA, which constitutively activates semaphorin signaling, increased AMPAR-mediates currents at hippocampal slices. In contrast, over-expression of dominant negative form of PlexA decreased AMPAR-mediates currents, suggesting that semaphorin signaling enhances AMPAR trafficking at hippocampal synapses. Next we focused on the learning-dependent AMPAR trafficking. We found that knock-down of PlexA4 exhibited impairment of learning-induced AMPAR trafficking, which consequently impairs learning performance. This phenotype is also seen by knock-down of Neulophilin-1 (NRP1). Since Sema3A preferentially binds to NRP1/PlexA4 complex, these results suggest that hippocampal learning activates Sema3A signaling, which induces synaptic delivery of AMPAR.
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