一般口演
シナプス
Synapses
座長：小山 隆太（東京大学大学院薬学系研究科 薬品作用学教室）・鶴田 文憲（筑波大学） |
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| 2022年6月30日 10:00～10:15 沖縄コンベンションセンター 会議場B3・4 第6会場
1O06m2-01
抗てんかんLGI1-ADAM22複合体を安定化する14-3-3-ADAM22相互作用
14-3-3 proteins stabilize LGI1-ADAM22 levels to prevent epilepsy in mice
*横井 紀彦(1,2)、深田 優子(1,2)、尾勝 圭(3)、山形 敦史(4)、劉 岩(3)、三宝 誠(5)、宮崎 裕理(1,2)、後藤 哲平(5)、平林 真澄(5)、深井 周也(3)、深田 正紀(1,2)
1. 生理研生体膜、2. 総研大院生命科学研究科、3. 京都大学大学院理学研究科、4. 理研BDR、5. 生理研遺伝子改変動物作製室
*Norihiko Yokoi(1,2), Yuko Fukata(1,2), Kei Okatsu(3), Atsushi Yamagata(4), Yan Liu(3), Makoto Sanbo(5), Yuri Miyazaki(1,2), Teppei Goto(5), Masumi Hirabayashi(5), Shuya Fukai(3), Masaki Fukata(1,2)
1. Div Membr Physiol, Natl Inst Physiol Sci, Okazaki, Japan., 2. Sch Life Sci, SOKENDAI, Okazaki, Japan., 3. Grad Sch Sci, Kyoto Univ, Kyoto, Japan, 4. RIKEN BDR, Yokohama, Japan, 5. Sect Mamm Transgen, Natl Inst Physiol Sci, Okazaki, Japan.
Keyword: epilepsy, protein complex, biogenesis, molecular precision medicine
What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, we found that ADAM22 is stoichiometrically phosphorylated in the mouse brain. In ADAM22 phosphorylation-deficient knock-in mice, the amounts of the ADAM22 and LGI1 proteins in the brain were decreased to about 40% and 55%, respectively, compared to that in the wild-type mouse brain without seizures. Phosphorylation-deficient ADAM22 abolished the phosphorylation-dependent interaction with 14-3-3 adaptor proteins, and subjected to AP2-mediated endocytosis for degradation. We demonstrated that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3 and forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we found that ~50% of LGI1 and ~10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment.
Ref)
Yokoi et al. Nat. Med. 21, 19-26 (2015)
Yamagata, Miyazaki, Yokoi et al. Nat. Commun. 9, 1546 (2018)
Yokoi et al. Cell Reports 37, 110107 (2021) | | 2022年6月30日 10:15～10:30 沖縄コンベンションセンター 会議場B3・4 第6会場
1O06m2-02
GABAergic-like dopamine synapses in the brain
*Jae-Ick Kim(1), Hyun-Jin Kim(1), Byungjae Hwang(1), Maria Reva(2,3,4), Jieun Lee(1), Byeong Eun Lee(1), Youngeun Lee(1), Eun Jeong Cho(1), Seung Eun Lee(7), Kyungjae Myung(1,5), Ja-Hyun Baik(6), Jung-Hoon Park(1)
1. Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea, 2. Institut Pasteur, Paris, France, 3. Sorbonne University, Paris, France, 4. École Polytechnique Fédérale de Lausanne (EPFL), Geneva, Switzerland, 5. Institute for Basic Science (IBS), Ulsan, Korea , 6. Korea University, Seoul, Korea, 7. Korea Institute of Science and Technology (KIST), Seoul, Korea
Keyword: Dopamine synapse, GABA co-transmission, GABAergic-like dopamine synapse, Parkinsonism
Dopaminergic axons originate in the midbrain and establish widely spread synapses throughout the brain. Synaptic transmission at these synapses plays a crucial role for volitional movement and reward-related behaviors, while the dysfunction of dopamine synapses causes various psychiatric and neurological disorders. Despite this significance, the true nature of brain-wide spatial and functional features of dopamine synapses remains poorly understood due to difficulties defining functional dopamine synapses at the molecular and physiological levels. Here we show that dopamine synapses are structured and function like GABAergic synapses in the mouse brain with marked regional heterogeneity. Dopamine transmission is strongly correlated with GABA co-transmission at dopamine synapses across the brain areas. In addition, functional dopamine synapses possess GABAergic postsynaptic markers and are unevenly distributed throughout the brain with distinct spatial clustering. In the dorsal striatum, GABAergic-like dopamine synapses are uniquely clustered on the dendrites and GABA transmission at dopamine synapses has disparate physiological characteristics. Importantly, knockdown of the inhibitory cell adhesion molecule in the postsynaptic striatal neurons unexpectedly did not weaken but instead facilitated GABA transmission at dopamine synapses. Lastly, the attenuation of GABA co-transmission precedes defects in dopaminergic transmission in an animal model of Parkinsonism. Our findings unravel distinct spatial and functional nature of GABAergic-like dopamine synapses in health and disease. Furthermore, the broader implication of our results is that GABAergic-like features of dopamine synapses can be utilized to better understand the real complexity of synaptic actions at dopamine synapses in regulating neural circuits. | | 2022年6月30日 10:30～10:45 沖縄コンベンションセンター 会議場B3・4 第6会場
1O06m2-03
海馬神経細胞アクティブゾーンにおける膜融合後に起こる小胞取り込みの蛍光イメージング
Fluorescent visualization of presynaptic endocytosed vesicles after exocytosis at individual hippocampal presynaptic active zones
*田中 洋光(1)、平野 丈夫(1)
1. 京都大学大学院理学研究科生物物理学教室
*Hiromitsu Tanaka(1), Tomoo Hirano(1)
1. Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
Keyword: Synaptic vesicle, Endocytosis, TIRF, Neuroligin
In presynaptic terminals, neurotransmitter is repetitively released depending on patterns of action potential firing. Synaptic vesicles need to be recycled efficiently so that multiple neurotransmitter releases can take place. However, mechanisms to regenerate synaptic vesicles are not fully understood. Especially, it remains unclear how synaptic vesicle proteins are retrieved after neurotransmitter release. To address this issue, we used total internal reflection fluorescence microscopy (TIRFM) that allows observation of fluorescent molecules with a high signal-to-noise ratio by limiting the depth of excitation field to approximately 100 nm. To visualize the fluorescent molecules at active zones with TIRFM, we induced formation of active zone-like membrane (AZLM) on the glass surface by coating Neuroligin, a synaptic adhesion molecule that can induce presynaptic differentiation through binding with Neurexin, another synaptic adhesion molecule. Rat hippocampal neurons were cultured on the Neuroligin-coated glasses and transfected with CAST-RFPt, SypHy and Neurexin. CAST-RFPt is a fusion protein of a red fluorescent protein TagRFPt and an active zone scaffold protein CAST, which was used as an AZLM marker. SypHy is a synaptic vesicle protein Synaptophysin tagged with super-ecliptic pHluorin (SEP), a pH-sensitive GFP variant. Endocytosis events were visualized by changing extracellular pH intermittently to an acidic condition using U-tube system. At pH 6.0, cell-surface SEP signals were quenched, and only signals could be detected from recently endocytosed vesicles in which acidification has not completed yet. We observed endocytosed SypHy signals 3 sec, 7 sec and 15sec after the onset of electrical field stimulation to trigger exocytosis of synaptic vesicles, and analyzed the spatial patterns of endocytosed signals at AZLM. The endocytosed signal intensity was positively correlated with the exocytosed intensity. We are going to show dynamics of a synaptic vesicle protein during repetitive exo- and endocytosis at a hippocampal presynaptic active zone revealed by a novel live-cell imaging method. | | 2022年6月30日 10:45～11:00 沖縄コンベンションセンター 会議場B3・4 第6会場
1O06m2-04
恐怖記憶の形成に関与するシナプスの検出法の開発
Development of a labeling method for fear memory synapses
*大西 泰地(1)、坂本 寛和(1)、大久保 洋平(2)、並木 繁行(1)、廣瀬 謙造(1)
1. 東京大学大学院医学系研究科、2. 順天堂大学医学部
*Taichi Onishi(1), Hirokazu Sakamoto(1), Yohei Okubo(2), Shigeyuki Namiki(1), Kenzo Hirose(1)
1. Grad Sch Med, Univ of Tokyo, 2. Sch Med, Juntendo Univ.
Keyword: synapse, memory, super-resolution microscopy
In the auditory cued fear conditioning, fear memory is stored in synapses between a sparse population of neurons in presynaptic auditory cortex and postsynaptic lateral amygdala. Such memory related neurons, often called engram cells, show increased activity and express c-fos during memory formation. Although it is important to examine the molecular anatomy of synapses between engram cells for understanding the molecular mechanisms of memory formation, there is no effective method for selective labeling of synapses between engram cells. In this study, we adopted an adeno associated virus-delivered c-fos promoter-driven Tet-On system to express tag-fused synaptophysin (tag-SYP) in auditory cortex neurons and FingR.PSD95, a PSD95 intrabody, in lateral amygdala neurons, in an activity-dependent manner. In this system, neuronal contacts between c-fos-expressing pre- and postsynaptic neurons were labeled with both tag-SYP and FingR.PSD95. We found that the number of neuronal contacts both positive for tag-SYP and FingR.PSD95 in the lateral amygdala was 15-fold higher in mice with cued fear conditioning than in control mice. Furthermore, using a super-resolution microscopy, we observed nanostructures of tag-SYP and FingR.PSD95 facing each other across a 50 nm cleft similar to endogenous synaptic proteins. Thus, we concluded that tag-SYP and FingR.PSD95 double positive neuronal contacts correspond to synapses between engram cells and called them engram synapses. Combining this method with immunohistochemistry, we showed that some endogenous synaptic molecules, including bassoon, Munc13-1 and PSD95, accumulated in the engram synapses. It is expected that our method enables comprehensive analysis of changes in the amount and the nanometer-scale spatial distribution of synaptic molecules induced by the fear memory formation. | |
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