一般口演（若手道場）
若手道場 シナプス・シナプス可塑性
Wakate Dojo: Synapses and Synaptic Plasticity
座長：長井 淳（理化学研究所）・服部 剛志（金沢大学 医薬保健研究域医学系 神経解剖学） |
|---|
| 2022年7月1日 9:00～9:15 沖縄コンベンションセンター 会議場A2 第7会場
2WD07m1-01
ショウジョウバエ視覚系において、膜タンパク質ファミリーであるBeat-Sideがシナプス特異性を制御する
Beat-Side transmembrane protein families regulate synaptic specificity in the Drosophila visual system
*小坂 二郎(1)、石井 愛莉沙(1)、王 旭(1)、大地 啓太(1)、羽毛田-鈴木 聡子(1)、鈴木 崇之(1)
1. 東京工業大学生命理工学院
*Jiro Osaka(1), Arisa Ishii(1), Xu Wang(1), Keita Oochi(1), Satoko Hakeda-Suzuki(1), Takashi Suzuki(1)
1. Grd Sch of Science and Technology, Tokyo Institute of Technology, Kanagawa, Japan
Keyword: synaptic specificity, Drosophila, cell adhesion molecule
All neurons have a surprising ability, namely, the synaptic specificity, to choose their specific synaptic partners out of the vast number of neurons, and to make synapses exclusively with the chosen one. Recent work proposed that all the neurons have the common machinery of synaptogenesis which is regulated by the unique set of cell adhesion molecules that establish the preference for their synaptic partner choice (Xu et al., 2019). However, the molecular basis remains unclear. Drosophila transmembrane interactome analysis revealed Beat superfamily and Side superfamily (Beat-Side superfamily) have heterophilic interaction (Ozkan et al., 2013, Li et al., 2017). Both Beat-Side proteins are transmembrane proteins with Immunoglobulin-like (Ig) domains on their extracellular. However, little is known about their function. Here we analyzed them as candidates of novel molecules that define synaptic specificity.
First, we tested if the Beat-Side superfamily works in the synaptic specificity of Drosophila lamina neurons, the secondary neuron of the photoreceptors. There are five types of lamina neurons (L1-L5), however, only the L2 and L4 neurons form the reciprocal connection. The single-cell RNAseq data indicated that there are several Beat-Side interactions between L2 and L4 neurons. They showed disrupted synapse formation pattern and connectivity change when all the Beat-Side interactions were interrupted. These results show that the Beat-Side superfamily is necessary for synaptic specificity of the lamina neurons.
Next, we tested if the Beat-Side interaction can artificially induce the synaptic connection. Photoreceptor R7 makes synaptic connections on the M4-6 layer of the medulla neuropil. We found that Beat2, a ligand for Side, specifically expressed on M1-2 and M9-10 layers. By mis-expressing Side, R7 mis-connected with M1-2 and M9-10 layers and showed ectopic synapse formation. This result indicates that Beat-Side interaction is sufficient to induce synaptic connection. Interestingly, the cytoplasmic domain of Side was dispensable for the synaptic connection, indicating the other molecule transduce the signal. We screened the molecules that work with Side and found that the IRM has genetic interaction with Side. Since IRM is reported to involve in synaptogenesis, Side and IRM may form a complex for establishing the synaptic formation and specificity. | | 2022年7月1日 9:15～9:30 沖縄コンベンションセンター 会議場A2 第7会場
2WD07m1-02
Emerging Roles of JAK/STAT signaling pathway in the Brain and Behavior
*Jeong-Kyu Han(1,2,3), Sang Jeong Kim(2,3)
1. Brain Science Institute, Korea Institute of Science and Technology, 2. Department of Physiology, College of Medicine, Seoul National University, 3. Department of Brain and Cognitive Sciences, Seoul National University
Keyword: JAK/STAT pathway, synaptic plasticity , neurodegenerative disease, psychiatric disorders
Brain has undergone a long evolution and has endogenously constructed molecular and cellular regulatory machinery to adapt. The brain cells subtly respond to external stimuli for survival, protection, regeneration, metabolic regulation, and communication. Therefore, it is possible that a lot of molecular and cellular machinery in the brain cells have their own role on generating multiple behaviors. Here, we suggest that Janus kinase/signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway can potentially influence a wide range of brain signals and behaviors. The JAK/STAT can manage synaptic plasticity, receptor regulation, and intracellular signaling in both physiological and pathophysiological conditions. The hallmark behavioral studies of JAK/STAT showed that JAK/STAT pathway has a direct impact on locomotive, feeding, pain, fear, and cognitive behaviors. In addition, JAK/STAT signaling is an important target for neurodegenerative disease and psychiatric disorders, such as Alzheimer’s and Parkinson's disease, post-traumatic stress disorder, and major depressive disorder. A series of studies of JAK/STAT implicates that JAK/STAT pathway has been evolutionarily invented in response to external stimuli, although more evidence should be added.
*Reference 1. Han et. al., Ablation of STAT3 in Purkinje cells reorganizes cerebellar synaptic plasticity in long-term fear memory network, eLife (2021)
*Reference 2. Kwon et. al., Dysfunction of microglial STAT3 alleviates depressive behavior via neuron-microglia interactions, Neuropsychopharmacology (2017) | | 2022年7月1日 9:30～9:45 沖縄コンベンションセンター 会議場A2 第7会場
2WD07m1-03
ヒト由来大脳皮質ニューロンにおいてCREBと共因子の時空間的な動態が神経活動依存的な遺伝子発現を制御する
Spatiotemporal dynamics of CREB with cofactors regulate activity-dependent gene expression in human cortical neurons
*渥美 友梨(1)、菅生 紀之(1)、岩⽥ 亮平(2)、Pierre Vanderhaeghen (2)、山本 亘彦(1)
1. 大阪大学大学院生命機能研究科、2. VIB-KU Leuven, Center for Brain & Disease Research, Leuven, Belgium
*Yuri Atsumi(1), Noriyuki Sugo(1), Ryohei Iwata(2), Pierre Vanderhaeghen (2), Nobuhiko Yamamoto(1)
1. Graduate school of Frontier Bioscience, Osaka University, 2. VIB-KU Leuven, Center for Brain & Disease Research, Leuven, Belgium
Keyword: Single-molecule imaging, Human cortical neurons, Activity-dependent gene regulations, Super enhancer
Activity-dependent transcriptional regulation plays a key role in neuronal development and plasticity, and its impairment leads to human brain disorders. A transcription factor, cAMP response element binding protein (CREB) is well characterized to promote gene expression by neuronal activity. Recently, the epigenetic regulation, including histone acetylation by CREB binding protein (CBP) which is known as Rubinstein-Taybi syndrome causal gene, has also been shown to be essential for the CREB activity. However, spatiotemporal aspects of activity-dependent CREB-DNA and CREB-protein interactions are almost unknown. To address this issue, we performed single-molecule imaging (SMI) of CREB in human-derived neuronal cells, manipulating or imaging other nuclear proteins. A Halo-tag human CREB expression vector was transfected in human ESC-derived cortical neurons, and time-lapse SMI was performed under highly inclined and laminated optical sheet (HILO) illumination. First, we found that the number of spots in which single-molecule CREB interacting with DNA for several seconds appear frequently (defined as hotspots) was markedly increased by KCl depolarization. Second, simultaneous SMI of Halo-tag CREB and SNAP-tag CBP molecules further demonstrated CREB and CBP were colocalized with a time delay. Furthermore, overexpression of a mutant CBP lacking histone acetyltransferase activity inhibited the long-lasting binding of CREB to DNA. Finally, we examined the spatial relationship between CREB and bromodomain containing 4 (Brd4), a super enhancer component with histone acetyltransferase activity, by live imaging of Halo-tag CREB and GFP-tagged Brd4. The result demonstrated that CREB hotspots accumulated in Brd4 clusters under KCl depolarization. Taken together, the present study suggests that neuronal activity promotes frequent binding of CREB to the specific DNA sites in highly acetylated chromatins to induce activity-dependent gene expression in human cortical neurons. | | 2022年7月1日 9:45～10:00 沖縄コンベンションセンター 会議場A2 第7会場
2WD07m1-04
軸索内微細構造解析のための新規蛍光電子相関顕微鏡技術の開発
Development of a Novel Correlative Light and Electron Microscopy Technique for the Intracellular Ultrastructural Analysis in In Vivo Axons
*柴山 光耀(1)、丸岡 久人(2)、岡部 繁男(2)、平林 祐介(1)
1. 東京大学大学院工学系研究科、2. 東京大学大学院医学系研究科
*Koyo Shibayama(1), Hisato Maruoka(2), Shigeo Okabe(2), Yusuke Hirabayashi(1)
1. School of Engineering, The University of Tokyo, 2. Graduate School of Medicine, The University of Tokyo
Keyword: Correlative light and electron microscopy, two-photon microscopy, Near-infrared branding, ascorbate peroxidase
Although recent studies have revealed that the intracellular ultrastructure is crucial for controlling the neural circuits and the synaptic transmission by determining neuronal properties, its roles in in vivo axons are poorly understood. While the functional imaging of neurons using light microscopy has the millimeter to micrometer-level resolution, analyses of the nanometer-scale neuronal ultrastructure requires the electron microscopy. Therefore, the technology to bridge the gap between these two microscopy techniques is an urgent need for the ultrastructural analysis of the neural circuit formation. Correlative Light and Electron Microscopy (CLEM) is a powerful technique that connects observation of the intracellular physiological functions and ultrastructure. However, due to the lack of precise correlation between light microscopy and electron microscopy observations, reliable high-throughput CLEM observation in vivo axons is yet to be achieved.
In the present study, we attempted to develop an innovative in vivo CLEM technique that links neural activity and intracellular ultrastructure in cortical axons and presynaptic boutons in an unprecedentedly precise and efficient manner. We introduced a novel axonal CLEM technique using Near-infrared branding (NIRB) and genetically-encoded ascorbate peroxidase (APEX2) localized at the plasma membrane. By optimizing the protein localization signals for APEX2 and brain sectioning method, electron microscopic re-identification of cortical axons, which was subjected to in vivo two-photon calcium imaging, became a feasible experiment. We will pursue physiological roles of the intracellular ultrastructure in regulating presynaptic properties using this novel axon CLEM technology. | |
|
|
