一般口演（若手道場）
若手道場 シナプスと回路
Wakate Dojo: Synapses and Networks
座長：木山 博資（名古屋大学医学系研究科）・河田 美穂（藤田医科大学） |
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| 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B3・4 第6会場
2WD06m1-01
神経活動に応じたAMPKの一過的な活性化が、海馬ニューロンにおけるミトコンドリア分裂と樹状突起形成を促進する。
Transient activation of AMPK in response to neural activity promotes mitochondrial fission and dendrite formation in hippocampal neurons.
*初田 茜(1)、藤島 和人(2)、栗栖 純子(5)、大野 伸彦(3,4)、見学 美根子(1,5)
1. 京都大学大学院生命科学研究科、2. 大阪医科薬科大学、3. 自治医科大学、4. 自然科学研究機構 生理学研究所、5. 京都大学 iCeMS
*Akane Hatsuda(1), Kazuto Fujishima(2), Junko Kurisu(5), Nobuhiko Ohno(3,4), Mineko Kengaku(1,5)
1. Graduate School of Biostudies, Kyoto Univerity, Kyoto, Japan, 2. Department of Anatomy & Cell Biology, Division of Life Sciences, Osaka Medical and Pharmaceutical University, Osaka, Japan, 3. Department of Anatomy, Division of Histology and Cell Biology, Jichi Medical University, Tochigi, Japan, 4. Division of Ultrastructural Research, National Institute for Physiological Sciences, Aichi, Japan, 5. Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan
Keyword: Mitochondrial fission, Dendrite development, AMPK
Activity-dependent dendrite formation is accomplished by extensive cytoskeletal remodeling and transcriptional changes, both of which accompany large ATP consumption. In order to constantly meet the high energy demands, mitochondrial homeostasis needs to be maintained. However, the molecular mechanisms of how neurons maintain mitochondrial homeostasis during dendrite development in response to neuronal activity is unclear. Recent studies have revealed that AMP-activated protein kinase (AMPK) serves as an energy sensor and sustains metabolic state by regulating mitochondrial dynamics. Here we show neuronal activity enhances mitochondrial fission via activation of AMPK during dendrite development in hippocampal neurons.
It has been shown that dissociated hippocampal neurons exhibit spontaneous firing during the first week in culture. We confirmed that dendrite complexity reduced by inhibition of neuronal activity by the treatment with TTX and APV. At the same time, we found that inhibition of neuronal activity also suppressed mitochondrial fission and caused mitochondrial elongation, while it did not affect mitochondrial transport in growing dendrites. Knockdown of AMPKa2 subunit recapitulated these phenotypes. Conversely, enhanced neuronal activity by high K+ or glutamate treatment, or pharmacological activation of AMPK, facilitated mitochondrial fission in dendrites. These data suggest that neuronal activity and AMPK activation enhances mitochondrial fission during dendrite development. Additionally, we visualized the spatiotemporal dynamics of AMPK and calcium influx by transfecting an AMPK-FRET probe and GCAMP6s in cultured hippocampal neurons. We found that AMPK activity showed irregular fluctuation that synchronized with Ca2+ spikes in the cell body and dendrites, which was suppressed by inhibition of neuronal activity or CaMKK2 knockdown. These data indicate that AMPK activity is dynamically regulated by spontaneous neuronal activity via CaMKK2. Furthermore, biochemical assay demonstrated that enhanced neuronal activity or AMPK activator treatment increased the phosphorylation of ULK1, a component of mitophagy complex, suggesting that AMPK activation by neuronal activity promotes the mitophagy pathway. Collectively, our results suggest that transient activation of AMPK by spontaneous neuronal activity enhances mitochondrial fission and mitophagy pathway, which may contribute to the mitochondrial quality control necessary for dendrite development. | | 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B3・4 第6会場
2WD06m1-02
神経回路形成因子LOTUSはBDNFにより発現増加し神経再生を促進する
The expression of LOTUS, a neural circuit formation factor, is increased by BDNF and promotes nerve regeneration.
*松林 潤平(1)、川口 祐生(1)、竹居 光太郎(1)
1. 横浜市立大学大学院 生命医科学研究科 生体機能医科学研究室
*Junpei Matsubayashi(1), Yuki Kawaguchi(1), Kohtaro Takei(1)
1. Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science, Yokohama, Japan
Keyword: LOTUS, BDNF, nerve regeneration
当研究室において発見されたLateral olfactory tract usher substance (LOTUS)はNogo receptor-1 (NgR1)の内在性アンタゴニストとして機能し、中枢神経の再生を促進する。しかしながら、LOTUSの発現量は神経障害や加齢に伴い減少することが報告されている。LOTUSの発現減少は中枢神経系の再生や可塑性が滞る一因だと推察されるが、LOTUSの発現制御機構については未だ明らかになっていない。そこで本研究では、LOTUSの発現増加を誘起する分子として同定したBrain-derived neurotrophic factor (BDNF)に着目し、BDNFによるLOTUSの発現制御機構の解明とLOTUSの発現上昇によるNgR1の機能抑制の亢進の検証を目的とする。
まず初めに、BDNFのLOTUS発現量に与える影響を解析した。マウス海馬初代培養神経細胞を用いて培養6日目にBDNFを添加し、24時間後のLOTUS発現量を調べた。その結果、BDNFを添加するとLOTUS発現量は有意に増加することが明らかとなった。次に、BDNFはTrkBに結合することから、Trk阻害剤であるk252aを用いて薬理学的な阻害実験を行った。その結果、BDNFによるLOTUSの発現上昇はk252aで処理することで抑制された。このことから、BDNFによるLOTUSの発現増加はTrkBを介することが示された。さらに、BDNFにより発現増加したLOTUSがNgR1の機能抑制を亢進するかを検証するために神経突起伸長アッセイを行った。野生型マウスとLOTUS遺伝子欠損マウスの海馬初代培養神経細胞を用いて培養1日目にBDNFおよびNogo66ペプチドを添加し、培養3日目の神経突起長を計測した。その結果、野生型およびLOTUS遺伝子欠損マウスの神経突起伸長はNogo66ペプチドの添加で阻害された。ここで、BDNFを同時に添加すると野生型マウスにおいては、Nogo66ペプチドの添加でみられた突起伸長阻害が抑制された。一方で、LOTUS遺伝子欠損マウスにおいては、BDNFを添加してもNogo66ペプチドによる突起伸長阻害作用が抑制されなかった。これらのことから、BDNFにより発現増加したLOTUSがNogo66による神経突起伸長阻害を抑制することが明らかとなった。
以上の結果から、LOTUSの発現増加を誘起する制御因子として新たにBDNFを同定した。また、BDNFによるLOTUSの発現上昇はNgR1の機能抑制を亢進することが示唆された。BDNFによる内在性LOTUSの発現制御に基づき、神経障害時におけるLOTUSの減少阻止や維持・亢進ができれば、内在性機序による新たな治療法の開発が期待できる。 | | 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B3・4 第6会場
2WD06m1-03
ゼブラフィッシュ迷走神経系が形成する反射神経回路における、特異的な神経結合の解析
Position-independent sensory-motor matching in the zebrafish vagus system
*兼子 拓也(1)、Cecilia B Moens(1)
*Takuya Kaneko(1), Cecilia B Moens(1)
1. Fred Hutchinson Cancer Research Center, Seattle, USA
Keyword: topographic map, vagus nerve, sensorimotor reflex, zebrafish
The vagus nerve (the 10th cranial nerve, conserved across vertebrates) is a bundle of sensory and motor axons that exit the brain and innervate diverse internal body parts, such as the throat, stomach, and heart. A main function of this nerve is to independently control individual body parts through distinct sensory-motor reflexes (e.g., the gag reflex for the throat, vagovagal reflex for the stomach, and baroreflex for the heart). Previous studies in our lab reported that, in larval zebrafish, vagus motor neurons innervating distinct body parts are continuously aligned on a coarse topographic map in the brain. Consequently, immediately adjacent motor neurons on the topographic map participate in distinct reflexes. It remains unanswered how these adjacent motor neurons with different roles are distinguished during development, so that they can be incorporated into separate reflex circuits. To address this, we study connectivity of individual motor neurons in larval zebrafish though calcium imaging. Our data show that local sensory stimuli induce reflexive calcium responses from specific subsets of motor neurons with little responses from other nearby motor neurons, demonstrating high specificity in sensory-motor connectivity. We then provide three lines of evidence supporting that this specific connectivity is not a simple outcome of topographic mapping during early development, but rather a product of subsequent circuit refinement. First, when the topographic map is just formed, local sensory inputs non-specifically activate diverse motor neurons, an indication of initially coarse connectivity. Second, when we inhibit neurotransmitter release from motor neurons, less motor neurons respond to sensory stimuli with reduced specificity. Thus, the refinement process may exclude functionally inappropriate motor neurons from the reflex circuit. Third, and most importantly, sensory-motor connectivity is resilient to topographic defects. When we force single motor neurons to innervate topographically incorrect targets, these mistargeted neurons exhibit calcium responses that are more closely correlated with the neurons innervating the same target than with the neurons that surround them. Taken together, our data support that circuit refinement provides each motor neuron with specific upstream connectivity that is appropriate for its motor function, independent of its topographic position. We are now examining what role neural activity has in this refinement. | | 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B3・4 第6会場
2WD06m1-04
Structural Embedding for Sensory Pathway based on Drosophila Adult
*Xiyang Sun(1), Fumiyasu Komaki(1,2)
1. RIKEN Center for Brain Science, 2. The University of Tokyo
Keyword: Structural embedding, sensory pathway, brain connectome
Animal behavior is highly influenced by nearly every sensory cue and thus understanding how sensory streams are transmitted and processed in neural circuits is vital. Here we systematically investigate network properties, structural organization, and their functional basis in the microscopic Drosophila (fruit fly) brain connectome. The overall degree and synaptic strength distribution are analyzed in this directed weighted network (FlyCiruit). Starting from the visual and olfactory sensory region, the organizations of their pre/post-synaptic neurons are clustered to show their hierarchical structures. And their neuronal numbers are depicted along the sensory pathway as a function of path length. Based on motif detection, the neuron triplets are also depicted in FlyCircuit to illustrate their significance. To further study the structural motif, we propose a structural embedding strategy while considering neuronal dendritic and axonal connections simultaneously. The hub and authority scores are assigned for each neuron based on dendritic and axonal connections respectively. After convergence, these scores, together with neuron and edge types, are represented via a low-dimensional embedding for the neuron’s neighborhood. Embedding space allows the unsupervised clustering to distinguish different neuron groups, and relations among them uncover their mesoscopic hierarchical structures. This structural embedding would provide a new framework for large-scale network analysis and open new directions for the design of neural architecture. | |
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