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座長：飛田 秀樹（名古屋市立大学医学研究科）・鶴田 文憲（筑波大学） |
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| 2022年7月2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
3O06m2-01
細胞非自律的なcGMP作用による線虫C. elegans のナビゲーション制御
A Non-Cell Autonomous Action of cGMP within Multi-Neuronal Network Regulates a C. elegans Navigation Behavior
*中野 俊詩(1)、森 郁恵(1)
1. 名古屋大学
*Shunji Nakano(1), Ikue Mori(1)
1. Nagoya University
Keyword: cGMP, neural network, C. elegans
cyclic GMP (cGMP) is a well-known intracellular second messenger and regulates the activities of diverse neuronal proteins, including ion channels and protein kinases. Previous studies highlighted the cell-autonomous action of cGMP, in which the change in the cGMP level resulted in the modulation of the target proteins in the same neurons. Here, we report that cGMP can act non-cell autonomously and controls a C. elegans navigation behavior within a thermal environment.
The temperature preference of C. elegans is plastic and determined by the past experience: animals cultivated at a constant temperature display their preference toward that cultivation temperature when placed on a thermal gradient. We conducted a forward genetic screen for mutants defective in thermotaxis and isolated a mutant of the gene rdl-1, which encodes the C. elegans ortholog of Retinal Degeneration 3 (RD3) protein. The mammalian RD3 protein participates in a cGMP signaling, in which RD3 regulates the activity and trafficking of guanylyl cyclases in photoreceptor cells. We observed that the C. elegans rdl-1 is expressed in multiple sensory neurons, including the major thermosensory neurons AFD. Expression of rdl-1 exclusively in the AFD neurons rescued the thermotaxis defect of rdl-1 mutants, indicating that rdl-1 functions in AFD to regulate thermotaxis. Surprisingly, the defect of rdl-1 mutants was also restored by expression of rdl-1 in four other sensory neurons, ADF, ASI, ASK or ASJ. These sensory neurons have not been associated with temperature sensing but rather with detecting chemicals, in particular nematode pheromones to measure the population density. Calcium imaging analysis showed that rdl-1 mutants are normal in temperature-evoked activity of the AFD thermosensory neurons. These observations suggest that the cGMP signaling is functionally shared among multiple sensory neurons, each of which is dedicated to detect different sensory modality, and we speculate that this cGMP network culminates in the regulation of the presynaptic outputs from the AFD thermosensory neurons.
We are currently monitoring the cGMP dynamics within the sensory neurons and examining whether rdl-1 regulates this process. The roles of this cGMP network in controlling the neural circuit dynamics and biological significance of this network will be discussed. | | 2022年7月2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
3O06m2-02
逆行性アデノ随伴ウイルスの遺伝子導入効率を向上させるチロシンキャプシド変異
The tyrosine capsid mutations on retrograde adeno-associated virus enhance gene transduction efficiency
*中浜 諒大(1)、植松 朗(1,2,3)、冨樫 和也(1)、榎本 和生(1,2,4)
1. 東京大学大学院
*Ryota Nakahama Nakahama(1), Akira Uematsu(1,2,3), Kazuya Togashi(1), Kazuo Emoto(1,2,4)
1. The University of Tokyo, 2. International Research Center for Neurointelligence (WPI-IRCN), 3. AMED-PRIME, 4. AMED-CREST
Keyword: Adeno-associated virus, Virus vector
Adeno-associated virus (AAV) vector has become a critical tool in neuroscience in part due to its durable transgene expression and safety profile. Among many serotypes of AAV vectors, retrograde-accessible AAV2-retro and anterograde-accessible AAV1 are often utilized for neural circuit dissection. However, a long term expression period is required to obtain enough expression level for use in neuroscience research. Introducing mutations to the AAV is a potential approach to enhance AAV gene transduction, as the mutations of AAV capsid tyrosine residues improve the transduction efficiency by evading host cell responses. The tyrosine mutant AAV has been rarely used for tracing and/or manipulating neural circuits so it has not been determined whether the mutation affects the neuronal transduction efficiency of AAV2-retro and AAV1. Thus we introduced the tyrosine mutations to AAV2-retro and AAV1 vectors and examined whether the tyrosine mutations could improve the transduction efficiency.
In this study, we considered that the gene transduction efficiency is composed of two components, the transfer efficiency and the expression efficiency. The transfer efficiency can be measured by the number of infected cells and the expression efficiency can be measured by the fluorescent intensity of virally expressed fluorescent protein. We first conducted in vitro assays to observe the time course change of the expression efficiency. We found that the expression efficiency of mutated AAV2-retro was significantly enhanced in the primary culture of cortical neurons at 1week post infection but there was no significant difference at 2week post infection, indicating the mutation accelerates gene expression of AAV2-retro. For in vivo application, we tested if the tyrosine mutation could enhance the expression efficiency in multiple neural circuits. We revealed that the expression efficiency of AAV2-retro was significantly enhanced with the mutation in cortico-cortical and subcortical circuits. To explore the possibility of the tyrosine mutation improving the retrograde transfer efficiency, we co-injected non-viral retrograde tracer and AAV2-retro to compare ratios of the labeled neuron number. The ratios exhibited no significant change in both circuits suggesting that the tyrosine mutation did not affect the retrograde transfer efficiency.
To assess the effect of the tyrosine mutation on anterograde monosynaptic transfer of AAV1, we injected AAV1-Cre into Cre-dependent tdTomato reporter mice. The labeled neuron number was decreased with the mutation. We conclude that the tyrosine mutant AAV2-retro, but not AAV1, would provide a more effective means of gene transduction that requires a shorter period of infection and dramatically boosts experimental efficiency. | | 2022年7月2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
3O06m2-03
アストロサイトにおける小胞体膜貫通型転写因子OASISを介した核膜ストレス応答機構
Molecular mechanism of nuclear envelope stress response mediated by ER-resident transcription factor OASIS in astrocytes
*上川 泰直(1)、齋藤 敦(1)、今泉 和則(1)
1. 広島大学大学院医系科学研究科
*Yasunao Kamikawa Kamikawa(1), Atsushi Saito(1), Kazunori Imaizumi(1)
1. Institute of Biomedical & Health Sciences, Hiroshima University
Keyword: Nuclear envelope, Astrocyte, OASIS
The nuclear envelope (NE) consists of double lipid bilayers and separates genomic DNA from the cytoplasm. The nuclear lamina, a dense meshwork underlaying the NE provides mechanical support to the NE. Accumulating evidence has demonstrated that a broad spectrum of cellular stresses, such as mechanical stress during neuronal migration in brain development, degenerates NE components and/or disrupts their functional interactions. These stresses are collectively called “NE stress”. NE stress has been proposed to be associated with the pathogenesis of several neurological disorders, such as Huntington’s disease and autosomal dominant leukodystrophy. However, the molecular mechanisms underlying cellular stress response against NE stress have not been elucidated.
Recently, we identified an endoplasmic reticulum membrane resident transcription factor, Old Astrocyte Specifically Induced Substance (OASIS), as key factor that responds to and attenuates NE stress (Kamikawa et. al., 2021 Cell Death Discov.). OASIS accumulates to damaged NE where the nuclear lamina is disrupted. Furthermore, primary cultured astrocyte derived from Oasis knockout mice exhibited nuclear deformation and increased DNA damage upon mechanical NE stress. OASIS co-localizes with several factors that are known to be involved in the repair of injured NE. In addition, proximity dependent labeling assay using a fusion protein of OASIS with a biotin ligase followed by mass spectrometry identified candidate proteins that are co-localized with OASIS at damaged NE. These findings suggest that OASIS plays crucial roles in NE stress response together with other key factors that mediate the repair of injured NE. Currently, the significance of interactions among OASIS and identified factors are under investigation. We will discuss overall molecular mechanism of cellular response to NE stress in astrocyte and its physiological impact. | | 2022年7月2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
3O06m2-04
ショウジョウバエ感覚ニューロンにおける神経回路リモデリングの分子機構
Molecular control of developmental neuronal remodeling in Drosophila neurons
*古澤 孝太郎(1)、石井 健一(1)、榎本 和生(1,2)
1. 東京大学大学院理学系研究科、2. ニューロインテリジェンス国際研究機構
*Kotaro Furusawa(1), Kenichi Ishii(1), Kazuo Emoto(1,2)
1. Grad Sch Sci, Univ of Tokyo, Tokyo, Japan, 2. International Research Center for Neurointelligence (WPI-IRCN)
Keyword: remodeling, elimination, pruning
Developmental neuronal remodeling is a process by which neurons initially extend their neurites to form synaptic connections, and later, unnecessary networks are eliminated to functionally refine the circuits during development. Neuronal remodeling is typically achieved in a compartmentalized manner, and thus is supposed to be spatio-temporarily regulated as defects in remodeling processes most likely lead to developmental disorders. Drosophila sensory neurons provide an excellent model system to investigate the mechanisms underlying developmental neuronal remodeling, as neural circuits are extensively remodeled during metamorphosis. For the past decade, we have studied cellular and molecular mechanisms of dendrite pruning in Drosophila sensory neurons during development (Yasunaga et al. 2010 Dev Cell; Kanamori et al. Science 2013; Kanamori et al. Nature Commun 2015; Kitatani et al. PLoS Genet 2020). Here, we show that presynapses/axons as well as dendrites are eliminated in Drosophila sensory neurons during metamorphosis. To understand underlying mechanisms of the presynapse/axon elimination, we performed a genetic screen and found that multiple degradation pathways including the ubiquitin proteasome system are required for the presynapse/axon elimination. Interestingly, presynapse/axon elimination and dendrite pruning seem to require different E3 ligases, suggesting a distinct control for each activity. We will discuss about how presynapse/axon elimination and dendrite pruning are distinctly but coordinately regulated in single neurons. | |
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