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清水 達太大学院医学研究科 医科学専攻助教
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■ 論文- The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp-His-Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA.2024年07月, PLoS genetics, 20(7) (7), e1011367, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.2023年12月, PLoS genetics, 19(12) (12), e1011089, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- ABSTRACT Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.The Company of Biologists, 2023年02月, Development, 150(4) (4)[査読有り]研究論文（学術雑誌）
- Chemical communication controls a wide range of behaviors via conserved signaling networks. Axon regeneration in response to injury is determined by the interaction between the extracellular environment and intrinsic growth potential. In this study, we investigated the role of chemical signaling in axon regeneration in Caenorhabditis elegans We find that the enzymes involved in ascaroside pheromone biosynthesis, ACOX-1.1, ACOX-1.2, and DAF-22, participate in axon regeneration by producing a dauer-inducing ascaroside, ascr#5. We demonstrate that the chemoreceptor genes, srg-36 and srg-37, which encode G-protein-coupled receptors for ascr#5, are required for adult-specific axon regeneration. Furthermore, the activating mutation in egl-30 encoding Gqα suppresses axon regeneration defective phenotype in acox-1.1 and srg-36 srg-37 mutants. Therefore, the ascaroside signaling system provides a unique example of a signaling molecule that regulates the regenerative pathway in the nervous system.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the EGL-30 Gqα-JNK MAP kinase cascade. However, it remains unclear what signals activate the EGL-30 pathway in axon regeneration. Here, we show that SRG-36 and SRG-37 act as upstream G-protein-coupled receptors (GPCRs) that activate EGL-30. C. elegans secretes a family of small-molecule pheromones called ascarosides, which serve various functions in chemical signaling. SRG-36 and SRG-37 are GPCRs for the dauer-inducing ascaroside ascr#5. Consistent with this, we found that ascr#5 activates the axon regeneration pathway via SRG-36/SRG-37 and EGL-30. Thus, ascaroside signaling promotes axon regeneration by activating the GPCR-Gqα pathway.2022年02月, The Journal of neuroscience : the official journal of the Society for Neuroscience, 42(5) (5), 720 - 730, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The post-injury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the non-muscle myosin light chain (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide-exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF-CDC42-MLC phosphorylation axis in a non-canonical Wnt signaling pathway that promotes axon regeneration.Significance Statement:Non-canonical Wnt signaling is mediated by Fz, Dsh, Rho-type GTPase, and non-muscle MLC phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a GEF for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the non-canonical Wnt signaling pathway controls axon regeneration via the CDK-14-EPHX-1-CDC-42-MLC phosphorylation axis.2021年08月, The Journal of neuroscience : the official journal of the Society for Neuroscience, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Axon regeneration is an evolutionarily conserved process essential for restoring the function of damaged neurons. In Caenorhabditis elegans hermaphrodites, initiation of axon regeneration is regulated by the RhoA GTPase-ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain phosphorylation signaling pathway. However, the upstream mechanism that activates the RhoA pathway remains unknown. Here, we show that axon injury activates TLN-1/talin via the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade and that TLN-1 induces multiple downstream events, one of which is integrin inside-out activation, leading to the activation of the RhoA-ROCK signaling pathway. We found that the non-receptor tyrosine kinase Src, a key mediator of integrin signaling, activates the Rho guanine nucleotide exchange factor (GEF) EPHX-1/ephexin by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.Significance StatementThe ability of axons to regenerate after injury is governed by cell-intrinsic regeneration pathways. We have previously demonstrated that the C. elegans RhoA GTPase-ROCK pathway promotes axon regeneration by inducing MLC-4 phosphorylation. In this study, we found that axon injury activates TLN-1/talin through the cAMP-Epac-Rap GTPase cascade, leading to integrin inside-out activation, which promotes axonal regeneration by activating the RhoA signaling pathway. In this pathway, SRC-1/Src acts downstream of integrin activation and subsequently activates EPHX-1/ephexin RhoGEF by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.2021年05月, The Journal of neuroscience : the official journal of the Society for Neuroscience, 41(22) (22), 4754 - 4767, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The hypothalamus plays a central role in homeostasis and aging. The hypothalamic arcuate nucleus (ARC) controls homeostasis of food intake and energy expenditure and retains adult neural stem cells (NSCs)/progenitor cells. Aging induces the loss of NSCs and the enhancement of inflammation, including the activation of glial cells in the ARC, but aging-associated alterations of the hypothalamic cells remain obscure. Here, we identified Sox2 and NeuN double-positive cells in a subpopulation of cells in the mouse ARC. These cells were reduced in number with aging, although NeuN-positive neuronal cells were unaltered in the total number. Diet-induced obesity mice fed with high-fat diet presented a similar hypothalamic alteration to aged mice. This study provides a new insight into aging-induced changes in the hypothalamus.FRONTIERS MEDIA SA, 2021年03月, FRONTIERS IN AGING NEUROSCIENCE, 12, 英語[査読有り]研究論文（学術雑誌）
- The breast cancer susceptibility protein BRCA1 and its partner BARD1 form an E3 ubiquitin ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1-BARD1 in post-mitotic cells, such as neurons, remain poorly defined. Here we report that BRC-1 and BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gqα-diacylglycerol (DAG) signaling pathway. This pathway is down-regulated by DAG kinase (DGK), which converts DAG to phosphatidic acid. We demonstrate that inactivation of DGK-3 suppresses the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1-BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1-BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1-BRD-1 complex regulates axon regeneration in concert with the Gqα-DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.Significance StatementBRCA1-BARD1 is an E3 ubiquitin ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1-BARD1 in post-mitotic cells, such as neurons, remain poorly defined. We show here that C. elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase DGK-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1-BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1-BARD1 in fully-differentiated neurons.2021年02月, The Journal of Neuroscience, 41(13) (13), 2842 - 2853, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- In Caenorhabditis elegans, axon regeneration is activated by a signaling cascade through the receptor tyrosine kinase (RTK) SVH-2. Axonal injury induces svh-2 gene expression by degradation of the Mad-like transcription factor MDL-1. In this study, we identify the svh-24/sdz-33 gene encoding a protein containing F-box and F-box associated domains as a regulator of axon regeneration in motor neurons. We find that sdz-33 is required for axon injury-induced svh-2 expression. SDZ-33 targets MDL-1 for poly-ubiquitylation and degradation. Furthermore, we demonstrate that SDZ-33 promotes axotomy-induced nuclear degradation of MDL-1, resulting in the activation of svh-2 expression in animals. These results suggest that the F-box protein is required for RTK signaling in the control of axon regeneration.SIGNIFICANCE STATEMENT:In C. elegans, axon regeneration is positively regulated by the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury via the Ets-like transcription factor ETS-4, whose transcriptional activity is inhibited by the Mad-like transcription factor MDL-1. Axon injury leads to the degradation of MDL-1, and this is linked to the activation of ETS-4 transcriptional activity. In this study, we identify the sdz-33 gene encoding a protein containing an F-box domain as a regulator of axon regeneration. We demonstrate that MDL-1 is poly-ubiquitylated and degraded through the SDZ-33-mediated 26S proteasome pathway. These results reveal that an F-box protein promotes axon regeneration by degrading the Mad transcription factor.2021年01月, The Journal of Neuroscience, 41(11) (11), 2373 - 2381, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Axon regeneration following nerve injury is a highly conserved process in animals. The nematode Caenorhabditis elegans is an excellent model for investigating the molecular mechanisms of axon regeneration. Recent studies using C. elegans have shown that the c-Jun N-terminal kinase (JNK) plays the important role in axon regeneration. Furthermore, many factors have been identified that act upstream of the JNK cascade after axotomy. This review introduces these factors and describes their roles during the regulation of axon regeneration.2020年02月, Journal of biochemistry, 167(5) (5), 433 - 439, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- In Caenorhabditis elegans, the JNK MAP kinase (MAPK) pathway is important for axon regeneration. The JNK pathway is activated by a signaling cascade consisting of the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury in a process involving the transcription factors ETS-4 and CEBP-1. Here, we find that svh-14/mxl-1, a gene encoding a Max-like transcription factor, is required for activation of svh-2 expression in response to axonal injury. We show that MXL-1 binds to and inhibits the function of TDPT-1, a C. elegans homolog of mammalian tyrosyl-DNA phosphodiesterase 2 [TDP2; also called Ets1-associated protein II (EAPII)]. Deletion of tdpt-1 suppresses the mxl-1 defect, but not the ets-4 defect, in axon regeneration. TDPT-1 induces SUMOylation of ETS-4, which inhibits ETS-4 transcriptional activity, and MXL-1 counteracts this effect. Thus, TDPT-1 interacts with two different transcription factors in axon regeneration.2019年10月, EMBO reports, 20(10) (10), e47517, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Axon regeneration following neuronal injury is an important repair mechanism that is not well understood at present. In Caenorhabditis elegans, axon regeneration is regulated by DDR-2, a receptor tyrosine kinase (RTK) that contains a discoidin domain and modulates the Met-like SVH-2 RTK-JNK MAP kinase signaling pathway. Here, we describe the svh-10/sqv-3 and svh-11 genes, which encode components of a conserved glycosylation pathway, and show that they modulate axon regeneration in C. elegans Overexpression of svh-2, but not of ddr-2, can suppress the axon regeneration defect observed in svh-11 mutants, suggesting that SVH-11 functions between DDR-2 and SVH-2 in this glycosylation pathway. Furthermore, we found that DDR-2 is N-glycosylated at the Asn-141 residue located in its discoidin domain, and mutation of this residue caused an axon regeneration defect. These findings indicate that N-linked glycosylation plays an important role in axon regeneration in C. elegans.2019年10月, Genetics, 213(2) (2), 491 - 500, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Axon regeneration is a conserved mechanism induced by axon injury that initiates a neuronal response leading to regrowth of the axon. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the JNK MAP kinase (MAPK) pathway. We have previously identified a number of genes affecting the JNK pathway using an RNAi-based screen. Analysis of these genes, called the svh genes, has shed new light on the regulation of axon regeneration, revealing the involvement of a signaling cascade consisting of a growth factor SVH-1 and its receptor, the tyrosine kinase SVH-2. Here, we characterize the svh-6/tns-1 gene, which is a homolog of mammalian tensin, and show that it is a positive regulator of axon regeneration in motor neurons. We demonstrate that TNS-1 interacts with tyrosine-autophosphorylated SVH-2 and the integrin β subunit PAT-3 via its SH2 and PTB domains, respectively, to promote axon regeneration. These results suggest that TNS-1 acts as an adaptor to link the SVH-2 and integrin signaling pathways.SIGNIFICANCE STATEMENT The Caenorhabditis elegans JNK MAPK pathway regulates the initiation of axon regeneration. Previously, we showed that a signaling cascade consisting of the HGF-like growth factor SVH-1 and its Met-like receptor tyrosine kinase SVH-2 promotes axon regeneration through activation of the JNK pathway. In this study, we show that the C. elegans tensin, TNS-1, is required for efficient regeneration after axon injury. Phosphorylation of SVH-2 on tyrosine mediates its interaction with the SH2 domain of TNS-1 to positively regulate axon regeneration. Furthermore, TNS-1 interacts via its PTB domain with the integrin β subunit PAT-3. These results suggest that TNS-1 plays a critical role in the regulation of axon regeneration by linking the SVH-2 and integrin signaling pathways.2019年07月, The Journal of Neuroscience, 39(29) (29), 5662 - 5672, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the mechanisms regulating axon regeneration are not well understood. Here, we identify the brc-2 gene encoding a homolog of the mammalian BRCA2 tumor suppressor as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that the RHO-1/Rho GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC-4/MLC) phosphorylation signaling pathway regulates axon regeneration. BRC-2 functions between RHO-1 and LET-502, suggesting that BRC-2 is required for the activation of LET-502 by RHO-1-GTP. We also find that one component that interacts with BRC-2, the ALP (α-actinin-associated LIM protein)/Enigma protein ALP-1, is required for regeneration and acts between LET-502 and MLC-4 phosphorylation. Furthermore, we demonstrate that ALP-1 associates with LET-502 and MLC-4. Thus, ALP-1 serves as a platform to activate MLC-4 phosphorylation mediated by the RHO-1-LET-502 signaling pathway.2018年08月, Cell Reports, 24(7) (7), 1880 - 1889, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Following axon injury, a cascade of signaling events is triggered to initiate axon regeneration. However, the mechanisms regulating axon regeneration are not well understood at present. In Caenorhabditis elegans, axon regeneration utilizes many of the components involved in phagocytosis, including integrin and Rac GTPase. Here, we identify the transthyretin (TTR)-like protein TTR-11 as a component functioning in axon regeneration upstream of integrin. We show that TTR-11 binds to both the extracellular domain of integrin-α and phosphatidylserine (PS). Axon injury induces the accumulation of PS around the injured axons in a manner dependent on TTR-11, the ABC transporter CED-7, and the caspase CED-3. Furthermore, we demonstrate that CED-3 activates CED-7 during axon regeneration. Thus, TTR-11 functions to link the PS injury signal to activation of the integrin pathway, which then initiates axon regeneration.2018年08月, Nature communications, 9(1) (1), 3099 - 3099, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- Organisms have developed many protective systems to reduce the toxicity from heavy metals. The nematode Caenorhabditis elegans has been widely used to determine the protective mechanisms against heavy metals. Responses against heavy metals can be monitored by expression of reporter genes, while sensitivity can be determined by quantifying growth or survival rate following exposure to heavy metals.BIO-PROTOCOL, 2017年06月, BIO-PROTOCOL, 7(11) (11), 英語[査読有り]研究論文（学術雑誌）
- The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2-JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration.2016年12月, PLoS genetics, 12(12) (12), e1006475, 英語, 国際誌[査読有り]研究論文（学術雑誌）
- 2019年, 日本分子生物学会年会プログラム・要旨集(Web), 42nd線虫C.elegansのF-boxタンパク質SVH-24はMad転写因子の分解により軸索切断依存的な遺伝子発現を活性化する
- 2019年, 日本分子生物学会年会プログラム・要旨集(Web), 42ndMax転写因子MXL-1はEts転写因子のSUMO化制御を介して軸索再生を促進する
- 2019年, 日本分子生物学会年会プログラム・要旨集(Web), 42ndCDK-14によるnon-canonical Wntシグナル経路を介した神経軸索再生の制御機構
- 2018年, 日本分子生物学会年会プログラム・要旨集(Web), 41st乳がん原因遺伝子BRCA1の線虫ホモログはGPCR-Gqシグナル経路を介して神経軸索再生を制御する
- 2018年, 日本分子生物学会年会プログラム・要旨集(Web), 41st細胞死認識経路による神経軸索再生促進機構
- 2018年, 日本分子生物学会年会プログラム・要旨集(Web), 41stRNA編集による線虫の神経軸索の再生制御機構
- 2017年, 日本生化学会大会(Web), 90th線虫EnigmaホモログによるRhoキナーゼシグナルを介した神経軸索再生制御
- 2016年, 日本分子生物学会年会プログラム・要旨集(Web), 39th乳癌原因遺伝子BRCA2の線虫ホモログBRC-2はRho-ROCK-MLCリン酸化経路で神経軸索再生を制御する
- 2015年, 日本生化学会大会(Web), 88th線虫のBRCA2はRhoシグナル-MLCリン酸化経路で神経軸索再生を制御する
- 日本学術振興会, 科学研究費助成事業, 若手研究, 神戸大学, 2021年04月01日 - 2023年03月31日視床下部タニサイトにおけるネクチン-1によるグルコース濃度の感知制御機構様々な生物の寿命は食事制限により延長するが、個体の老化・寿命の制御機構の多くが未解明である。視床下部では、第三脳室の側壁を覆うタニサイトが脳脊髄液中のグルコース濃度を感知し、その情報を摂食行動制御の中枢である弓状核へ伝達する。近年、視床下部は老化・寿命を司る上位中枢として注目されており、弓状核で産生される摂食制御ホルモンは老化や寿命との関連が示唆されている。このホルモン産生制御にはタニサイトの正常なグルコース感知機能が重要と考えられるが、老化過程におけるこの維持機構は不明である。研究代表者はこれまでにタニサイトにおいて、細胞接着分子ネクチン-1が特徴的な発現パターンを示すことを明らかにしている。そこで本研究では、ネクチン-1とタニサイトのグルコース感知機能および弓状核ホルモン産生制御との関係について検討する。 当初はネクチン-1遺伝子欠損マウスを使用して解析を進める予定であったが、ネクチン-1はタニサイトだけでなく、神経細胞やアストロサイトなどでも発現しており、タニサイトだけでの機能解析が難しいことが明らかになった。そこで、ネスチンプロモーターでCreERT2を発現させ、タモキシフェン投与により成体でタニサイトを含む幹細胞でネクチン-1を欠損させることができるコンディショナルノックアウトマウスを作成した。このマウスではタモキシフェン投与後2週間で約70%のネクチン-1陽性タニサイトでネクチン-1を欠損させることができた。現在このマウスを用いて解析を進めているが、すでにネクチン-1の欠損により弓状核のホルモン産生が変化することを見出している。加えて、このマウスでは摂食行動なども変化する傾向を見出しており、今後詳細な解析を行う。
- 日本学術振興会, 科学研究費助成事業, 研究活動スタート支援, 神戸大学, 2020年09月11日 - 2022年03月31日α2タニサイトの構造・機能とネクチン１の発現様々な生物の寿命は食事制限により延長するが、個体の老化と寿命を制御する機構の大部分は未解明である。視床下部の第三脳室の側壁を覆うタニサイトは脳脊髄液中のグルコース濃度を感知するほか、成体でも神経幹/前駆細胞機能を持つ。近年、タニサイトが個体の寿命延長に関与することが示唆されているが、加齢時の神経幹/前駆細胞機能の維持機構は不明である。本研究では、神経幹細胞機能の高いタニサイトで特異的に発現する細胞接着分子ネクチンを同定し、神経幹細胞機能の維持に関与する可能性を見出した。また、摂食行動を制御する弓状核に幹細胞マーカーSox2陽性の神経細胞の存在を見出し、加齢により細胞数が減少することを報告した。
- 日本学術振興会, 科学研究費助成事業, 特別研究員奨励費, 名古屋大学, 2019年04月25日 - 2021年03月31日ＲＮＡ編集による神経軸索再生制御機構の解明神経軸索再生は線虫から哺乳動物まで種を超えて保存された機構であるが、その詳細にはまだ不明な点が多く残されている。線虫C. elegansでは、分泌因子SVH-1が頭部神経から分泌され、増殖因子として受容体チロシンキナーゼSVH-2を介してJNK型MAPK経路を活性化し神経軸索再生を制御する。また、SVH-1はプロテアーゼとして受容体非依存的に幼虫期の生育も制御する。そのため、SVH-1が増殖因子として神経軸索再生を制御するためにはプロテアーゼ活性が不活性化される必要があるが、そのメカニズムは不明であった。本研究ではSVH-1機能変換の制御因子として、シチジンデアミナーゼSVH-17に着目した。まず、SVH-17が神経軸索再生に関与することを見出し、SVH-1 -SVH-2経路で機能することを明らかにした。次にSVH-17は切断神経でなく、svh-1が特異的に発現する頭部神経で機能することを明らかにした。さらに、svh-17変異体の軸索再生率低下はSVH-1のプロテアーゼ活性に必要な触媒三残基の1つであるHis-755(CAU)をチロシン(UAU)へ置換したsvh-1(H755Y)の発現で抑圧されることを明らかにし、実際にsvh-1 mRNAでHis-755をコードするC2263が低頻度にUへ変換されることを示した。興味深いことにC2263周辺はステムループ構造を形成する。この構造にアミノ酸が変化しないように変異を導入したところ、svh-1変異体の幼虫致死の表現型はレスキューしたのに対し、軸索再生率低下はレスキューしなかった。ここでさらにHis-755をチロシンへ置換することで、軸索再生率低下はレスキューされ、幼虫致死の表現型はレスキューしなかった。加えて、svh-17は幼虫期では発現せず、成虫期で発現するようになることを示した。以上の結果から、SVH-17は成虫期にsvh-1 mRNAのC2263周辺のステムループ構造を認識し、C2263を特異的にUへ変換することでSVH-1のプロテアーゼ活性を不活性化し、神経軸索再生を制御することが示唆された。