2021 Fiscal Year Research-status Report
Demonstration of the vertebrate CMP-sialic acid synthetase as a novel regulatory protein of neural cell apoptosis
| Project/Area Number |
21K15040
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| Research Institution | Nagoya University |
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Principal Investigator |
呉 迪 名古屋大学, 生物機能開発利用研究センター, 助教 (10817547)
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| Project Period (FY) |
2021-04-01 – 2023-03-31
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| Keywords | CSS / neurogenesis / apoptosis / medaka |
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| Outline of Annual Research Achievements |
CMP-sialic acid synthetase (CSS) is a key enzyme for the expression of sialic acid (Sia)-containing
glycoconjugates on the cell surface. N-domain of CSS is catalytic domain, which contained the active site and 5 conserved motifs; however, medaka with a point mutation in the N-domain of CSS (named MuN) was lethal at early developmental stage due to neural cell apoptosis without affecting the sialylation state. These results suggest that CSS plays a critical role in neurogenesis, not only as a sialylation-involved enzyme but also as an apoptosis related protein. To clarify how CSS regulates the neural cell apoptosis, focusing on proteins that interact with CSS in neural system, I executed the following experiments: (1) To determine the protein, which is interacted with mouse CSS (mCSS) in mouse neuroblastoma cell line Neuro2A, the proximity labeling technique (Turbo ID) were used. As a result, mCSS was interacted with various proteins in Neuro2A cell and one of them is Fragile X related protein (FXRP). The interaction of mCSS and FXRP was confirmed by immunoprecipitation experiments. (2) To characterize the interaction between FXRP and CSS at the molecular level, the interaction of FXRP with wild-type, MuN CSS and R188H CSS (mutation found from patients of intellectual disability) were confirmed in Neuro2A cells. However, the interaction with FXRP was not significantly different between wild-type CSS and mutant CSSs. (3) To clarify the significance of the CSS and FXRP interaction at the animal level, the neurogenesis and the heart development of MuN medaka were observed.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
This project is progressing more smoothly than initially planned because of the following reasons:
(1)The proximity labeling experiment for the first time confirmed that various proteins interacted with CSS in Neuro2A cell. The interaction with FXRP was confirmed by the immunoprecipitation experiment, indicating that the interaction of FXRP and CSS possibly plays important role in neural development and myogenesis, because FXRP is known to mediate transport of specific mRNAs to different intracellular compartments and inhibit translation of their target mRNAs,
(2)The interaction of FXRP with MuN and R188H mutant CSS was compared with that with wild-type CSS using proximity labeling and immunoprecipitation. Since no obvious difference was observed between wild-type CSS and mutant CSSs in Neuro2A cells, the interaction should be confirmed at the animal level.
(3)Our previous study showed that MuN mutant induced apoptosis in telencephalon and optic tectum of brain in medaka. To confirmed whether the apoptosis in MuN medaka was induced by ER stress, the CCAAT-enhancer-binding protein homologous protein (CHOP) expression was quantified by real-time PCR. Since the expression of CHOP was obviously increased in MuN medaka at 8 dpf, it can be concluded that MuN mutant-induced ER stress results in apoptosis at the animal level. Furthermore, the heart development of MuN medaka was observed from 0 to 8 dpf to confirm the reason for lethality. As a result, the abnormal heart development was observed in MuN medaka, which is the similar phenotype to that of FXRP knock-down zebrafish.
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| Strategy for Future Research Activity |
The following experiments should be executed:
(1)Not only FXRP but also other candidates of interacting proteins of CSS were confirmed by proximity labeling technique. It is necessary to confirm all the interacting proteins by mass spectrometric analysis (MS) for further study. Furthermore, the important domain for the interaction of CSS protein will be determined by the chimeras technique.
(2)To confirm the difference of the interaction with FXRP between wild-type CSS and MuN CSS at the animal level, firstly, the expression profiles of CSS and MuN CSS during neurogenesis are established by live imaging using knock-in technique; Secondly, the FXRP expression in MuN medaka and wild-type medaka are quantified and compared by the real-time PCR method or in situ technique; thirdly, the interaction differences between wild-type CSS and MuN CSS are confirmed at the animal level by affinity-purification and western blotting using specific antibody.
(3)To clarify the significance of the interaction between FXRP and CSS during neurogenesis and myogenesis at animal level, the neural system-specific and muscle-specific CSS-KO medaka strain will be generated using CRISPR/Cas9 nickase-mediated knock-in technique.
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Research Products
(5 results)
