2020 Fiscal Year Research-status Report
Investigation of the role of radial glia in regulating LysoPtdGlc required for nervous system development
| Project/Area Number |
20K06884
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| Research Institution | Kyoto University |
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Principal Investigator |
GUY ADAM・TSUDA 京都大学, 生命科学研究科, 准教授 (30634779)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | Lipid biology / Neurochemistry |
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| Outline of Annual Research Achievements |
I developed and utilized a strategy of primary cell culture followed by digital PCR to quantify the exact amount of mRNA for each of the ten isoforms of secreted phospholipase A2 (sPLA2) enzyme in different cell types and tissues in developing embryonic nervous system. Each isoform of sPLA2 was absolutely quantified as copy number per 10ng cDNA. I discovered that radial glia in spinal cord specifically express one isoform of secreted phospholipase A2 enzyme: sPLA2 V, and supported this finding by analyzing spinal cord tissue derived from embryos genetically lacking sPLA2 V. Nociceptive dorsal root ganglion (DRG) sensory neurons expressed sPLA2 IB and III. Non-radial glial cells in the spinal cord expressed a complex mixture of isoforms: sPLA2 IB, IIF, III, X and XIIA.
Using antibody staining with specific monoclonal antibodies and confocal microscopy of reverse open-book preparations of spinal cord, I conducted measurement and quantification of TrkA domain in developing spinal cord of wildtype and sPLA2 V knockout mice, and performed statistical analyses of these data. I discovered that KO mice possessed an abnormally large TrkA domain in the spinal cord, that was statistically significant. This was true for every level of cervical, thoracic and lumbar DRG examined, although the biggest effect observed was in the lower regions of the spinal cord. According to my hypothesis this should be specific to nociceptive axons, therefore control data are needed for other modalities of sensory neurons.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
My ability to conduct research has been disrupted by temporary shutdown of laboratory workplace and other coronavirus precautions such as remote work.
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| Strategy for Future Research Activity |
I plan to directly measure the concentration of lysophosphatidylglucoside (LysoPtdGlc) in the embryonic spinal cord. Then compare this with the concentration of LysoPtdGlc in spinal cord tissue derived from embryos genetically lacking sPLA2 V. According to my hyothesis, spinal cord cells in knockout mice should have an impaired ability to hydrolyse the phospholipid precursor phosphatidylglucoside (PtdGlc) into LysoPtdGlc, therefore should presnet a much lower concentraion of LysoPtdGlc when measured by mass spectrometry.
I will carry out control experiments to prove that the defects in TrkA domain formation are specific to TrkA and do not involve proprioceptive TrkC-expressing axon tracts.
I aim to perform behavioural analyses in adult mice, looking for deficits in nociception.
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