2018 Fiscal Year Research-status Report
Identification and characterization of PLA2 enzyme required for sensory nervous system development
| Project/Area Number |
17K14970
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
GUY ADAM・TSUDA 国立研究開発法人理化学研究所, 脳神経科学研究センター, 研究員 (30634779)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | Molecular Biology / Cell Biology / Neuroscience / Lipid Biology |
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| Outline of Annual Research Achievements |
I am studying two knockout (KO) mouse lines that have genetic deletion of specific secreted PLA2 genes: pla2g5 and pla2g10. I have found that embryos of both KO mice lines show defects in the spinal cord projections of nociceptive sensory afferents. Specifically, the nociceptive afferents project to inappropriate medial and/or deep domains inside the spinal cord, from where they are absent in wild type. In the normal situation, only proprioceptive afferents project to these areas.
I have developed a "reverse open-book" method combined with confocal microscopy for efficient quantitative anaylsis of nociceptive axon TrkA expression in whole spinal cord (instead of multiple transverse sections) including all the DRGs from cervical to sacral levels. Both pla2g5 and pla2g10 show an abnormal thickening of the TrkA domain in the mediolateral aspect reflecting a defect in axon guidance. pla2g10 showed a stronger phenotype than pla2g5. This is consistent with my hypothesis that in the absence of secreted PLA2-generated LysoPtdGlc, axon guidance of nociceptive spinal cord afferents is perturbed and the nociceptive axons occupy abnormal territory from which they are usually absent.
These findings corroborate my previous findings using DiI labelling and transverse sections.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Progress in analyses of KO embryos is good for pla2g5 and pla2g10 KO mice embryos. However, I have found that analysis of pla2g3 KO is technically very hard to achieve, due to a spermatocyte mobility defect in adult mice. Therefore, my analysis of pla2g3 embryos will have to be reconsidered.
Mass spectrometry analyses of LysoPtdGlc concentration in spinal cord tissues is ongoing and requires further time. There is a slight technical difficulty in distinguishing LysoPtdGlc and LysoPhosphatidylinositol in whole tissue.
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| Strategy for Future Research Activity |
I will carry out the following research plans:
1) Analyse nociceptive afferent axon patterning in spinal cord of WT and pla2g5 and pla2g10 KO mouse embryos using "reverse open book" method combined with confocal microscopy for quantitative analysis of spinal cord projection of sensory axons.
2) Mass spectrometry analyses of WT and pla2g5 and pla2g10 KO spinal cord concentration of LysoPtdGlc.
3) Culture spinal cord astrocytes from WT or KO spinal cord, and assess their ability to produce or hydrolyse LysoPtdGlc.
4) If time permits, carry out behavioural analyses of adult KO mice, in particular, tests of nociceptive sensory perception, as I hypothesise that these mice possess defects in nociceptive circuitry.
I will present my latest data at JNS annual meeting Neuro2019. My abstract has been peer-selected as an oral presentation.
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