| 研究課題/領域番号 |
15K10556
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| 研究機関 | 滋賀医科大学 |
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研究代表者 |
J・P Bellier 滋賀医科大学, 神経難病研究センタ－, 助教 (80346022)
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| 研究分担者 |
林 維光 滋賀医科大学, 医学部, 助教 (80242973)
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| 研究期間 (年度) |
2015-04-01 – 2018-03-31
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| キーワード | Tetrahydrobiopterin / Serotonin / GTP cyclohydrolase 1 / Folate pathway / Pain |
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| 研究実績の概要 |
Tetrahydrobiopterin (BH4) is a cofactor required for the biosynthesis of monoamine neurotransmitters (such as serotonin or dopamine) and nitric oxide. Those neurotransmitters are believed to modulate the nociceptive transmission. Therefore, regulate BH4 synthesis might be a lever to control pain signalling. GTP cyclohydrolase 1 (GCH1; EC 3.5.4.16) is a key enzyme for the de novo synthesis of BH4 in the biopterin metabolic pathway. BH4 may also be salvaged from its oxidized form BH2, via a metabolic route involving methylenetetrahydrofolate reductase (MTHFR), a enzyme of the folate metabolic pathway. GCH1 and MTHFR has been shown to be involved in the variability of the response to pain in human and rodents.
Modulation of the de novo BH4 synthesis and BH4 salvage were explored and the regulatory aspect of monoaminergic neurotransmission was investigated. A monoclonal neutralizing antibody against regulated form of GCH1 to modulate the de novo synthesis of BH4 was produced. Several clones appear to be suitable and subcloning is under process. In the same context, a new polyclonal antiserum against GCH1 was raised that allow high anatomical resolution of GCH1 in the enteric nervous system, and might allow to assess how BH4 be involved in visceral pain. In addition, serotonin modulation of sensory and nociceptive neurotransmission was investigated in a invertebrate model, and might simplify nociception assessment. The BH4 salvage pathway was investigated, especially the properties of the intermediate enzymes between choline metabolic and biopterin metabolic pathways.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The experiments were implemented as planned, however some aspects of the project encountered delay inherent to experimental research processes, while others are progressing more smoothly than expected.
Production of a new monoclonal antibody appear to succeed but final subcloning step is taking slightly more time than expected. The reason of this delay is due to the relatively large numbers of positive clones to screen, moreover additional screening techniques were used to further validate more efficiently the clones actually suitable for subsequent purposes. Examination of serotoninergic pathway in an invertebrate animal model followed an unexpectedly smooth progress. It indicates that similar to the situation in vertebrate; serotonin may also modulate sensory neurotransmission in invertebrate. It also suggest that invertebrate model might be a useful and simple model to assess nociceptive neurotransmission. This aspect of the work is considered as finished and will be submitted for publication. Study of the BH4 salvage pathway was almost steadily implemented as planned. The examination of the choline metabolic pathway progressed rather more smoothly than expected, while the examination of folate metabolic pathway is more challenging.
Rather unexpected promising results was obtained with one antiserum raised against GCH1 in the enteric nervous system, which might open the possibility to study the impact of GCH1 in visceral pain and eventually provide new opportunity to understand visceral pain.
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| 今後の研究の推進方策 |
The future implementation of the project will follow what was initially planned, with a few modifications.
The production and characterization of a new monoclonal antibody will be achieved. Its neutralizing properties against regulated form of GCH1 will be tested on protein extract and on cultivated cell line. GCH1 activity and BH4 production will be assessed using HPLC with electrochemical detection. The Choline and MTHFR metabolism will be investigated in dorsal root ganglion of animal pain model, using immunohistochemical and biochemical approaches. Selected drugs that might potentially inhibit enzymes of those pathway will be tested. HPLC and enzymatic assay will be used to assess change in metabolic flux. Finally, in regard of promising results obtained with GCH1 antibody in the enteric nervous system and the possibility of involvement of GCH1 in visceral pain, further experiment will be performed to confirm and validate the antibody.
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| 次年度使用額が生じた理由 |
Almost all reagents described in the experimental design were bought as initially planned. However because of slight delay in the monoclonal antibody production, all planned incurring amount was not used.
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| 次年度使用額の使用計画 |
This fiscal year, remaining incurring amount of the previous fiscal year, will be used according to its initial purpose : to finish the production of a monoclonal antibody.
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