2016 Fiscal Year Research-status Report
Therapeutic potential of the metabolic relationship between the product of two genetic markers for pain (gch1 and mthfr)
| Project/Area Number |
15K10556
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
J・P Bellier 滋賀医科大学, 神経難病研究センター, 助教 (80346022)
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| Co-Investigator(Kenkyū-buntansha) |
林 維光 滋賀医科大学, 医学部, 助教 (80242973)
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| Project Period (FY) |
2015-04-01 – 2018-03-31
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| Keywords | Tetrahydropbiopterin / Serotonin / GTP Cyclohydrolase 1 / Pain / Immunotherapy / Inflammatory pain |
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| Outline of Annual Research Achievements |
GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the biosynthetic pathway for tetrahydrobiopterin (BH4), and is known as a marker for neuropathic pain. While GCH1 regulate availability in BH4, it may also regulate the availability in serotonin and nitric oxide, which are believed to modulate the nociceptive neurotransmission. Therefore, regulation of BH4 synthesis might be a lever to control pain signalling. This project explores the modulation of BH4 synthesis through targeting GCH1 both by immunotherapeutic. We produced a specific monoclonal antibody against GCH1 for use in immunotherapy strategy. Biochemical and immunohistochemical analyses showed that the antibody recognizes only the regulated form of GCH1, which is merely located in the sensory system. Together, these results indicate that the antibody fulfilled two important conditions for the development of immunotherapy strategy against pain: specificity against GCH1 and specificity to the sensory system (that might reduce side effect). In addition, serotonin modulation of sensory and nociceptive neurotransmission was investigated in a invertebrate model, it indicates that regulation of sensory process match essentially with those of the vertebrate. Finally, during the course of development of the GCH1 antibody, we also obtained incidentally a unique antiserum that stain most the enteric nervous system, opening potential new opportunity for the treatment of visceral pain.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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.
Application of the newly produced monoclonal antibody for immunotherapy is currently under investigation in animal model of neuropathic pain. In the same context, a newly raised polyclonal antiserum against GCH1 has been shown to be useful for delineating the enteric nervous system. It suggests that GCH1 involvement in pain might be also involved in visceral pain. Therefore we are currently investigating the involvement of GCH1 in visceral pain, and the potential therapeutic use of the antiserum that we have produced.
Examination of serotoninergic pathway in an invertebrate animal model followed an unexpectedly smooth progress, and indicate serotonin may also modulate sensory neurotransmission in invertebrate, indicating that the strategy used by invertebrate to cope against nociceptive stimuli might have a usage in the vertebrate.
Study of the alternative BH4 recycling pathway was implemented as planned and steadily. choline metabolism and its relation with pain is currently under investigation in a rat model of neuropathic pain. An number of potential inhibitor and agonist to the choline metabolism is currently under investigation. However, involvement in the nociceptive system is not demonstrated yet.
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| Strategy for Future Research Activity |
The future implementation of the project will follow what was initially planned, with a few modifications that aim at investigate inflammatory for visceral pain. We will continue to investigate the neutralizing properties of the newly generated monoclonal against regulated form of GCH1 on cultivated cell line. GCH1 activity and BH4 production will be assessed using HPLC with electrochemical detection. The antibody will be tested in vivo in a model of neuropathic pain and a model of inflammatory pain. While initially we have planned to use a model of inflammatory pain induced by intraplantar injection of Carrageenan solution, we decided to move to a model of visceral inflammation. The decision is taken because of unexpected interesting result obtained with the polyclonal antibody above described, which might provide interesting results in such model. We will continue to investigate 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 enzyme of those pathway will be tested. HPLC and enzymatic assay will be used to assess change in metabolic pathways.
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| Causes of Carryover |
Almost all reagents described in the experimental design were bought as initially planned. We took advantage of commercial campaign that resulted in a substantial save. In addition, we published our data in journal without publication fee.
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| Expenditure Plan for Carryover Budget |
This fiscal year, remaining incurring amount of the previous fiscal year, will be used according to its initial purpose, and to pursue the goal described in the proposed project. Additional usage plan include open access for journal and promotion of our result by attending conferences.
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