Research Projects -
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Development of neuroprotective therapy for amyotrophic lateral sclerosis using serotonergic reagent
Grant number：24659431 2012.04 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
ASANUMA Masato, MIYAZAKI Ikuko
Authorship：Coinvestigator(s)
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
We examined neuroprotective effects of serotonin-1A receptor agonist 8-OH-DPAT on progressive motor neuron degeneration in amyotrophic lateral sclerosis (ALS) model mice and in primary culture cells. The pre-treatments of 8-OH-DPAT prior to onset of motor symptoms delayed disease onset. The chronic post-treatments of the drug after the onset significantly inhibited disease progression and showed protective effects on motor neuron loss in the cervical and lumber spinal cords in the ALS models. Furthermore, we revealed that the neuroprotective effects are based, in part, on expression and release of metallothionein in/from astrocytes via serotonin-1A receptors and sequent activation of transcription factor Nrf2.
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A new regulative mechanism for monoamine transporter expression in plasma membrane : The participation of histamine H3 receptor
Grant number：22592065 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
SOGAWA Norio, OHYAMA Kazumi, SOGAWA Chiharu, MIYAZAKI Ikuko, KITAYAMA Shigeo
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
To study the participation of histamine H3 receptor (H3R) on the transition of monoamine transporters into plasma membrane which is necessary to exert their function as transporters, we investigated the relation between H3R and noradrenaline transporter (NET) expression by using coexpressing cell system for both H3R and NET. The results which obtained from this experiment were suggested that there was a mechanism by which H3R inhibited the NET expression into the plasma membrane by binding to NET and making it fit tightly in the cell.
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Grant number：21591082 2009 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ASANUMA Masato, MIYAZAKI Ikuko
Authorship：Coinvestigator(s)
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
We revealed that induction and sequent release of glutathione or metallothionein in astroglia play an antioxidative protective role against neurotoxicity induced by dopamine neuron-specific oxidative stress in this study. Using neuron-astroglia coculture and parkinsonian model mice, we also proposed a possible therapeutic strategy for Parkinson's disease that a serotonin receptor agonist can protect dopamine neurons via activation of dopamine quinone-sensing molecules in astroglia.
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Studies on dopamine neuron-specific dmenerative factor dopamine quinone in the pathogenesis of Parkinson's disease.
Grant number：17590878 2005 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ASANUMA Masato, MIYAZAKI Ikuko
Authorship：Coinvestigator(s)
Grant amount：\3700000 （ Direct expense: \3400000 、 Indirect expense：\300000 ）
To clarify the possible roles of dopamine neuron-specific degenerative factor dopamine quinone in the pathogenesis of Parkinson's disease and to investigate therapeutic application of the dopamine quinone-regulating agents, we performed following experiments using dopaminergic neuronal cultured cells and hemi-parkinsonian model mice.
1. Screening of interacting molecules with dopamine or dopamine quinone
By electron spin resonance method using in vitro dopamine quinone generating system, we identified metallothionein-1, some dopamine agonists and certain drug as dopamine quinone-interacting molecules.
2. Dopamine quinone-induced toxicity in cultured neuronal cells and effects of regulation of quinone-quenching system
In dopaminergic CATH.a cells, dopamine quinone generated from excess free dopamine exerts neurotoxic effects. However, the dopamine-quinone-induced neurotoxicity was prevented by quenching of dopamine quinone, e.g. quinone reductase inducer, metallothionein inducer and a drug which enhances up-take of the substrate of glutathione synthesis in astroglial cells.
3. Changes in quinone-quenching system and effects of its regulation in parkinsonian animal models
Several molecules of quinone-quenching system were activated in the lesioned striatum of hemi-parkinsonian mice. The drug, which interacts with dopamine quinone, enhanced astroglial proliferation, up-take of glutathione substrate in astroglial cells, and increased glutathione levels in the striatum. Furthermore, the drug ameliorated dopaminergic neuronal loss in the substantia nigra and inhibited repeated L-DOPA injections-induced elevation of protein-bound quinone (quinoprotein) in the striatum. Repeated L-DOPA administration markedly elevated striatal quinoprotein levels and reduced the dopaminergic nerve terminals specifically on the lesioned side in metallothionein-knockout parkinsonian mice, suggesting that intrinsic cysteine-rich molecule metallothionein protects against L-DOPA-induced dopamine quinone neurotoxicity in parkinsonian mice by its quinone-quenching property.
These experimental results suggest a therapeutic potency of dopamine quinone-quenching agents for the patients of Parkinson's disease, and clarify that neuro-glial interaction on up-take of the substrate of glutathione synthesis plays a role in neuroprotective strategies against progressive dopaminergic neurodegeneration. -
Studies on tyrosinase-producing cell transplantation into parkinsonian models.
Grant number：14570596 2002 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ASANUMA Masato, MIYAZAKI Ikuko
Authorship：Coinvestigator(s)
Grant amount：\3200000 （ Direct expense: \3200000 ）
To investigate the possible effects of tyrosinase-producing cell transplantation in parkinsonian models, we performed intrastriatal transplantation of melanocytes or melanoma cells, as tyrosinase-producing cells, into hemi-parkinsonian model animals, as follows :
1.Transplantation of melanoma cells into the striatum of parkinsonian models
Intrastriatal transplantation of B16-F1 melanoma cells, as tyrosinase-producing cells, into hemi-parkinsonian rats lesioned by 6-OHDA markedly ameliorated apomorphine-induced rotation behavior towards contralateral side up to 40 days after the transplantation of melanoma cells, coinciding with expression of dopamine-positive signals around survived transplant.
2.Primary cultured melanocvtes from the back of newborn mice
We established the procedure of primary culture for melanocytes from the back of newborn C57BL mice, which showed higher tyrosinase activity than melanocytes from albino mice.
3.Transplantation of primary_cultured melanocytes into the striatum of parkinsonian models
The transplantation of primary cultured melanocytes from the back of newborn black mice into the lesioned striatum of hemi-parkinsonian mice markedly and continuously ameliorated the apomorphine-induced contralateral rotation behavior up to 3 months after the melanocyte transplantation. In the transplanted group with melanocytes, the striatal transplant melanocytes survived coinciding with dopamine- and tyrosinase-positive signals along the transplant in the striatum 3 months after the transplantation.
4.Primary cultured melanocytes from the adult mice
We tried to establish the culture of melanocytes from the back skin of adult C57BL mice for future auto-transplantation of melanocytes into parkinsonian models, but failed to do it because of contamination of the folliculi.
5.Distribution of tyrosinase-positive cells in the brain of parkinsonian models
Tyrosinase constitutively expressed mainly on oligodendrocytes in the corpus callosum, striatum and substantia nigra. In the parkinsonian model mice, tyrosinase-positive oligodendrocytes increased in the striatum, whereas it decreased in the substantia nigra.
These experimental results suggest a therapeutic potency of tyrosinase-producing cells such as melanocytes which may complement tyrosine hydroxylase when dopaminergic neurons are degenerated in Parkinson's disease, and may normalize L-DOPA-induced abnormal dopamine turnover