| 若手道場10 |
|---|
| 2021/10/1 13:00～14:00 ZOOM 若手道場
WD10-1
神経細胞における排出トランスポーターMRP5/ABCC5の機能発現と臨床薬による阻害
Functional expression and inhibition by clinically used drugs of efflux transporter MRP5/ABCC5 in neuronal cells
○八木 寛史, Takahiro Ishimoto, Yusuke Masuo, Yukio Kato
金沢大学医薬保健総合研究科創薬専攻
○Hirofumi Yagi, Takahiro Ishimoto, Yusuke Masuo, Yukio Kato
Faculty of Pharmacy, Kanazawa University
Multidrug resistance associated protein 5 (Mrp5/Abcc5) was reported to be highly expressed in cortical neurons compared with other Abcc family members, although its physiological role remains unclear. Mrp5 pumps out cyclic nucleotides cGMP and cAMP, and these nucleotides are known to regulate cognitive function at least partially via control of neuronal differentiation. Therefore, Mrp5 may regulate such neuronal function via efflux of the cyclic nucleotides. The aim of the present study was to clarify functional expression and inhibition by clinically used drugs of Mrp5 in neuronal cell line Neuro2a. We established an assay to check efflux activity of Mrp5 in Neuro2a using a compound CMFDA, which is metabolized to the fluorescent Mrp5 substrate after transport into the cells. Neuro2a transfected with siRNA for Mrp5 showed higher intensity of intracellular fluorescence of CMFDA metabolite compared with that transfected with control siRNA. In addition, exposure to Mrp5 inhibitors probenecid and zaprinast increased the fluorescent intensity. These results suggest that Mrp5 is functionally expressed in Neuro2a. The knockdown of Mrp5 by siRNA significantly increased number of differentiated cells with long neurites, suggesting that down-regulation of Mrp5 may lead to enhancement of neuronal differentiation. Therefore, we conducted a screening for Mrp5 inhibitors among clinically used drugs acting in the central nervous system using the established assay. Among 93 drugs examined, perampanel, biperiden, midazolam, and nortriptyline increased the fluorescent intensity of the CMFDA metabolite. Further studies are required to evaluate whether these Mrp5 inhibitor drugs may promote neuronal differentiation, and analyze its mechanisms underlying. | | 2021/10/1 13:00～14:00 ZOOM 若手道場
WD10-2
知的障害責任分子RAC3の大脳発達における生理機能と分子病態機構の解明
RAC3 variants in neurodevelopmental disorders and their pathophysiological significance
○西川 将司, Hidenori Ito, Hidenori Tabata, Koh‑ichi Nagata
Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan
○Masashi Nishikawa, Hidenori Ito, Hidenori Tabata, Koh‑ichi Nagata
Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan
Variants in RAC3, encoding a small GTPase which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a neurodevelopmental disorder (NDD) with structural brain anomalies and facial dysmorphism. We analyzed the pathophysiological mechanisms underlying the development of RAC3-related brain malformations analyzing the role of pathogenic RAC3 variants in the development of mouse cerebral cortex. In vitro analyses revealed that all tested variants resulted in an active status of RAC3, with each mutated protein exhibiting a spectrum of different affinities to downstream effectors. We then focused on the 4 variants affecting the Switch II region, which is supposed to be a hot spot common to other RHO family genes, such as RAC1 and CDC42. Acute expression of the 4 variants using in utero electroporation caused defects in cortical neuron morphology and migration during corticogenesis. The defects were rescued by a dominant negative version of PAK1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes. | | 2021/10/1 13:00～14:00 ZOOM 若手道場
WD10-3
腹側被蓋野にチロシン水酸化酵素野生型あるいはS40E変異体を過剰発現させたマウスにおける生化学的および行動学的変化
Biochemical and behavioral alterations of mice overexpressing wild-type or S40E mutant of tyrosine hydroxylase in the ventral tegmental area
○志村 武信1,2
1.東京工業大学 生命理工学院 生命理工学系 一瀬研究室, 2.Addictive Substance Project, Tokyo Metropolitan Institute of Medical Science, 3.Jichi Medical University
○Takenobu Shimura1,2
1.School of Life Science and Technology, Tokyo Institute of Technology, 2.Addictive Substance Project, Tokyo Metropolitan Institute of Medical Science, 3.Jichi Medical University
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine. There are two major mechanisms for regulation of the TH activity. One is a feedback inhibition by the binding of dopamine to its active site, while the dopamine-bound enzyme is stable. The other is the phosphorylation at the N-terminal region of TH. Phosphorylation at the 40th serine can rescue TH from the inhibition by dopamine and re-activate it. A mutant, TH_S40E, mimics TH phosphorylated at the 40th serine. Here, we examined the effects of overexpression of TH in the mesolimbic dopaminergic neurons in the mice using an adeno-associated virus (AAV) vector and examined the dopamine metabolism in the nucleus accumbens and the effects of methamphetamine (METH) administration.
We injected AAV harboring wild-type TH (TH_wt), TH_S40E, or AcGFP, a fluorescent protein derived from Aequorea coerulescens, in the ventral tegmental area, and four-weeks after the AAV-injection the mice were administered METH intraperitoneally. We found that the TH protein levels were significantly increased in the nucleus accumbens in the TH_wt but not in the TH_S40E administered mice. Although the dopamine contents were not significantly different among three groups, the amounts of the dopamine metabolites, DOPAC and HVA, were significantly elevated only in the TH_S40E mice. These data suggest an enhanced dopamine metabolism in the TH_S40E mice. We investigated the locomotive activities after administration of METH in these mice. The TH_S40E mice showed a greater locomotive activity than the mice administered with TH_wt or AcGFP. Our data suggest that alteration in the TH activity should affect the responses to METH and the modification of the TH activity might be a target for the treatment to drug-abuse.
| | | |
|
|
