STUDY OF THE PHENOTYPES AND FUNCTIONS OF TRP FAMILY MEMBERS EXPRESSED IN NEURONS

TRP家族成员神经元表达的表型和功能研究

• 批准号: 15590059
• 负责人: KANEKO Shuji
• 金额: $ 2.3万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590059/
• 关键词: TRPC5; TRPC4; TRPM2; siRNA; Xenopus oocytes; primary culture neuron; cerebral cortex; hydrogen peroxide; ADPリボース; 受容体活性化型カチオンチャネル; Ca^<2+>流入; RNA干渉法; TRPチャネル; 活性酸素; 神経細胞死; カルシウム; ラジカルストレス; RNAi

(1) A brief exposure to hydrogen peroxide (H_2O_2) induces severe deterioration of primary cultured neurons in vitro. We have investigated a possible link between the H_2O_2-induced neuronal death and Ca^<2+>-permeable TRPM2 channels that are supposed to be regulated by ADP-ribose (ADPR) and/or H_2O_2. In most of cultured cerebral cortical neurons from fetal rat, TRPM2 proteins were detected at cell bodies immunocytochemically. Application of H_2O_2 to cultured neurons elicited an increase in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) caused by Ca^<2+> influx and subsequent neuronal death in a similar concentration range. Molecular cloning of rat TRPM2 cDNA revealed several differences in amino acid sequences within Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H_2O_2-induced Ca^<2+> influx and H_2O_2-induced cell death were elicited in human embryonic kidney cells heterogeneously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA (siRNA) against rat TRPM2 caused decrease in immunoreactive TRPM2 content and the H_2O_2-induced Ca^<2+> influx. Moreover, H_2O_2-induced neuronal death was significantly inhibited by the siRNA treatment. These results suggest a critical role of TRPM2 in regulation of neuronal survival as well as the intracellular Ca^<2+> homeostasis.(2)In cultured cerebral cortical neurons from feral rat, TRPC1,3,4,5 and 6 proteins are detected by immunocytochemistry. In Fura-2 loaded neurons, Ca^<2+> influx was observed after P2Y receptor stimulation with ATP, which was inhibited by La^<3+>, Zn^<2+> and SKF96365. Both TRPC3 immunoreactivity and the receptor-activated Ca^<2+> influx were decreased in the neurons treated with anti-TRPC3 antisense oligodeoxynucleotide. There results suggest that TRPC3 channels are involved in the receptor-operated Ca^<2+> influx in immature cerebral cortical neurons.

(1)过氧化氢（H_2O_2）短时间暴露于体外原代培养的神经元可引起严重的损伤。我们研究了H_2O_2诱导的神经元死亡与Ca^<2+>透过性TRPM 2通道之间的可能联系，该通道被认为是受ADP-核糖（ADPR）和/或H_2O_2调节的。在培养的胎鼠大脑皮质神经元中，TRPM 2蛋白免疫细胞化学染色可见于胞体。将H_2O_2应用于培养的神经元，在相似的浓度范围内，引起细胞内Ca^<2+浓度（[Ca^<2+]_i）增加，这是由于Ca^<2+内流和随后的神经元死亡引起的。大鼠TRPM 2 cDNA的分子克隆揭示了与人和小鼠TRPM 2相比，在营养盒区域内的氨基酸序列的几个差异。在异源表达大鼠TRPM 2的人胚肾细胞中，ADPR诱导的电流反应、H_2O_2诱导的Ca^<2+>内流和H_2O_2诱导的细胞死亡均被诱发。用针对大鼠TRPM 2的小干扰RNA（siRNA）处理培养的神经元，可使TRPM 2的免疫反应性含量和H_2O_2诱导的Ca^<2+>内流减少。此外，siRNA处理显著抑制H_2O_2诱导的神经元死亡。这些结果表明TRPM 2在调节神经元存活以及细胞内Ca^2+稳态中具有关键作用。(2)In用免疫细胞化学方法检测原代培养的大鼠大脑皮质神经元中TRPC 1、3、4、5和6蛋白的表达。在Fura-2负载的神经元中，ATP刺激P2 Y受体后可观察到Ca^2+内流，La^<3+>、Zn^<2+>和SKF 96365可抑制Ca ^2+内流。TRPC 3反义寡核苷酸处理的神经元TRPC 3免疫反应性和受体激活的Ca^2+内流均降低。这些结果表明TRPC 3通道参与了未成熟大脑皮层神经元中受体操纵的Ca^2+内流。

项目成果

Nicotinic acetylcholine receptor-mediated neuroprotection by donepezil against glutamate neurotoxicity in rat cortical neurons

• DOI: 10.1124/jpet.103.050104
• 发表时间: 2003-08-01
• 期刊: JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
• 影响因子: 3.5
• 作者: Takada, Y;Yonezawa, A;Akaike, A
• 通讯作者: Akaike, A

Serofendic acid prevents acute glutamate neurotoxicity in cultured cortical neurons.

Serofendic Acid 可防止培养的皮质神经元中的急性谷氨酸神经毒性。

• 发表时间: 2003
• 期刊: Eur.J.Pharmacol. 477
• 作者: H.Toyohara et al.;M.Kinoshita-Kawada et al.;Haruhiko Toyohara et al.;Mariko Kinoshita-Kawada et al.;M.Kinoshita-Kawada et al.;H.Toyohara et al.;F.Osakada et al.;S.Fujimoto et al.;Shinji Fujimoto et al.;Feng Wang et al.;Fujimoto et al.;F.Osakada et al.;N.Sakka et al.;R.Taguchi et al.;Y.Takada et al.;Noriko Sakka et al.;Yuki Takada et al.;Haruki Shibata et al.;Ryota Taguchi et al.
• 通讯作者: Ryota Taguchi et al.

Neuroprotective effects of α-tocopherol on oxidative stress in rat striatal cultures

• DOI: 10.1016/s0014-2999(03)01495-x
• 发表时间: 2003-03
• 期刊: European Journal of Pharmacology
• 影响因子: 5
• 作者: Fumitaka Osakada;A. Hashino;T. Kume;H. Katsuki;S. Kaneko;A. Akaike

Identification of scallop DMT as a metal transporter responsible for cadmium accumulation

鉴定扇贝 DMT 作为负责镉积累的金属转运蛋白

• 发表时间: 2005
• 期刊: FEBS Letters (In press)
• 作者: H.Toyohara et al.;M.Kinoshita-Kawada et al.;Haruhiko Toyohara et al.;Mariko Kinoshita-Kawada et al.;M.Kinoshita-Kawada et al.;H.Toyohara et al.
• 通讯作者: H.Toyohara et al.

N-methyl-D- aspartate receptors contribute to the maintenance of dopaminergic neurons in rat midbrain slice cultures.

N-甲基-D-天冬氨酸受体有助于维持大鼠中脑切片培养物中的多巴胺能神经元。

• 发表时间: 2003
• 期刊: Neurosci.Lett. 341
• 作者: Katsuki H;Shibata H;Takenaka C;Kume T;Kaneko S;Akaike A.
• 通讯作者: Akaike A.