Analyses of the activity dependent presynaptic function forming

活动依赖性突触前功能形成的分析

• 批准号: 14580740
• 负责人: KATAOKA Masakazu
• 金额: $ 1.98万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580740/
• 关键词: Complexin II; axonal localization; neuronal primary culture; soluble proteins; SNAP-25; protein phosphorylation; mutant mice; chimeric mice

The mechanisms of specific localization of synaptic proteins at synaptic site of central nervous system (CNS) have been unclear. Little is known how the soluble proteins functioning in synapses localize at synapse in particular. Complexin (CPX) is a soluble pre-synaptic regulatory protein, and is thought to regulate exocytosis of neurotransmitters release from pre-synapse. Here we show the initial study to elucidate the localization mechanism of soluble synaptic proteins in synapse by using CPX as a model molecule.The myc-tagged CPX (mCPX) construct was transfected into rat primary hippocampus neuron with liposome reagent, and the localization of expressed protein was evaluated by the fluorescent intensity analysis of images up taken by laser confocal scanning microscopy. In the dendrite, fluorescence intensities from mCPX and EGFP, a nonspecific soluble protein control, decreased at periphery of the dendrite. In the axon, the mCPX was transferred to periphery of the axon although the … More EGFP showed the same tendency with that at dendrite, indicating that CPX has a specific mechanism to be transferred to the axon.To identify the domain required for the specific transfer to axon, the deletion analysis was conducted. Fluorescent analyses indicated that the deletion of 19 amino acid residues, G71-P89, within the "central core" resulted the loss of the specific axonal localization of CPX derivatives. The EGFP derivative fused with the G71-P89 peptide showed axonal localization, indicating that the G71-P89 region conditions the axonal transport of CPX.SNAP-25, a member of SNARE proteins, is essential for the secretory vesicle fusion with plasma membrane and phosphorylated at the Ser187 residue by the phorbor-ester stimulation in PC12 cells. The pharmacological study is important to elucidate the function of the protein phosphorylation in cellular function. In addition, to know how the phosphorylatin state in living organ is also important for understanding the biological system. Here we show the mode of the SNAP-25 at Ser187 in rat brain by using anti-phosphorylated SNAP-25 antibody as a probe. Western blotting of rat brain lysates indicated that SNAP-25 is phosphorylated in rat brain. The developmental patterns of expression and the SNAP-25 phosphorylation was not pararell. The SNAP-25 expression is detected from E18 embryonic brain and the expression continues to increase lineally up to P25. The phosphorylation of SNAP-25 cannot be detected until P9 and then the phosphorylation increment continue to P25. Thus the SNAP-25 phosphorylation is regulated developmentally in rat brain. The time course study using the rat hyppocampal culture showed similar phosphorylation pattern of SNAP-25, implying the SNAP-25 phosphorylation is genetically programmed under the interaction between neuronal cells and glial cells. In the investigation of the effect of environmental stimulation that confers the neural plasticity, the SNAP-25 phosphorylation shows small decrement by in vivo long-term-potentiation. The decrement of the SNAP-25 phosphorylation is also observed in the rat brain introduced epilepsy by kainate. These results clearly exhibit that the phosphorylation state of SNAP-25 at Ser187 is altered by developmental stage and environmental change, thus the SNAP-25 phospohorylation takes important roles in higher brain function(s). Less

突触蛋白在中枢神经系统(CNS)突触部位的特异性定位机制尚不清楚。突触中的可溶性蛋白如何在突触中发挥作用，特别是在突触中的定位，目前还知之甚少。复合蛋白(CPX)是一种可溶性的突触前调节蛋白，被认为调节突触前释放的神经递质的胞吐。本研究以CPX为模型分子，通过脂质体将Myc标记的CPX(MCPX)载体导入原代培养的大鼠海马神经元，通过激光共聚焦扫描显微镜对表达蛋白进行荧光强度分析，初步探讨了突触中可溶性突触蛋白的定位机制。在树突中，mCPX和EGFP(一种非特异性的可溶性蛋白对照)的荧光强度在树突周围减弱。在轴突中，mCPx被转移到轴突的外围，尽管…更多的EGFP显示出与树突相同的趋势，表明CPX具有特定的转移到轴突的机制。为了确定特定的结构域转移到轴突所需的区域，进行了缺失分析。荧光分析表明，“中心核”中19个氨基酸残基G71-p89的缺失导致了CPX衍生物的特异性轴突定位的丧失。与G71-p89多肽融合的EGFP衍生物显示出轴突定位，表明G71-p89区域决定了CPX的轴突运输。SNAP-25是SNARE蛋白中的一员，是PC12细胞分泌囊泡与质膜融合所必需的，并在磷酸-酯刺激下被Ser187残基磷酸化。药理学研究对于阐明蛋白质磷酸化在细胞功能中的作用具有重要意义。此外，了解活体器官中的磷酸化状态对于理解生物系统也很重要。在这里，我们用抗磷酸化的SNAP-25抗体作为探针，展示了SNAP-25在大鼠脑内Ser187位的模式。大鼠脑组织裂解产物的Western blotting结果表明SNAP-25在大鼠脑内被磷酸化。SNAP-25的表达和磷酸化的发育模式不是平行的。SNAP-25的表达是从E18胚胎脑中检测到的，并且表达持续线性增加，直到P25。SNAP-25的磷酸化直到P9才能检测到，然后磷酸化增加持续到P25。因此，SNAP-25的磷酸化在大鼠脑内是发育调节的。用大鼠下丘脑培养的时间进程研究显示SNAP-25的磷酸化模式相似，这表明SNAP-25的磷酸化是在神经细胞和神经胶质细胞相互作用下的基因编程。在研究环境刺激赋予神经可塑性的作用中，SNAP-25的磷酸化在体内长期增强后表现出轻微的下降。海人酸致癫痫大鼠脑组织中SNAP-25的磷酸化水平也明显降低。这些结果清楚地表明，SNAP-25在Ser187处的磷酸化状态会随着发育阶段和环境的变化而改变，因此SNAP-25的磷酸化在高级脑功能中起着重要作用(S)。较少

项目成果

M.Takahashi, M.Kataoka, ほか: "New Aspects of Neurotransmitter Release and Exocytosis : Regulation of Neurotransmitter Release by Phosphorylation"J.Pharmacol.Sci. 93. 41-45 (2003)

M.Takahashi、M.Kataoka 等：“神经递质释放和胞吐作用的新方面：通过磷酸化调节神经递质释放”J.Pharmacol.Sci 93. 41-45 (2003)

Shoji-Kasai Y, Itakura M, Kataoka M, Yamamori S, Takahashi M.: "Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells."Eur J Neurosci.. 15. 1390-1394 (2002)

Shoji-Kasai Y、Itakura M、Kataoka M、Yamamori S、Takahashi M.：“蛋白激酶 C 介导的分泌囊泡向质膜的易位以及 PC12 细胞神经递质释放的增强。”Eur J Neurosci.. 15. 1390-

Y.Shoji-Kasai, M.Kataoka ほか: "Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells"Eur J Neurosci. 15. 1390-1394 (2002)

Y. Shoji-Kasai，M. Kataoka 等人：“蛋白激酶 C 介导的分泌囊泡向膜质的易位和 PC12 细胞神经递质释放的增强”Eur J Neurosci. 15. 1390-1394 (2002)。

Takahashi M, Itakura M, Kataoka M.: "New Aspects of Neurotransmitter Releasee and Exocytosis : Regulation of Neurotransmitter Release by Phosphorylation."J. Pharmacol. Sci.. 93. 41-45 (2003)

Takahashi M、Itakura M、Kataoka M.：“神经递质释放和胞吐作用的新方面：通过磷酸化调节神经递质释放。”J。

M.Takahashi: "New aspect of neurotransmitter release and exocytosis : regulation of neurotransmitter release by phosphorylation"J.Pharmacol.Sci.. 93. 41-45 (2003)

M.Takahashi：“神经递质释放和胞吐作用的新方面：通过磷酸化调节神经递质释放”J.Pharmacol.Sci.. 93. 41-45 (2003)