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Analysis of the Functions in Synapse of Amyloid Precursor Protein

淀粉样前体蛋白突触功能分析

基本信息

批准号：
11680755
负责人：
NIINOBE Michio
金额：
$ 1.66万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

We studied on physiological functions of amyloid precursor protein (APP695) in neural cells, and found following results. (1) Postsynaptic responses evoked by a presynaptic action potentials decreased gradually, when the antibody to Cterminal peptide (25 amino acid residues) of APP695 or C-terminal peptide was injected into presynaptic neurons of cholinergic synapses formed between rat sympathetic neurons in culture. The same results were also observed with injection of C-terminal small peptide (8 amino acid residues) relevant to endocytosis. These results suggested that inhibition of the synaptic transmission with the antibody or the C-terminal peptide resulted from inhibition of re-uptake of synaptic vesicles. (2) We analyzed effects of APP695 over-expressed with adenovirus vector into human postmitotic neural cells (NT-2). The results revealed that, in immunohistochemical and biochemical approaches, activated caspase-3 was detected in NT-2 cells at 48 hours after the transfection. Further apoptotic degeneration of NT-2 cells was observed at 72 hours. But the degeneration was blocked by addition of caspase-3 inhibitor into the culture medium. These results suggest that APP695 over-expressed into NT-2 cells induced apoptotic degeneration with activation of caspase-3 by some mechanism. (3) We screened the binding proteins to the extracellular domain in the extract of adult mouse brain using GST-fusion protein or biotinylated peptide as ligands. The results revealed that some proteins with high molecullar weight were found using GST-fusion protein of the extracellular domain (amino acid sequence 1-596) as a ligand, and that mouse homologue of serine-threonine kinase like protein of Caenorhabditis elegans was found using biotinylated peptide (amino acid sequence 66-81) as a ligand.

我们对淀粉样前体蛋白（APP 695）在神经细胞中的生理功能进行了研究，发现：(1)将抗APP 695 C端肽（25个氨基酸残基）的抗体或C端肽注射到培养的大鼠交感神经元之间形成的胆碱能突触的突触前神经元，突触前动作电位诱发的突触后反应逐渐减弱。注射与内吞作用相关的C-末端小肽（8个氨基酸残基）也观察到相同的结果。这些结果表明，用抗体或C-末端肽抑制突触传递是由于抑制突触囊泡的再摄取。(2)我们分析了用腺病毒载体过表达的APP 695对人有丝分裂后神经细胞（NT-2）的影响。结果显示，转染后48 h，NT-2细胞中caspase-3的表达明显增加，caspase-3的活性明显增强。72 h后NT-2细胞进一步凋亡变性。但加入caspase-3抑制剂可阻断细胞的这种变性。这些结果表明，APP 695过表达到NT-2细胞诱导凋亡变性与caspase-3激活通过某种机制。(3)我们用GST融合蛋白或生物素化肽作为配体，从成年小鼠脑提取液中筛选与胞外区结合的蛋白。结果表明，以胞外区GST融合蛋白（氨基酸序列1-596）为配体，发现了一些高分子量的蛋白质，以生物素化肽（氨基酸序列66-81）为配体，发现了秀丽隐杆线虫丝氨酸-苏氨酸激酶样蛋白的小鼠同源物。