Analysis of the Functions in Synapse of Amyloid Precursor Protein

我们研究了淀粉样蛋白前体蛋白（APP695）在神经细胞中的生理功能，结果如下：(1)在培养大鼠交感神经元间形成的胆碱能突触的突触前神经元中注射APP695的c端肽（25个氨基酸残基）抗体或c端肽抗体后，突触前动作电位引起的突触后反应逐渐减弱。注射与内吞作用相关的c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，抗体或c端肽抑制突触传递是由于抑制突触囊泡的再摄取。(2)分析了用腺病毒载体过表达APP695对人有丝分裂后神经细胞（NT-2）的影响。结果显示，免疫组织化学和生化方法显示，转染后48小时NT-2细胞中检测到活化的caspase-3。72h观察NT-2细胞进一步凋亡变性。在培养液中加入caspase-3抑制剂可阻断细胞的退化。提示APP695过表达至NT-2细胞，通过激活caspase-3，通过一定机制诱导凋亡变性。(3)以gst融合蛋白或生物素化肽为配体筛选成年小鼠脑提取物的胞外结构域结合蛋白。结果表明，以胞外结构域的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；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。

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.