Analysis of the Functions in Synapse of Amyloid Precursor Protein

我们对淀粉样前体蛋白(APP695)在神经细胞中的生理功能进行了研究，取得了以下结果。(1)向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内注射抗APP695终末多肽(25个氨基酸残基)或C端肽的抗体后，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末端小肽(8个氨基酸残基)也能观察到同样的结果。这些结果表明，抗体或C端肽对突触传递的抑制是由于抑制了突触小泡的再摄取。(2)分析腺病毒载体过表达APP695对人有丝分裂后神经细胞(NT-2)的影响。结果表明，在免疫组织化学和生化方法中，转染后48h，在NT-2细胞中检测到活化的caspase-3。72小时后，NT-2细胞出现进一步的凋亡性变性。但在培养液中加入caspase-3抑制剂可阻止细胞的变性。以上结果提示，APP695过表达可通过激活caspase-3诱导NT-2细胞的细胞凋亡。(3)以GST融合蛋白或生物素化多肽为配体，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)为配基，发现了一些高相对分子质量的蛋白质；以生物素化的多肽(氨基酸序列66-81)为配基，发现了秀丽线虫丝氨酸-苏氨酸激酶样蛋白的小鼠同源蛋白。

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.