Kim S H, Suh Y H
Department of Pharmacology, College of Medicine, Seoul National University, Korea.
J Neurochem. 1996 Sep;67(3):1172-82. doi: 10.1046/j.1471-4159.1996.67031172.x.
We have previously shown that a recombinant carboxyl-terminal 105-amino-acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non-selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 microM)- and time (from 48 h)-dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid beta protein (A beta). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, A beta increased LDH release only slightly even at 50 microM but significantly inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self-aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas A beta impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and A beta, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.
我们之前已经表明，淀粉样前体蛋白（APP）的重组羧基末端105个氨基酸片段（CT105）在非洲爪蟾卵母细胞中诱导出强烈的非选择性内向电流。在此，我们研究了CT105肽对培养的哺乳动物细胞的毒性作用。CT105肽以浓度（从10微摩尔起）和时间（从48小时起）依赖性方式诱导培养的大鼠皮质神经元和PC12细胞中显著的乳酸脱氢酶（LDH）释放。CT105的毒性作用比淀粉样β蛋白（Aβ）的任何片段都更强。然而，CT105肽并不影响U251人胶质母细胞瘤细胞的活力。与CT105相反，即使在50微摩尔时，Aβ也仅轻微增加LDH释放，但在亚微摩尔浓度下显著抑制3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐还原。在测试的各种神经保护药物中，只有改变膜流动性的胆固醇能够显著减弱CT105的细胞毒性。CT105肽在溶解时形成多个自聚集体。用亚致死浓度的CT105预处理并未显著改变细胞对过氧化氢和谷氨酸的敏感性。内源性CT肽不仅存在于细胞裂解物中，也存在于PC12细胞的条件培养基中。这些结果表明，CT肽可能通过形成孔道或非选择性离子通道直接攻击细胞膜，而Aβ首先损害细胞内代谢途径。因此，据认为在APP加工过程中形成的CT和Aβ可能通过不同机制参与阿尔茨海默病中的神经元变性。
