Rohn T T, Ivins K J, Bahr B A, Cotman C W, Cribbs D H
Institute for Brain Aging and Dementia, Department of Neurology, University of California, Irvine, CA 92697-4540, USA.
J Neurochem. 2000 Jun;74(6):2331-42. doi: 10.1046/j.1471-4159.2000.0742331.x.
Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.
尽管有大量证据表明β-淀粉样前体蛋白（APP）代谢改变及其β-淀粉样片段的积累是阿尔茨海默病（AD）的关键特征，但APP的正常生理功能仍不清楚。我们使用与人类、大鼠或小鼠APP细胞外结构域结合的单克隆抗体22C11，研究了APP在神经元中的潜在作用。将皮质神经元暴露于22C11会诱导形态学变化，包括神经突退化、核浓缩和核小体间DNA裂解，这些变化与神经元凋亡死亡一致。数据表明，22C11与纯化的APP或包含22C11表位的合成肽（APP(66 - 81)）预孵育后，可显著减轻22C11诱导的神经元损伤，这支持了22C11通过与APP结合介导凋亡的作用。小鼠IgG或单克隆抗体P2 - 1（对人类而非大鼠APP的氨基末端具有特异性）均无明显作用的数据进一步支持了22C11的特异性。此外，用22C11处理皮质神经元后，表明凋亡的生化特征是血影蛋白形成120 kDa和150 kDa的降解产物。在用通用的半胱天冬酶抑制剂N - 苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸（O - 甲基） - 氟甲基酮预处理神经元后，22C11诱导的形态学和生化变化均被阻止。用谷胱甘肽乙酯（GEE，一种细胞可渗透形式的谷胱甘肽）预先孵育皮质神经元，可使其完全免受22C11的损伤，因此提示氧化途径参与22C11介导的神经元变性。γ-谷氨酰半胱氨酸合成酶抑制剂丁硫氨酸亚砜胺预先处理神经元会增强22C11的毒性作用，这一观察结果进一步支持了上述观点。最后，利用海马神经元的分隔培养，还证明了选择性应用22C11会导致局部神经突退化，而在神经突区添加GEE可阻止这种退化。因此，单克隆抗体与APP的结合最初会引发神经突退化，随后在神经元培养物中发生半胱天冬酶依赖性凋亡，这说明了该蛋白在神经元中的一种新特性，可能与AD相关的严重神经元细胞死亡有关。
