Yatin S M, Varadarajan S, Link C D, Butterfield D A
Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington 40506-0055, USA.
Neurobiol Aging. 1999 May-Jun;20(3):325-30; discussion 339-42. doi: 10.1016/s0197-4580(99)00056-1.
The amyloid beta-peptide (A beta)-associated free radical oxidative stress model for neuronal death in Alzheimer's disease (AD) brain predicts that neuronal protein oxidation is a consequence of A beta-associated free radicals [8]. In this study we have used both in vitro and in vivo models of beta-amyloid (A beta) toxicity to detect free radical induced oxidative stress by the measure of protein carbonyl levels. These model systems employed cultured hippocampal neurons exposed to exogenous synthetic A beta(1-42) and transgenic Caenorhabditis elegans (C. elegans) animals expressing A beta(1-42). We also investigated the importance of the A beta(1-42) Met35 residue for free radical formation in peptide solution and for peptide-induced protein oxidation and neuronal toxicity in these model systems. A beta(1-42) in solution yielded an EPR spectrum, suggesting that free radicals are associated with this peptide; however, neither the reverse [A beta(42-1)] nor methionine-substituted peptide [A beta(1-42)Met35Nlc] gave significant EPR spectra, suggesting the importance of the methionine residue in free radical formation. A beta(1-42) addition to cultured hippocampal neurons led to both neurotoxicity (30.1% cell death, p < 0.001) and increased protein oxidation (158% of controls, p < 0.001). and both of those effects were not observed with reverse or Met35Nle substituted peptides. C. elegans transgenic animals expressing human A beta(1-42) also had significantly increased in vivo protein carbonyls (176% of control animals, p < 0.001), consistent with our model. In contrast, transgenic animals with a Met35cys substitution in A beta(1-42) showed no increased protein carbonyls in vivo, in support of the hypothesis that methionine is important in A beta-associated free radical oxidative stress. These results are discussed with reference to the A beta-associated free radical oxidative stress model of neurotoxicity in AD brain.
阿尔茨海默病（AD）脑神经元死亡的淀粉样β肽（Aβ）相关自由基氧化应激模型预测，神经元蛋白氧化是Aβ相关自由基作用的结果[8]。在本研究中，我们使用了β淀粉样蛋白（Aβ）毒性的体外和体内模型，通过测量蛋白羰基水平来检测自由基诱导的氧化应激。这些模型系统采用了暴露于外源性合成Aβ（1-42）的培养海马神经元和表达Aβ（1-42）的转基因秀丽隐杆线虫（C. elegans）动物。我们还研究了Aβ（1-42）的Met35残基在肽溶液中自由基形成以及在这些模型系统中肽诱导的蛋白氧化和神经元毒性方面的重要性。溶液中的Aβ（1-42）产生了电子顺磁共振（EPR）光谱，表明自由基与该肽相关；然而，反向肽[Aβ（42-1）]和甲硫氨酸取代肽[Aβ（1-42）Met35Nle]均未给出明显的EPR光谱，这表明甲硫氨酸残基在自由基形成中具有重要作用。向培养的海马神经元中添加Aβ（1-42）会导致神经毒性（细胞死亡30.1%，p < 0.001）和蛋白氧化增加（为对照组的158%，p < 0.001）。而反向或Met35Nle取代肽则未观察到这两种效应。表达人Aβ（1-42）的转基因秀丽隐杆线虫体内的蛋白羰基也显著增加（为对照动物的176%，p < 0.001），这与我们的模型一致。相比之下，Aβ（1-42）中Met35被半胱氨酸取代的转基因动物在体内未显示蛋白羰基增加，这支持了甲硫氨酸在Aβ相关自由基氧化应激中很重要的假说。本文结合AD脑Aβ相关自由基氧化应激神经毒性模型对这些结果进行了讨论。
