Aksenov M Y, Aksenova M V, Butterfield D A, Geddes J W, Markesbery W R
Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536, USA.
Neuroscience. 2001;103(2):373-83. doi: 10.1016/s0306-4522(00)00580-7.
In this study we used immunohistochemistry and two-dimensional fingerprinting of oxidatively modified proteins (two-dimensional Oxyblot) together to investigate protein carbonyl formation in the Alzheimer's disease brain. Increased protein oxidation was detected in sections from the hippocampus and parahippocampal gyrus, superior and middle temporal gyri of six Alzheimer's disease and six age-matched control human subjects, but not in the cerebellum. In two brain regions severely affected by Alzheimer's disease pathology, prominent protein carbonyl immunoreactivity was localized in the cytoplasm of neurons without visual pathomorphological changes and degenerating neurons, suggesting that intracellular proteins might be significantly affected by oxidative modifications. Following two-dimensional electrophoresis the positions of some individual proteins were identified using specific antibodies, and immunoblot analysis for protein carbonyls was performed. These studies demonstrated the presence of protein carbonyl immunoreactivity in beta-tubulin, beta-actin and creatine kinase BB in Alzheimer's disease and control brain extracts. Protein carbonyls were undetectable in spots matching glial fibrillary acidic protein and tau isoforms. Specific protein carbonyl levels in beta-actin and creatine kinase BB were significantly higher in Alzheimer's disease than in control brain extract. beta-Tubulin did not demonstrate a significant increase in specific protein carbonyl content in Alzheimer's disease brains. We suggest that oxidative stress-induced injury may involve the selective modification of different intracellular proteins, including key enzymes and structural proteins, which precedes and may lead to the neurofibrillary degeneration of neurons in the Alzheimer's disease brain.
在本研究中，我们联合使用免疫组织化学和氧化修饰蛋白的二维指纹图谱法（二维氧印迹法）来研究阿尔茨海默病大脑中蛋白质羰基的形成情况。在6例阿尔茨海默病患者及6例年龄匹配的对照者的海马、海马旁回、颞上回及颞中回切片中检测到蛋白质氧化增加，但在小脑切片中未检测到。在受阿尔茨海默病病理严重影响的两个脑区，显著的蛋白质羰基免疫反应定位于神经元细胞质中，未见明显的病理形态学改变及神经元变性，提示细胞内蛋白质可能受到氧化修饰的显著影响。二维电泳后，使用特异性抗体鉴定了一些单个蛋白质的位置，并进行了蛋白质羰基的免疫印迹分析。这些研究表明，在阿尔茨海默病及对照脑提取物中，β-微管蛋白、β-肌动蛋白和肌酸激酶BB中存在蛋白质羰基免疫反应。在与胶质纤维酸性蛋白和tau异构体匹配的斑点中未检测到蛋白质羰基。阿尔茨海默病患者β-肌动蛋白和肌酸激酶BB中的特异性蛋白质羰基水平显著高于对照脑提取物。在阿尔茨海默病大脑中，β-微管蛋白的特异性蛋白质羰基含量未显示出显著增加。我们认为，氧化应激诱导的损伤可能涉及不同细胞内蛋白质的选择性修饰，包括关键酶和结构蛋白，这在阿尔茨海默病大脑中神经元神经原纤维变性之前发生，并可能导致其发生。
