Vincent I, Rosado M, Davies P
Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Cell Biol. 1996 Feb;132(3):413-25. doi: 10.1083/jcb.132.3.413.
The mechanism(s) leading to widespread hyper-phosphorylation of proteins in Alzheimer's disease (AD) are unknown. We have characterized seven new monoclonal antibodies recognizing independent phospho-epitopes in the paired helical filament proteins (PHF) found in AD brain. These antibodies show pronounced immunoreactivity with cultured human neuroblastoma cells that are in the M phase of cell division, but have no discernible reactivity with interphase cells. Immunoreactivity with these antibodies does not localize to the microtubule spindles or chromosomes in M phase, but is confined to the surrounding cytoplasm. Similar staining in M phase is observed with cultured cells of various tissue types and species. Cells arrested in M phase with the microtubule depolymerizing agent, nocodazole, show marked increases in immunoreactivity with the antibodies by immunofluorescence staining, ELISA, and immunoblotting. In neuroblastoma cells, the appearance of the TG/MC phospho-epitopes coincides with activation of mitotic protein kinases, but not with the activity of the neuronal specific cyclin-dependent kinase, cdk5. These data suggest that the TG/MC epitopes are conserved mitotic phospho-epitopes produced as a result of increased mitotic kinase activity. To investigate this possibility in AD, we examined the staining of human brain tissue with MPM-2, a marker antibody for mitotic phospho-epitopes. It was found that MPM-2 reacts strongly with neurofibrillary tangles, neuritic processes, and neurons in AD but has no staining in normal human brain. Our data suggest that accumulation of phospho-epitopes in AD may result from activation of mitotic posttranslational mechanisms which do not normally operate in mature neurons of brain.
导致阿尔茨海默病（AD）中蛋白质广泛过度磷酸化的机制尚不清楚。我们已鉴定出七种新的单克隆抗体，它们可识别AD脑内成对螺旋丝蛋白（PHF）中的独立磷酸化表位。这些抗体与处于细胞分裂M期的培养人神经母细胞瘤细胞呈现出明显的免疫反应性，但与间期细胞没有明显反应。这些抗体的免疫反应性并不定位于M期的微管纺锤体或染色体，而是局限于周围的细胞质。在各种组织类型和物种的培养细胞中均观察到M期的类似染色。用微管解聚剂诺考达唑使细胞停滞在M期，通过免疫荧光染色、酶联免疫吸附测定（ELISA）和免疫印迹法可发现，这些抗体的免疫反应性显著增加。在神经母细胞瘤细胞中，TG/MC磷酸化表位的出现与有丝分裂蛋白激酶的激活一致，但与神经元特异性细胞周期蛋白依赖性激酶cdk5的活性无关。这些数据表明，TG/MC表位是由于有丝分裂激酶活性增加而产生的保守有丝分裂磷酸化表位。为了在AD中研究这种可能性，我们用MPM-2（一种有丝分裂磷酸化表位的标记抗体）检测了人脑组织的染色情况。结果发现，MPM-2与AD中的神经原纤维缠结、神经突和神经元有强烈反应，但在正常人类大脑中无染色。我们的数据表明，AD中磷酸化表位的积累可能是由于有丝分裂后翻译机制的激活所致，而这种机制在大脑成熟神经元中通常并不起作用。
