Sihag R K, Wang L W, Cataldo A M, Hamlin M, Cohen C M, Nixon R A
Laboratories for Molecular Neuroscience, McLean Hospital, Belmont, MA 02178.
Brain Res. 1994 Sep 5;656(1):14-26. doi: 10.1016/0006-8993(94)91361-7.
The formation of neurofibrillary tangles (NFTs) and paired-helical filaments (PHFs) in Alzheimer's disease (AD) reflects a major disorganization of the cytoskeleton. The role of the neuronal membrane skeleton in the development of these abnormalities has not previously been investigated. In this study, we used 9 antibodies raised against the erythrocyte membrane skeleton protein 4.1 (P4.1) for immunocytochemical and immunoblot analyses to investigate whether or not the brain homologues of this protein were constituents of NFTs or PHFs. Our results show that 7 of the 9 monospecific antibodies against the human and pig erythrocyte P4.1 stained NFTs in the prefrontal cortex and hippocampus of AD brains. The P4.1 antibodies used here did not cross-react with tau protein isolated from AD brain, and preabsorption of these antibodies with tau protein did not cause loss of NFT staining. In age-matched control brains, these P4.1 antibodies stained neuronal cell bodies or nuclei. Six of the antibodies also stained isolated NFTs but the SDS-insoluble NFTs were immunostained only by two of the P4.1 antibodies. By using inositol hexaphosphate affinity chromatography and immunoblot analysis, we identified a 68-kDa protein as the most likely brain analogue of P4.1. When SDS-extracted proteins from the isolated NFTs were immunoblotted, a 50-kDa band was immunostained. The 68-kDa and 50-kDa proteins were not stained by tau protein and neurofilament subunit NF-H antibodies, that strongly stained NFTs. We conclude that brain protein 4.1 isoform(s) are constituents of NFTs in AD.
阿尔茨海默病（AD）中神经原纤维缠结（NFTs）和双螺旋丝（PHFs）的形成反映了细胞骨架的严重紊乱。此前尚未研究神经元膜骨架在这些异常发展中的作用。在本研究中，我们使用了9种针对红细胞膜骨架蛋白4.1（P4.1）产生的抗体进行免疫细胞化学和免疫印迹分析，以研究该蛋白的脑同源物是否为NFTs或PHFs的组成成分。我们的结果表明，9种针对人和猪红细胞P4.1的单特异性抗体中有7种可对AD脑前额叶皮质和海马体中的NFTs进行染色。此处使用的P4.1抗体与从AD脑分离的tau蛋白不发生交叉反应，用tau蛋白对这些抗体进行预吸附不会导致NFT染色消失。在年龄匹配的对照脑中，这些P4.1抗体对神经元细胞体或细胞核进行染色。其中6种抗体也对分离出的NFTs进行染色，但SDS不溶性NFTs仅被两种P4.1抗体免疫染色。通过使用肌醇六磷酸亲和层析和免疫印迹分析，我们鉴定出一种68 kDa的蛋白为最可能的P4.1脑同源物。当对从分离出的NFTs中提取的SDS蛋白进行免疫印迹时，一条50 kDa的条带被免疫染色。68 kDa和50 kDa的蛋白未被tau蛋白和神经丝亚基NF-H抗体染色，而这些抗体可强烈染色NFTs。我们得出结论，脑蛋白4.1异构体是AD中NFTs的组成成分。
