Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
FASEB J. 2011 Nov;25(11):3896-905. doi: 10.1096/fj.11-181297. Epub 2011 Aug 9.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by brain pathology of intracellular neurofibrillary tangles (NFTs) and extracellular amyloid plaques. NFTs contain aberrantly hyperphosphorylated Tau as paired helical filaments (PHFs). Although NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. Here, we provide direct evidence on the identity of NFs in NFTs by immunochemical and mass spectrometric analysis. We have purified sarkosyl-insoluble NFTs and performed liquid chromatography/tandem mass spectrometry of NFT tryptic digests. The phosphoproteomics of NFTs clearly identified NF-M phosphopeptides SPVPKSPVEEAK, corresponding to Ser685, and KAESPVKEEAVAEVVTITK, corresponding to Ser736, and an NF-H phosphopeptide, EPDDAKAKEPS*KP, corresponding to Ser942. Western blotting of purified tangles with SMI31 showed a 150-kDa band corresponding to phospho-NF-M, while RT97 antibodies detected phospho-NF-H. The proteomics analysis also identified an NF-L peptide (ALYEQEIR, EAEEEKKVEGAGEEQAAAK) and another intermediate filament protein, vimentin (FADLSEAANR). Mass spectrometry revealed Tau phosphopeptides corresponding to Thr231, Ser235, Thr181, Ser184, Ser185, Thr212, Thr217, Ser396, and Ser403. And finally, phosphopeptides corresponding to MAP1B (corresponding to Ser1270, Ser1274, and Ser1779) and MAP2 (corresponding to Thr350, Ser1702, and Ser1706) were identified. In corresponding matched control preparations of PHF/NFTs, none of these phosphorylated neuronal cytoskeletal proteins were found. These studies independently demonstrate that NF proteins are an integral part of NFTs in AD brains.
阿尔茨海默病（AD）是一种神经退行性疾病，其特征是脑内存在神经原纤维缠结（NFTs）和细胞外淀粉样斑块的病理学变化。NFTs 包含异常过度磷酸化的 Tau 作为双螺旋丝（PHFs）。尽管 NFTs 的神经丝已经通过免疫组织化学显示为 NFTs 的一部分，但一直存在争议，即 NFTs 中的 NF 蛋白的身份是由于磷酸化 NF 抗体与磷酸化 Tau 的交叉反应。在这里，我们通过免疫化学和质谱分析提供了 NFTs 中 NF 的确切证据。我们已经纯化了 Sarkosyl 不溶性 NFTs，并对 NFT 胰蛋白酶消化物进行了液相色谱/串联质谱分析。NFT 的磷酸化蛋白质组学清楚地鉴定了 NF-M 磷酸肽 SPVPKSPVEEAK，对应于 Ser685，以及 KAESPVKEEAVAEVVTITK，对应于 Ser736，以及 NF-H 磷酸肽 EPDDAKAKEPS*KP，对应于 Ser942。用 SMI31 对纯化的缠结进行 Western 印迹显示对应于磷酸化 NF-M 的 150 kDa 带，而 RT97 抗体检测到磷酸化 NF-H。蛋白质组学分析还鉴定了 NF-L 肽（ALYEQEIR，EAEEEKKVEGAGEEQAAAK）和另一种中间丝蛋白 vimentin（FADLSEAANR）。质谱揭示了对应于 Thr231、Ser235、Thr181、Ser184、Ser185、Thr212、Thr217、Ser396 和 Ser403 的 Tau 磷酸肽。最后，鉴定了对应于 MAP1B（对应于 Ser1270、Ser1274 和 Ser1779）和 MAP2（对应于 Thr350、Ser1702 和 Ser1706）的磷酸肽。在相应的 PHF/NFT 匹配对照制剂中，没有发现这些磷酸化神经元细胞骨架蛋白。这些研究独立地证明 NF 蛋白是 AD 大脑中 NFTs 的一个组成部分。
