Identification of the components of paired helical filaments

阿尔茨海默病（AD）的标志之一是不寻常的神经纤维（称为成对螺旋丝（PHF））的进行性积累。由于PHF在大脑皮层中的浓度与痴呆的严重程度密切相关，因此人们一直致力于阐明PHF的性质。然而，PHF的成分分析一直进展缓慢，因为显着的不溶性PHF。免疫细胞化学研究，使用抗体的细胞骨架蛋白提供了相互矛盾的数据PHF的组成部分，仅仅是由于免疫交叉反应。为了避免这种模糊性，我们开发了一种蛋白质化学方法来鉴定PHF组分。PHF经甲酸处理后，用赖氨酰内肽酶消化，产生的肽用PHLC分离。分析所有主峰的氨基酸组成和序列。从PHF蛋白酶，泛素，tau和p ...更多信息 对蛋白质进行测序。泛蛋白在PHF中似乎是缀合形式，而其靶蛋白仍未鉴定。Tau整合到PHF的羧基第三位。蛋白质片段的存在最好解释为由于PHF制剂中淀粉样蛋白丝的污染。因此，泛素和tau蛋白是PHF的两个明确的组成部分。我们还重新检查了AD脑切片使用抗体的人类tau蛋白，PHF的明确组成部分之一。tau蛋白免疫染色显示，除了老年斑和神经元缠结外，整个AD皮质存在广泛的网状结构。在联合皮质的第3层和第5层，网状结构最密集，表明与锥体细胞关系密切。进一步观察发现，该网状结构由无数长5-30 μ m的异常卷曲纤维组成，其一端经常肿胀。卷曲纤维似乎来自锥体细胞体及其树突。这些形态学特征强烈表明，这些卷曲的纤维代表体树突发芽。这一假说可以解释为什么几种胎儿抗原在AD脑中表达。这也与最近的一项意外发现相一致，即与正常对照相比，AD脑中的神经生长活动增加。少

One of the hallmarks of Alzheimer's disease (AD) is the progressive accumulation of unusual neuronal fibers, termed paired helical filaments (PHF). Since the concentration of PHF in cerebral cortex was suggested to well correlated with the degree of dementia, much effort has been concentrated to the elucidation of the nature of PHF. However, the component analysis of PHF has been only slowly progressing because of remarkable insolubility of PHF.Immunocytochemical studies using antibodies to cytoskeletal proteins provided conflicting data on the components of PHF due solely to immunological cross-reactivities. To avoid such ambiguity, we developed a protein chemical approach to the identification of the PHF components. After treatment with formic acid, PHF were digested with lysylendopeptidase and the produced peptides were separated by PHLC. All major peaks were analyzed for their amino acid compositions and sequences. From the PHF digests, proteolytic fragments of Ubiquitin, tau and p … More rotein were sequenced. ubiquitin appears to be of a conjugated form in PHF, while its target protein remains unidentified. Tau is integrated into PHF at its carboxyl third. the presence of protein fragments is best interpreted as being due to contamination of amyloid filaments in the PHF preparation. Thus, ubiquitin and tau are the two definite components of PHF.We also re-examined AD brain sections using antibodies to human tau, one of the definite components of PHF. The tau immunostaining revealed extensive meshworks throughout AD cortex in addition to senile plaques and neurofibrillary tangles. The meshwork was most dense in layers 3 and 5 of the association cortex, suggesting a close relationship with pyramidal cells. Further observations showed that the meshwork consists of innumerable abnormaly curly fibers which were 5-30 um long and often swollen at the one end. The curly fibers appears to come from pyramidal cell bodies and their dendrites. These morphological features strongly suggest that these curly fibers represent somatodendritic sprouting. This hypothesis could explain why several fetal antigens are expressed in the AD brain. This is also compatible with a recent unexpected finding that nerve growth activities are increased in AD brain compared with normal controls. Less