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Characterization of the inhibitory activity against aggregation of amyloid β protein in the extract of human cerebellum

人小脑提取物中β淀粉样蛋白聚集抑制活性的表征

基本信息

批准号：
09670638
负责人：
TAMAOKA Akira
金额：
$ 1.79万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670638/
关键词：
Alzheimer's disease amyloid β protein cerebellum insulin degrading enzyme diffuse plaque aggregation Thioflavin T 瀰漫性老人斑

项目摘要

To compare the degrading and the aggregating activity between Alzheimer's disease (AD) and normal control brains, we homogenized cerebral cortices from AD and control brains with Tris-saline buffer, extracted soluble fractions, which were incubated at 37 ゜C with added synthetic amyloid β (Aβ) 1-42 peptides. After different incubation times, these samples were immunoblotted with anti-Aβ1-42 antibody by ECL method. Aβ monomer with 〜4kDa molecular weight was usually decreased after 12 hours incubation in control samples, but only after 24 hours in samples from AD brains. In addition, smear patterns with higher molecular weights were getting more remarkable in AD samples as compared with controls. These findings suggest that Aβ-degrading and -aggregating actives may be decreased and increased respectively in AD brains in comparison with controls.To investigate Aβ-degradating activities, we measured insulin degrading enzyme (IDE) activities, previously reported to degrade Aβ, in soluble fra … More ctions from AD and control brains. IィイD1125ィエD1-labeled insulin was added in soluble fractions and precipitated with trichloroacetic acid (TCA). Radioactivities recovered in supernatants after TCA precipitation were measured as IDE activities, which did not show any significant differences between AD and control brains, indicating that IDE could not be involved in the pathogenesis of AD.To evaluate amounts of Aβ species in extracted fractions from human cerebellum, we measured them using sandwich ELISA, and revealed that cerebellar cortices contain more Aβ42 than Aβ40 and the ratios of full-length Aβ1-42 were lower as the total Aβ amounts were smaller. These findings suggest that in diffuse plaques, which represent main plaques in cerebellar cortices, Aβx-42 containing amino-terminal truncated fragments deposits in earlier stage and then Aβ1-42 and Aβ40 sunsequantly accumulate when total Aβamounts increase. From the fact that Congo red staining did not show birefringence in cerebellar sections, Aβ deposited in cerebellar cortices scarcely form amyloid fibrils.Finally to analyze inhibitory activities against Aβ-aggregation, we employed cerebellar cortices, which has been revealed to mainly contain non-fibrillar Aβ deposition. Cerebellar cortices were separated from normal human autopsied brains, homogenized with phosphate buffer, and ultracentrifuged to get supernatants. Synthetic Aβ1-42 peptides were solubilized in deionized water to make Aβ solution. Cerebellar supernatant and Aβ solution were mixed and incubated for different durations, and Aβ-aggregation were assessed using Thiflavin T binding assay, which confirmed that cerebellar extractions contain inhibitory activities against Aβ-aggregation. Less

为了比较阿尔茨海默病 (AD) 和正常对照大脑之间的降解和聚集活性，我们用 Tris-盐水缓冲液匀浆 AD 和对照大脑的大脑皮层，提取可溶性级分，将其在 37°C 下与添加的合成淀粉样蛋白 β (Aβ) 1-42 肽一起孵育。经过不同的孵育时间后，通过 ECL 方法用抗 Aβ1-42 抗体对这些样品进行免疫印迹。在对照样本中，分子量约为 4kDa 的 Aβ 单体通常在孵育 12 小时后下降，但在 AD 脑样本中仅在 24 小时后下降。此外，与对照相比，AD 样品中具有较高分子量的涂片图案变得更加显着。这些发现表明，与对照组相比，AD 大脑中的 Aβ 降解和聚集活性可能分别减少和增加。为了研究 Aβ 降解活性，我们测量了 AD 和对照大脑可溶性部分中的胰岛素降解酶 (IDE) 活性，此前报道可降解 Aβ。 IィイD1125ィエD1 标记的胰岛素添加到可溶性级分中，并用三氯乙酸 (TCA) 沉淀。 TCA沉淀后上清液中回收的放射性被测量为IDE活性，这在AD和对照大脑之间没有显示出任何显着差异，表明IDE不能参与AD的发病机制。为了评估人小脑提取组分中Aβ种类的含量，我们使用夹心ELISA对其进行测量，结果发现小脑皮质含有比Aβ40更多的Aβ42和比率随着总 Aβ 量的减少，全长 Aβ1-42 的含量也降低。这些发现表明，在代表小脑皮质主要斑块的弥漫性斑块中，含有氨基末端截短片段的Aβx-42在早期沉积，然后当总Aβ量增加时，Aβ1-42和Aβ40随后积累。由于刚果红染色在小脑切片中未显示双折射，因此沉积在小脑皮质中的 Aβ 几乎不形成淀粉样原纤维。最后，为了分析对 Aβ 聚集的抑制活性，我们采用了小脑皮质，已发现小脑皮质主要含有非纤维状 Aβ 沉积。从正常人尸检大脑中分离出小脑皮质，用磷酸盐缓冲液匀浆，并超速离心以获得上清液。将合成的 Aβ1-42 肽溶解在去离子水中以制备 Aβ 溶液。将小脑上清液和 Aβ 溶液混合并孵育不同时间，并使用 Thiflavin T 结合测定法评估 Aβ 聚集，证实小脑提取物具有 Aβ 聚集抑制活性。较少的