|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1999 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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
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