Alzheimer’s Disease (AD) is a serious disease for elderly in the world. The etiology of AD is very complex. Until now no one can provide a full picture for this disease. Amyloid-beta (Aβ) peptide (Aβ40 and Aβ42) aggregation is one of the main pathological features of AD. Thus, the fribrillogenic properties of these peptides have been a focal point of interest in AD research. However, intermediates of the fibrillogenic process are unstable species and their biological roles are difficult to define....[ Read more ]

Alzheimer’s Disease (AD) is a serious disease for elderly in the world. The etiology of AD is very complex. Until now no one can provide a full picture for this disease. Amyloid-beta (Aβ) peptide (Aβ40 and Aβ42) aggregation is one of the main pathological features of AD. Thus, the fribrillogenic properties of these peptides have been a focal point of interest in AD research. However, intermediates of the fibrillogenic process are unstable species and their biological roles are difficult to define.

To facilitate study of the Aβ amyloidosis, fusion of green fluorescent protein (GFP) to the N-terminus of amyloid peptides is designed to stop the fibrillogenic process. GFPuv is a bulky fluorophores, they will provide a more convenient way to detect Aβ peptides. Based on experimental evidences, fibrillogenesis is stopped when b beta-amyloid peptide is fused with GFPuv.

Purification of Aβ peptides from E. coli and their characterization are the first objectives for my project. Aβ fusion protein was expressed in the E. coli under appropriate conditions. The high purity fusion protein with a histidine tag was successfully obtained through metal-chelating column chromatography. However, it was found that the Aβ fusion protein from the crude cytosolic extract was mainly in a truncated form. The correct form of Aβ fusion proteins was only present in the cell debris. High purity of histidine-tagged Aβ peptide was also expressed and purified in similar conditions from cell debris. Purified proteins were compared to the synthetic peptides without fusion. Circular dichroism spectrum provided data to study the secondary structure of protein. Polarized light microscopy and transmission electron microscopy showed the morphology of protein aggregates. The results from these experiments indicated that purified and synthetic proteins are similar.

Fibrillogenesis of the amyloid proteins was our main concern. It was found that the fusion protein oilgomers are quite stable even under harsh conditions, for examples, the high temperature and high concentration of guanidine.HCl. In addition, calcium was found to greatly increase the rate of fibrillogenesis. Moreover, other cations in salts, e.g. NaCl, KCl, NH₄Cl and (NH₄)₂SO₄, also increase the rate of fibrillogenesis to a lower extent in comparison with calcium. These results provided more insights into beta-amyloid fibrillogenesis.

It is of special importance to find a compound that could stop the aggregation of Aβ peptide. In order to achieve this, an assay was developed to search for inhibitors. The fluorescence of fusion protein was found to become weaker and weaker when it aggregates. Fluorescent strength of fusion protein was thus used as a simple and convenient assay for its fibrillogenic extent. Among 592 different samples screened, eight were found to inhibit Aβ amyloidosis while two of them showed much higher activity than the others. A pure compound, clioquinone, was also found to inhibit the aggregation.