Molecular mechanisms of Ab amyloid deposition in Alzheimer disease

Sammanfattning: The general aim of this study was to investigate the mechanisms underlying amyloid b-peptide (Ab) amyloid deposition in Alzheimer disease (AD) brain. Primary structure of intact Ab variants in amyloid partially purified from brains of AD patients and non-demented controls was determined. In all cases analyzed, three principal Ab variants, Ab1-40, Ab1-42, and Ab11-42, were detected. A significant difference in the Ab1-40/Ab1-42 ratio was found between AD patients and non-demented controls. Using a novel reverse phase liquid chromatography system, it was also demonstrated that a substantial part of the Ab molecules deposited in AD amyloid was either truncated or post-translationally modified. By purifying amyloid with regard to Ab-binding proteins, it was found that apolipoprotein E (apoE) had formed unusually stable complexes with polymers of the Ab peptide in AD brain. The apoE moiety of the complexes is a C-terminal fragment of apoE, and genotyping of the AD patients showed that the complexes could include both the apoE3 and apoE4 isoform. Complexes formed in vitro appeared to be morphologically similar, if not identical, to the Ab-apoE complexes purified from AD brain. Further in vitro studies were aimed at defining the region in the Ab molecule that is responsible for its polymerization into fibrils. Electron microscopy showed that Ab17-40 could form fibrils, albeit with a different morphology than that of Ab1-40. These fibrils were not stained by thioflavin T, suggesting that residues N-terminal to Leul7 are important for true amyloid formation. In a solid-phase interaction system, the shortest peptide fragment retaining Ab1-40-binding capacity was KLVFF (Ab16-20). Residues Lysl6, Leul7, and Phe20 were critical in mediating this Ab1-40 binding. An Ab1-28 derivative, where Lysl6, Leul7, and Phe20 were exchanged for alanine, remained soluble and did not form fibrils. Furthermore, it was demonstrated that a peptide sharing the KLVFF sequence inhibited fibril formation of full-length Ab1-40. Protease resistance of Ab amyloid was studied in an in vitro system, and it was shown that Ab1-40, upon polymerization, becomes resistant to tryptic degradation. Other Ab fragments, Abl-28, Ab12-28, and Ab25-35, were also tested for their polymerization-induced resistance towards tryptic cleavage. Both Ab12-28 and Ab25-35 formed atypical Ab fibrils but still developed protease resistance, arguing against fibril formation per se as being the sole determinant and cause of the resistance. In conclusion, the present study demonstrates that AD amyloid consists of a complex mix of intact, truncated, and post-translationally modified Ab variants. A substantial portion of these Ab molecules can be found in extremely avid complexes with apoE. Moreover, a discrete motif that is crucial for polymerization and fibril formation was identified between residues 16-20 in the Ab molecule. The study also provides some insight into the molecular basis for Ab protease resistance.