FIBRIL FORMATION BY PRIMATE, RODENT, AND DUTCH-HEMORRHAGIC ANALOGS OF ALZHEIMER AMYLOID BETA-PROTEIN

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation on fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. Beta-sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40). At pH 2.5, beta-sheet formation also occurred though at greatly reduced rates for both Pm(1-40) and Du(1-40), indicating the importance of both hydrophobic and electrostatic interactions in amyloid formation. At high pH, the beta-conformation persisted much more in Du(1-40) than in Ro(1-40) and not at all in Pm(1-40). The single substitution of Glu22-->Gln in Du (1-40) enhanced fibril stability under electron microscopy conditions as demonstrated by visualization of fibrils in the absence of any stabilizing pretreatment such as glutaraldehyde fixation. A decrease in electrostatic potential and a possible enhancement of interactions between uncharged side chains adjacent to the core region of beta/A4 (residues 17-21) could account for the greater stability of Du(1-40) fibrils. C(1-40) peptide, which contains numerous substitutions, assembled into fibrils that differed only slightly in morphology from naturally occurring beta/A4 proteins; and Pm*(40-1) only formed amorphous aggregates, with no fibrillar structure identifiable. X-ray diffraction of the various assemblies formed by Pm(1-40), Ro(1-40), Du(1-40), and C(1-40) indicated the cross-beta conformation typical for amyloid. Correlation of our findings with the neurotoxicity data suggests tha beta/A4 neurotoxicity may be related not only to the correct sequence but also to the proper folding into a cross-beta fibril.