Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity.

The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3-18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Circular dichroism indicates that none of the peptides displays beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

The non-Abeta component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms beta-sheet structures and fibrils [El-Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem. 258, 157-163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 degrees C the full-length peptide and fragments NAC(3-18) and NAC(1-18) scrambled sequence [NAC(1-18 s)] were toxic, whereas fragments NAC(1-13) and NAC(6-14) were not. CD indicates that NAC(3-18) and NAC(1-18 s) exhibit beta-sheet secondary structure in aqueous solution, whereas NAC(1-13) and NAC(6-14) do not. NAC(3-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1-18 s) it does not come out of solution to a significant degree. Fragments NAC(1-13) and NAC(6-14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form beta-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Abeta peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying beta-sheet. These results suggest that an intermediate, which exhibits beta-sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.

The N-terminal region of non-A beta component of Alzheimer's disease amyloid is responsible for its tendency to assume beta-sheet and aggregate to form fibrils.

Examination of the N-terminal sequence of non-A beta component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid beta peptide (A beta), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N-terminal half was likely to form a beta-structure whereas the C-terminal half was likely to form an alpha-helix. NAC in solution altered from random coil to beta-sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC-(1-18)-peptide and NAC-(19-35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1-18)-peptide became congophilic and aggregated to form fibrils of diameter 5-10 nm, whereas NAC-(19-35)-peptide did not bind Congo Red and remained in solution. Circular dichroism spectroscopy was used to study the secondary structure of NAC and the two fragments. In trifluoroethanol/water mixtures, NAC and NAC-(19-35)-peptide adopted alpha-helical structure but NAC-(1-18)-peptide did not. NAC-(1-18)-peptide and NAC formed beta-sheet in acetonitrile/water mixtures more readily than did NAC-(19-35)-peptide. CD spectra of NAC or NAC-(1-18)-peptide in aqueous solution indicate the formation of beta-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles A beta, and other amyloidogenic proteins, in that aggregation is dependent upon beta-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.