Examination of the N-terminal sequence of non-Aβ 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 β peptide (Aβ), 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 β-structure whereas the C-terminal half was likely to form an A-helix. NAC in solution altered from random coil to β-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-(1Ϫ18)-peptide and NAC-(19Ϫ35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1Ϫ18)-peptide became congophilic and aggregated to form fibrils of diameter 5Ϫ10 nm, whereas NAC-(19Ϫ35)-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-(19Ϫ35)-peptide adopted A-helical structure but NAC-(1Ϫ18)-peptide did not. NAC-(1Ϫ18)-peptide and NAC formed β-sheet in acetonitrile/water mixtures more readily than did NAC-(19Ϫ35)-peptide. CD spectra of NAC or NAC-(1Ϫ18)-peptide in aqueous solution indicate the formation of β-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles Aβ, and other amyloidogenic proteins, in that aggregation is dependent upon β-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.
The non-beta-amyloid (Ab) component of Alzheimer's disease amyloid (NAC) and its precursor a-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and a-synuclein both form b-sheet structures upon ageing, aggregate to form ®brils, 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 b-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce b sheet, fragments NAC(8±18) and NAC(8±16) both form b-sheet structure. Only NAC(8±18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms ®brils, as determined by electron microscopy. These ®ndings indicate that residues 8±16 of NAC, equivalent to residues 68±76 in a-synuclein, comprise the region crucial for toxicity.
Background: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB.
Fibrillar deposits of alpha-synuclein occur in several neurodegenerative diseases. Two mutant forms of alpha-synuclein have been associated with early-onset Parkinson's disease, and a fragment has been identified as the non-amyloid-beta peptide component of Alzheimer's disease amyloid (NAC). Upon aging, solutions of alpha-synuclein and NAC change conformation to beta-sheet, detectable by CD spectroscopy, and form oligomers that deposit as amyloid-like fibrils, detectable by electron microscopy. These aged peptides are also neurotoxic. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified. NAC(8-18) is the smallest fragment that aggregates, as indicated by the concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. Fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Hence residues 8-16 of NAC comprise the region crucial for toxicity. Toxicity induced by alpha-synuclein, NAC and NAC(1-18) oligomers occurs via an apoptotic mechanism, possibly initiated by oxidative damage, since these peptides liberate hydroxyl radicals in the presence of iron. Molecules with anti-aggregational and/or antioxidant properties may therefore be potential therapeutic agents.
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