Modulation of aggregation and structural polymorphisms of ß-amyloid fibrils in cellular environments by pyroglutamate-3 variant cross-seeding.

Amyloidogenic deposition of ß-amyloid (Aß) peptides in human brain involves not only the wild-type Aß (wt-Aß) sequences, but also posttranslationally modified Aß (PTM-Aß) variants. Recent studies hypothesizes that the PTM-Aß variants may trigger the deposition of wt-Aß, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aß variants, the pyroglutamate-3-Aß (pyroE3-Aß) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aß drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aß on wt-Aß in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aß40 leads to accelerated extracellular and intracellular aggregation of wt-Aß40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aß40 or the static presence of pyroE3-Aß40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aß40 also modulates the molecular level structural polymorphisms of the resultant wt-Aß40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aß40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aß40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

Cross-Seeded Fibrillation Induced by Pyroglutamate-3 and Truncated Aß40 Variants Leads to Aß40 Structural Polymorphism Modulation and Elevated Toxicity.

The pathological amyloid plaques in Alzheimer's disease (AD) patients contain not only the wild-type ß-amyloid (wt-Aß) peptide sequences but also a variety of post-translationally modified variants. The pyroglutamate-3 Aß (pyroE3-Aß), which is generated from its truncated precursors ΔE3-Aß, shows the highest abundance among all modified Aß variants. Previous works have shown that pyroE3-Aß and/or ΔE3-Aß, compared with the wild-type sequences, led to a more rapid fibrillation process and final fibrils with higher neuronal cytotoxicity levels. However, much less is known about how the formation of pyroE3/ΔE3-Aß fibrils would affect the amyloid deposition of wt-Aß peptides, which are the main pathological events in AD. We show in the present work that the pyroE3/ΔE3-Aß40 fibrils differ significantly from the wt-Aß40 fibrils in terms of their molecular structures. When added into monomeric wt-Aß40 peptides, these variant fibrils can cross-seed the formation of wt-Aß40 fibrils with fibrillation kinetics that are greater than the self-seeded fibrillation of wt-Aß40. Furthermore, the cross-seeding process modulates the molecular structures of the yielded wt-Aß40 fibrils, which show similar features as their variant seeds. The cross-seeded fibrillation process also induces higher cytotoxicity levels compared with the self-seeded fibrillation of wt-Aß40. Overall, our results support the hypothesis that pyroE3 and ΔE3-Aß40 variants may serve as triggering factors of the pathological amyloid aggregation of wt-Aß40 and may underlie the pathological significance of pyroE3/ΔE3-Aß40 variants on the structural polymorphism of Aß deposits.

N-Terminal Modified Aß Variants Enable Modulations to the Structures and Cytotoxicity Levels of Wild-Type Aß Fibrils through Cross-Seeding.

Post-translational modifications (PTMs) of ß-amyloid (Aß) peptides are considered as triggering factors in sporadic Alzheimer's disease. However, studies to show the influence of pre-existing PTM-Aß fibrils on wild-type Aß peptides, which directly mimic the triggering scenarios, are rare. Here we show that three types of pathologically relevant PTM-Aß variants with modifications in a particular segment (from D7 to V12) of the primary sequence lead to distinct impacts on the fibrillization of wild-type Aß peptides. In general, the triggering effects are observed through cross-seeding between the PTM-Aß seeds and wild-type peptides, which consequently induce modulations in the resultant wild-type fibril structures and elevations in the fibrillar cytotoxicity levels. Modifications with a similar chemical nature, such as the S8-phosphorylation and Y10-nitration, both of which introduce additional side-chain negative charges, show comparable structural-modulation and cytotoxicity-elevation effects. The results imply the biological influences of PTM-Aß variants on the formation of amyloid deposits through cross-seeded fibrillization.