Ultrastructural evidence for self-replication of Alzheimer-associated Aß42 amyloid along the sides of fibrils.

The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).

Chiral Selectivity of Secondary Nucleation in Amyloid Fibril Propagation.

Chirality is a fundamental feature of asymmetric molecules and of critical importance for intermolecular interactions. The growth of amyloid fibrils displays a strong enantioselectivity, which is manifested as elongation through the addition of monomers of the same, but not opposite, chirality as the parent aggregate. Here we ask whether also secondary nucleation on the surface of amyloid fibrils, of relevance for toxicity, is governed by the chirality of the nucleating monomers. We use short amyloid peptides (Aß20-34 and IAPP20-29) with all residues as L- or all D-enantiomer in self and cross-seeding experiments with low enough seed concentration that any acceleration of fibril formation is dominated by secondary nucleation. We find a strong enantio-specificity of this auto-catalytic process with secondary nucleation being observed in the self-seeding experiments only. The results highlight a role of secondary nucleation in strain propagation.

Autocatalytic amplification of Alzheimer-associated Aß42 peptide aggregation in human cerebrospinal fluid.

Alzheimer's disease is linked to amyloid ß (Aß) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aß aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aß42 in human CSF through kinetic experiments at several Aß42 monomer concentrations (0.8-10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.

Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloid-ß peptide.

Mapping free-energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in a range of disorders. Here, we generalize the strategy used to probe free-energy landscapes in protein folding to determine the activation energies and entropies that characterize each of the molecular steps in the aggregation of the amyloid-ß peptide (Aß42), which is associated with Alzheimer's disease. Our results reveal that interactions between monomeric Aß42 and amyloid fibrils during fibril-dependent secondary nucleation fundamentally reverse the thermodynamic signature of this process relative to primary nucleation, even though both processes generate aggregates from soluble peptides. By mapping the energetic and entropic contributions along the reaction trajectories, we show that the catalytic efficiency of Aß42 fibril surfaces results from the enthalpic stabilization of adsorbing peptides in conformations amenable to nucleation, resulting in a dramatic lowering of the activation energy for nucleation.

Secondary nucleation in amyloid formation.

Nucleation of new peptide and protein aggregates on the surfaces of amyloid fibrils of the same peptide or protein has emerged in the past two decades as a major pathway for both the generation of molecular species responsible for cellular toxicity and for the autocatalytic proliferation of peptide and protein aggregates. A key question in current research is the molecular mechanism and driving forces governing such processes, known as secondary nucleation. In this context, the analogies with other self-assembling systems for which monomer-dependent secondary nucleation has been studied for more than a century provide a valuable source of inspiration. Here, we present a short overview of this background and then review recent results regarding secondary nucleation of amyloid-forming peptides and proteins, focusing in particular on the amyloid ß peptide (Aß) from Alzheimer's disease, with some examples regarding α-synuclein from Parkinson's disease. Monomer-dependent secondary nucleation of Aß was discovered using a combination of kinetic experiments, global analysis, seeding experiments and selective isotope-enrichment, which pinpoint the monomer as the origin of new aggregates in a fibril-catalyzed reaction. Insights into driving forces are gained from variations of solution conditions, temperature and peptide sequence. Selective inhibition of secondary nucleation is explored as an effective means to limit oligomer production and toxicity. We also review experiments aimed at finding interaction partners of oligomers generated by secondary nucleation in an ongoing aggregation process. At the end of this feature article we bring forward outstanding questions and testable mechanistic hypotheses regarding monomer-dependent secondary nucleation in amyloid formation.