Significance: Alzheimer’s disease affects a rapidly growing number of individuals worldwide. Key unresolved questions relate to the onset and propagation of the disease, linked to the self-assembly of amyloid β peptide into fibrillar and smaller aggregates. This study investigates the propagation of aggregates of amyloid β peptide and asks whether hydrophobic molecular features observed on the fibril surface correlate with its ability to catalyze the formation of new aggregates. This question is motivated by the associated formation of intermediate forms that are toxic to neuronal cells. The results imply that surface catalysis is independent of surface details but requires that the monomers that form the new aggregate can adopt the structure of the parent aggregate without steric clashes.

Alzheimer’s disease is a neurodegenerative condition which involves heavy neuronal cell death linked to oligomers formed during the aggregation process of the amyloid β peptide 42 (A β 42). The aggregation of A β 42 involves both primary and secondary nucleation. Secondary nucleation dominates the generation of oligomers and involves the formation of new aggregates from monomers on catalytic fibril surfaces. Understanding the molecular mechanism of secondary nucleation may be crucial in developing a targeted cure. Here, the self-seeded aggregation of WT A β 42 is studied using direct stochastic optical reconstruction microscopy (dSTORM) with separate fluorophores in seed fibrils and monomers. Seeded aggregation proceeds faster than nonseeded reactions because the fibrils act as catalysts. The dSTORM experiments show that monomers grow into relatively large aggregates on fibril surfaces along the length of fibrils before detaching, thus providing a direct observation of secondary nucleation and growth along the sides of fibrils. The experiments were repeated for cross-seeded reactions of the WT A β 42 monomer with mutant A β 42 fibrils that do not catalyze the nucleation of WT monomers. While the monomers are observed by dSTORM to interact with noncognate fibril surfaces, we fail to notice any growth along such fibril surfaces. This implies that the failure to nucleate on the cognate seeds is not a lack of monomer association but more likely a lack of structural conversion. Our findings support a templating role for secondary nucleation, which can only take place if the monomers can copy the underlying parent structure without steric clashes or other repulsive interactions between nucleating monomers.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A β 42, which is associated with Alzheimer’s disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD β-strands grafted onto a scaffold protein. Each construct was expressed as wild-type and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first β-strand. We investigated the stability, oligomerization, anti-amyloid activity, and affinity for amyloid-β (Aβ42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form co-aggregates with Aβ, the CTD constructs instead bind to Aβ42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two β-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aβ peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aβ42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

The self-assembly of the amyloid β 42 (Aβ42) peptide is linked to Alzheimer's disease, and oligomeric intermediates are linked to neuronal cell death during the pathology of the disease. These oligomers are produced prolifically during secondary nucleation, by which the aggregation of monomers is catalyzed on fibril surfaces. Significant progress has been made in understanding the aggregation mechanism of Aβ42; still, a detailed molecular-level understanding of secondary nucleation is lacking. Here, we explore the role of four hydrophobic residues on the unstructured N-terminal region of Aβ42 in secondary nucleation. We create eight mutants with single substitutions at one of the four positions─Ala2, Phe4, Tyr10, and Val12─to decrease the hydrophobicity at respective positions (A2T, A2S, F4A, F4S, Y10A, Y10S, V12A, and V12S) and one mutant (Y10F) to remove the polar nature of Tyr10. Kinetic analyses of aggregation data reveal that the hydrophobicity at the N-terminal region of Aβ42, especially at positions 10 and 12, affects the rate of fibril mass generated via secondary nucleation. Cryo-electron micrographs reveal that most of the mutants with lower hydrophobicity form fibrils that are markedly longer than WT Aβ42, in line with the reduced secondary nucleation rates for these peptides. The dominance of secondary nucleation, however, is still retained in the aggregation mechanism of these mutants because the rate of primary nucleation is even more reduced. This highlights that secondary nucleation is a general phenomenon that is not dependent on any one particular feature of the peptide and is rather robust to sequence perturbations.