Ultrastructural evidence for self-replication of Alzheimer-associated Aβ42 amyloid along the sides of fibrils

Abstract

<jats:p>The nucleation of Alzheimer-associated Aβ peptide monomers can be catalyzed by preexisting Aβ 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 Aβ42 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 Aβ42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).</jats:p>

Identification of on- and off-pathway oligomers in amyloid fibril formation

“…Oligomers have variously been dened in the literature as species having smaller size, lower growth rate, less ordered structure, distinct surface properties or higher toxicity compared to brils 8,11,12,[14][15][16][17][18][19] (Fig. 2).…”

“…The examples shown illustrate oligomers formed on fibril surfaces during the fibril assembly process (left) and end state fibrils (right) (images adapted from ref. 14), or using solid-state NMR, in which case oligomers produce less well-resolved spectra (left, adapted from ref. 15) than fibrils (right, adapted from ref.…”

“…(d) Differences in surface properties, for example hydrophobicity, may be detected using fluorescence probes such as 8-anilino-1-naphthalenesulfonic acid (ANS), Nile red, 17 or by cryo-EM, in which case oligomers may have a higher tendency than fibrils to adsorb to the grid surface (adapted from ref. 14). (e) Toxicity differences can be detected using for example Ca 2+ influx assays, 18 or measurements of Long Term Potentiation (LTP) in ratsa process related to memory and learning (adapted from ref.…”

Nanoscale Hyperspectral Imaging of Amyloid Secondary Structures in Liquid

“…This mechanism of secondary nucleation is thus most powerful and the driving force of fibril aggregation of Aβ 1‐42 at least under the conditions studied [28] . Only recently, ultrastructural evidence for the existence of time‐intermediate growing aggregates along the sides of fibrils was observed using cryo‐electron microscopy (EM) and special conditions [29] . The TERS studies strengthen these findings and further highlight that these surface‐attached entities are composed of anti‐parallel β‐sheet secondary structures as the primary seeds.…”

Secreted Chaperones in Neurodegeneration

“…Immunogold electron microscopy showed that formation of Abeta fibrils in vitro was drastically delayed in the presence of recombinant Bri2 and proSP-C BRICHOS domains, likely due to the fact that BRICHOS domain binds to Abeta fibrils, shielding it from further surface nucleation ( Willander et al, 2012 ). More recently, cryo-EM studies showed that the process of Abeta fibrillation consisted of both formation of free floating protofibrils as well as fibrils with surface nucleation; in the presence of the BRICHOS domain, a higher number of free-floating Abeta protofibrils were found, while in its absence, secondary nucleation was favored ( Tornquist et al, 2020 ). This supports the idea that the possible mechanism of action is by reducing secondary nucleation rather than prevention of de novo fibril formation.…”