. Abundant Aβ fibrils in ultracentrifugal supernatants of aqueous extracts from Alzheimer’s disease brains. BioRxiv, October 18, 2022 bioRxiv

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  1. Although a prominent role of aberrant Aβ in the pathogenesis of Alzheimer's disease (AD) is no longer in reasonable doubt, how the structure of Aβ assemblies is linked to disease remains uncertain. Soluble, low-molecular weight assemblies of Aβ are diffusible and especially toxic, whereas long fibrils of polymeric Aβ, which constitute the insoluble amyloid core of Aβ plaques, are considered less noxious.

    The operational definition of soluble Aβ, i.e., the Aβ that remains in the supernatant following ultracentrifugation of brain homogenates, has been a source of ambiguity. What, exactly, are the soluble Aβ entities, and how are they involved in AD? Monomeric and oligomeric Aβ are established components of the soluble fraction, but in this report, Stern and colleagues describe the additional presence of short, diffusible Aβ-amyloid fibrils in ultracentrifugation supernatants from AD brain homogenates. The Aβ in conventionally defined “soluble” fractions of these homogenates thus includes a greater diversity of aggregates than had been previously recognized.

    The researchers employed the relatively gentle method of aqueous soaking to extract diffusible Aβ from AD brain tissue samples. Using cryo-EM, they found that the molecular structure of the diffusible Aβ fibrils is the same as that of insoluble fibrils obtained from AD brain homogenates with the aid of the detergent sarkosyl. Sarkosyl thus appears not to alter the basic molecular architecture of the fibrils, a reassuring affirmation of findings from prior studies in which the detergent was used in the fiber-extraction protocol.

    Another implication of this research is that it is risky to ascribe the functional characteristics of “soluble” tissue extracts solely to low-molecular-weight oligomers. How the diffusible Aβ fibrils contribute to the bioactivity of soluble supernatants, and to the pathobiology of AD, remains to be determined.

    There are translational advantages to investigations of human tissues, but detailed structural and mechanistic analyses of diffusible fibrils might profitably be undertaken in animal models of Aβ deposition (if such fibrils occur in animal models).

    The discovery of diffusible fibrils could facilitate the application of cryo-EM more broadly to the question of proteopathic strains in different brain areas and different subjects. For instance, do the fibrils differ in quantity or quality in arctic APP-mutant AD cases, in which most Aβ deposits are diffuse in nature? Finally, is the structure of diffusible fibrils linked to that of pre-fibrillar, low-molecular-weight Aβ oligomers? The molecular architecture of these oligomers, currently a technically intractable problem, remains a salient question.

    View all comments by Lary Walker
  2. This may be an extremely important discovery. The findings of the Selkoe lab suggest that they finally identified the neurotoxic Aβ assembly that everybody has been looking for, for decades (Haass and Selkoe, 2007). These are not small soluble oligomers, but rather large Aβ assemblies consisting of numerous Aβ molecules. These fibers, which are floating in aqueous solution, can be precipitated by ultracentrifugation. Strikingly, the cryo-EM structure of the fibers resembles that of the previously described Aβ fibers isolated from amyloid plaques (Yang et al., 2022). Even more strikingly, lecanemab, an anti-amyloid antibody that reduces amyloid plaque load and even cognitive decline, selectively binds to this previously unknown assembly. In contrast, bapineuzumab, which binds monomeric and aggregated Aβ, also detects soluble Aβ in the supernatants after ultracentrifugation. These findings may have implications for rational antibody design.

    Moreover, perhaps small molecules can be designed that specifically inhibit aggregation of these Aβ assemblies. Finally, the TREM2 agonistic antibody 4D9 seems to clear the halo of Aβ-surrounding plaques (Schlepckow et al., 2020). This could suggest that such antibodies selectively remove the newly identified Aβ fibers, which could be liberated from amyloid plaques and mediate neurotoxicity. This suggests that combinatorial treatments with anti-Aβ antibodies, such as lecanemab, together with TREM2-modulating antibodies, may efficiently and selectively clear neurotoxic Aβ fibers. Finally, one may also test if such Aβ assemblies have seeding activity in mouse models.

    References:

    . Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol. 2007 Feb;8(2):101-12. PubMed.

    . Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Mol Med. 2020 Apr 7;12(4):e11227. Epub 2020 Mar 10 PubMed.

    . Cryo-EM structures of amyloid-β 42 filaments from human brains. Science. 2022 Jan 14;375(6577):167-172. Epub 2022 Jan 13 PubMed.

    View all comments by Christian Haass
  3. Where and when do Aβ and tau interact?

    One of the most important questions left to answer in the puzzle of Alzheimer’s disease is when and where do aggregating forms of Aβ and tau interact to give rise to extracellular Aβ amyloid and intracellular tau-reactive neurites and tangles? Does Aβ ever come into direct contact with tau, and if so, in which cellular compartment? Most studies to date have used end-stage AD brain, in which the aggregates are firmly established, and are “cemented” into their respective extra- and intracellular locations.

    This report by Stern et al. confirmed the protofilament fold of Aβ fibrils extracted from minced/chopped (not homogenized) end-stage AD brain tissue using 25 mM Tris and 150 mM NaCl, a cocktail of protease inhibitors, and a 30-minute “soaking” technique, followed by sufficient ultracentrifugal force. The extraction method appears important. Previously (Yang et al., 2022), Aβ fibrils were only extracted using a modified sarkosyl protocol (adding sarkosyl following homogenization), which provided “abundant Aβ filaments alongside other amyloids”, whereas the more standard sarkosyl extraction (adding sarkosyl at later stages) yields, surprisingly, “only tau filaments.” Now Stern et al. report that “fibrils account for a substantial proportion of high-molecular weight Aβ aggregates in soaking extracts.” The “soaking” technique was also used recently for soluble Aβ oligomer extraction, “at least some of which displayed fibrillar character (Stern et al., 2022). It seems that the elusive Aβ fibrils are not substantially enriched by any of these extraction techniques. Even though the authors made some attempts to minimize potential sources of ex vivo aggregation, a concern remains that these Aβ fibrillar formations might have been triggered by complex extraction procedures/conditions, while predominantly enriching non-fibrillar Aβ aggregates of various molecular weights and co-aggregates of other polypeptides and macromolecules.

    Such non-fibrillar Aβ aggregates (including “oligomers”) may be the most “preferred” and expected neurotoxic state of Aβ in AD brains. For example, recent reports suggest that amorphous aggregates of positively charged polypeptides could sequester aggregation-prone Aβ and suppress the primary nucleation and elongation of Aβ fibrils, thereby modulating Aβ toxicity, despite the local increase in their concentration in biomolecular condensates (Küffner et al., 2021). For example, TDP-43 inhibits Aβ fibrillization at the initial and oligomeric stages of Aβ (Shih et al., 2020). It has also been shown that α-synuclein phase-separates into liquid bio-condensates by electrostatic complex coacervation with positively charged polypeptides, such as tau (Gracia et al., 2022). Condensates undergo either fast gelation, or coalescence followed by slow amyloid aggregation, depending on the conditions, e.g., increasing salt concentration can abolish the inhibitory effect due to charge screening and initiate Aβ fibrillization (Küffner et al., 2021). Importantly in this context, Stern et al. concede that “it remains possible that lecanemab’s target may also include non-fibrillar aggregates because we could not exclude their presence.”

    We have recently revisited end-stage AD brain sarkosyl‐extraction to characterize tau and Aβ by biochemical assays and electron microscopy. Immunogold labelling revealed non‐filamentous, globular co‐aggregates of tau and Aβ, along with filamentous tau in the sarkosyl‐insoluble fractions (Mukherjee et al., 2022). Quantitative proteomics showed equimolar amounts of Aβ and R1 tau, while the insoluble fraction was rich in the microtubule-binding region 3R/4R mixed tau isoforms with multiple post‐translational modifications. Aβ and tau may form heterogeneous globular aggregates (whether before or/and during extraction is unknown, but they are decorated by aducanumab and anti-N-terminal tau antibodies), possibly through biomolecular condensation pathways driven by multivalent interactions (hydrophobic, electrostatic, etc.) from which filamentous Aβ may emerge. This fibrilization may be modulated by cofactors and/or even extraction conditions (see figure below). We note that there were also many globular shapes in the negative stain immunoelectron microscopy images of Stern et al.

    [Courtesy of Mukherjee et al., J. Neurochemistry, 2022.] 

    Maybe the time has come to move away from using end-stage AD brain and begin to concentrate on the earliest possible sites of Aβ/tau interactions in the precuneus of persons dying with preclinical AD (Ruwanpathirana et al., 2022)? 

    References:

    . Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of alpha-synuclein and tau. Nat Commun. 2022 Aug 6;13(1):4586. PubMed.

    . Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation. Chem Sci. 2021 Feb 18;12(12):4373-4382. PubMed.

    . Quantitative proteomics of tau and Aβ in detergent fractions from Alzheimer's disease brains. J Neurochem. 2022 Oct 21; PubMed.

    . Mapping the association between tau-PET and Aβ-amyloid-PET using deep learning. Sci Rep. 2022 Aug 30;12(1):14797. PubMed.

    . TDP-43 interacts with amyloid-β, inhibits fibrillization, and worsens pathology in a model of Alzheimer's disease. Nat Commun. 2020 Nov 23;11(1):5950. PubMed.

    . A calcium-sensitive antibody isolates soluble amyloid-β aggregates and fibrils from Alzheimer's disease brain. Brain. 2022 Jan 27; PubMed.

    . Cryo-EM structures of amyloid-β 42 filaments from human brains. Science. 2022 Jan 14;375(6577):167-172. Epub 2022 Jan 13 PubMed.

    View all comments by Colin Masters

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