07 Jul 2022

It’s easy to blame soluble oligomers for whatever ails neurons, but it’s hard to isolate and characterize them. Now, scientists report a way to capture these nefarious species from biological fluids. As described in the July 7 Nature Chemistry, the method relies on a dual-affinity probe called Capture Molecule for Amyloid Precipitation. CAP-1 comprises two Pittsburgh Compound B-like molecules tied to biotin. The PiBs bind β-sheet structures, and the biotin draws them out of solution when it latches onto streptavidin-laced magnetic beads added to the biofluid. The authors call this process “amyloid precipitation” (AP). In test experiments, CAP-1 pulled α-synuclein oligomers, but not monomers, from cerebrospinal fluid.

“This AP method enables an array of molecular and cellular techniques, ranging from single-molecule imaging to cytotoxicity studies, to be performed to characterize the structural and functional properties of protein aggregates,” wrote first author Margarida Rodrigues, UK Dementia Research Institute, University of Cambridge, U.K., and colleagues. The study was co-led by David Klenerman and Steven Lee at Cambridge, Sonia Gandh at University College London, and Thomas Snaddon, Indiana University, Bloomington.

Soluble oligomers are thought to be the most toxic species of amyloid. They take the blame for poisoned synapses, abnormally permeable membranes, damaged mitochondria, and other mayhem in neurodegenerative pathogenesis (Aug 2019 news; De et al., 2019; Jan 2017 news). Alas, in trying to isolate these entities from the brain or the cerebrospinal fluid, scientists have been stymied by the minuscule concentrations and the oligomers' heterogeneity. For a while, the lack of success was such that the oligomers' very existence was called into question (e.g., Oct 2011 webinar).

Antibody- and aptamer-based methods have proven useful but are limited to specific epitopes that might be hidden or modified in some oligomers. Rodrigues and colleagues based their method on the structure of oligomers rather than their sequence, believing it would capture a range of oligomer species associated with neurodegeneration.

CAP-1 mimics the typical overall Y-shape of antibodies, but with PiB moieties for binding oligomers and a biotin tail for easy purification (see image above). It bound tightly to α-synuclein oligomers and fibrils, with a dissociation constant of about 14 nM, but not to monomers, in keeping with the affinity of PiB for β-sheets. Binding to Aβ was similar, with a Kd of 17 nM, or ~130 times stronger than Thioflavin T, on which PiB is based. Streptavidin beads pulled down CAP-1 complexes containing α-synuclein, as judged by fluorescence, atomic force microscopy, and mass spectrometry after treatment with trypsin. While the authors used α-synuclein throughout the study, they note that they found similar results with both Aβ oligomers and small aggregates of tau.

To test if this method would work in biological fluids, Rodrigues spiked human CSF with α-synuclein monomers, or with monomers plus less than 5 percent synthetic oligomers, then added CAP-1. The affinity probe pulled down almost no monomer but half of the added oligomers, even though their concentration was much lower.

To test if CAP-1 bound toxic oligomers, the scientists tested how well spiked CSF was able to perforate plasma membranes. They added CSF to liposomes and measured how much calcium got into them. Clearing the same CSF with CAP-1 reduced calcium permeability by about 80 percent.

Next, Rodrigues tried the same experiment with CSF from a person who had Parkinson’s. CAP-1 treatment cut liposome permeability in half.

“Overall, this structure-based approach will pave the way to understanding the exact molecular species responsible for neurodegeneration in humans and consequently hasten development of simple and robust early diagnosis methods,” wrote the authors.—Tom Fagan

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References

News Citations

1. Aβ Dimers Block Glutamate Uptake, Fire Up Synapses 9 Aug 2019
2. Sweat the Small Stuff: Teeniest Aβ Oligomers Wreak Most Havoc 12 Jan 2017

Webinar Citations

1. Clearing the Fog Around Aβ Oligomers 14 Oct 2011

Paper Citations

1. De S, Wirthensohn DC, Flagmeier P, Hughes C, Aprile FA, Ruggeri FS, Whiten DR, Emin D, Xia Z, Varela JA, Sormanni P, Kundel F, Knowles TP, Dobson CM, Bryant C, Vendruscolo M, Klenerman D. Different soluble aggregates of Aβ42 can give rise to cellular toxicity through different mechanisms. Nat Commun. 2019 Apr 4;10(1):1541. PubMed.

Further Reading

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