20 May 2022

In the brains of people with Alzheimer’s disease, toxic oligomers and protofibrils of Aβ are thought to spark a detrimental cascade. Scientists have been trying to develop drugs that extinguish these embers before they become a wildfire. In the May 17 Cell Reports Medicine, Dieter Willbold, Forschungszentrum Jülich, Germany, and colleagues reported that one such peptide, RD2, broke down Aβ oligomers extracted from people who had had AD. Ditto for Aβ aggregates obtained from three mouse models of amyloidosis. Notably, people and mice had almost identical patterns of Aβ oligomers in their brains, as judged by an assay that distinguishes these polymorphs from Aβ monomers. These findings support the translational potential of RD2, which has just completed a Phase 1 trial in people who have mild cognitive impairment. Topline results will be presented at the Alzheimer’s Association International Conference in August.

To better understand the landscape of Aβ oligomers in AD, first author Bettina Kass used surface-based fluorescence intensity distribution analysis. In sFIDA, Aβ oligomers are captured by one antibody, then labeled with two fluorescent antibodies against the N-terminus. These detection antibodies are quantified using laser scanning microscopy or fluorescence correlation spectroscopy (Aug 2009 conference news). The antibodies fluoresce at different wavelengths, and only pixels that emit both are counted. Because the two detection antibodies must bind to the same epitope, the assay does not detect monomers. However, it cannot discriminate oligomer size. “Bigger oligomers may bind more antibodies and be brighter, but the limitations of microscopy resolution make it difficult to quantify the size of each particle,” Willbold told Alzforum.

Hence, to profile the mass and concentration of oligomers in the brain, Kass fractionated proteins by size from brain tissue of six AD cases and four age-matched controls. She found oligomers of all sizes reached a maximum of around 1 pM in fractions from controls. Concentrations varied widely in AD (see image below). The highest, around 1,800 pM, occurred in the fractions containing oligomers of around 400 kDa. Smaller oligomers between 60 to 150 kDa reached about 11 pM. Both large and small oligomers have been reported to be toxic (Jan 2017 news; Sehlin et al., 2012).

The researchers saw a similar distribution of Aβ oligomers in whole brain homogenates from three mouse models of familial AD: APP/PS1 with the Swedish APP mutation and PSEN lacking exon 9; APP-SW DI carrying the Swedish, Dutch, and Iowa APP variants; and APPlon with the London mutation. All were 18 or 24 months old, at least one year after amyloid plaques had started forming.

Different Species—Same Profile. Brain tissue homogenates from people who had had AD (orange) contained more Aβ oligomers of various sizes (x axis) than did homogenates from age-matched controls (yellow). A similar distribution was seen in brain tissue from aged APP/PS1 mice (blue) compared to wild-type animals (gray). Concentrations are shown in a Log₁₀ scale. [Courtesy of Kass et al., Cell Reports Medicine, 2022.]

Previously, Willbold had used sFIDA to detect aggregates in CSF from 14 people who had AD and, as a proof of concept, synthetic Aβ oligomers added to plasma from five healthy people (Wang-Dietrich et al., 2013; Kravchenko et al., 2017). He said they have since tested sFIDA in more than 800 CSF samples, though the data has not yet been published. Whether sFIDA will be a useful clinical assay remains to be seen. Exploratory data from the Phase 1 study of RD2 may help address this question.

Others believe the assay is not robust or sensitive enough to work as a routine diagnostic. One problem is that Aβ oligomers in the CSF hover in the femtomolar range. Willbold and colleagues have pushed the lower limit of detection below this threshold by automating the assay (see Herrmann et al., 2017; reviewed by Kulawik et al., 2018).

But for now, sFIDA remains an in-house assay at FZ Jülich, and some, including Dietmar Thal, KU Leuven, Belgium, see no reason to use it. Thal thinks that commercially available ELISAs are sufficient to measure fluid Aβ oligomers in a clinical research setting (Feb 2014 news; Jul 2012 conference news; Apr 2010 news). “When you have a cheap method that works easily and you can trust, there is no reason to change it unless you have a much better method that is not too expensive,” he said. Still, no oligomer test is routinely used in research studies. Willbold has founded attyloid GmbH to continue clinical sFIDA assay development.

Busting Up Oligomers
Knowing that Aβ oligomer profiles are similar in humans and mice, Willbold wondered if RD2, aka PRI-002, a peptide that disrupts Aβ protofibrils and plaques in mice, would do the same for human oligomers (see image below, Zhang et al., 2019; Aug 2009 conference news). Comprising D- and not the natural L-amino acids, this dodecamer dodges destruction by proteases in the body and provokes no immune response, making it an attractive drug candidate.

Kass and colleagues tested RD2 ex vivo on Aβ aggregates from mouse and human brain tissue. They added 20 or 50 μM RD2 to the protofibril-rich tissue fraction from 18-month-old APP/PS1 mice, then measured Aβ oligomer concentration six times over 20 hours. RD2 time- and dose-dependently cleared aggregates, with the low dose removing two-thirds of oligomers and the high cutting 81 percent.

Did RD2 also break down Aβ oligomers in human brain tissue? The researchers added 5 or 20 μM RD2 to tissue homogenates from four AD cases. After 23 hours, the lower dose shrank Aβ oligomer load by 70 percent, while 20 μM slashed 90 percent of aggregates.

To the authors, RD2’s ability to clear aggregates within the complex milieu of human and mouse brain homogenates supports the peptide’s therapeutic potential. Previously, they reported that RD2 readily crossed the blood-brain barrier in mice, reaching similar levels in the brain as in the blood (Apr 2015 conference news; Leithold et al., 2016). Eighteen-month-old APP/PS1 mice fed RD2 for 12 weeks had fewer Aβ protofibrils in brain homogenates measured via sFIDA, fewer cortical amyloid plaques, and better learning and memory (Schemmert et al., 2018).

RD2 has just completed a Phase 1 trial in 20 older adults with mild cognitive impairment due to AD. Willbold said topline data will be presented at this year’s AAIC. A Phase 2 trial is slated to begin in mid-2023.—Chelsea Weidman Burke

Comments

1. • Dieter Willbold
     Heinrich Heine University Düsseldorf; Forschungszentrum Jülich; and Priavoid GmbH, Düsseldorf, Germany
   • Posted: 06 Feb 2023

   Reading this contribution, one may obtain the impression that there are "cheap" assays available that quantitate oligomers with the required sensitivity. As a comment, I just would like to add that I am unaware of another assay (besides sFIDA) that is reliably and robustly able to quantify protein aggregates, regardless of their particular conformation, down to the low femtomolar range. Recent examples for the usefulness and the reliability of sFIDA are the publications by Blömeke et al., 2022, and Schaffrath et al., 2023.

   References:

   Blömeke L, Pils M, Kraemer-Schulien V, Dybala A, Schaffrath A, Kulawik A, Rehn F, Cousin A, Nischwitz V, Willbold J, Zack R, Tropea TF, Bujnicki T, Tamgüney G, Weintraub D, Irwin D, Grossman M, Wolk DA, Trojanowski JQ, Bannach O, Chen-Plotkin A, Willbold D. Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting. NPJ Parkinsons Dis. 2022 Jun 2;8(1):68. PubMed.

   Schaffrath A, Schleyken S, Seger A, Jergas H, Özdüzenciler P, Pils M, Blömeke L, Cousin A, Willbold J, Bujnicki T, Bannach O, Fink GR, Willbold D, Sommerauer M, Barbe MT, Tamgüney G. Patients with isolated REM-sleep behavior disorder have elevated levels of alpha-synuclein aggregates in stool. NPJ Parkinsons Dis. 2023 Feb 2;9(1):14. PubMed.

   View all comments by Dieter Willbold

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References

News Citations

1. Vienna: New Tack to See Amyloid Oligomers in Body Fluids 3 Aug 2009
2. Sweat the Small Stuff: Teeniest Aβ Oligomers Wreak Most Havoc 12 Jan 2017
3. Test Closes in on Oligomers, May Distinguish Alzheimer’s Patients From Controls 26 Feb 2014
4. New Assays for Aβ Oligomers in CSF Claim Femtogram Sensitivity 24 Jul 2012
5. A Diagnostic Test for Large Aβ Oligomers? 13 Apr 2010
6. Vienna: Can a D-Peptide Turn Tiger Into Pussycat? 4 Aug 2009
7. D-peptides as Drugs? Protein Therapy Approaching Phase 1 Trials 10 Apr 2015

Research Models Citations

1. APPswe/PSEN1dE9 (C57BL6)
2. Tg-SwDI (APP-Swedish,Dutch,Iowa)
3. APP(V717I)

Therapeutics Citations

1. PRI-002

Paper Citations

1. Sehlin D, Englund H, Simu B, Karlsson M, Ingelsson M, Nikolajeff F, Lannfelt L, Pettersson FE. Large aggregates are the major soluble Aβ species in AD brain fractionated with density gradient ultracentrifugation. PLoS One. 2012;7(2):e32014. PubMed.
2. Wang-Dietrich L, Funke SA, Kühbach K, Wang K, Besmehn A, Willbold S, Cinar Y, Bannach O, Birkmann E, Willbold D. The Amyloid-β Oligomer Count in Cerebrospinal Fluid is a Biomarker for Alzheimer's Disease. J Alzheimers Dis. 2013 Jan 1;34(4):985-94. PubMed.
3. Kravchenko K, Kulawik A, Hülsemann M, Kühbach K, Zafiu C, Herrmann Y, Linnartz C, Peters L, Bujnicki T, Willbold J, Bannach O, Willbold D. Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay. Biol Chem. 2017 Apr 1;398(4):465-475. PubMed.
4. Herrmann Y, Kulawik A, Kühbach K, Hülsemann M, Peters L, Bujnicki T, Kravchenko K, Linnartz C, Willbold J, Zafiu C, Bannach O, Willbold D. sFIDA automation yields sub-femtomolar limit of detection for Aβ aggregates in body fluids. Clin Biochem. 2017 Mar;50(4-5):244-247. Epub 2016 Nov 5 PubMed.
5. Kulawik A, Heise H, Zafiu C, Willbold D, Bannach O. Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases. FEBS Lett. 2018 Feb;592(4):516-534. Epub 2018 Feb 8 PubMed.
6. Zhang T, Gering I, Kutzsche J, Nagel-Steger L, Willbold D. Toward the Mode of Action of the Clinical Stage All-d-Enantiomeric Peptide RD2 on Aβ42 Aggregation. ACS Chem Neurosci. 2019 Dec 18;10(12):4800-4809. Epub 2019 Nov 22 PubMed.
7. Leithold LH, Jiang N, Post J, Ziehm T, Schartmann E, Kutzsche J, Shah NJ, Breitkreutz J, Langen KJ, Willuweit A, Willbold D. Pharmacokinetic Properties of a Novel D-Peptide Developed to be Therapeutically Active Against Toxic β-Amyloid Oligomers. Pharm Res. 2016 Feb;33(2):328-36. Epub 2015 Sep 17 PubMed.
8. Schemmert S, Schartmann E, Zafiu C, Kass B, Hartwig S, Lehr S, Bannach O, Langen KJ, Shah NJ, Kutzsche J, Willuweit A, Willbold D. Aβ Oligomer Elimination Restores Cognition in Transgenic Alzheimer's Mice with Full-blown Pathology. Mol Neurobiol. 2018 Jul 12; PubMed.

Other Citations

1. PRI-002

Further Reading

Papers

1. van Groen T, Schemmert S, Brener O, Gremer L, Ziehm T, Tusche M, Nagel-Steger L, Kadish I, Schartmann E, Elfgen A, Jürgens D, Willuweit A, Kutzsche J, Willbold D. The Aβ oligomer eliminating D-enantiomeric peptide RD2 improves cognition without changing plaque pathology. Sci Rep. 2017 Nov 24;7(1):16275. PubMed.

News

1. Elusive or Not, Aβ Oligomers Are in BioPharma Crosshairs 13 Dec 2017
2. San Diego: Tweakers and Tweezers—Grappling with Novel Aβ Therapies 14 Dec 2010