Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life

Abstract

Amyloid-β (Aβ) deposits are a relatively late consequence of Aβ aggregation in Alzheimer‘s disease. When pathogenic Aβ seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aβ seeds before Aβ deposition becomes detectable in Aβ precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aβ assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aβ deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aβ seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer‘s disease—currently defined as Aβ deposition without clinical symptoms—may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

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Fig. 1: Targeting Aβ seeds at the pre-amyloid stage.
Fig. 2: Brain Aβ assemblies recognized by the different antibodies.
Fig. 3: Removal of higher-molecular-weight Aβ assemblies in cmAducanumab-treated mice.
Fig. 4: Pharmacokinetics and target engagement of antibodies at the pre-amyloid stages.
Fig. 5: Targeting pre-amyloid Aβ seeds leads to long-lasting reduction of cerebral β-amyloidosis.
Fig. 6: Targeting pre-amyloid Aβ seeds reduces p-Tau+ neuronal dystrophy and microglial activation.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank G. Salvadori, M. Lambert, L. Häsler, J. Odenthal and all other members of our departments for experimental help. This work was supported by the EC Joint Programme on Neurodegenerative Diseases under the grants JPND-NewTargets and JPND-REfrAME (to M.J.), from the EU/EFPIA/Innovative Medicines Initiative [2] Joint Undertaking (IMPRiND consortium grant no. 116060 to M.J.), the Alexander von Humboldt Foundation (to L.C.W.) and National Institutes of Health grant nos. P50 AG025688 and ORIP/OD P51OD011132 (to L.C.W.).

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Authors

Contributions

R.E.U., J.S., S.K.F., L.Y., U.O., M.S. and M.J. designed and performed the passive immunization and seeding experiments. C.R., J.R., C.B., A.B., F.B., R.A., N.B., S.A.K., U.O., M.S. and M.J. designed and performed the biochemical and histological work. R.E.U., E.M.U.G., A.B. and M.S. designed and performed the pharmacokinetics work. A.S. contributed to data acquisition and data analysis. S.C., F.K., J.B.S., J-U.R., H.C., F.Q., P.H.W. and T.B. contributed the antibodies and provided experimental input. M.S., L.C.W. and M.J. designed the overall study; together with R.E.U., C.R., J.R. and E.M.U.G., they wrote the manuscript. All other coauthors edited the manuscript.

Corresponding author

Correspondence to Mathias Jucker.

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Competing interests

S.C., F.K. and J.B.S. are current or former employees of Lundbeck. F.Q., P.H.W. and T.B. are current employees and/or shareholders of Biogen. J.-U.R and H.C. are former employees of Probiodrug AG; M.S. is a former employee of Novartis. The other authors declare no competing interests.

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Peer review information Nature Neuroscience thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Semi-native agarose gel electrophoresis of synthetic Aβ.

Semi-native agarose gel electrophoresis was performed using monomeric, oligomeric and fibrillar Aβ, in the same fashion as the first steps of the ARPA method, to determine Aβ distribution among fractions. Monomeric Aβ was prepared using Aβ1-40 (Bachem) in DMSO. The oligomeric preparation followed a protocol for Aβ-derived diffusible ligands (Ryan, D. A., Narrow, W. C., Federoff, H. J. & Bowers, W. J. An improved method for generating consistent soluble amyloid-beta oligomer preparations for in vitro neurotoxicity studies. J. Neurosci. Methods 190, 171–179, 2010) using Aβ1-42, and fibrils were prepared by incubating 100 μM Aβ1-42 at 37 °C for 24 hours. Agarose lanes were cut as described in Fig. 2a and the Methods section; agarose gel pieces were melted followed by denaturing immunoblotting and then probed for Aβ with antibody 6E10. Chemiluminescent signal was captured with Amersham Hyperfilm ECL (in contrast to the chemiluminescent imager used for quantitative analysis in Fig. 2). See also Methods for details. Note that (presumed) monomeric Aβ runs in fractions 6 and 7, whereas oligomeric Aβ species are additionally seen in fractions 4 and 5, and Aβ fibrils are in fractions 1 to 3. This experiment was done independently twice with similar results. Source data

Extended Data Fig. 2 Aβ assemblies from AD brain, ARPA and seeding activity.

a, PBS-homogenates from the frontal cortex of three AD subjects (Braak stage VI) were pooled to get a representative sample. ARPA (see Fig. 2 for a description) is shown for the various antibodies. Immunoblots were probed with Aβ-antibody 6E10. The experiment was repeated 3 times with similar results. b, AD brain fractions (F)1-7 and their dilutions were injected into the hippocampus of young, pre-depositing 2- to 3-month-old male APP23 host mice. Brains were immunohistochemically analyzed for Aβ deposition 8 months later. c, Results for F2 and F5 (both undiluted) is shown and reveal a high seeding activity for F2 and no seeding for F5. Scale bar: 500 μm. d, Number of mice with induced Aβ deposition/total mice per group at each dilution from the various fractions (initially all groups had 6-7 mice, of which 4 treated with the undiluted F2 fraction died). e, SD50 for the different fractions was defined as the negative log10 of the brain extract dilution at which 50% of the host mice showed induced Aβ deposition (see Methods). The specific seeding activity (SD50/total Aβ) for each fraction is shown and indicates a peak for F4. Source data

Extended Data Fig. 3. Semiquantitative comparison of signals obtained by ARPA in young, predepositing APP23 mice.

ARPA was performed in PBS homogenates from young, 6-month-old male APP23 brains as presented in Fig. 2d. For semiquantitative analysis, densitometric values of fractions (F) 6 and 7 obtained from the chemiluminescence imager were normalized to the time of exposure. The signal per second was calculated from the five longest exposure times or the five exposure times before signal saturation and the mean was taken. The obtained signal per second for Ctrl1/2 was considered as background, and therefore subtracted from the values calculated for all other antibodies, which were in total set equal to 100%. Relative values for each fraction were plotted. Beta1 and mC2 show similar signal-to-second values. The highest signal-per-second was obtained from m266, whereas the lowest signals were calculated for cmAdu and mE8 (see insert). All data are represented as means (n = 3 experiments) ± SEMs. For details see Fig. 2d.

Extended Data Fig. 4 Lack of detectable Aβ antibody titers in normal and control antibody-treated APP23 mice.

Plasma was taken for analysis from randomly selected 6-month-old male APP23 tg mice, either non-treated (tg, n = 5) or treated for 5 consecutive days with Ctrl1 or Ctrl2 antibody and analyzed 6 weeks later (n = 5/group; same mice as presented in Fig. 1). As a positive control, plasma from the Beta1- and cmAdu-injected mice one day after the injection were included (n = 5 each) together with a pool of non-tg mice as a further negative control. Two different ELISA setups were used and performed on two consecutive days, one optimized to measure Beta1 titers a, and another one optimized to measure cmAdu titers b–d, (see Methods). Data are represented as group means ± SEMs. Results reveal that the injections of Ctrl antibodies into APP23 tg or non-tg mice did not induce Aβ antibody titers. Further, no detectable titers were found in untreated APP23 tg or non-tg mice.

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Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots Fig. 3a.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots Fig. 2a.

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Uhlmann, R.E., Rother, C., Rasmussen, J. et al. Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life. Nat Neurosci 23, 1580–1588 (2020). https://doi.org/10.1038/s41593-020-00737-w

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