Decreased amyloid-β and increased neuronal hyperactivity by immunotherapy in Alzheimer's models

Abstract

Among the most promising approaches for treating Alzheimer´s disease is immunotherapy with amyloid-β (Aβ)-targeting antibodies. Using in vivo two-photon imaging in mouse models, we found that two different antibodies to Aβ used for treatment were ineffective at repairing neuronal dysfunction and caused an increase in cortical hyperactivity. This unexpected finding provides a possible cellular explanation for the lack of cognitive improvement by immunotherapy in human studies.

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Figure 1: Anti-Aβ antibody 3D6 reduces amyloid pathology but aggravates neuronal dysfunction.
Figure 2: Worsening of neuronal dysfunction by anti-Aβ antibodies can occur independently of the effects on Aβ pathology.

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Acknowledgements

We thank H. Adelsberger, P. Apostolopoulos, T. Starc, K. Kratz and C. Karrer for excellent technical assistance. We thank Janssen Alzheimer Immunotherapy for providing the PDAPP mice and the 3D6 antibodies. We thank F. Bard for advice and comments on the manuscript. The work was funded by an Advanced ERC grant to A.K., the EU FP7 program (Project Corticonic) and the Deutsche Forschungsgemeinschaft (IRTG 1373 and SFB870). M.A.B. was supported by the Hans und Klementia Langmatz Stiftung.

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Authors

Contributions

M.A.B. and A.K. designed the study. M.A.B., C.G. and A.D.K. performed the in vivo experiments. U.N. performed the quantification of Aβ levels in Tg2576 mice. B.S. performed Aβ-plaques staining in brain slices. M.A.B., C.G., A.D.K., M.S., H.F. and A.K. performed the data analysis. M.A.B. and A.K. wrote the manuscript.

Corresponding authors

Correspondence to Marc Aurel Busche or Arthur Konnerth.

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Competing interests

U.N. is, and M.S. was, an employee and shareholder of Novartis Pharma AG, Basel, Switzerland.

Integrated supplementary information

Supplementary Figure 1 PDAPP mice exhibit abnormal neuronal hyperactivity.

(a,b) Top: Layer 2/3 cortical neurons imaged in vivo in WT (a) and PDAPP (b) mouse and activity maps, where hue is determined by the frequency of spontaneous Ca2+ transients. Bottom: Ca2+ transients of neurons marked in top panel. (c) Cumulative distribution of Ca2+ transients in WT (black) was significantly different from PDAPP (orange) mice (n = 510 neurons in WT vs. 548 neurons in PDAPP; Kolmogorov Smirnov test, P < 0.001). Average frequency: WT = 1.6 ± 0.3 transients per min vs. PDAPP = 3.4 ± 0.2 transients per min, n = 4 mice each; two-sample t-test, t = 4.77, d.f. = 6, P = 0.003. (d) Frequency distributions show elevated fractions of hyperactive neurons (red) in PDAPP mice. (e) Pie charts show the fractions of silent (blue; 12.0 ± 1.9% in WT vs. 11.3 ± 0.7% in PDAPP, n = 4 mice each; two-sample t-test, t = −0.34, d.f. = 6, P = 0.748), normal (white) and hyperactive (red; 3.5 ± 1.6% in WT vs. 17.0 ± 1.2% in PDAPP, n = 4 mice each, two-sample t-test, t = 6.84, d.f. = 6, P = 0.001) neurons in WT (left) and PDAPP (right) mice.

Supplementary Figure 2 Anti-Aβ antibody 3D6 reduces amyloid pathology but aggravates neuronal dysfunction in 15-months-old PDAPP mice.

(a) Amyloid burden is significantly reduced in 3D6-treated (green) as compared to control (black) PDAPP mice. Each dot represents an individual animal, and the horizontal bar represents the mean with s.e.m. (control = 27.5 ± 6.9%, n = 8 mice, vs. 3D6 = 5.1 ± 1.4%, n = 8 mice; two-sample t-test, t = 3.21, d.f. = 7.57, P = 0.013). (b) Frequency of Ca2+-transients in control (gray) and 3D6-treated (green) PDAPP mice. Same conventions as in Figure 1. The difference between the groups is significant (control = 2.9 ± 0.4 transients per min vs. 3D6 = 6.9 ± 1.5 transients per min, n = 7 mice each; two-sample t-test, t = 2.66, d.f. = 6.78, P = 0.034). (c) Frequency distributions and pie charts show higher fractions of hyperactive neurons (red) in 3D6-treated (50.7 ± 11.8%, n = 7 mice) when compared with isotype-treated control (12.0 ± 3.0%, n = 7 mice) PDAPP mice (two-sample t-test, t = 3.19, d.f. = 6.79, P = 0.016). Asterisks indicate significance (P < 0.05).

Supplementary Figure 3 Aβ-levels were unchanged in Tg2576 mice after treatment with β1-antibodies.

(a,b) Summary of the amount of soluble (TBS, a) and total (formic acid extracted, b) Aβ-40 (left) and Aβ-42 (right) in forebrains of isotype-treated (black, n = 7 mice) and β1-treated (blue, n = 10 mice) Tg2576 mice. Each dot represents an individual animal, and the horizontal bar represents the mean with s.e.m. There were no significant differences in the Aβ-levels between the groups (TBS: Aβ-40 = t = 0.529, d.f. = 15, P = 0.605, Aβ-42 = t = 0.314, d.f. = 15, P = 0.758; Formic acid: Aβ-40 = t = 0.25, d.f = 15, P = 0.806, Aβ-42 = t = −0.001, d.f. = 15, P = 0.999; all P values were determined by two-sample t-test). NS, not significant.

Supplementary Figure 4 No significant contribution of inflammatory processes to the aggravation of neuronal hyperactivity.

(a) Pie charts showing similar fractions of hyperactive neurons (red) in β1-treated (59.5 ± 8.9, n = 10 mice) Tg2576 mice as well as Tg2576 mice that were treated with β1 plus dexamethasone (55.1 ± 10.5, n = 5 mice; two-sample t-test, t = −0.30, d.f. = 13, P = 0.768). The average frequency of Ca2+-transients in β1-treated (10.6 ± 2.2 transients per min, n = 10 mice) and β1 plus dexamethasone-treated (8.1 ± 1.5 transients per min, n = 5 mice) Tg2576 mice was also similar (two-sample t-test, t = −0.94, d.f. = 12.99, P = 0.365). (b) Representative in vivo experiment showing Ca2+-transients from six layer 2/3 cortical neurons in a Tg2576 mouse before (control, left), during and after (wash-out, right) the local intracortical application of lipopolysaccharides (LPS) dissolved in extracellular saline (1 mg/ml, delivery by pressure application over 60 s). Normal neurons are marked in black and hyperactive neurons in red. (c) Summary graph showing the results from all experiments with local LPS application in Tg2576 mice. Same conventions as in Figure 1. Extreme point is marked individually. Frequency (top panel) was not significantly different between baseline control recording (6.2 ± 0.8 transients per min), LPS application (5.0 ± 0.7 transients per min) and wash-out (5.5 ± 0.9 transients per min; n = 16 trials in 4 mice; ANOVA, F(2,41) = 0.592, P = 0.558). In addition, the fractions of hyperactive neurons (bottom panel) were similar under all three conditions (control = 33.3 ± 12.5%, LPS = 29.0 ± 10.2%, wash-out = 35.9 ± 10.7%, n = 4 mice each; ANOVA, F(2,9) = 0.097, P = 0.909). NS, not significant.

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Supplementary Figures 1–4 and Supplementary Table 1 (PDF 817 kb)

Supplementary Methods Checklist (PDF 400 kb)

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Busche, M., Grienberger, C., Keskin, A. et al. Decreased amyloid-β and increased neuronal hyperactivity by immunotherapy in Alzheimer's models. Nat Neurosci 18, 1725–1727 (2015). https://doi.org/10.1038/nn.4163

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