Aβ seeds resist inactivation by formaldehyde

Abstract

Cerebral β-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated β-amyloid (Aβ) into young, pre-depositing Aβ precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aβ itself. Here we report that the β-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aβ are maintained in fixed tissues. The resistance of Aβ seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aβ aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Fig. 1

Formaldehyde-fixed brain tissue from AD patients induces cerebral β-amyloidosis in APP23 transgenic mouse hosts. a, b Young 3–4 month-old APP23 transgenic (tg) mice were intracerebrally inoculated with extracts of formaldehyde-fixed tissue from two AD cases (AD1 and AD2) or from a non-demented human control (Ctr) case. Four months after intrahippocampal extract injection (2.5 μl), Aβ-deposition was induced in the hippocampus of APP23 tg mice receiving AD extract (a). No Aβ-deposits were induced by the control brain extract (b). c Stereological quantification of the percent area of the hippocampus occupied by immunoreactive Aβ (Aβ load) induced by extract from the two AD cases and the control case (n = 3–6 mice/group, mean ± SEM, scale bar: 200 μm).

Fig. 2

Formaldehyde-fixed brain tissue from an aged APPPS1 mouse donor induces cerebral β-amyloidosis in APP-tg mouse hosts. a Schematic diagram of the tissue preparation protocol in which hemibrains from aged APPPS1 tg mice and non-tg wildtype mice (WT) underwent formaldehyde fixation (“fixed”) or were fresh-frozen (“fresh”). b Immunoblot analysis with an antibody specific to human Aβ (6E10) reveals approximately 20-fold less recoverable (monomeric/monomerized) Aβ in the brain extract from the fixed tg hemisphere compared to the fresh-frozen tg hemisphere. c–f In vivo seeding capacity of fresh-frozen or fixed brain extracts following intrahippocampal injection into pre-depositing 3–4 month-old APP23 tg mice. Brains were immunohistologically analyzed 4 months later. Robust Aβ deposition was found in the hippocampal formation after inoculation with extract from the fresh-frozen (1:20 diluted) tg brain (c). Aβ deposition also was induced by brain extract from the formaldehyde-fixed tg mouse brain (d). Extracts from both fresh-frozen (e) and fixed (f) WT brains did not induce Aβ deposits. g Stereological quantification of the percent area of the hippocampus occupied by immunoreactive Aβ (Aβ load) revealed that the deposition induced by extract from the fixed tg hemibrain was about half of that achieved with the 1:20-diluted tg extract from the fresh-frozen hemibrain. ANOVA (genotype of donor material × tissue preparation) revealed a significant effect of the donor genotype (F(1, 16) = 33.64; p<0.001) but no significant effect of the tissue preparation (F(1, 16) = 3.282; p = 0.089) or interaction (F(1, 16) = 3.582; p = 0.077) (n = 4–6 mice/group; mean ± SEM, scale bar: 200 μm).

Fig. 3

Formaldehyde-fixed brain material from APP-transgenic mice harbors in vitro seeding capacity. Fibrillization kinetics of recombinant Aβ1–40 were monitored by incorporation of Thioflavin T (ThT). Lag times were determined as measures of the seeding capacity of the extracts. Both APP23 brain tissue and APPPS1 brain tissue show reduced in vitro seeding activity in response to formaldehyde fixation (n = 4/group). Note that the fresh-frozen extracts were not diluted in this experiment (compare also to Fig. 2). Extracts of both fixed APP23 and fixed APPPS1 mouse brains still show strong seeding activity compared to fixed (or fresh-frozen) wildtype (WT) brain extracts (n = 3/group). Each data point represents the mean lag time of an individual brain extract determined by measurement of eight technical replicates, with seeding by all of the extracts measured twice, with the exception of extracts from three donor mice that were measured only once. The mean of each group is indicated by a black line. ANOVA (genotype of donor material × tissue preparation with matched fixed vs. fresh values) revealed a significant effect of the donor genotype (F(2, 8) = 124.8; p<0.001), tissue preparation (F(1, 8) = 759.9; p<0.001) and interaction (F(2, 8) = 256.1; p<0.001). Subsequent Tukey’s multiple comparison tests revealed that lag times associated with both fixed APP23 and fixed APPPS1 mouse brain extracts were shorter compared to lag times in the presence of fixed WT brain extracts (all ps<0.001). Similarly, the lag times yielded by fresh-frozen APP23 and APPPS1 mouse brain extracts were significantly shorter compared to those associated with fresh-frozen WT brain extracts (all ps<0.001).

Fig. 4

Formaldehyde fixation preserves strain-like properties of seeded Aβ plaques. Brain extracts from aged APPPS1 or APP23 tg donor mice (the extracts were from two donor mice each) were intracerebrally injected into pre-depositing, 3–4 month-old APP23 tg host mice. Brains were analyzed 4 months after inoculation using Aβ immunostaining. a–d The extract of the fresh-frozen APP23 brain tissue induced a more diffuse pattern of Aβ deposition (a), whereas the extract of the fresh-frozen APPPS1 brain tissue induced a more punctate pattern of Aβ deposition (b). Injection of extracts from formaldehyde-fixed brain tissue induced a more punctate pattern for both APP23 (c) and APPPS1 (d) donor mice (scale bar: 200 μm). e Spectral properties of the induced Aβ deposits using luminescent conjugated oligothiophenes (pFTAA). For quantitative analysis, the ratio of the intensity of the emitted light at 492 nm and 599 nm was calculated (see Methods). Each dot represents one Aβ plaque. The mean and SEM are indicated for each animal (n = 3–4/group). ANOVA (genotype of donor material × tissue preparation) revealed a significant effect of the donor genotype (F(1, 9) = 165.6; p<0.001) but no significant effect of the tissue preparation (F(1, 9) = 4.856; p = 0.055) or interaction (F(1, 9) = 3.947; p = 0.078).