Aβ aggregation is independent of PrPC expression
APP-PS1 mice overexpress APP encoding the Swedish mutation plus PS1 with deletion of exon 9 [15, 16]. In contrast, J20 mice overexpress only APP with Swedish and Indiana mutations . In order to assess the role of PrPC in both AD mouse models, we crossed the APP-PS1 and J20 mice with a PrPC knock-out (KO) mouse line. Wild-type or PrPC KO littermates generated without expressing APP or PS1 transgenes were used as controls (Fig 1A). PrPC expression did not alter the expression of APP, nor did the overexpression of the mutated genes APP or APP/PS1 induce any changes in PrPC levels (Fig 1B and 1C).
Fig 1. Knockout of PrPC does not affect total APP levels in wild type or AD-model mice.
A) Total brain homogenate from 12-month old mice across 8 genotypes were analysed via western blot. APP was labelled using the N-terminal monoclonal antibody 22C11, and PrPC labelled using ICSM18. n = 5. B) Quantification of APP expression levels determined by western blot. J20 mice showed significantly higher total APP expression than APP-PS1 mice irrespective of PrPC status (APP-PS1 vs J20, p = 0.024), however no significant differences were observed between the PrPC +/+ and -/- variants of any APP genotype. n = 5. C) Quantification of PrPC expression levels determined by western blot. No significant differences in PrPC expression were observed between APP genotypes. Deletion of PrPC resulted in a significant difference on PrPC expression levels for all the lines used in this study, when compared to their respective controls (WT vs PrPC KO, p = 0.036; APP-PS1 vs APP-PS1 PrPC KO, p = 0.019; WT vs PrPC KO, p = 0.019; J20 vs J20 PrPC KO, p = 0.026). n = 5.
For the four mouse lines (APP-PS1, J20, APP-PS1 PrPC KO and J20 PrPC KO) and their respective control littermates (WT and PrPC KO) we collected brain samples at 3, 6 and 12 months of age and, then examined Aβ species and aggregation using immunohistochemical and biochemical techniques.
Deposition of Aβ in the brains of J20 mice is visible after 6 months of age with plaques concentrated in the hippocampus, corpus callosum and cortex. Minimal plaques appear in the cerebellum even after 12 months of age. By contrast, plaques in APP-PS1 mice are more widely spread over the brain, being readily detected in cerebral cortex, olfactory bulb, hippocampus, corpus callosum and cerebellum. APP-PS1 whole brain sagittal sections exhibit twice as many plaques at 12 months than the J20 mice (APP-PS1 median: 2815 plaques, J20 median: 1315 plaques, p = 0.014) whilst brain area plaque coverage is comparable between lines (APP-PS1 median: 1.16%, J20 median: 0.88%, p = 0.75) (Fig 2A–2C). Ablation of PrPC did not alter the number, location or area covered by plaques in either AD mouse model (Fig 3A–3C), in agreement with previously published results in APP-PS1 mice .
Fig 2. Progressive brain deposition of Aβ plaques in APP-PS1 and J20 mice.
A) Representative images of APP-PS1 and J20 12-month old mice stained with 82E1b anti-Aβ antibody. Scale bar: 2 mm. B) Quantification of Aβ plaques area during aging. n = 5–8. C) Quantification of Aβ plaques number. APP-PS1 mice exhibited a higher number of plaques at 12 months of age (APP-PS1 vs J20, p = 0.014). n = 5–8.
Fig 3. Deposition of Aβ plaques in APP-PS1 and J20 mice is independent of PrPC expression.
A) Representative images showing hippocampus, cortex and corpus callosum at 12 months of age of the four mouse lines studied. Scale bar: 700 μm. B) Quantification of the area covered by plaques on the above mentioned areas. For all the APP mutant lines there was a significant difference in area covered when compared to their respective controls (WT vs APP-PS1, p = 0.02; PrPC KO vs APP-PS1 PrPC KO, p = 0.0005; WT vs J20, p = 0.0006; PrPC KO vs J20 PrPC KO, p = 0.037) and, similar amounts when compared APP mutant lines to their respective ablated PrPC line (APP-PS1vs APP-PS1 PrPC KO, p = 0.75; J20 vs J20 PrPC KO, p>0.99). n = 5–9 C) Quantification of the number of plaques on the above mentioned areas. Transgenic mice exhibited a higher number of plaques compared to their respective wild-type littermates (WT vs APP-PS1, p = 0.005; PrPC KO vs APP-PS1 PrPC KO, p = 0.002; WT vs J20, p = 0.0008; PrPC KO vs J20 PrPC KO, p = 0.031) and no significant difference when compared to their respective ablated PrPC line (APP-PS1vs APP-PS1 PrPC KO, p>0.99; J20 vs J20 PrPC KO, p>0.99). n = 5–9.
Total Aβ peptides in whole brain homogenates collected at 6 and 12 months were quantified by immunoassay. At 12 months old, APP-PS1 mice had significantly more Aβ42 than J20 mice (for Aβ42 APP-PS1 median: 223 ng/mg, J20 median: 55 ng/mg, p = 0.04), but did not reach significance for the Aβ40 peptide (for Aβ40 APP-PS1 median: 113 ng/mg, J20 median: 16 ng/mg, p = 0.14); with PrPC having no impact on Aβ peptide levels in either model (Fig 4). The amount of Aβ40 and Aβ42 peptides in APP-PS1 mice was almost 90% lower at 6 months versus 12 months of age (Aβ42 APP-PS1 median: 19 ng/mg, Aβ40 APP-PS1 median: 11 ng/mg, at 6 months).
Fig 4. Immunoassays demonstrate that total Aβ peptide levels in APP-PS1 and J20 mouse brain are independent of PrPC expression.
Total brain homogenates from mice at 12 months of age were analysed by multiplex Aβ peptide panel (6E10) immunoassay from MSD. A) APP-PS1 expressing or not PrPC presented higher levels of Aβ42 than J20 samples (APP-PS1 vs J20, p = 0.04; APP-PS1 PrPC KO vs J20 PrPC KO, p = 0.004). n = 5–8. B) Quantification of Aβ4o levels revealed no changes due to ablation of PrPC in any of the mouse lines. Levels of Aβ4o were not significantly different between APP-PS1 and J20 mice (APP-PS1 vs J20, p = 0.14). n = 5–8.
Similarly, when the levels of Aβ oligomers were quantified using the 1C22 immunoassay we found that APP-PS1 mice had significantly higher levels than their wild-type littermates, whereas there was no detectable significant difference between the J20 mice and their wild-type littermates, at 12 months of age (WT vs APP-PS1, p = 0.035; WT vs J20, p = 0.25). PrPC expression had no impact on the levels of 1C22-reactive Aβ oligomers in either APP-PS1 or J20 transgenic lines (Fig 5). Analysis of the levels of Aβ oligomers capable of binding PrPC revealed no significant differences between J20 mice and their respective wild type controls. In contrast, APP-PS1 samples contained significantly higher levels of these Aβ oligomers, with no differences detected between mice with Prnp +/+ and Prnp -/- backgrounds (Fig 6). We then characterised the conformation of the Aβ oligomers, using the OC antibody which recognises parallel, in register fibrils (distinct from the A11 antibody, which binds to anti-parallel Aβ structures [25, 26]. A11 and OC antibodies recognise mutually exclusive epitopes and it has been suggested that A11 binds prefibrillar amyloid material, which could change conformation and aggregate into fibrils, while the OC fibrillar oligomers are protofibrils, transient intermediates, that ultimately become fibrils. These OC Aβ oligomers may represent fibril nuclei which are the minimal stable aggregate that is capable of elongating by recruiting additional monomers . Interestingly, only APP-PS1 mice but not J20 mice, had a significant amount of these OC-Aβ oligomers (WT vs APP-PS1, p = 0.012; WT vs J20, p = 0.87) (Fig 7). It has been suggested that Aβ oligomers that are able to bind to PrPC have an OC conformation [7, 27]. Levels of OC-Aβ oligomers did not change after ablation of PrPC in either mouse line (Fig 7). Next, we hypothesised that the total level of Aβ oligomers in APP-PS1 would directly correlate to the amount of Aβ oligomers capable of binding PrPc. Interestingly, only APP-PS1 mice, and not J20 mice, showed a positive and significant correlation between total amount of Aβ oligomers and oligomers that bind to PrPC (Fig 8). This is in agreement with previous studies which showed that J20 mice produce mainly A11-Aβ oligomers . Given the low number of samples available in the current study, it would be helpful to further confirm this correlation with a bigger group size. Collectively, these results demonstrate that APP-PS1 mice produce more Aβ oligomers than J20 mice and that the oligomers from APP-PS1s are conformationally distinct and bind PrPC.
Fig 5. 1C22-detected Aβ oligomers levels in APP-PS1 and J20 mouse brain are not altered by PrPC ablation.
Total brain homogenates were assayed using 1C22 anti-Aβ oligomer antibody (WT vs APP-PS1, p = 0.035; APP-PS1 vs APP-PS1 PrPC KO, p>0.99; WT vs J20, p = 0.25; J20 vs J20 PrPC KO, p>0.99). n = 4–7.
Fig 6. Aβ oligomers that bind to PrPC are present in APP-PS1 at higher levels than in J20 mouse brain, but do not change due PrPC expression.
Total brain homogenates were analysed by DELFIA immunoassay to detect PrPC-binding Aβ species (APP-PS1 vs J20, p = 0.037; WT vs APP-PS1, p = 0.009; APP-PS1 vs APP-PS1 PrPC KO, p>0.99; WT vs J20, p = 0.12; J20 vs J20 PrPC KO, p>0.99). n = 6–9.
Fig 7. Aβ oligomers present in APP-PS1 and J20 mouse brain have different conformations, independent of PrPC expression.
Total brain homogenates were quantified by dot blot using OC antibody (WT vs APP-PS1, p = 0.012; APP-PS1 vs APP-PS1 PrPC KO, p>0.99; WT vs J20, p = 0.87; J20 vs J20 PrPC KO, p>0.99). n = 5–8.
Fig 8. Positive correlation of Aβ oligomers that bind PrPC with total amount of Aβ oligomers in APP-PS1, but not in J20 mice.
A) Data from Figs 5 and 6 showed a positive correlation for APP-PS1 brain samples (Spearman r = 0.9, p = 0.04). n = 5. B) No correlation was found for values obtained using J20 brain samples (Spearman r = -0.8, p = 0.17). n = 4.