Representative animals from different groups were studied histopathologically for spongiform brain degeneration after hematoxilin-eosin staining (A), reactive astroglyosis by GFAP staining (B) and Aβ deposition by immunohistochemistry using the 4G8 anti-Aβ antibody (C) and staining with the amyloid specific die thioflavin S (D). It is important to emphasize that the prion deposits in mice affected by RML prions are not thioflavin S positive, but are rather diffuse pre-fibrillar aggregates. The pictures in panel A and B correspond to the medulla, panel C to the hippocampus or cortex as indicated, and panel D to the cortex. (See also supplementary figures 2 and 3).

A: Sections from the cortex of animals in different groups were stained with fluorescent antibodies against Aβ (4G8, green) and PrP (6H4, red) and co-localization evaluated by confocal microscopy. White arrows point to the typical Aβ amyloid plaques seen in transgenic mice and AD patients; yellow arrows point to the typical diffuse accumulations of PrP^Sc aggregates in the brain of TSE affected individuals. B: Co-immunoprecipitation experiments. Aliquots from the brain of animals from different groups were immunoprecipitated by 4G8 antibody, which specifically recognize Aβ. The immunoprecipitated material was separated by electrophoresis and analyzed by western blot using the 6H4 anti-prion antibody and as control the AKT antibody.

A: To assess the effect of AD neuropathology in the onset of prion disease we inoculated i.p. Tg2576 mice with RML prions at 365 (red line) and 45 (green line) days old. As control, age matched WT mice (non-transgenic littermates) were inoculated with the same stock and quantity of the infectious agent (black line). WT animals infected at 45 or 365 days old developed prion disease at very similar times and thus the data for these two groups are presented together in the graph. Clinical signs were assessed as described in Methods. When animals were definitively diagnosed with prion disease they were sacrificed to avoid further pain. The data show that both groups of Tg2576 inoculated mice develop prion disease at a shorter time compared to age matched WT controls. In addition, we performed a second infectivity passage in WT mice by inoculating infectious material from the brain of a sick Tg2576 animal injected at 365 days old (blue line). B: Average of incubation periods of the different groups showed in panel A, including the statistical comparison between each experimental group with the WT control mice. The statistical analysis was done using the student-t test.

A: Brain homogenates from clinically sick Tg2576 mice inoculated with prions at 45 or 365 days old or as a control Tg2576 mice non-inoculated with prions (right panel) were PK digested and Western blotted in order to analyze PrP^Sc burden. The result shown corresponds to one animal representative of all mice in the group. The animal of the 45 days group shown in the figure was sacrificed at 190 days post-inoculation (dpi) and the animal of the 365 days at 159 dpi. B: For comparison, we measured the PrP^Sc levels in the brain of WT mice challenged with RML prions and sacrificed at 140, 169, 193 and 225 dpi. Only the animal at 225 dpi was exhibiting signs of prion disease. Numbers at the top of each gel represent brain dilution. Brain dilutions were performed from a 10% brain homogenates and various dilutions are shown to facilitate the comparisons. C: Densitometric analysis of PrP^Sc quantity in different animals. Western blot data as those shown in panels A and B were evaluated densitometrically to estimate the extent of the PrP^Sc signal. The data represent the average and standard error of 5 different animals in each group. As seen, Tg2576 mice inoculated with prions at 45 and 365 days old accumulate a similar quantity of PrP^Sc as WT mice but at a much shorter time. (See also Supplementary Table 1).

A: The effect of purified PrP^Sc on Aβ aggregation was measured overtime by sedimentation followed by sensitive ELISA. Seed-free soluble Aβ1-42 (0.01 mg/ml) was incubated with different concentrations of purified PrP^Sc seeds or PBS (control). The concentration of PrP^Sc is expressed as a percentage of oligomers per Aβ monomer and was calculated assuming that a PrP^Sc oligomer has an average molecular weight of 7,700 KDa. The latter was based on data coming from flow field fractionation of PrP^Sc and corresponds to the fraction with the highest concentration of PrP^Sc (Silveira et al., 2005). Samples were incubated at 25°C with shaking for the indicated times. Thereafter soluble and aggregated Aβ were separated by centrifugation at 14,000 rpm for 10 min and the quantity of peptide in the supernatant was measured by ELISA. Experiment was done by triplicate and results represent the average ± standard error. Analysis by two-ways ANOVA (using condition and time as the variables) show that the kinetic of Aβ aggregation in the presence of PrP^Sc is highly significantly different from the control (P<0.0001). B: The effect of Aβ aggregates on PrP misfolding was studied by incubating 10 μg of recombinant mouse PrP in the presence of increasing concentrations of preformed Aβ fibrils. Fibrils were prepared as indicated in Methods and aliquots corresponding to 0.14% (1.2 μg of total Aβ), 0.28% (2.4 μg of total Aβ), 0.56% (4.8 μg of total Aβ) and 1.1% (9.6 μg of total Aβ) were added to monomeric recPrP (lines 1, 2, 3, and 4 respectively). The concentration of Aβ fibrils is expressed as a molar percentage per recPrP monomer and was calculated assuming that the average molecular weight of Aβ fibrils is 2,000 KDa, as estimated by a combination of size-exclusion chromatography, atomic force microscopy and electron microscopy (Goldsbury et al., 2000). The mixture was incubated for 30 h at 37°C in an Eppendorf® Thermomixer with cycles of 1 minute agitation at 1500 rpm and 1 minute incubation. To assess PrP misfolding, samples were incubated at 37°C with 7 ug/mL of PK and PrP^res signal analyzed by western blot. Line 5 is the control with the same quantity of recPrP incubated in the absence of Aβ fibrils. Line 6 corresponds to recPrP non-treated with PK to display the migration of the full length protein. The numbers at the right side correspond to the molecular weight standards.