In order to assess the in vivo
interaction between the pathological processes implicated in AD and TSEs, we inoculated prions intra-peritoneally (i.p.) into Tg2576 mice at different stages of AD progression. One group of animals was inoculated at 45 days old when Aβ accumulation is not yet detectable and a second group was inoculated at the age in which amyloid deposition begin in these animals (365 days old). Age-matched wild type mice (non-transgenic littermates) were treated in the same way. Evaluation of the onset of prion disease showed that Tg2576 mice develop clinical symptoms significantly faster than WT littermates (). Interestingly, the acceleration of the disease depended on the stage of AD-like pathology, since transgenic mice inoculated at 365 days old showed a substantially shorter incubation period than animals inoculated at 45 days old (). These differences are not an effect of the animal age, because WT mice inoculated at 45 or 365 days old did not show any statistically significant difference in the onset of prion symptoms. To assess whether the accelerated disease produced in Tg2576 animals kept the infectious characteristics, we inoculated WT mice with brain homogenate of sick animals from the group of Tg2576 injected at 365 days old. The results of this second passage showed an average incubation period of 202.4 days, characteristic of RML prions inoculated i.p. and similar to the incubation time observed in our first passage in WT animals (). Additional biochemical (glycosylation profile and electrophoretical mobility of PrPSc
) and pathological (spongiform degeneration and astroglyosis) studies further showed that the RML-Tg2576 properties were the same as expected for RML prions (supplementary figure 1
). These results indicate that the strain characteristics of RML prions are likely maintained after passage in AD transgenic mice.
Alzheimer’s pathology accelerates prion disease in mice models
Histopathological analyses of brains from Tg2576 prion infected mice showed the coexistence of both TSE and AD pathologies. The brain exhibited extensive spongiform degeneration (), reactive astrogliosis (), Aβ deposition () and PrPSc accumulation (see below). Conversely, Aβ deposits were not detected in the brains of WT mice inoculated with prions and no vacuolation or PrPSc accumulation was seen in old non-infected Tg2576 mice ().
Interaction between Aβ and PrP in the brain
The degree of spongiform degeneration in animals with the double pathology did not differ from those affected only by TSE (). The extent of vacuolation in diverse areas of the brain is widely used to characterize TSEs. Indeed, different prion strains often produce a distinct pattern of spongiform degeneration (Morales et al., 2007
). Evaluation of the lesion profile in different brain areas of Tg2576 mice infected with prions showed a similar pattern in all inoculated groups (Supplementary Fig. 2
). Vacuolation profiles were not statistically significantly different between Tg2576 and WT prion infected mice, indicating that spongiform degeneration did not change due to the presence of Aβ deposits.
Conversely, it is clear that both brain inflammation () and Aβ deposition () were substantially higher on animals bearing the double pathology. The increase on brain inflammation may be an additive result, since both pathologies are associated with astrogliosis (Diedrich et al., 1987
) (). More remarkably is the dramatic increase on Aβ deposition observed in the Tg2576 inoculated mice compared with non-infected animals. Indeed, some of the Tg2576 mice (2 out of 8) inoculated at 45 days old and sacrificed when prion disease was evident (around 185 days later), showed Aβ diffuse amyloid deposits at an age (~230 days) when these animals never show amyloid lesions (). These deposits were recognized with anti-Aβ specific antibodies in both hippocampus and cortex (), but were negative for thioflavin S (). The later is not surprising since early, diffuse, pre-amyloid deposits in Alzheimer’s brain are usually not detectable by this amyloid-binding die (Tagliavini et al., 1998
). However, a more detailed study of thioflavin S staining at higher magnification showed that these lesions were indeed slightly stained by thioflavin S (Supplementary Fig 3
). Moreover, the size, number and maturity of Aβ plaques in the Tg2576 group inoculated at 365 days old was dramatically higher than in the age-matched control inoculated with PBS (). To quantitatively analyze the extent of Aβ aggregation in prion infected Tg2576 mice, we determined both the percentage of brain area covered by thioflavin S positive Aβ aggregates and the number of plaques between 365 days old prion infected and age-matched non-infected animals. The results showed that Aβ plaque area as well as the number of plaques was significantly higher in Tg2576 mice infected with prions (P<0.01). Indeed, infected animals have more than 2-fold higher number of plaques and strikingly more than 10-fold greater plaque area than non-infected Tg2576 (data not shown). These data strongly supports an interaction between the prion and AD pathologies, leading to a dramatic increase on the misfolding, aggregation and cerebral accumulation of Aβ in the presence of PrPSc
To evaluate whether PrPSc
accumulation was also increased in the presence of AD pathology, we measured the quantity of PrPSc
in the brain by Western blot analysis (). Infected Tg2576 mice were sacrificed when clinical signs of scrapie were confirmed, in average 186 and 165 days post-inoculation (d.p.i.) in animals injected at 45 and 365 days old, respectively. As shown in , the quantity of PrPSc
in the brain was high and similar in these two groups (one representative animal is shown in each group). No PrPSc
was detected in AD transgenic mice non-infected with prions. As comparison we analyzed by Western blot the levels of PrPSc
in 365 days old prion inoculated WT mice at different time points after i.p. inoculation ( and supplementary table 1
). In animals sacrificed at 140, 153 and 161 d.p.i. no PrPSc
was detected even in concentrated samples (). In contrast, at these times most of the Tg2576 mice inoculated at 365 days old showed clear clinical signs and strong deposition of PrPSc
in their brains ( and ). At 169 d.p.i. only one out of three WT mice showed a very faint PrPSc
signal (in absence of clinical signs) (). The quantity of PrPSc
in the brain of infected WT mice became high and similar to the one in Tg2576 groups after around 225 days post-inoculation (). These results indicate that PrPSc
formation and accumulation in the brain is accelerated in mice simultaneously affected by AD brain pathology.
PrPSc levels in Tg2576 or WT mice inoculated with prions
One putative explanation for the acceleration of AD and TSE pathologies in animals affected by both diseases could be a direct interaction between Aβ and PrP misfolded proteins leading to speeding up the process of misfolding and aggregation. To assess this possibility, we studied the colocalization of both proteins in pathological aggregates. In Tg2576 mice injected with prions at 365 days old we detected signals reactive against Aβ and PrP antibodies in both compacted amyloid plaques, typical of AD, and large diffuse deposits, characteristics of prion affected animals (). Conversely, no colocalization was observed in non-infected Tg2576 or WT infected with RML, where only the characteristics aggregates associated to AD or prion disease were observed, respectively (). These results suggest that misfolded proteins interact in the brain when both pathological processes are occurring simultaneously. To further assess the interaction between Aβ and PrP in the brain we performed co-immunoprecipitation studies. As shown in , immunoprecipitation using the 4G8 antibody that specifically recognize Aβ, co-immunoprecipitated a large amount of PrP in T2576 animals inoculated with prions. A fainter band was also observed in prion infected WT animals, but no signal was observed in transgenic or WT mice non-infected with prions. A negative control developed with the antibody against the AKT protein showed no co-immunoprecipitation ().
To evaluate whether the interaction between Aβ and PrP may have functional consequences for the processes of protein misfolding and aggregation, we performed in vitro
aggregation experiments in mixtures of both proteins. It is widely accepted that proteins misfold and aggregate by a seeding-nucleation mechanism, in which the formation of an oligomeric nuclei is the key step that control the kinetic of aggregation (Jarrett and Lansbury, 1993
; Soto et al., 2006
). It has been described that seeds composed of one protein can accelerate a second misfolding process through a process known as cross-seeding (Harper and Lansbury, 1997
; O’Nuallain et al., 2004
). To test the possibility that Aβ and PrP may cross-seed each other to accelerate protein misfolding and aggregation, we evaluated the seeding capability of purified PrPSc
in the aggregation of synthetic Aβ (). Addition of various small quantities of prion seeds produced a clear and dramatic acceleration of Aβ aggregation, measured as a shortening of the lag phase for polymerization. The acceleration of Aβ aggregation was directly proportional to the quantity of PrPSc
seeds added to the sample (). These results may explain in part the large increase of Aβ deposition observed in animals inoculated with murine prions. In order to evaluate whether Aβ aggregates alter PrP misfolding, we studied the aggregation of recombinant prion protein (recPrP) in the presence of different quantities of Aβ fibrils. The formation of misfolded recPrP was studied by the standard proteinase K (PK) degradation assay followed by western blot. In the absence of Aβ fibrils, only faints bands of PK resistance recPrP were observed with molecular weights of 17 and 12 KDa, similar to previously reported results (Atarashi et al., 2008
) (). However in the presence of various quantities of Aβ aggregates, a prominent PK-resistant band was observed with an apparent molecular weight of around 17KDa. The switch on the molecular weight is indicative of bona-fide
PrP conversion and is similar to the size expected for the unglycosylated PK resistant PrPSc
core. We are currently testing whether misfolded recPrP produced upon incubation with Aβ fibrils is infectious to animals. Interestingly, the extent of PK-resistant recPrP was directly proportional to the quantity of Aβ fibrils added to the reaction (). This data provides evidence for a functional interaction between Aβ and PrP resulting in mutual acceleration of protein misfolding and aggregation by cross-seeding.
Cross-seeding of PrP and Aβ misfolding and aggregation in vitro