Characterization of AD/LBD, AD, PA, and NDC cases
In this study, we selected a subset of AD, PA, and NDC donors that were previously analyzed (13) for examination of seeding activity in APP, APP/tau, and tau transgenic mice (Table 1, AD 1 and 2, PA 1,2,3,4, and NDC 1 and 2). All 8 of these donors exhibited some degree of Lewy body pathology (LBD). Two of the donors selected for the study were sub-categorized with cerebral amyloid angiopathy (CAA); PA 3 had a high abundance of vascular amyloid with a CAA score of 2+-3+ while AD 1 had moderate levels of vascular Aβ deposition with a score of 1+ (Table 1). As expected, the two AD brains had significant accumulation of tau, indicated by Braak staging (5.5 and 6) (Table 1). The PA and NDC brains had lower abundance of tau than the AD brains, but were similar to each other (2, 2.5, 3, 2, and 2, 3, respectively) (Table 1). In this set of cases, all also exhibited incidental, diffuse, αSyn pathology. To these 8, we added 6 additional donors that included two AD (no LBD), two with Lewy-body variant AD (AD/LBD), and 2 additional controls that were free of all types of pathology (Table 1, AD 3 and 4, AD/LBD 1 and 2, and NDC 3 and 4). These brains were used in seeding APP, APP/α-synuclein (αSyn), and αSyn mice so that we would be able to compare AD and NDC that lacked αSyn pathology to AD/LBD brains with high levels of αSyn pathology.
To characterize the pathology in the AD and PA brains used to seed the APP/tau, APP, and tau mice, sections were stained with a pan-Aβ antibody (Fig. S1a), Thioflavin S (Thio-S) (Fig. S1b), and with a phospho-specific tau antibody (Fig. S1c). We observed widespread Aβ deposition of both diffuse and compact amyloid in both AD and PA (Fig. S1a, b), with a subset showing striking CAA. PA 3 had numerous, Thio-S positive, vascular amyloid deposits, consistent with the assessment of CAA as 2+-3+ (Fig. S1a, b). Similarly, we observed Thio-S positive amyloid staining surrounding several vessels within AD 1, consistent with a CAA score of 1+ (Fig. S1a, b). AD 2 contained several cored Thio-S positive deposits while the compact deposits in PA 1,2, and 4 had little to no Thio-S staining. As expected, AD 1 and 2 contained both substantial phosphorylated tau in form of dystrophic neurites, neuropil threads and neuronal inclusions while all four of the PA were negative for tau deposits (Fig. S1c). NDC 1 and 2 were negative for Aβ and tau deposits by both immunostaining and Thio-S staining.
Cerebral injection of brain lysates into newborn APP/tau and APP mice results in widespread, robust amyloid deposition
To gain a better understanding of the type of Aβ pathology that unseeded PrP.APPsi mice produce, we harvested breeder mice at various ages to assess phenotypic variation (Online Resource, Fig. S2a and b) (27). At 12 months of age, when deposition begins to occur in the brains of PrP.APPsi mice, any given section through the cortex and hippocampus may exhibit 1 or 2 diffuse tufted deposits and/or cored deposits (score +/-). At this age, Aβ deposition also begins to appear in the meninges surrounding the cerebellum (Online Resource 2, Fig. S2a). Approximately 50% of PrP.APPsi mice at 11-13 months of age exhibit no Aβ pathology or show only meningeal deposition in the cerebellum (Online Resource 2, Fig. S2a). To concurrently examine whether tau pathology could be augmented by seeding from these lysates, the PrP.APPsi mice were crossed to a line of mice that express human tau P301L (iTau-P301L) (Table 2) (28, 37). The iTau-P301L mice express mutant human tau at levels 2-3 fold higher than nontransgenic mice (Fig. S3).
To compare the relative ability of homogenates prepared from AD and PA cases to seed Aβ deposition in these mice, lysates from the pre-frontal cortex of the AD, PA, and NDC were injected into the cerebral ventricles of newborn mice at P0 (20, 32, 33). We confirmed that the lysates contained Aβ by immunoblotting and ELISA (Fig. S1d and Table 1). Aβ was detectable by immunoblot in each of the lysates used for injection (Fig. S1d), and enzyme-linked immunosorbent assay (ELISA) measurements confirmed high levels of SDS-soluble and formic acid (FA) soluble Aβ42 in each brain lysate (Table 1). We hypothesized that injection of these lysates in P0 mice would result in widespread dispersion of the Aβ or tau seeds, and potentially offer the best chance to extensively alter the type of pathology that would be induced by seeding.
To assess amyloid pathology, hemibrains of seeded mice were stained with a pan-Aβ antibody and Thio-S. In mice injected with either AD or PA lysates, we observed significant induction of Aβ deposition, comprised primarily of diffuse deposits throughout the cortex and hippocampus, with some vascular deposition in the pia surrounding the cortex and within the hippocampal fissure (Fig. 1b, c). Mice injected with homogenates from NDC 1 and 2, and mice that were not injected, had little Aβ pathology by 12 months of age (Fig. 1b, c). Examples of cases with the most severe pathology are shown in Figure 1, while images of animals with the least pathology are shown in Figure S4. In all cases, however, the induced Aβ pathology exhibited a diffuse, Thio-S negative morphology (Fig. S5). The levels of induced Aβ pathology in mice that co-expressed APPsi and Tau-P301L were similar to that of mice that expressed only APPsi (Fig. 1b versus c, S4a versus b). Mice that were transgenic for only Tau-P301L, or were non-transgenic, showed no evidence of Aβ deposition (data not shown). To compare the data across all of the mice examined, Aβ pathology was qualitatively scored by three independent observers (Fig. 1d, e). At 12 months, only PA 4, was scored as having a level of Aβ pathology that approached mice injected with the AD cases; both of which exhibited widespread, diffuse pathology (Fig. 1b, c). Aβ pathology in mice seeded by PA 1, 2, and 3 was scored as less abundant than in mice seeded by the AD cases (Fig. 1d, e). We confirmed the high seeding activity of AD 1 and 2, and PA 4 by assessing Aβ pathology at 9 months post-injection. Although the severity of pathology varied, multiple animals injected with each of these lysates exhibited Aβ deposition (Fig. S6a). Notably, dilution of the brain lysates by 10-fold prior to injection greatly diminished seeding activity (Fig. S6b). To further examine the relative seeding activity of PA lysates, we injected newborn PrP.APPsi mice with each of the 4 PA lysates and NDC 1, and analyzed pathology at 18 months post-injection. Although the mice injected with the PA lysates appeared to have more severe Aβ deposition, we also observed some significant Aβ pathology in animals injected with the NDC lysate (Fig S7). Whether this outcome was due to some small amount of Aβ seed in the NDC 1 will require further study. Collectively, these studies demonstrated that AD and PA brains contain misfolded forms of Aβ that preferentially seed diffuse Aβ pathology.
Two of the donor cases were identified as having CAA pathology, AD 1 and PA 3, and were scored 1+ and 2+-3+, respectively (Table 1). To determine if CAA pathology was seeded in the recipient mice, we stained sections with Thio-S and searched for evidence of vascular amyloid. In mice seeded with PA 3, we observed Thio-S positive, vascular pathology in 6 of the 7 recipient mice however, the incidence of this pathology was limited (Fig. S8). Although AD 1 also contained CAA pathology, we did not observe Thio-S positive, vascular pathology in mice seeded by this homogenate. These findings suggest that it may be possible to selectively increase CAA using seeding, but enhancing such pathology may require purification of cerebral vessels before preparation of the seeds.
We biochemically confirmed our histological data by analyzing Aβ levels from sequentially extracted brain lysates using C-terminal specific antibodies in sandwich ELISAs to measure Aβ40 and Aβ42 specifically. In our Aβ ELISAs the detection limit was approximately 0.04 pmol/g. In the brains of all seeded mice, the levels of Aβ40, both SDS-soluble and FA were 10-100 fold lower than that of Aβ42 (Fig. 2a, b). Consistent with the presence of abundant diffuse Aβ pathology, we detected elevated levels of Aβ42 in SDS-soluble fractions from mice seeded with AD 1 and 2, and PA 4 (Fig. 2c). Somewhat surprisingly, the levels of Aβ42 in the FA-soluble fractions were similar to that of the SDS fraction despite the absence of cored Aβ deposits (Fig. 2c and d). Notably, there was considerable variation in the levels of Aβ in these seeded animals, which rendered relatively few indications of statistical differences (Fig. 2e-h). For Aβ40 measures, the only instance in which the seeded mice had levels that were higher than uninjected controls, or mice injected with NDC lysate, were mice seeded by AD 1 (Fig. 2e, f). For measurements of SDS-soluble Aβ42, the brains of mice seeded with AD 1, 2, and PA 4 were the only examples in which the levels were statistically higher than the levels in mice seeded with NDC 1 and 2, or uninjected mice (Fig. 2g). In the FA-soluble fractions, only the AD lysates possessed higher levels of Aβ42 than the brains of mice seeded with the two NDC lysates or uninjected mice (Fig. 2h). Collectively, these findings demonstrated that both AD and PA brains have the potential to seed diffuse Aβ pathology, with the AD brains appearing to be slightly more potent.
To determine whether the injection of these lysates into the brains of newborn PrP.APPsi/iTau-P301L mice also induced tau pathology, we stained the brains of the seeded animals with antibodies specific for phosphorylated tau (AT8) and misfolded tau (MC1). Immunostaining with the antibody CP27, which is specific for human tau, confirmed the presence of human P301L tau in the bigenic mice, but we observed no obvious reactivity with AT8 or MC1 antibodies (Fig. S9).
To determine if the induction of amyloid would secondarily induce αSyn pathology, we examined the seeding activity of individuals with AD and LBD pathology. To characterize the pathology of these AD, AD/LBD and NDC brains, we stained sections with a pan-Aβ antibody (Fig. 3a), Thio-S (Fig. 3b), αSyn antibody (Fig. 3c), and a phospho-specific tau antibody (Fig. 3d). Both AD 3 and 4 contained numerous amyloid deposits that were compact and Thio-S positive (Fig. 3a, b), had widespread tau positive inclusions (Fig. 3d), and lacked αSyn pathology (Fig. 3c). AD 3 showed striking Thio-S positive amyloid staining surrounding vessels, consistent with a CAA score of 3+ (Fig. 3b, Table 1). We also observed CAA staining with AD/LBD 1 (Fig. 3b). The AD/LBD cases showed modest Aβ pathology with some αSyn pathology and sparse tau deposition (Fig. 3a, c, d). NDC 3 contained some amyloid pathology, but both lacked αSyn and tau pathology (Fig. 3).
PrP.APPsi/M20 and PrP.APPsi mice injected with AD brains developed widespread, robust Aβ pathology by 12 months post-injection (Fig. 4a and b). Aβ deposition was primarily diffuse and Thio-S negative (Fig S10). There was no difference in Aβ seeding capacity between PrP.APPsi/M20 and PrP.APPsi mice. Interestingly, AD/LBD 1 and 2 also promoted deposition of Aβ in mice expressing APPsi with and without αSyn (Fig. 4), indicating that the AD/LBD brains contained considerable levels of Aβ seeds despite a much lower burden of Aβ pathology (Fig. 3). None of the injected animals developed appreciable αSyn pathology (data not shown). Although two of the donor cases contained significant CAA pathology, CAA was not seeded in the recipient mice (Fig S10).