Puncta point the way Amyloid plaques composed of Aβ peptides and neurofibrillary tangles composed of aberrant tau proteins are the key pathological hallmarks in the Alzheimer’s disease (AD) brain. However, understanding which conformers of Aβ and tau play a pathological role at each step of AD pathogenesis has been difficult to elucidate. Aoyagi et al. have developed sensitive cellular assays that detect aberrant Aβ and tau in postmortem brain homogenates from patients with AD or other neurodegenerative diseases. Using fluorescent puncta as a readout, these assays now reveal that patients with AD who died at an older age have lower Aβ and tau pathological conformers than do patients who died at a younger age.

Abstract The hallmarks of Alzheimer’s disease (AD) are the accumulation of Aβ plaques and neurofibrillary tangles composed of hyperphosphorylated tau. We developed sensitive cellular assays using human embryonic kidney–293T cells to quantify intracellular self-propagating conformers of Aβ in brain samples from patients with AD or other neurodegenerative diseases. Postmortem brain tissue from patients with AD had measurable amounts of pathological Aβ conformers. Individuals over 80 years of age had the lowest amounts of prion-like Aβ and phosphorylated tau. Unexpectedly, the longevity-dependent decrease in self-propagating tau conformers occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the ability of postmortem AD brain homogenates to induce misfolded tau in the cellular assays showed an exponential decrease with longevity, with a half-life of about one decade over the age range of 37 to 99 years. Thus, our findings demonstrate an inverse correlation between longevity in patients with AD and the abundance of pathological tau conformers. Our cellular assays can be applied to patient selection for clinical studies and the development of new drugs and diagnostics for AD.

INTRODUCTION The plaques and tangles of Alzheimer’s disease (AD) were first described more than a century ago (1). In 1984, the amyloid-β (Aβ) peptide was described (2), and the tau protein in neurofibrillary tangles (NFTs) was reported soon thereafter (3–6). Both Aβ and tau proteins adopt pathogenic conformations that spread through the brain (7, 8) in a manner similar to the prion protein (PrPSc), which causes Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker (GSS) disease, fatal familial insomnia, and kuru (9). Because our knowledge of prions has expanded with the identification of physiological nonpathogenic prions, a more inclusive definition has emerged: Prions are composed of host-encoded proteins that adopt alternative conformations, which are self-propagating (10). Here, we refer to Aβ and tau as “prion-like” proteins. Although the vast majority of AD cases are sporadic, an important but small number of familial cases have been instructive. In 1989, one of us reported genetic linkage of the P102L mutation in the PrP open reading frame that causes familial GSS (11). Two years later, familial AD was linked to the V717I mutation in the amyloid precursor protein (APP) open reading frame (12). The importance of Aβ was further elucidated through investigations of AD pathogenesis based on genetic linkage studies between inherited AD and mutations in either APP or its processing enzymes (13, 14). Sequential cleavage of APP yields Aβ40 and Aβ42 as the major peptide isoforms (14). Aβ42 can polymerize into oligomers or fibrils and ultimately forms amyloid deposits in the brain (14). The role of tau in AD was clarified in 1998 when three different point mutations in the MAPT gene encoding tau protein isoforms were shown to cause familial Pick’s disease (15, 16). In familial cases of cerebral amyloid angiopathy (CAA) caused by mutations in the Aβ coding region of APP, death generally occurs in the fourth or fifth decade of life and in the absence of substantial accumulation of insoluble tau (17, 18). In contrast to CAA, misfolded tau spreads through many brain regions in both sporadic and familial AD, resulting in cognitive decline. Hypotheses about the role of Aβ abound: Aβ deposition is considered inconsequential by some (19, 20), or Aβ is thought to transiently initiate tau misfolding and polymerization into NFTs by others (21–24). Aβ might also have transient toxicity due to the accumulation of Aβ oligomers, which peak in the early, prodromal phase of AD progression (25, 26). Alternatively, an early steady progression of Aβ oligomerization, deposition, and spreading has been proposed. The latter hypothesis is supported by the correlation of increasing AD severity with the spread of Aβ throughout the brain (27, 28) and decreased Aβ42 in cerebrospinal fluid (CSF) (29). In this view, early and progressive formation of prion-like Aβ conformers leads to dysfunction in the central nervous system (CNS), including an inability to clear misfolded tau molecules leading to the accumulations of NFTs. To address this hypothesis, assays are needed to measure and rapidly compare Aβ and tau prion-like activities in the postmortem brain tissue of deceased patients with AD. However, current methods for the measurement of pathogenic Aβ conformations rely on either time-consuming transgenic mouse models or in vitro biophysical methods that are performed at superphysiological peptide concentrations. Earlier studies showed that AD patient brain–derived Aβ and synthetic Aβ injected into a transgenic mouse brain behaved in a prion-like manner (30–34). Current clinical imaging ligands measure insoluble amyloids, the role of which in disease is uncertain. These imaging methods fail to measure biologically active neuroinvasive prion-like Aβ and tau proteins. In contrast, highly reproducible and rapid cell-based methods have been devised to measure prion-like tau and α-synuclein proteins expressed as fusions with fluorescent proteins in mammalian cells (35–38). Here, we describe the development of an analogous cellular assay for Aβ, allowing Aβ and tau to be compared in postmortem brain tissue samples from patients with AD or other neurodegenerative diseases. Prion-like Aβ and tau activity decreased with longevity despite the presence of increasing NFTs. This decrease in tau prion-like activity paralleled similar decreases in tau phosphorylation. Thus, the greatest tau prion-like activities were found in individuals who died at relatively young ages, despite having the lowest abundance of total insoluble tau. It is the relative abundance of biologically active, phosphorylated prion-like tau and not the total amount of inert insoluble tau that correlated with longevity.

DISCUSSION A wealth of evidence argues that both Aβ and tau adopt pathological conformations leading to prion-like spreading throughout the brain during AD pathogenesis. The findings reported here establish the presence of both prion-like Aβ and prion-like tau proteins in the brains of patients who died of either sporadic or inherited AD. Moreover, studies by us and others in cellular and transgenic mouse models have previously demonstrated prion-like tau in the brains of patients who died of FTLD-tau (36, 38, 59–61). Our results extend those of earlier studies by demonstrating that postmortem brain tissue from patients with FTLD-tau was devoid of both prion-like Aβ and α-synuclein proteins (Fig. 3). The linear trends in prion-like Aβ and tau with longevity were observed over a range of sporadic and familial forms of AD. Thus, what had appeared to be a set of disparate disorders can now be seen as a continuous spectrum, with the defining feature being the spreading of prion-like Aβ and tau through the CNS. Our findings from earlier molecular genetic studies and those described here begin to create a more complete view of the chemical processes that feature in the pathogenesis of AD, because they allow a clear definition between inactive inert tau and Aβ versus their active prion-like forms. Both human and animal studies argue that pathologically misfolded Aβ initiates formation of prion-like tau (18, 21, 62, 63) in AD. Presumably, the formation of prion-like Aβ begins in one or more brain regions and then spreads to others. The movement of PrPSc prions and prion-like proteins such as α-synuclein, tau, and, possibly, Aβ42 from one CNS region to another argues for trans-synaptic spread (64–69). The apparent spread of prion-like proteins in the CNS is reflected by their regional distribution and has been well documented in neuropathological studies (70, 71). The identification of distinct prion-like Aβ conformers in AD brain samples with different etiologies has been particularly informative (72–75). It remains to be determined which of these conformers are related to distinct disease phenotypes or which are associated with selective prion-like tau formation. Using the cellular assays reported here, we can now correlate the presence of a given conformer with prion-like tau or prion-like Aβ activities across a variety of phenotypic manifestations of AD. Recent studies report that the minimal size of a biologically active tau aggregate may range from a monomer (76, 77) to linear aggregates of ~100 nm in length (78). Pentameric or smaller tau aggregates were unable to support prion-like tau activity (79). Additionally, tau phosphorylation may also contribute to the conformation of prion-like tau as shown in previous studies where immunodepletion of p-tau in brain extracts used as inocula abolished prion-like tau activity in recipient cells (80, 81) or in animal models (79). These findings are consistent with our data, demonstrating a relationship between prion-like tau activity and the extent of tau phosphorylation in brain samples from patients with AD obtained at different ages of death (Figs. 5 and 6). Development of a new cellular assay for prion-like Aβ peptides has permitted us to compare prion-like Aβ generated using synthetic Aβ fibrils versus transgene-encoded Aβ preparations. Our cellular assay is useful for measuring Aβ prion-like activities generated by allele-specific Aβ subtypes. In addition, it has enabled parallel quantification of prion-like Aβ and prion-like tau activities, providing a direct quantitative comparison of these actively propagating species, rather than comparisons of inert protein deposits. Our data also show that the patients with AD with greatest longevity had lower concentrations of both prion-like Aβ and prion-like tau at the time of death compared to patients who died at younger ages from AD-related disease. Previous studies showed that the abundance of NFTs correlated well with the extent of brain atrophy and cognitive decline in AD (29, 82). These studies focused only on total insoluble tau. By examining the age of death as a variable, we found that low prion-like tau activity correlated with greater longevity. Both the extent of prion-like Aβ activity and the abundance of APP, Aβ40, and Aβ42 decreased with longevity in a roughly synchronous manner. This finding is consistent with the hypothesis that prion-like Aβ features early during the formation of pathological tau tangles. Moreover, measurable prion-like Aβ activity was found in the oldest patients, suggesting that it continues to participate throughout AD pathogenesis. However, the R2 values that we found ranged from 0.12 to 0.2, indicating that many factors (from the methods of sample collection to genetic components) appeared to have a sizable influence on the observed correlation. Clearly, genetic factors such as the APOE ε4 allele and TREM2 variants, which have been implicated in Aβ metabolism and clearance, can strongly increase the risk of AD (56, 83). Although we found interesting trends with respect to the APOE ε4 genotype and gender, we will need to perform larger studies that carefully sample all of the different APOE genotypes. The strong associations among longevity, prion-like tau activity, and tau phosphorylation are particularly intriguing. Such findings are consistent with the greater contribution of tau versus Aβ protein misfolding to the AD phenotype as measured by neurological dysfunction and neuropathological lesions. One particularly notable result is the accumulation of insoluble tau that increases with a greater age at death in contrast to insoluble p-tau, which decreases with increasing longevity (Figs. 4 and 5). Although these relationships were clear from examining the extent of prion-like tau formation and phosphorylation per gram of total brain protein, these findings became more notable when the data were normalized according to the abundance of insoluble tau (Fig. 6). Our findings argue that all insoluble tau is not equally neurotoxic and that biochemical events such as phosphorylation influence the formation of prion-like tau or modulate tau toxicity. It remains to be determined whether the low abundance of prion-like tau in long-lived patients with AD is a result of a slowly replicating tau conformer or is due to host factors that more readily clear prion-like conformers or shift prion-like tau toward a more inert amyloid state (e.g., total insoluble tau). Thus, future work aimed at the development of diagnostic reagents and effective therapeutics for AD will need to focus on prion-like tau activity and its associated posttranslational modifications rather than total insoluble tau. Measuring both prion-like Aβ and prion-like tau abundance in postmortem brain samples is likely to have many applications. Antemortem detection of Aβ and tau activities in the CSF or blood of patients with AD, as shown for PrP prions (84–86), may provide more informative diagnostic tools to stage disease and measure efficacy of putative therapeutics. Also, our findings may help to illuminate both the successes and failures of pharmaceutical approaches that target Aβ and the Aβ-tau axis. The availability of paired cellular assays for measuring prion-like Aβ and prion-like tau should contribute to future drug discovery programs for AD.

MATERIALS AND METHODS Study design The aim of our study was to develop a rapid, quantitative cell-based assay to measure biologically active Aβ derived from postmortem human brain tissue. We first engineered HEK293T cell lines that were sensitive to Aβ40 and Aβ42 and validated the assay using synthetic Aβ fibrils that had previously been shown to be transmissible when injected into transgenic mouse brains (33, 34). We tested our cell lines with these Aβ fibril preparations in three independent experiments. The sample size and time points (including end points) were chosen for cell and animal experiments based on our previous work (34, 37, 38, 75). We used the minimum number of animals required to obtain a significant difference based on the expected variability, and all mice were randomly assigned and gender balanced. Deidentified human postmortem brain tissue was collected from several brain bank repositories located in the United States, Europe, and Australia (table S1). We replaced the identity of the samples with an internal code, and the investigator performing the experiments was blinded to the sample identity during testing and analysis. Experimental replicates for each experiment are listed in the figure legends. Cell line development Constructs encoding human WT and mutant Aβ42 or Aβ40 fused with YFP at the N or C terminus were introduced into the pIRESpuro3 vector (Clontech), were transfected to HEK293T cells (American Type Culture Collection), and monoclonal cell lines were generated and maintained as described (37). Cell lines expressing full-length human α-synuclein with the A53T mutation or human tau containing the repeat domain of 4R tau with the mutations P301L and V337 M fused with YFP were generated as described (37, 38). Development of the cellular assay The cellular bioassay for Aβ was developed as previously described (37, 38). Briefly, cells were plated in a 384-well plate at a density of 3000 cells per well (70 μl per well) with Hoechst 33342 (0.1 μg/ml) (Thermo Fisher). A mixture of Lipofectamine 2000 (1.5% final volume, Thermo Fisher), OptiMEM (78.5% final volume, Thermo Fisher), and sample (20% final volume) was incubated at room temperature for 2 hours and plated in four replicate wells (10 μl per well). Plates were then incubated and imaged every 24 hours on the IN Cell Analyzer 6000 (GE Healthcare) for 3 to 4 days. Images of both the DAPI (4′,6-diamidino-2-phenylindole) and FITC (fluorescein isothiocyanate) channels were collected from four different regions in each well. The images were analyzed using the IN Cell Developer software with an algorithm developed to identify intracellular aggregates only in live cells. Preparation of synthetic Aβ fibrils Preparation of synthetic Aβ fibrils was performed as described (34). Briefly, the WT Aβ40 and WT Aβ42 peptides were purchased from Bachem. Lyophilized peptides were dissolved to hexafluoroisopropanol (5 mg/ml) and separated in 200-μg aliquots. Hexafluoroisopropanol was evaporated in a SpeedVac and stored at −20°C. For conversion, the dried peptide film was solubilized in 20 μl of dimethyl sulfoxide and diluted with 980 μl of aqueous buffer solution containing 10 mM sodium phosphate. Samples were incubated at 37°C for 72 hours in 1.5-ml centrifugation tubes under constant agitation at 900 rpm. The resulting samples were spun down for 1 hour at 100,000g, and the pellet was resuspended in 100 μl of PBS at 2 mg/ml. Samples were further analyzed or diluted, snap frozen in liquid nitrogen, and stored at −80°C. PTA precipitation of Aβ peptides and tau proteins in postmortem brain samples PTA precipitation of human postmortem brain samples was performed as described (48, 87). Briefly, 10% brain homogenate was incubated in 2% sarkosyl and 0.5% benzonase (Sigma) at 37°C with constant agitation (1200 rpm) in an orbital shaker for 2 hours. Sodium PTA was dissolved in double-distilled H 2 O (ddH 2 O), and the pH was adjusted to 7.0. PTA was added to the solution to a final concentration of 2%, which was then incubated overnight under the same conditions. The sample was centrifuged at 16,100g for 30 min at room temperature, and the supernatant was removed. The resulting pellet was resuspended in 2% sarkosyl in PBS and 2% PTA in ddH 2 O (pH 7.0). The sample was again incubated for at least 1 hour before a second centrifugation. The supernatant was again removed, and the pellet was resuspended in PBS using 10% of the initial starting volume and stored at −80°C. To establish cellular assays for measuring prion-like Aβ and tau loads, we prepared a dilution series (e.g., 0.01, 0.03, and 0.1×) of all PTA-precipitated brain samples to perform the initial experiments. Once we established the dilution factor that best suited the majority of all samples, we performed subsequent experiments with only one or two dilution factors to conserve sample stocks. Using this approach, we ensured that our aggregation-inducing activity measurements were well within the dynamic range of the bioassay. Formic acid extraction of insoluble proteins in postmortem brain tissue for ELISA Fifty microliters of formic acid was added to 25 μl of 10% brain homogenate and placed in an ultracentrifuge tube. The samples were vortexed, sonicated for 20 min at 37°C in a water-bath sonicator, and then centrifuged at 100,000g for 1 hour. We removed 50 μl of the supernatant and neutralized it with 950 μl of neutralization buffer in a low-binding tube. The neutralization buffer consisted of 1 M tris base and 500 mM dibasic sodium phosphate with no pH adjustment. (If a very small pellet or layer of lipids formed at the top of the supernatant, then we aspirated the sample from the middle of the supernatant to maximize the protein in the extract.) Samples were aliquoted into low-binding tubes and flash frozen in liquid nitrogen. The following ELISA kits from Thermo Fisher Scientific were used according to the manufacturer’s protocol: APP (KHB0051), Aβ40 (KHB3481), Aβ42 (KHB3441), total tau (KHB0041), p-tau S396 (KHB7031), p-tau S199 (KHB7041), and p-tau T231 (KHB8051). The Aβ43 ELISA kit was from IBL-America (27710). Each sample was analyzed in duplicate. We adjusted the raw ELISA values to total brain protein (grams) in the clarified 10% brain homogenate as determined by bicinchoninic acid assay (Pierce/Thermo Fisher Scientific). Transgenic mice TgAPP23 mice, which express human APP (751–amino acid isoform) containing the Swedish mutation under the control of the Thy-1.2 promoter, were maintained on a C57BL/6 background. TgCRND8 mice, which express human APP (695–amino acid isoform) with the Swedish and Indiana mutations under the control of the hamster Prnp promoter, were maintained on a mixed B6/C3 background. TgGfap-luc mice, which express firefly luciferase under the control of the murine Gfap promoter, were a gift from Caliper Life Sciences and were maintained on an FVB/N background. To create bigenic mice, TgAPP23 and TgCRND8 mice were crossed with TgGfap-luc animals and were screened for the presence of both transgenes. Aged homozygous Tg0N4Rtau*P301S (“Tg2541+/+”) and Tgα-Syn*A53T (“TgM83+/+”) mice were used to provide control brain samples (50, 51). Animals were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. Bioluminescence imaging Bioluminescence imaging of the brains of bigenic TgAPP23:Gfap-luc mice and TgCRND8:Gfap-luc mice was performed as previously described (32). Isoflurane-anesthetized, head-shaved mice were imaged (60-s exposure) after receiving an intraperitoneal injection of 50 μl of d-luciferin potassium salt solution (30 mg/ml; Gold Biotechnology) that was prepared in PBS (pH 7.4) (a dose of ~60 mg/kg). Brain bioluminescence values were calculated from images displaying surface radiance using circular regions of interest and then were converted to total photon flux (photons per second) using Living Image software version 4.4 (PerkinElmer). Statistical analysis Statistical analyses were performed with GraphPad Prism version 7. Data are shown as means ± SEM. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) with post hoc Dunnett’s test. We used linear regression analysis for correlation plots. For two-group (frequency distribution) comparisons, we used a nonparametric Mann-Whitney U test. A value of P < 0.05 was considered significant. The exponential decay equation model (one-phase decay) used the least-squares (ordinary) fitting method with no constraints applied.

SUPPLEMENTARY MATERIALS stm.sciencemag.org/cgi/content/full/11/490/eaat8462/DC1 Fig. S1. Development of YFP-Aβ fusion HEK293T cell lines: Synthetic Aβ fibril inocula- and puncta-inducing kinetics. Fig. S2. Development of YFP-Aβ fusion HEK293T cell lines: Individual clones. Fig. S3. Amyloid plaque pathology, astrogliosis, and prion-like Aβ abundance during disease progression in TgAPP23 and TgCRND8 mice. Fig. S4. APOE ε4 status, gender, and brain region influence the extent of prion-like Aβ and tau abundance in AD postmortem brain tissue. Fig. S5. Correlation of different Aβ and tau species with age at death of patients with AD. Table S1. Source of postmortem human brain tissue samples. Data file S1. Source data.

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