Aqueous extractable nonfibrillar and sarkosyl extractable fibrillar Alzheimer's disease tau seeds have distinct properties

Abstract

Pathological tau fibrils in progressive supranuclear palsy, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer's disease each have unique conformations, and post-translational modifications that correlate with unique disease characteristics. However, within Alzheimer's disease (AD), both fibrillar (sarkosyl insoluble (AD SARK tau)), and nonfibrillar (aqueous extractable high molecular weight (AD HMW tau)) preparations have been suggested to be seed-competent. We now explore if these preparations are similar or distinct in their in vivo seeding characteristics. Using an in vivo amplification and time-course paradigm we demonstrate that, for AD HMW and AD SARK tau species, the amplified material is biochemically similar to the original sample. The HMW and SARK materials also show different clearance, propagation kinetics, and propagation patterns. These data indicate the surprising co-occurrence of multiple distinct tau species within the same AD brain, supporting the idea that multiple tau conformers - both fibrillar and nonfibrillar- can impact phenotype in AD.

Keywords: Alzheimer; Kinetics; Seeding; Spreading; Tau.

Fig. 1

PBS extractable AD HMW and insoluble AD SARK tau seeds have different pathology distribution patterns over time. (a) Representative images of AT8-positive immunostaining at 1 day, 1 week, 1 month, and 3 months after injection in PBS, AD HMW Tau- and AD SARK Tau-injected hTau mice. Sparse AT8-positive cells (arrows) and extensive AT8 tau pathology (arrowheads) are observed in both tau-injected groups 1 and 3 months after injection, respectively. Scale bar = 500 μm. (b) Representative images of AT8-positive immunostaining (arrowheads) in the peri-/entorhinal cortex at 1 day, 1 week, 1 month, and 3 months after injection in PBS, AD HMW Tau- and AD SARK Tau-injected hTau mice. Scale bars = 100 μm. c-d. Distribution (%) of AT8 pathology across brain regions 1 month and 3 months after injection between AD HMW and AD SARK tau-injected animals. Data represented as mean ± SEM, n = 4–6/group, individual t-test for each brain region. HIPP = hippocampus, ATN = anterior thalamus nuclei, DG = dentate gyrus, MBs = mamillary bodies, OCx = hippocampus overlaying cortex (motor and somatosensory), PER/ENT = peri-/entorhinal cortex, RSP = retro-splenial cortex, SUB = subiculum

Fig. 2

Tau seeds’ characteristics are maintained after in vivo amplification. a-d. Quantification of seeding activity over time in PBS and sarkosyl extracts of hippocampal and entorhinal cortex homogenates from injected hTau mice. At each time point, seeding activity was corrected to the corresponding PBS-injected group. The graph shows the average of 3 independent seeding assay. Data represented as mean ± SEM, n = 4–6/group, Two-way ANOVA. e. Representative images of ThioS-positive staining (arrowheads) at 3 months after injection in PBS, AD HMW Tau- and AD SARK Tau-injected hTau mice. Scale bars = 100 μm. f. Quantification of the ThioS-positive cells density in different brain regions. Data represented as mean ± SEM, n = 5/group, individual t-test for each brain region. PYR = hippocampal pyramidal layer, SUB = subiculum, PER/ENT = peri-/entorhinal cortex, OCx = hippocampus overlaying cortex (motor and somatosensory). g-h. Quantification of seeding activity in PBS and sarkosyl extracts of hippocampal homogenates from injected hTau and APPxhTau mice. The graph shows the average of 3 independent seeding assay. Data represented as mean ± SEM, n = 3/group, Two-way ANOVA

Fig. 3

Endogenous Tau phosphorylation profiles differ according to tau pathology stage. (a) Quantification of various pTau epitopes (normalized to total tau) over time in PBS extracts of hippocampal homogenates from injected hTau mice. (b) Quantification of various pTau epitopes (normalized to total tau) over time in PBS extracts of entorhinal cortex homogenates from injected hTau mice. (c) Quantification of various pTau epitopes (normalized to total tau) 3 months after injection in sarkosyl extracts of hippocampal homogenates from injected hTau mice. (d) Quantification of various pTau epitopes (normalized to total tau) 3 months after injection in sarkosyl extracts of entorhinal cortex homogenates from injected hTau mice. At each time point, data was normalized to the corresponding PBS-injected group. Phosphorylation at S195 (pS195) was measured using Tau1 antibody which recognizes de-phosphorylated tau S195, and the inverse reported on the graph for visualization purposes. Data represented as mean ± SEM, n = 4–6/group, individual t-test for each pTau

Fig. 4

Microglial phenotype varies over time and with the nature of the exogenous Tau seed. (a) Representative images of the morphology of Iba1-positive microglia in the CA1 of the hippocampus of PBS, AD HMW, and AD SARK tau-injected mice over time. Rod-like microglia are observed at different time-points (yellow arrows). (b) Quantification of the percentage of rod-like Iba1-positive cells normalized to PBS-injected animals at each time point. Data represented as mean ± SEM, n = 4–6/group, Two-way ANOVA

Fig. 5

Human AD brain contains multiple distinct tau pathologies. The human AD brain contains various tau species that can be differentially extracted based on their solubility: PBS-extractable AD HMW tau and sarkosyl-insoluble AD SARK tau. In vivo in hTau mice, both species start inducing pathology 1 month after injection (red colors), but the spatial distribution of tau pathology differs across injection groups. Heavier tau loads (bright red vs. light red) are observed in AD HMW tau-injected mice in the subiculum, cingulate, and perirhinal region compared to AD SARK-injected animals. Within 3 months after injection (blue colors), AD SARK tau-injected animals also present tau pathology in the peri-/entorhinal region, and all AD tau-injected mice start getting tau pathology in the mammillary bodies. All tau pathology-bearing brain regions are either directly or indirectly, via the subiculum, connected to the injection site in the dorsal CA1 (purple dot). Importantly, AD HMW tau seeds amplify as PBS-extractable tau species, while AD SARK tau gives rise to sarkosyl-insoluble tau. Considering the definition of prion strains [17], these different observed characteristics suggest that AD HMW tau and AD SARK tau are distinct substrains of AD tau. CA1: CA1 field of the hippocampus, SUB: subiculum, DG: dentate gyrus, SS: somatosensory cortex, CING: cingulate, RSP: retrosplenial cortex, ATN: anterior thalamic nuclei, PER: perirhinal cortex layer V, ENT: entorhinal cortex layer II, MM: mammillary bodies. The images were generated using the Scalable Brain Atlas composer