Abstract
BackgroundAlthough several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity.MethodsTau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors.ResultsWe observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients.ConclusionsThis study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.
Highlights
Several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood
From our in-house antibody collection, PT76, developed by immunization with human Alzheimer’s disease (AD) brain-derived Paired-helical filament (PHF) as first described in Vandermeeren et al [13] was found to bind a Tau epitope encompassing residues 249–258 which is localized N-terminal from the microtubule binding domain (MTBD)
To translate our findings made with K18 fibrils to in vivo-produced Tau aggregates from human AD brains, we show that sonication improves the seeding potency of these fibrils and creates smaller Molecular weight (MW) aggregates, which is indicative of a similar phenomenon as observed for K18 (P301L fibrils)
Summary
Several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. As Tau pathology has been suggested to progress through a defined pattern over time [5], it is plausible that an extracellular component is contributing to the spreading of the disease [6]. While the pattern of propagation and the brain regions affected by Tau pathology are identified, it is not completely understood how this mechanism of Tau spreading occurs. Seeding experiments with Tau species derived from interstitial fluid and cerebrospinal fluid collected from AD individuals, suggested the presence of seedingcompetent Tau species in extracellular fluids [8]
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