Abstract
Tau protein forms self-replicating assemblies (seeds) that may underlie progression of pathology in Alzheimer’s disease (AD) and related tauopathies. Seeding in recombinant protein preparations and brain homogenates has been quantified with “biosensor” cell lines that express tau with a disease-associated mutation (P301S) fused to complementary fluorescent proteins. Quantification of induced aggregation in cells that score positive by fluorescence resonance energy transfer (FRET) is accomplished by cell imaging or flow cytometry. Several groups have reported seeding activity in antemortem cerebrospinal fluid (CSF) using various methods, but these findings are not yet widely replicated. To address this question, we created two improved FRET-based biosensor cell lines based on tau expression, termed version 2 low (v2L) and version 2 high (v2H). We determined that v2H cells are ~ 100-fold more sensitive to AD-derived tau seeds than our original lines, and coupled with immunoprecipitation reliably detect seeding from samples containing as little as 100 attomoles of recombinant tau fibrils or ~ 32 pg of total protein from AD brain homogenate. We tested antemortem CSF from 11 subjects with a clinical diagnosis of AD, 9 confirmed by validated CSF biomarkers. We used immunoprecipitation coupled with seed detection in v2H cells and detected no tau seeding in any sample. Thus we cannot confirm prior reports of tau seeding activity in the CSF of AD patients. This next generation of ultra-sensitive tau biosensors may nonetheless be useful to the research community to quantify tau pathology as sensitively and specifically as possible.
Highlights
Intracellular aggregates of the microtubule-associated protein tau define Alzheimer’s disease (AD) and related neurodegenerative tauopathies
Construction of tau repeat domain (RD)(P301S) version 2 high (v2H) biosensor cells Upon sequencing of the plasmid used to express tau RD (P301S)-CFP/YFP in the original biosensor line, we found an error in the Kozak sequence that could reduce translation efficiency
With AD-derived tau, we detected seeding from 1 ng total protein equivalent with v1 cells, but 100 pg and 10 pg with version 2 low (v2L) and v2H cells, respectively (Fig. 2c, f ), representing an increased sensitivity of tenfold for v2L and 100-fold for v2H over the original line [21]
Summary
Intracellular aggregates of the microtubule-associated protein tau define Alzheimer’s disease (AD) and related neurodegenerative tauopathies. In AD, tau progressively accumulates in defined patterns that involve brain networks [6]. This has been hypothesized to occur via formation of aggregate “seeds” in a single neuron or group of neurons that exit and gain entry to neighboring or synaptically connected cells. We have previously used the original biosensor cells to detect tau seeding activity in mouse and human brain prior to neurofibrillary tangle pathology. This indicates that seed formation is perhaps the earliest detectable tau-related pathological event [16, 24], and could be an excellent disease biomarker. Reliable detection of seeding activity in peripheral fluids such as CSF or blood from living subjects has not yet been established, but could improve specific diagnosis of tauopathy
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