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Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF - PubMed

  • ️Fri Jan 01 2021

Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF

Brian D Hitt et al. Acta Neuropathol Commun. 2021.

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.

Keywords: Alzheimer’s disease; Biomarkers; Cerebrospinal fluid; Seeding; Tau.

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Figures

Fig. 1
Fig. 1

v2H biosensors express more tau-fusion protein. Fluorescent micrographs of P301S v1 (a, d), v2L (b, e), and v2H (c, f) cells demonstrate an increase in fluorescence of both v2 cell lines over first-generation biosensor cells, and of v2H over v2L when examined under the YFP channel. d–f show cells treated with synthetic tau fibrils delivered by Lipofectamine 2000. Western blot against tau (g) and GFP (h) demonstrates higher levels of tau-fusion protein in v2H biosensor cells compared to v2L cells. Negative control (−) represents lysate from naïve HEK293 cells and positive control ( +) represents lysate HEK293 cells with a single incorporation of the tau RD-mClover construct. The slightly higher molecular weight of the tau-YFP fusion in v1 cells is due to a longer linking sequence in the original fusion protein

Fig. 2
Fig. 2

P301S v2H cells exhibit increased sensitivity to AD-derived tau seeds. The FRET assay was performed with successive dilutions of synthetic tau fibrils mixed with Lipofectamine 2000 (a) and without transfection reagent (b) (log scale), and of immunopurified tau from an AD brain with Lipofectamine 2000 (c). d–f) are expansions of the dose response curves of in (a–c), respectively. Lower limit of detection (LLD—defined as the lowest quantity of tau fibrils that produces a signal of FRET positivity statistically distinguishable from background, T-test, p < 0.05) with Lipofectamine-mediated synthetic fibril seeding was 10 amoles for all cell lines. For naked synthetic fibril seeding the LLD was 0.59 pmol for all cell lines, and with AD brain protein it was 1 ng for v1, 100 pg for v2L, and 10 pg for v2H (represent pre-IP quantities of total protein). Error bars are SEM of 3 technical replicates over which each sample was divided

Fig. 3
Fig. 3

High sensitivity detection of tau seeding from biological sources. a Dose response curves of P301S v2H cells with protein from PS19 transgenic, wild-type, and tau knock-out mouse brain. b Dose response curves using protein from frontal lobe tissue of 5 AD cases. c, d Expansions of (a, b), respectively. LLD was 316 pg for PS19 brain, 305 pg for AD1, 153 pg for AD2, 1.2 ng for AD3, 78 ng for AD4, and 1.2 ng for AD5. Error bars are the SEM of 3 technical replicates over which each sample was divided

Fig. 4
Fig. 4

Efficient purification of tau seeds from CSF. a 10 ng of protein from frontal cortex of case AD1 was spiked into control CSF or PBS. FRET positivity resulting from IP followed by seeding assay of spiked samples did not differ between CSF and PBS, or with volume of IP. b 1 ml aliquots of control CSF were spiked with a serial dilution of protein from brain AD1. c 1 ml aliquots of control CSF were spiked with a serial dilution of recombinant tau fibrils. Seeding was detected from these spiked samples down to 31.6 pg of total AD brain protein and 100 attomoles of recombinant fibrils (monomer equivalent). Pre-IP shows FRET positivity from direct treatment with the same amount of protein spiked into the corresponding sample. Error bars are SEM of 3 technical replicates over which each sample was divided

Fig. 5
Fig. 5

Absent CSF seeding in v2H biosensor cells. Results are from immunopurification from 1 ml CSF. The left end of the graph shows FRET positivity from a serial dilution of synthetic tau fibrils. * LLD = 10 attomoles of tau. Error bars are SEM of 3 technical replicates over which each sample was divided

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