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Continuous Monitoring of Tau-Induced Neurotoxicity in Patient-Derived iPSC-Neurons - PubMed

  • ️Fri Jan 01 2021

Continuous Monitoring of Tau-Induced Neurotoxicity in Patient-Derived iPSC-Neurons

Derek H Oakley et al. J Neurosci. 2021.

Abstract

Tau aggregation within neurons is a critical feature of Alzheimer's disease (AD) and related tauopathies. It is believed that soluble pathologic tau species seed the formation of tau aggregates in a prion-like manner and propagate through connected neurons during the progression of disease. Both soluble and aggregated forms of tau are thought to have neurotoxic properties. In addition, different strains of misfolded tau may cause differential neurotoxicity. In this work, we present an accelerated human neuronal model of tau-induced neurotoxicity that incorporates both soluble tau species and tau aggregation. Using patient-derived induced pluripotent stem cell (iPSC) neurons expressing a tau aggregation biosensor, we develop a cell culture system that allows continuous assessment of both induced tau aggregation and neuronal viability at single-cell resolution for periods of >1 week. We show that exogenous tau "seed" uptake, as measured by tau repeat domain (TauRD) reporter aggregation, increases the risk for subsequent neuronal death in vitro These results are the first to directly visualize neuronal TauRD aggregation and subsequent cell death in single human iPSC neurons. Specific morphologic strains or patterns of TauRD aggregation are then identified and associated with differing neurotoxicity. Furthermore, we demonstrate that familial AD iPSC neurons expressing the PSEN1 L435F mutation exhibit accelerated TauRD aggregation kinetics and a tau strain propagation bias when compared with control iPSC neurons.SIGNIFICANCE STATEMENT Neuronal intracellular aggregation of the microtubule binding protein tau occurs in Alzheimer's disease and related neurodegenerative tauopathies. Tau aggregates are believed to spread from neuron to neuron via prion-like misfolded tau seeds. Our work develops a human neuronal live-imaging system to visualize seeded tau aggregation and tau-induced neurotoxicity within single neurons. Using an aggregation-sensing tau reporter, we find that neuronal uptake and propagation of tau seeds reduces subsequent survival. In addition, human induced pluripotent stem cell (iPSC) neurons carrying an Alzheimer's disease-causing mutation in presenilin-1 undergo tau seeding more rapidly than control iPSC neurons. However, they do not show subsequent differences in neuronal survival. Finally, specific morphologies of tau aggregates are associated with increased neurotoxicity.

Keywords: Alzheimer's disease; FTLD; MAPT; PSEN1 L435F; iPSC; neurotoxicity; tau aggregation; tau seeding.

Copyright © 2021 Oakley et al.

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Figures

Figure 1.
Figure 1.

TauRD-YFP reporter and Aβ expression. A, Unstained fluorescent image of control and PSEN1 iPSC colonies showing partial expression of TauRD reporter. Scale bar, 100 µm. B, Density distribution of neuronal soma YFP fluorescence intensity measured by live imaging of iPSC neurons at the end of differentiation (day 15): control = 255 YFP-positive neurons; PSEN1 = 228 YFP-positive neurons. This analysis includes all YFP-expressing neurons visible on epifluorescent imaging. C, Western blot results demonstrating equivalent expression of the TauRD-YFP construct in PSEN1 L435F and control iPSC-derived neuronal cultures (n = 3 independent neuronal differentiations, day 28, mean ± SD). D, E, Secreted Aβ43 species and Aβ42/Aβ40 and Aβ43/Aβ40 ratios are elevated in PSEN1 L435F iPSC neurons (mean ± SD). D, Absolute Aβ levels in media for control and PSEN1 mutant iPSC neurons (D28 media, ELISA, ± SD). E, Aβ42/Aβ40 and Aβ43/Aβ40 ratios (±SD; Extended Data Figures 1-1, 1-2).

Figure 2.
Figure 2.

TauRD-YFP reporter expression in iPSC neurons and aggregation in response to misfolded tau seeds. Control and PSEN1 mutant iPSC neurons as indicated with and without exposure to tau seeds for 7 d before fixation and immunohistochemistry for DAPI (blue), YFP (cyan), and phospho-tau (PHF1, red). Following exposure to tau seeds, iPSC neurons develop aggregates of TauRD-YFP reporter (arrowheads), shown at higher magnification in inset. Scale bar, 50 μ

m

(Extended Data Figure 2-1).

Figure 3.
Figure 3.

Live imaging of neurite seeding and cell death in iPSC neurons. A, Graphs depict the average number of TauRD-YFP reporter seeds within neurites per well for 7 d following the addition of either seed-containing lysate (rTg4510) or control lysate (±SE). n = 3 independent neuronal differentiations for all experiments except control neurons paired with 1ug control lysate where n = 2. Values represent the sum of two technical replicates per experiment. Subtracted baseline is the overall average number of events detected in the no-lysate condition for each line (quantification noise). B, Mean area under the curve for neurite seeding assay in A plotted for each dosage of seed-containing lysate, grouped by cell line. *p ≤ 0.05, **p ≤ 0.01. Two-way ANOVA results at top are for the interaction of cell line and dosage (n.s., p = 0.9). C–F, Following the addition of tau seeds at 0 h (C), TauRD-YFP aggregation occurs beginning in neurites (arrows; D), concentrating in cell body (E), and preceding neuronal death, highlighted by increased red signal from nuclear dye (arrowhead; F). The cell body disappears following cell death. Cyan, TauRD-YFP; white, Tau-YFP aggregates (saturated, pseudocolored); red, NucSpot 650 showing maximal signal at cell death. PSEN1 mutant iPSC neurons used in this example. Scale bar, 25 μ

m

(Extended Data Figures. 3-1, 3-2).

Figure 4.
Figure 4.

Accelerated aggregate formation in PSEN1 mutant iPSC neurons. A, PSEN1 L435F mutant neurons show a higher percentage of TauRD-YFP cell soma aggregates compared with control at both 48 h and 7 d after addition of 10 µg seed material (p = 0.0009 and p = 0.0232 respectively; n = 4 independent neuronal differentiations, ±SE). B, Time course of soma aggregate formation in control and PSEN1 L435F mutant iPSC neurons (p < 0.0001, plots are ±95% confidence interval). C, Onset of TauRD reporter aggregation does not correlate with expression levels. The onset of TauRD reporter aggregation is plotted against normalized fluorescence intensity of the TauRD reporter per cell at the first imaging time point (t0). Pearson correlation is plotted for each cell line. n = 3 independent differentiations. In total, 591 control neurons and 441 PSEN1 mutant neurons are depicted. Normalized t0 values correspond to the average TauRD fluorescence intensity in a region of interest centered over the tracked neuron divided by the average fluorescence intensity of all tracked neurons in each replicate per cell line. Neurons that do not develop TauRD aggregates are excluded from this analysis (Extended Data Figure 4-1).

Figure 5.
Figure 5.

TauRD aggregate toxicity in control and familial AD iPSC neurons. A, B, Reduced survival in control (A) and PSEN1 L435F (B) neurons that form aggregates within 48 h after addition of seeds (Early) compared with all other neurons in the same well (Others), including cells that develop aggregates at later time points [p < 0.0001 for both (+) vs (–) aggregate comparisons; n.s. for line comparison by log-rank test; n = 4 independent neuronal differentiations; plots are ±95% confidence interval]. Crosses indicate censored cells. The number at risk for each time point is presented below. Time is represented in hours after seed addition. C, Survival curves for early aggregate forming neuronal populations (<48 h after seed material addition) from control and PSEN1 mutant cells. D, Survival curves for all aggregate-forming neurons from both cell lines beginning from the time of aggregate formation. Censor crosses are frequent in D because of censoring at the experimental end point, which occurs at a variable time with respect to aggregate formation in each cell (Extended Data Figures. 5-1, 5-2, 5-3).

Figure 6.
Figure 6.

TauRD aggregate morphologies correlate with neurotoxicity. A, Example images of observed TauRD aggregate morphologies (control neurons, n = 322; and PSEN1 L435F neurons, n = 267). B, Relative proportions of each aggregate morphology for control and PSEN1 cell lines ±SE. C–F, Boxplots. C, Neuronal life span in control and PSEN1 iPSC neurons with aggregates, grouped by aggregate morphology (Ct, control, PS1, PSEN1). D–F, Comparison of neuronal life span (D), Onset of aggregation (E), and aggregate life span (F), respectively, for cells with disordered and ordered aggregates, grouped by cell line. In B and C, p values are for pairwise comparison between the two cell lines. In D–F, p values are for pairwise comparison between aggregate types.

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