Internalized Tau Oligomers Cause Neurodegeneration by Inducing Accumulation of Pathogenic Tau in Human Neurons Derived from Induced Pluripotent Stem Cells

Abstract

Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer's disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau. However, the molecular and cellular mechanisms of pathogenic tau transmission remain unknown. The studies described herein investigate tau pathology propagation using human neurons derived from induced pluripotent stem cells. Neurons were seeded with full-length human tau monomers and oligomers and chronic effects on neuronal viability and function were examined over time. Tau oligomer-treated neurons exhibited an increase in aggregated and phosphorylated pathological tau. These effects were associated with neurite retraction, loss of synapses, aberrant calcium homeostasis, and imbalanced neurotransmitter release. In contrast, tau monomer treatment did not produce any measureable changes. This work supports the hypothesis that tau oligomers are toxic species that can drive the spread of tau pathology and neurodegeneration.

Significance statement: Several independent studies have implicated tau protein as central to Alzheimer's disease progression and cell-to-cell pathology propagation. In this study, we investigated the ability of different tau species to propagate pathology in human neurons derived from induced pluripotent stem cells, which to date has not been shown. We demonstrated that tau oligomers, but not monomers, induce accumulation of pathological, hyperphosphorylated tau. This effect was accompanied with neurite degeneration, loss of synapses, aberrant calcium homeostasis, imbalanced neurotransmitter release, and ultimately with neuronal death. This study bridges various tau pathological phenotypes into a single and relevant induced pluripotent stem cell neuronal model of human disease that can be applied to the discovery of the mechanisms of tau-induced neurodegeneration.

Keywords: Alzheimer's disease; hiPSC neurons; neurodegeneration; pathology propagation; tau oligomer seeds.

Figure 1.

Characterization of tau oligomers. A, Graph represents a time course of formation of tau oligomers based on change of FP. Results are expressed as a change of FP (Δmp) between oligomerization reaction (with heparin) and negative control reaction (without heparin) at indicated time points. B, Fluorescence intensity of ThT bound to T40-tau during the aggregation process of 15 d in presence of heparin that led to a tau fibril formation. All values are expressed as mean ± SEM. C, High-mass MALDI-TOF MS comparing cross-link-stabilized (red) and untreated (blue) tau oligomers demonstrates that our preparations consist of a heterogeneous, multimeric population of tau monomers, dimers, trimers, and tetramers that include noncovalent binding with heparin. D, Immunoblot probed with total tau antibody shows the presence of tau monomers, dimers, and trimers in our oligomerization reaction of 4 h. E, Images of atomic force microscopy of 4 h oligomer preparation show the formation of majority spherical oligomeric structures or elongated tau threads. Tau monomers (from the preparation that contains no heparin and therefore does not cause oligomerization) do not bind to mica surface.

Figure 2.

Internalization of tau seeds by hiPSC neurons. A, Representative live-cell images of Cy3-tau conjugate (red) show the internalization of tau monomers and oligomers, but not fibrils (10 d preparation), 1 h after addition. Hoechst was used to stain nuclei (blue). Scale bar, 50 μm. B, Images demonstrate the ability of background suppressor to efficiently suppress the fluorescence of Cy3-tau oligomers added to a well with a media, at the same concentration used in the uptake assay. C, Graph shows the decrease in the cellular intensity of Cy3-tau oligomers after heparinase III treatment. hiPSC neurons pretreated for 3 h with heparinase III were seeded with Cy3-tau oligomers for 1 h (Student's t test, ***p < 0.0001). D, Number of healthy nuclei per well with and without heparinase III treatment (n = 6 wells per condition, 9 fields, Student's t test, p = 0.8). All values are expressed as mean ± SEM. E, Images of the microfluidic device show the localization of Cy5-tau fluorescence signal in compartment 1 of the device where the Cy5-tau was added, but not in the microgrooves, demonstrating that the fluidic isolation creates independent microenvironment in microfluidic compartments. F, Images of hiPSC neurons growing in microfluidic device with somas and dendrites localized to a somal compartment and axons crossing the microgrooves. Cy5-tau monomers or oligomers were added to somal compartment for 2 h and images were taken 18 h after treatment. Fluorescence of Cy5-fluorophore in axons shows the uptake and trafficking of both tau monomers and oligomers.

Figure 3.

Tau oligomers induce accumulation of pathological tau. A–D, High-content image analyses of the hiPSC neurons 7 d after seeding with tau monomers and oligomers, and stained for pathological tau using MC1 antibody (A), ThS staining (B), and anti-phospho T231 tau (C) and anti-phospho S396/404 tau antibody (D). All graphs represent the percentage of cells positive for markers of pathological tau expressed as fold change versus vehicle. Representative images show cells positive for MC1 (A, red), ThS (B, green), and phospho-tau staining (C, D, green). Hoechst was used to stain nuclei (blue). Scale bar, 50 μm (n = 6 wells per condition, 9 fields, ANOVA with post hoc Dunnett's test vs vehicle, *p < 0.05, **p < 0.005, ***p < 0.0001). E, Images of Western blot analysis of sarkosyl-soluble and sarkosyl-insoluble fractions. PHF1 antibody (anti-phospho S396/404) was used to detected phospho-tau; GAPDH and Coomassie blue protein stain were used as a loading control. Molecular weight in kilodaltons is indicated along the blot. Western blots were quantified by densitometric analysis (graphs; n = 3, ANOVA with post hoc Dunnett's test vs vehicle; insoluble fraction, *p < 0.05; soluble fraction, p = 0.19). F, Graph represents the percentage of cells positive for MC1 staining 7 d after seeding after treatment with rapamycin. Rapamycin was added 24 h before tau seeding. DMSO was used as a vehicle (n = 6 wells per condition, 9 fields, ANOVA with post hoc Dunnett's test, ^###p < 0.0001 vs vehicle **p < 0.005 vs oligomer). G, Graph shows the number of LC3-positive autophagosomes per cell after rapamycin pretreatment (n = 3 wells per condition, 9 fields, Student's t test, *p < 0.05). All values are expressed as mean ± SEM.

Figure 4.

Exogenous tau oligomers recruit endogenous tau protein. A, Illustration of tau protein tagged with Flag-6xHis and mutated at the binding site of the HT7 antibody. B, Illustration of AlphaLISA assay principle; interaction between exogenous (Flag detected) and endogenous (HT7 detected) tau proteins brings acceptor and donor beads in very close proximity, which enables energy transfer from one bead to another, generating luminescence signal. C, Graph represents the Flag-HT7 AlphaLISA luminescence signal (fold change vs vehicle) from hiPSC-neuronal lysates seeded with two concentrations (50 and 250 nm) of tau monomers and oligomers. Lysates were harvested 4 d after seeding (n = 3 per condition, ANOVA with post hoc Dunnett's test, *p < 0.05 vs vehicle, **p < 0.005 vs vehicle). D, Graph shows sandwich ELISA signal with HT7 and total tau antibody used to detect HT7-mutant tau and wild-type (wt) tau. RU, Relative units. All values are expressed as mean ± SEM. E, High-resolution confocal images show the colocalization of Flag (exogenous tau) and HT7 (endogenous tau) staining in hiPSC neurons 4 d after treatment with tau-Flag oligomers. Higher magnification shows three-dimensional reconstruction of the Z-stacked images processed with Volocity program. Scale bar, 50 μm.

Figure 5.

Tau oligomers cause neurite retraction and neurotoxicity. A, Image demonstrates the masks of enlarged nuclei, representing cell body (yellow arrow) and neurites that extend from the cell body, detected by Harmony 3.1.1 (PerkinElmer) image-analysis software, based on fluorescence from Hoechst dye and tau immunostaining. Neurite segments (red arrows) are calculated as parts of the neurites between neurite intersections (red dots). B, Quantification of percentage of neurons MC1-positive for pathological tau 7 d after the treatment with increased concentrations of tau monomers or oligomers. Oligomer treatment resulted in concentration-dependent increase in MC1-positive cells, compared with monomer treatment that showed no effect, only the background of the MC1 staining (<0.5% MC1-positive cells; n = 6 wells per condition, 9 fields, for oligomers ANOVA with post hoc Dunnett's test, ***p < 0.0001 vs vehicle, Student's t test, ^###p < 0.0001 vs vehicle, for monomers, p = 0.93). C, Graphs represent high-content image analyses of neurite outgrowth 7 d after seeding with different concentrations of tau oligomers. The graph on the left shows tau oligomers' dose-dependent decrease of total neurite length per cell, while the graph on the right shows number of neurite segments per cell. Neurites were detected using total tau staining. D, Graph shows the effect of increasing concentration of tau oligomers on the number of healthy nuclei (detected with Hoechst dye) 7 d after seeding (C and D: n = 6 wells per condition, 9 fields, ANOVA with post hoc Dunnett's test vs vehicle, ***p < 0.0001). E, High-content image analyses of the intensity of live-cell dye 7 d after seeding with 50 nm tau oligomers. Average fluorescence intensity of cells per well was expressed in graph as a percentage of vehicle-treated cells (n = 6 wells per condition, 9 fields, Student's t test, ***p < 0.0001 vs vehicle). F, LDH activity detected in the medium of cells treated with tau oligomers (50 nm) and vehicle-treated cells (n = 3 per condition, Student's t test, **p < 0.005). G, Graphs present high-content image analyses of neurite outgrowth 7 d after seeding with different concentrations of tau monomers. H, Graph shows the number of healthy nuclei (detected with Hoechst dye) 7 d after treatment with increasing concentration of tau monomers (G and H: n = 6 wells per condition, 9 fields, ANOVA with post hoc Dunnett's test vs vehicle, no significant change). All values are expressed as mean ± SEM.

Figure 6.

Time-lapse imaging revealed progressive neurite degeneration in neurons treated with tau oligomers. A, Representative image illustrates the definition marks of neurites (pink) and cell bodies (yellow) defined with NeuroTrack algorithm based on phase contrast. B, Graphs show the total neurite length (left) and number of branching points (right) per square millimeter, averaged from four imaged fields. Results were obtained from time-lapse live-cell imaging of the neurons treated with 50 nm tau seeds. DPS, Days postseeding. C, Graph presents the long-term quantification of the number of cell bodies in neuronal cultures treated with tau seeds. Arrow indicates plate removal from the imaging system for the treatment and washout of tau seeds (all graphs: n = 6 wells per condition, 4 fields, ANOVA with post hoc Dunnett's test vs vehicle, ***p < 0.0001). All values are expressed as mean ± SEM. D, Bright-field images acquired with IncuCyte ZOOM demonstrating the reduced neurite network of the hiPSC neurons 7 d after seeding with tau oligomers. Scale bar, 50 μm.

Figure 7.

Accumulation of pathological tau causes neuronal degeneration. A, High-content image analyses of neurite outgrowth expressed in graphs as total neurite length (in micrometers) per cell (left) and number of segments per cell (right), during a time course of 3 weeks after seeding with 50 nm tau oligomers, monomers, and vehicle-treated hiPSC neurons. Results are expressed as a percentage change from the vehicle within each time point. Representative images (right) demonstrate the neurite network reduction of hiPSC neurons treated with tau oligomers compared with vehicle-treated cells 14 d after seeding. Neurites were detected with total tau immunostaining (red). Hoechst was used to stain nuclei (blue). B, C, Graphs represent the reduction in neurite length and reduction in number of segments of neurons treated with tau oligomers 14 d after seeding, when neurites were detected with TUJ1 (B) and MAP2 immunostaining (C; n = 6 wells per condition, 9 fields, Student's t test, *p < 0.05, **p < 0.005, ***p < 0.0001 vs vehicle). Representative images of neurite network detected with TUJ1 (B, red) and MAP2 (C, red) immunostaining. Nuclei were detected with Hoechst stain (blue). D, E, Graphs present high-content image analyses of total neurite length of MC1-positive and non-MC1-positive cells 14 d after treatment with tau oligomers and vehicle, when neurites were detected with tau (D) and MAP2 (E) immunostaining. Representative images of MC1/tau (D, right) and MC1/MAP2 (E, right) double staining that demonstrate the reduction of neurite network (red) in neuronal population positive for MC1 staining (green) 14 d after tau oligomer seeding. Higher-magnification images (60×) show the localization of MC1 pathological tau on neuronal processes and somas, and also on the degenerating neurites (arrows). Nuclei were detected with Hoechst stain (blue). Scale bar, 50 μm. F, Graph shows the percentage of the number of healthy nuclei stained with Hoechst in neuronal cultures treated with tau seeds compared with vehicle at indicated time points. Representative images of MC1 (red) and Hoechst nuclear stain (blue) 14 d after treatment with tau oligomers showing a reduction in the number of Hoechst-positive nuclei in tau oligomer-treated neurons compared with vehicle. Scale bar, 100 μm (A, D–F: n = 6 wells per condition, 9 fields, ANOVA with post hoc Dunnett's test vs vehicle, *p < 0.05, **p < 0.005, ***p < 0.0001). G, Number of MC1-positive cells compared with number of non-MC1 cells during a time course of 3 weeks after seeding with 50 nm tau oligomers (***p < 0.0001 vs 7 d MC1-positive cells). All values are expressed as mean ± SEM.

Figure 8.

Pathogenic tau oligomers cause loss of synapses and disrupt intracellular calcium levels and neurotransmitter release. A, Graphs represent the quantification of number of puncta positive for the synaptic markers synapsin I and synaptophysin in hiPSC neurons 14 d after treatment with tau oligomers (n = 6 wells per condition, 9 fields, Student's t test, *p < 0.05). Representative images (to the right of graphs) of synapsin and synaptophysin staining (green) and Hoechst nuclear stain (blue). B, Graphs show the levels of released neurotransmitter GABA and glutamate 14 d after tau oligomer seeding (n = 6 wells per condition, Student's t test, *p < 0.05). C, High-content image analyses of the Ca²⁺-dye intensity per cell 14 d after tau oligomer seeding (n = 5 wells per condition, 9 fields, Student's t test, ***p < 0.0001). Images show an increased intracellular fluorescence intensity of Ca²⁺ dye (green) in cells treated with tau oligomers compared with vehicle-treated cells. Treatment with 30 μm NMDA was used to validate the increase of intracellular Ca²⁺ dye after neuronal activation. Nuclei were stained with Hoechst (blue). Scale bar, 50 μm.