Fate and propagation of endogenously formed Tau aggregates in neuronal cells

Abstract Tau accumulation in the form of neurofibrillary tangles in the brain is a hallmark of tauopathies such as Alzheimer's disease (AD). Tau aggregates accumulate in brain regions in a defined spatiotemporal pattern and may induce the aggregation of native Tau in a prion‐like manner. However, the underlying mechanisms of cell‐to‐cell spreading of Tau pathology are unknown and could involve encapsulation within exosomes, trans‐synaptic passage, and tunneling nanotubes (TNTs). We have established a neuronal cell model to monitor both internalization of externally added fibrils, synthetic (K18) or Tau from AD brain extracts, and real‐time conversion of microtubule‐binding domain of Tau fused to a fluorescent marker into aggregates. We found that these endogenously formed deposits colabel with ubiquitin and p62 but are not recruited to macroautophagosomes, eventually escaping clearance. Furthermore, endogenous K18‐seeded Tau aggregates spread to neighboring cells where they seed new deposits. Transfer of Tau aggregates depends on direct cell contact, and they are found inside TNTs connecting neuronal cells. We further demonstrate that contact‐dependent transfer occurs in primary neurons and between neurons and astrocytes in organotypic cultures.


EMBO Molecular Medicine
Patricia Chastagner et al.
A CAD cells were transfected with FL Tau 1N4R P301S-YFP encoding plasmid for 6 h, and then challenged or not with non-labeled K18 fibrils (sonicated, with or without Lipofectamine 2000 as indicated) and left o/n before trypsinization and replating for an additional 24 h. Cells were finally fixed, labeled with WGA, and analyzed by confocal microscopy (40× magnification). Pictures of cells containing aggregates are shown for conditions 2 and 3, representative of the results. The arrows point to cells containing fibrils; scale bars are 10 lm. B The plot shows the percentage with SEM of transfected cells where FL Tau appeared as inclusions (1, 9.8, and 55, respectively, for conditions 1, 2, and 3). Statistically significant differences are compared to the control conditions (1, Lipofectamine without fibrils) by one-way ANOVA and Tukey's post hoc test (****P = 1.07E-08 for 1 vs 3, 3.65EÀ10 for 2 vs 3). The efficiency of transfection was 47%, and the numbers of cells containing green aggregates counted were 222, 693, and 526, respectively, for conditions 1, 2, and 3, over three independent experiments. C Representative confocal pictures (63× with 1.6 zoom, one Z-stack in the 2D picture, orthogonal views covering 5.6 lm in 17 stacks) of CAD cells treated with K18-ATTO 594 fibrils as in (A). Upper left panel is a cell without red or green aggregates, and the three other panels are a cell where FL Tau-YFP is aggregated. Green is FL Tau-YFP, and red is the fibrils; scale bars are 10 lm. D Below the schematics of the experiment are representative confocal images (40× objective) of donor CAD cells (transfected with FL Tau 1N4R P301S-YFP expression vector), challenged or not with non-labeled K18 fibrils (respectively, second and first lane panels), acceptor cells with conditioned medium from K18-challenged donor cells (acceptor cells + SN, third lane panels), and coculture of donor (with K18) and acceptor cells in the bottom panels. The images are representative Z-stacks, except from the bottom panel which is a maximal projection covering five upper stacks (1.4 lm in total, allowing to visualize TNTs, not attached to the dish). In the merged images, white is WGA, green is YFP, red is mCherry, and nuclei are stained in blue. Arrows point to FLTau puncta inside acceptor cells and the arrowhead shows a green dot inside a TNT, which is indicated with a bracket. Insets are threefold enlargements of the boxed regions in the lower panels. Scale bars are 10 lm.

EMBO Molecular Medicine
Patricia Chastagner et al.
A RD-YFP SH cells were challenged with non-labeled K18 fibrils for 2 days before fixation, saponin permeabilization, and staining with antibodies recognizing, respectively, TOM 20, EEA1, Furin + Giantin, vimentin, alpha-tubulin, or WGA. Representative deconvoluted confocal images are presented, and blue staining is DAPI in the merged pictures; and scale bars are 10 lm. B Representative confocal images (of three independent experiments) of RD-YFP aggregates overlapping with p62 and ubiquitin, induced either by synthetic K18 fibrils or by AD-derived extracts as indicated on the left. RD-YFP SH cells were challenged with non-labeled K18 fibrils for 2 days or AD XT for 4 days before fixation, saponin permeabilization, and staining with antibodies recognizing all types of ubiquitin chains (Ub) or K63-linked ubiquitin chains (Ub [K63]) and p62 (white). Insets are threefold enlargements of the boxed regions, showing colocalizations of Ub, p62, and RD-YFP; scale bars are 10, 2 lm in insets. C Ubiquitination of RD-YFP aggregates. Frozen cell pellets of RD-YFP SH cells treated with K18 fibrils and grown for 4 days were thawed on ice and cells were lysed in PBS-Triton X-100 0.05%, and next total extracts (tot) were ultracentrifugated at 100,000 g to separate soluble material (S) from pellets (P) corresponding to insoluble material, including aggregates, shown by brackets in the WB of the left panel (4-12% gel in MES buffer, denaturation in 1% Laemmli without reducing agent). Tot and P fractions were loaded again, and the same membrane was blotted consecutively with antibodies against ubiquitin (right panel) and RD-YFP (GFP). Ubiquitinated material and aggregates are shown with brackets, and the arrow points to monomeric RD-YFP. MW (kDa) is indicated for each gel. D Quantification of the colocalization between RD-YFP and LC3 in various conditions. Cells were treated as in Fig 3A, except that antibody recognizing LC3 was used, and quantifications were performed as in 3B. The graph represents the mean percentage (+ SEM) of green material overlapping with LC3, and the number of cells analyzed was 14, 18, 14, and 24, respectively, for each condition over two experiments. PCC is indicated below the graph. Statistically significant differences were compared to the soluble conditions (one-way ANOVA and Tukey's post hoc test, [****P = 1.34EÀ07, 5.79EÀ10, 8.17EÀ07]). E Representative confocal images of RD-YFP SH cells, challenged with non-labeled K18 fibrils for 2 days. LysoTracker (red) was added to the culture 30 min before fixation, and scale bars are 10 lm. F Representative picture of RD-YDP SH cells, 14 days after challenging with non-labeled fibrils and processed for immunofluorescence as in Fig 3A. Insets are threefold enlargements of the boxed region; white arrows point to colabelings; scale bars are 10 lm. G Quantification of colocalization of RD-YFP material with p62-positive structures after 7 or 14 days of culture. Confocal pictures were analyzed in 3D with Imaris software, as in Fig 3B. Below the graph (mean percentage with SEM) is indicated the corresponding Pearson's correlation coefficient (PCC). The number of cells analyzed in each condition over three independent experiments was 39, 81, and 65, respectively. Statistically significant differences are compared to the soluble conditions (one-way ANOVA and Tukey's post hoc test [****P = 6.13EÀ07 and 7.85EÀ08 for NT and Baf, respectively]). Note that the differences between agg NT and BafA1 are not significant, for percentage of overlapping and for PCC. H Analysis of the number of green dots per cell among the population of cells containing RD-YFP aggregates after treatment with bafilomycin A1 or bortezomide. Cells were treated and imaged as described in Fig 3A, and analysis of 63× images was performed using spot detector wizard (scale 3, threshold 80) under Icy software, with a total number of analyzed cells over three independent experiments of 95, 87, and 39 for each condition, respectively. Each analyzed cell is represented on the dot plot, and the bars indicate the means AE SEM (respectively, 106, 120, and 126). Statistical analysis was performed by one-way ANOVA and Tukey's post hoc test, and all the pairwise comparisons were not significant (ns). Note that the difference in mean aggregate number compared to Fig 4F is

EMBO Molecular Medicine
Patricia Chastagner et al.
A Conditioned medium of cells treated with K18 fibrils is devoid of K18 fibrils. Twenty-four-hour conditioned media (1 ml) of cells (SH-SY5Y or RD-YFP SH) treated or not with K18 as indicated, providing from independent experiments, were ultracentrifuged at 100,000 g for 1 h at 4°C. Pellets containing insoluble material were solubilized in 1% SDS-containing Laemmli without reducing agent and analyzed by WB for the presence of K18 (detected with anti-V5 antibody). As positive control, the same volume of fibril-containing medium, collected at the end of the 6-h incubation on cells (i.e., containing fibrils that were not uptaken by cells), was processed the same way (lane 7). The K18 ladder corresponding to the fibrils is indicated by brackets. Right is overexposure of the lanes 1-6 of the membrane. B Quantification of the percentage of RD-YFP SH acceptor cells with insoluble RD-YFP, depending on the condition (12.5% for donor cells and 0.37% for donor SN) in the experiment described in Fig 4C. Analysis was performed using ICY software, and data represent the number of aggregate-containing cells over the total number of green cells without red nuclei + SEM (the total number of acceptor cells analyzed over two independent experiments was 1,788 for coculture and 1,028 for SN) with statistical analysis by two-tailed unpaired t-test (***P = 0.0006). C Visualization of the direct transfer of endogenously formed RD-YFP aggregates to SH-SY5Y cells. Above the pictures is a schematic representation of the experiment.
Donor RD-YFP SH cells expressing aggregates obtained 2 days after treatment with non-labeled K18 fibrils were cocultured for 24 h with SH-SY5Y cells first transfected with H2B-mCherry expression vector. The images are Z-stack projections covering nine slices (each 0.43 lm), and similar experiments were performed three times. Transferred RD-YFP aggregate is indicated by the white arrow, and single-channel pictures are grayscale images; and scale bar is 10 lm.  Figure EV4.

EMBO Molecular Medicine
Patricia Chastagner et al.
A Two-day coculture of SH-SY5Y cells expressing RD-YFP and nls-Red (acceptor cells) with DS9 cells (donor cells). Below the schematic representation of the experiment are representative confocal images showing maximum intensity projections of six z-slices (covering 2 lm of thickness). In the merged images, white is WGA labeling, green is RD-YFP, red is nls-Red nuclei, and nuclei are stained in blue with DAPI; scale bars are 10, 5 lm in the enlarged boxes. Below each image are twofold enlargements of the corresponding boxed areas. B Quantification of experiment described in (A), giving the percentage of converted nls-Red-expressing RD-YFP SH cells after coculture with DS9 cells or SN (donor cells or donor SN). Analysis was performed using ICY software, data represent the number of converted acceptor cells over the total number of nls-Red-expressing RD-YFP SH cells + SEM, and means are 3.04 (donor cells) and 0.03 (donor SN). The total number of RD-YFP SH cells analyzed over three independent experiments was 1,923 for coculture and 1,654 for SN, statistical analysis by two-tailed unpaired t-test (**P = 0.0075). C Representative confocal image showing a TNT connecting two DS9 cells (green arrow) or connecting one RD-YFP nls-Red SH cell and one DS9 cell (white arrow). To improve visualization of the TNTs, cells were incubated for 1 min with trypsin just before PFA fixation. Unstacked Z-stack images of donor CAD cells and acceptor primary cortical neurons after 24h in coculture allow appreciating that CAD cells (CTG) and neurons (MAP-2) are on different spatial planes. The images of the series cover a range from 0.33 to 4.02 lm (numbered 1-12), with 0.33-lm plane thickness. While red arrowheads point to Tau puncta in donor cells (D), and yellow arrowheads point to Tau puncta detected in the cell body and neurites of an acceptor neuron (A). Scale bars represent 10 lm.