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Fate and propagation of endogenously formed Tau aggregates in neuronal cells

2020; Springer Nature; Volume: 12; Issue: 12 Linguagem: Inglês

10.15252/emmm.202012025

1757-4684

Patricia Chastagner, Frida Loría, Jessica Vargas, Josh Tois, Marc I. Diamond, George Okafo, Christel Brou, Chiara Zurzolo,

Neuroscience and Neuropharmacology Research

Article12 November 2020Open Access Transparent process Fate and propagation of endogenously formed Tau aggregates in neuronal cells Patricia Chastagner Patricia Chastagner Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Frida Loria Frida Loria Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Jessica Y Vargas Jessica Y Vargas Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, France Search for more papers by this author Josh Tois Josh Tois Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, France Search for more papers by this author Marc I Diamond Marc I Diamond Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author George Okafo George Okafo GlaxoSmithKline, Stevenage, UK Search for more papers by this author Christel Brou Christel Brou Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Chiara Zurzolo Corresponding Author Chiara Zurzolo [email protected] orcid.org/0000-0001-6048-6602 Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Patricia Chastagner Patricia Chastagner Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Frida Loria Frida Loria Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Jessica Y Vargas Jessica Y Vargas Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, France Search for more papers by this author Josh Tois Josh Tois Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, France Search for more papers by this author Marc I Diamond Marc I Diamond Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author George Okafo George Okafo GlaxoSmithKline, Stevenage, UK Search for more papers by this author Christel Brou Christel Brou Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Chiara Zurzolo Corresponding Author Chiara Zurzolo [email protected] orcid.org/0000-0001-6048-6602 Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, FranceThese authors contributed equally to this work Search for more papers by this author Author Information Patricia Chastagner1, Frida Loria1,4, Jessica Y Vargas1, Josh Tois1, Marc I Diamond2, George Okafo3, Christel Brou1 and Chiara Zurzolo *,1 1Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, Paris, France 2Center for Alzheimer's and Neurodegenerative Diseases, Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA 3GlaxoSmithKline, Stevenage, UK 4Present address: Laboratorio de Apoyo a la Investigación, Hospital Universitario Fundación Alcorcón, Madrid, Spain *Corresponding author. Tel: +33 0145688277; E-mail: [email protected] EMBO Mol Med (2020)12:e12025https://doi.org/10.15252/emmm.202012025 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. SYNOPSIS Using a neuronal cell reporter system, this study shows that exogenous and endogenous Tau fibrils seed Tau misfolding. Endogenously formed Tau aggregates block their own degradation through the autophagic pathway and are transferred through tunneling nanotubes (TNTs) to neighboring cells. A neuronal biosensor allows measuring the rates and kinetics of seeding of endogenous Tau aggregates in living cells. Synthetic and Alzheimer's disease (AD) patient-derived fibrils seed endogenous aggregates following the same kinetics in the biosensor cell line. Endogenously formed aggregates escape autophagy and proteasome degradation. Tau aggregates are propagated mainly in a cell-contact-dependent manner, and are found inside TNTs. All these events may play key roles in the pathobiology of AD and other tauopathies. The paper explained Problem In Alzheimer disease (AD) as in other tauopathies, Tau neurofibrillary tangles are able to seed abnormal conformations on normal proteins, initiating a self-amplifying cascade, and can spread from their initial production site to other areas in the brain, following well-defined pathways. However, the underlying events that explain these features are still a matter of debate, as the fate and behavior of endogenously formed aggregates have not been assessed. Results We established a neuronal reporter cell system, allowing to record in real time the conversion of endogenously expressed Tau RD domain from soluble state to aggregates upon addition of external fibrils, either synthetic or from AD patient brain extract. We show that seeding is not rate-limiting and follows the same kinetics with both sources of fibrils. Further, the endogenously formed aggregates are recognized as autophagic cargoes, but fail to be transferred to lysosome for degradation as the autophagy flux is partly blocked. Both exogenous and endogenous aggregates, including those composed of full-length Tau, are transmitted between cells in a contact-dependent manner and found inside TNTs in neuronal cell lines. Tau fibrils are also transferred through direct cell contact to primary neurons and 3D organotypic cultures, where recipient cells were identified to be neurons and/or astrocytes. Impact This work gives an original and comprehensive picture of the pathobiology of AD and other tauopathies by analyzing the intracellular events that lead to the formation and spreading of Tau aggregates. It provides the groundwork for future intervention therapies specifically designed to improve clearing of Tau fibrils and to block their propagation throughout the brain. Introduction The progressive accumulation of aggregated misfolded proteins is a common phenotype observed in several neurodegenerative disorders. In Alzheimer's disease (AD), the hallmark proteins are extracellular amyloid-beta deposits (senile plaques) and intracellular inclusions (neurofibrillary tangles), which consist of microtubule-associated protein Tau, hereafter named Tau protein. These ordered assemblies have properties similar to amyloid fibrils and may propagate throughout the brain in a prion-like manner. Like prions, Tau fibrils act as templates for conversion of native protein to a fibrillar form, initiating a self-amplifying cascade, and can spread from their initial production site to other areas in the brain, following well-defined pathways (Jucker & Walker, 2013). In AD brains, phosphorylated Tau accumulates first at the noradrenergic locus coeruleus (Braak et al, 2011; Grinberg & Heinsen, 2017), with seeding first detected at entorhinal/limbic areas (Kaufman et al, 2018), before it spreads in a stereotypical manner to interconnected neocortical regions (Jucker & Walker, 2011). Tau accumulation is detected in the brain at least one decade before the appearance of the clinical symptoms of AD, by which time the proteins have spread progressively throughout patients' brains (Holtzman et al, 2011). The degree of tauopathy in the brain correlates with the cognitive decline in AD (Braak & Braak, 1991), suggesting that spreading of Tau deposits could be associated with disease progression. More recently, it has been shown that the prion-like activity of insoluble Tau, rather than its bulk accumulation, was inversely correlated with longevity of patients, highlighting the importance of understanding how Tau aggregates are generated and spread (Aoyagi et al, 2019). Moreover, dominantly inherited mutations in the microtubule-associated protein Tau (MAPT) gene, which encodes Tau protein, cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T). Dysfunction of Tau is also involved in multiple disorders linked to neurodegeneration and dementia, collectively termed tauopathies (Goedert & Spillantini, 2017). Tunneling nanotubes (TNTs) represent a newly discovered mechanism of cell-to-cell spreading of different cargoes, including abnormal proteins. TNTs were first observed in 2004 (Rustom et al, 2004) and are comprised of F-actin-containing channels that connect cells over large distances. They represent a novel mechanism for long-range intercellular communication operating in different cell types and in many diseases (Abounit & Zurzolo, 2012; Baker, 2017). Unlike other filamentous bridges (e.g., filopodia, cytonemes), TNTs directly connect the cytoplasm of distant cells (Sartori-Rupp et al, 2019) and selectively transfer a wide variety of cellular materials, e.g., cytoplasmic molecules, miRNA, vesicles, and organelles. In addition, TNTs can be hijacked by various pathogens, such as bacteria, viruses, and protein aggregates (Gousset & Zurzolo, 2009; Abounit et al, 2016a; Victoria & Zurzolo, 2017; Ariazi et al, 2017). Previous research has suggested that exogenously added Tau fibrils in neuronal cocultures can propagate through TNTs (Tardivel et al, 2016; Abounit et al, 2016b). However, data supporting the dynamics of spreading and subsequent seeding are still missing, particularly as the protein aggregates used in those studies were not formed endogenously. Here, using neuronal cell lines, as well as primary neurons and organotypic cultures, we analyzed the subcellular compartment where fibrils could be processed and eventually propagated. Then, we created a neuronal biosensor cell model (Holmes et al, 2014) expressing the microtubule-binding domain of Tau fused to a fluorescent marker (RD-YFP) to extract quantitative parameters regarding time and efficiency of new aggregates seeding, induced by either synthetic or natural AD-derived Tau fibrils. We demonstrate that both synthetic and natural fibrils can seed the formation of endogenous Tau aggregates. The latter escape degradation by both autophagy and proteasome. Finally, we show that endogenously formed Tau aggregates, composed of RD-YFP or of full-length Tau, and appearing when cells were challenged by synthetic fibrils, use a cell contact-dependent manner, possibly TNTs, to propagate in different neuronal culture models. Results Tau fibril transfer between neuronal cells via cell-to-cell contact To study how Tau fibrils propagate between cells, we generated fluorescently labeled Tau fibrils from monomeric K18 proteins (containing the aa 244–372 of human Tau protein, also called RD domain), shown to have similar structural and physicochemical properties regarding fibril formation compared with full-length Tau (Ait-Bouziad et al, 2017). Purified K18 monomers were assembled into insoluble aggregates in the presence of heparin, purified by ultracentrifugation, and labeled with ATTO 594. The quality of the assembled fibrils was checked by SDS–PAGE and Coomassie staining, as well as by thioflavin T assay as shown before (Fig EV1A) (Li & Lee, 2006). Click here to expand this figure. Figure EV1. Spreading of K18-ATTO 594 fibrils and of DID Quality control of K18 fibrils. After incubation in the presence (+) or absence (−) of heparin, fibrils were purified by ultracentrifugation. Supernatant (SN) and pellet were controlled by SDS–PAGE (18%) followed by Coomassie staining (upper panel), and by thioflavin T assay (graph below) where fluorescence intensity was monitored (Ex 450, Em 510 nm, integration time 200 ms). On the right side of the gel are the apparent molecular weights (kDa), and the white lane indicates that intervening lanes from the same gel have been spliced out. This experiment is representative of three independent preparations of fibrils. Uptake of K18 fibrils by cells. Three representative confocal pictures (one Z-stack in the 2D picture, orthogonal views covering 17 μm in 17 stacks) of CAD cells, first challenged with K18-ATTO 594 fibrils, trypsinized 24 h later, and replated for an additional 24 h, in the conditions used for all coculture experiments. White is WGA staining, and red is the fibrils; scale bars are 10 μm. Transfer of DiD in CAD cells. Left, quantification by flow cytometry of the relative percentage of DiD-loaded acceptor cells upon treatment with CK666 during the coculture. Data represent the means (+ SD), normalized to non-treated coculture arbitrarily set at 100%, of three independent experiments, with statistical analysis by two-tailed unpaired t-test (mean + CK666=120%, ***P = 0.0008). Right is the same analysis when the cell were cultured in sparse conditions, not favoring direct cell contacts (mean = 45%, three independent experiments, *P = 0.011). Spreading of K18-ATTO 594 fibrils in SH-SY5Y cells. Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after coculture of donor and acceptor cells (total), or culture of acceptor cells with donor-conditioned medium for 24 h (secretion). The total transfer was arbitrarily set at 100%, and cell-to-cell contact transfer was calculated by subtracting secretion transfer from total transfer. Data represent the means (+ SD) of four experiments, with statistical analysis by two-tailed unpaired t-test (****P = 7.17E−10). Download figure Download PowerPoint Mouse neuron-like CAD cells challenged with the fibrils by cationic lipid-mediated transfection (Lipofectamine) exhibited red puncta inside the cells. These puncta were resistant to trypsin treatment, showing that the fibrils were internalized by approximately 50% of the cells and were not stuck on the cell surface (Fig 1A (top left) and Fig EV1B). Since exogenously added fibrils from full-length Tau have been previously shown to transfer between cells in various cell lines including CAD cells, and were observed inside TNTs (Abounit et al, 2016b), we assessed whether our K18-ATTO 594 fibrils could be transferred between CAD cells in a classical coculture assay. Here, CAD donor cells, first challenged overnight with K18-ATTO 594 fibrils, were trypsinized and replated in coculture with a population of acceptor cells expressing H2B-GFP (Fig 1A top right and bottom left panels). This protocol assured that there were no residual fibrils stuck on the cell surface of donor cells and no residual Lipofectamine 2000 (Fig EV1B). After 18 h of coculture, we performed immunofluorescence (Fig 1A) and flow cytometric analysis (Fig 1B) to quantify the H2B-GFP-expressing acceptor cells positive for K18-ATTO 594 (Abounit et al, 2015). Acceptor cells (cultured in parallel) were also challenged for 18 h with conditioned media from donor cells (named donor SN, for supernatant in Fig 1A bottom right panel), to directly monitor the amount of K18-ATTO 594 fibrils transferred via a secretory mechanism only (i.e., not dependent on cell-to-cell contact; named secretion in Fig 1B). Figure 1A is a representative immunofluorescence, where after coculture (which allowed cell-to-cell contact), K18-ATTO 594-positive spots were visualized in acceptor cells (indicated by arrows in the bottom left panel). In contrast, very few red puncta were detected when acceptor cells were challenged with the supernatant from donor cells (bottom right panel). Quantitation of the data was performed by flow cytometric analysis, which showed that cell-to-cell contact-dependent transfer accounted for 94% of the total transfer, while the percentage of transfer through supernatant was negligible (Fig 1B). To assess whether this cell contact-dependent transfer could be correlated with a TNT-dependent mechanism, we analyzed whether compounds or culture conditions that modulate TNT formation affected Tau fibril transfer. When the cells in coculture were treated with CK666 (an Arp2/3 inhibitor known to decrease filopodia formation and to increase TNT formation (Delage et al, 2016; Swaney & Li, 2016; Keller et al, 2017; Sartori-Rupp et al, 2019)), we observed a 30% increase in K18 transfer (Fig 1C), whereas the small amount of transfer mediated by the supernatant (termed secretion) was not affected by this treatment (4.8 and 4.3% of total transfer in the absence or presence of CK666, respectively, indicated by the hatched area of the bars in Fig 1C). Inversely, when plating cocultured cells in sparse conditions, where inter-cell distance inhibits TNT growth (Abounit et al, 2016a; Zhu et al, 2018), the percentage of acceptor cells containing K18 fibrils dropped significantly (Fig 1C, right graph). As a control, we monitored in parallel cocultures the transfer of DiD-labeled vesicles from donor cells to H2B-GFP-expressing acceptor cells, which we have previously shown to be transferred predominantly through TNTs (Gousset et al, 2013; Abounit et al, 2015, 2016a; Delage et al, 2016). We observed similar variations in the number of acceptor cells positive for DiD or for Tau depending on the coculture conditions (CK666 treatment or sparse cells, compare Fig 1C to Fig EV1C). Overall, the absolute percentage of acceptor cells containing K18 fibrils was around 10%. Considering the internalization efficiency of K18-ATTO 594 fibrils in donor cells (which was in a range of 30–60% of cells, depending on the experiment), the transfer efficiency of the fibrils was comparable to the transfer efficiency of DiD-labeled vesicles (where labeling occurred in almost 100% of the donor cells, and around 25% of acceptor cells were positive for DiD after coculture). Together, these results suggested that both DiD-labeled vesicles and K18-ATTO 594 fibrils could be transferred between cells following similar paths considering their efficiency and their response to actin regulators. To verify that these results were not specific of the CAD model system, we repeated the experiments in human neuroblastoma SH-SY5Y cells, obtaining similar results as those with CAD cells (Fig EV1D). Overall, these results confirmed that K18-ATTO 594 fibrils spread intercellularly in vitro via contact-dependent mechanisms, which were affected by conditions perturbing/increasing TNT formation. To confirm that the transfer could occur through TNTs, we analyzed whether K18-ATTO 594 fibrils were found within TNTs, identified as WGA-positive protrusions, non-adherent to the plate, and connecting distant cells (Fig 1D, compare z10 to z3, attached to the substrate). We observed that K18 fibrils could be found inside TNTs in CAD cells (arrows in Fig 1D, and orthogonal views showing that red puncta are surrounded by membrane labeled with WGA), indicating that this could be a predominant way of intercellular spreading. Figure 1. Spreading of K18-ATTO 594 fibrils in CAD cells Transfer of K18-ATTO 594 fibrils from donor cells to H2B-GFP-expressing acceptor cells. Representative confocal images of each population are in the upper panels, and below are pictures after 24 h of coculture of the two populations (left) and of acceptor cells treated with conditioned medium from donor cells for 24 h (SN, right). In white is the cell membrane labeling with WGA (wheat germ agglutinin) coupled to Alexa 647. The arrows point to acceptor cells containing fibrils, scale bars are 10 μm. Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after coculturing donor and acceptor cells (total), or culturing acceptor cells with donor-conditioned medium for 24 h (secretion). The total transfer is arbitrarily set at 100%, and cell-to-cell contact transfer is calculated by subtracting secretion transfer from total transfer. Data represent the means (+ SD) of four independent experiments, with statistical analysis by two-tailed unpaired t-test (****P = 4.64E-08). Quantification by flow cytometry of the relative percentage of K18-ATTO 594-positive acceptor cells upon treatment with CK666 during the coculture. Left, data represent the means (132%) + SD, normalized to non-treated coculture arbitrarily set at 100%, of three independent experiments, with statistical analysis by two-tailed unpaired t-test (*P = 0.015). The hatched area of each bar represents the part due to secretion (respectively, 4.8 and 4.3% in the absence and presence of CK666). Right is the same analysis when the cells were cultured in sparse conditions, not allowing cell-to-cell contacts (mean = 47.7%, three independent experiments, *P = 0.020). Representative confocal images (40×) of CAD cells treated with 1 µM K18-ATTO 594 fibrils, 24 h after fibril addition, and fixed and stained with WGA-Alexa 488 (green) and DAPI (blue in the merge panels). Bottom panels are a bottom slice corresponding to the substrate-attached surface of cells (z3), upper panels correspond to slice 10 of the same picture (z10), not attached to the substrate. On the right and below z10 pictures are the orthogonal views (xz and yz) of the same region covering 27 slices over 11 μm in total. The arrows point to red fibrils into a WGA-positive TNT. Scale bars are 10 μm. Download figure Download PowerPoint Monitoring Tau fibril seeding and propagation over cell cultures We next asked whether K18 Tau fibrils were able to seed new aggregates in neuronal cells after intercellular spreading. We first confirmed that K18 fibrils were able to efficiently seed aggregates in CAD cells transiently expressing full-length Tau 1N4R P301S fused to YFP (named FLTau hereafter). The appearance of FLTau aggregates was dependent on the presence of K18 fibrils (Fig EV2A and B), which partially overlapped within the cell (Fig EV2C). Then, in order to determine whether the endogenously formed FL aggregates were able to spread to neighbor cells we performed a coculture experiment, where donor FLTau-expressing CAD cells first challenged with K18 fibrils were put in contact with acceptor cells expressing H2B-mCherry for 24 h before fixation and confocal imaging. Again, as control for secretion-mediated transfer we challenged acceptor cells with the supernatant of donor cells grown in separate dishes. As shown in Fig EV2D, no transfer was observed from supernatant, whereas green dots were observed in acceptor cells in coculture conditions, and were also detected inside TNTs (see insets of bottom panels). Quantitative analysis of the number of acceptor cells containing green dots (Fig EV2E) showed that FLTau transfer was strictly cell contact-dependent and on the same order of magnitude as DID transfer in these cells. Click here to expand this figure. Figure EV2. Entry of K18 fibrils, seeding, and transfer of full-length Tau in CAD cells 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 μm. 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, 3.65E−10 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. Representative confocal pictures (63× with 1.6 zoom, one Z-stack in the 2D picture, orthogonal views covering 5.6 μm 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 μm. 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 μm 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 μm. Quantification of the percentage of FLTau-positive acceptor cells after coculturing donor and acceptor cells (cells), or culturing acceptor cells with donor-conditioned medium for 24 h (SN). In the scatter dot plot, each symbol is a tile of four fields of acquisition, and bars are means (0.72 and 24.3 respectively) ± SEM. The total number of acceptor cells counted over two independent experiments was 229 for SN and 286 for cell coculture. Statistical analysis was unpaired t-test, P = 0.0107 (*). Download figure Download PowerPoint These data indicated that FLTau can be seeded by exogenously added fibrils and form endogenous FL aggregates that in turn spread mainly through a cell-to-cell contact-mediated mechanism. This is important to understand the propagation of endogenously formed fibrils; however, this system is not suitable to further investigate the underlying mechanisms. Being fluorescently labeled and recapitulating Tau aggregation, YFP-tagged Tau RD (P301L) has been previously shown to be good surrogate to investigate Tau pathology (Sanders et al, 2014). Thus, to follow and further investigate the seeding and transfer processes in living cells, we established a new biosensor cell line based on SH-SY5Y cells stably expressing YFP-tagged Tau RD (P301L) via lentiviral transduction (this cell line is named RD-YFP SH hereafter). We compared RD-YFP SH cells to previously established similar biosensor in non-neuronal HEK 293 cells stably expressing YFP-tagged Tau RD (LM: P301L/V337M) either in the soluble form (DS1 cells), or in the form of inherited inclusions (DS9 cells) (Sanders et al, 2014). In accordance with previous results (Sanders et al, 2014), DS1 cells were converted to inclusion-expressing cells after 2 days of exposure to the K18 fibrils, demonstrating seeded aggregation of Tau RD-YFP biosensors in a dose-dependent manner (Fig 2A). Next, we challenged the RD-YFP SH neuronal biosensor cell with the ATTO 594-labeled K18 fibrils. Also in this case, we obtained a significant proportion of RD-YFP SH cells containing green aggregates, sometimes overlapping the red fibrils (Fig 2B, and see below), confirming the ability of the K18 fibrils to seed de novo RD-YFP aggregates. Overall, we observed around 25% of inclusion-expressing RD-YFP SH cells 2 days after exposure to 1 μM K18 fibrils (Fig 2B). To monitor seeding and spreading in a time-dependent manner in a quantitative assay, we took advantage of the IncuCyte-automated incubator microscope system, which allowed recording the conversion of the sensor cells upon K18 treatment. The cells were automatically imaged inside the incubator every 30 min over 3 days, and real-time quantitative live-cell and fluorescence analysis was performed. By this assay, we could quan