Tunneling nanotubes spread fibrillar α‐synuclein by intercellular trafficking of lysosomes
2016; Springer Nature; Volume: 35; Issue: 19 Linguagem: Inglês
10.15252/embj.201593411
ISSN1460-2075
AutoresSaïda Abounit, Luc Bousset, Frida Loría, Seng Zhu, Fabrice de Chaumont, Laura Pieri, Jean‐Christophe Olivo‐Marín, Ronald Melki, Chiara Zurzolo,
Tópico(s)Alzheimer's disease research and treatments
ResumoArticle22 August 2016free access Transparent process Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes Saïda Abounit Saïda Abounit Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Luc Bousset Luc Bousset Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France Search for more papers by this author Frida Loria Frida Loria Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Seng Zhu Seng Zhu Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Fabrice de Chaumont Fabrice de Chaumont Laboratoire d'Analyse d'Images Quantitative, Institut Pasteur, Paris Cedex 15, France Search for more papers by this author Laura Pieri Laura Pieri Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France Search for more papers by this author Jean-Christophe Olivo-Marin Jean-Christophe Olivo-Marin Laboratoire d'Analyse d'Images Quantitative, Institut Pasteur, Paris Cedex 15, France Search for more papers by this author Ronald Melki Ronald Melki Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France Search for more papers by this author Chiara Zurzolo Corresponding Author Chiara Zurzolo [email protected] Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Saïda Abounit Saïda Abounit Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Luc Bousset Luc Bousset Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France Search for more papers by this author Frida Loria Frida Loria Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Seng Zhu Seng Zhu Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Fabrice de Chaumont Fabrice de Chaumont Laboratoire d'Analyse d'Images Quantitative, Institut Pasteur, Paris Cedex 15, France Search for more papers by this author Laura Pieri Laura Pieri Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France Search for more papers by this author Jean-Christophe Olivo-Marin Jean-Christophe Olivo-Marin Laboratoire d'Analyse d'Images Quantitative, Institut Pasteur, Paris Cedex 15, France Search for more papers by this author Ronald Melki Ronald Melki Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France Search for more papers by this author Chiara Zurzolo Corresponding Author Chiara Zurzolo [email protected] Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France Search for more papers by this author Author Information Saïda Abounit1, Luc Bousset2,‡, Frida Loria1,‡, Seng Zhu1,‡, Fabrice Chaumont3, Laura Pieri2, Jean-Christophe Olivo-Marin3, Ronald Melki2,‡ and Chiara Zurzolo *,1,‡ 1Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France 2Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France 3Laboratoire d'Analyse d'Images Quantitative, Institut Pasteur, Paris Cedex 15, France ‡These authors contributed equally to this work ‡These authors are co-senior authors *Corresponding author. Tel: +33 01 45 68 82 77; E-mail: [email protected] The EMBO Journal (2016)35:2120-2138https://doi.org/10.15252/embj.201593411 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 Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. Synopsis Misfolded α-synuclein fibrils propagate in cell culture by transferring between neurons through tunneling nanotubes (TNTs) inside lysosomes, indicating a possible role of TNTs and lysosomes in the spreading and propagation of Parkinson's pathology. α-synuclein fibrils are targeted to cell lysosomes for degradation. α-synuclein fibrils enhance formation of TNTs between neighbouring cells, possibly though increasing oxidative stress. Lysosomes overloaded with α-synuclein fibrils transfer from donor cells to neighbouring (acceptor) cells inside TNTs connecting the two populations. Once in acceptor cells, α-synuclein fibrils are able to seed the aggregation of endogenous soluble cytosolic α-synuclein, conceivably by escaping lysosomes. Introduction Alpha-synuclein (α-synuclein) is a 140 amino acid protein widely expressed in the brain, localized principally in the cytosol and at presynaptic terminals in association with vesicles. Its exact function is still unclear, although it appears to be involved in neurotransmitter release (Bendor et al, 2013; Burré et al, 2013). Misfolded α-synuclein aggregates into intraneuronal inclusions called Lewy bodies (LB) and intraglial inclusions, associated with neuronal and glial loss in specific regions of the brain. These inclusions represent the histopathological hallmark of synucleinopathies that include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are characterized by a chronic and progressive decline in motor, cognitive, behavioural and autonomic functions (McCann et al, 2014). Considerable efforts have been made to understand the progression of synucleinopathies. Braak and colleagues were the first to show that α-synuclein neuropathology progresses throughout the brain in a specific and predictable manner, suggesting that the progression of the disease is associated with the propagation of a neurotropic infectious entity (Braak et al, 2003a,b). Recent data indicate that this neurotropic agent might be aggregated α-synuclein. Pioneering studies supporting this hypothesis evidenced the presence of LB in grafted foetal mesencephalic dopaminergic neurons in the brain of PD subjects over 10 years after transplantation (Kordower et al, 2008; Li et al, 2008). Since then, intercellular α-synuclein transfer in vitro and in vivo has been reported (Desplats et al, 2009; Hansen et al, 2011; Kordower et al, 2011; Angot et al, 2012; Luk et al, 2012a,b; Mougenot et al, 2012; Masuda-Suzukake et al, 2013, 2014; Bae et al, 2014; Recasens et al, 2014). Furthermore, it has been shown that fibrillar α-synuclein seeds the aggregation of the soluble form of the protein in a prion-like manner (Bousset et al, 2013; Aulić et al, 2014; Volpicelli-Daley et al, 2014). Knowledge of the precise mechanism of α-synuclein propagation from one cell to another is therefore needed for better understanding the mechanism of disease progression. Although secretion was shown to be a possible mechanism of α-synuclein intercellular transfer (Desplats et al, 2009; Lee et al, 2010, 2013; Hansen et al, 2011; Freundt et al, 2012; Konno et al, 2012; Brahic et al, 2016), the predictable and specific patterns of spreading of α-synuclein inclusions in the brains of PD patients (Braak et al, 2003b) together with in vitro and in vivo studies (Freundt et al, 2012; Rey et al, 2013; Holmqvist et al, 2014; Peelaerts et al, 2015) suggest a mechanism of spread following neuronal circuits. Here, we developed a robust cell model to study in vitro the mechanism of cell-to-cell transfer of structurally and functionally characterized fluorescent human α-synuclein fibrils previously shown to recapitulate α-synuclein pathogenicity by being toxic and inducing seeding in neuronal cells and in animal models (Pieri et al, 2012; Bousset et al, 2013; Peelaerts et al, 2015). We specifically investigated the role of tunneling nanotubes, F-actin containing membranous bridges that connect the cytoplasm of remote cells (Abounit & Zurzolo, 2012), allowing direct transfer of various cargoes (Austefjord et al, 2014) and thereby implicated in several physiological processes (Marzo et al, 2012). TNTs have been found in tissues of various origins (Chinnery et al, 2008; Lou et al, 2012; Seyed-Razavi et al, 2013; Ady et al, 2014), and recently TNT-like connections have been observed in human glioblastoma tumours implanted in mouse brains (Osswald et al, 2015). Of interest, data showing TNT-mediated HIV transfer from infected to uninfected T cells (Sowinski et al, 2008) and transfer of prions and other pathogens (Onfelt et al, 2004; Gousset & Zurzolo, 2009; Gousset et al, 2009) support the role of TNTs as a general conduit used by pathogens for spreading. In the present study, we found that efficient transfer of α-synuclein fibrils between neuron-like cells and primary neurons in culture relied on tunneling nanotubes (TNTs). Following TNT-mediated transfer, α-synuclein fibrils were able to induce the aggregation of soluble, endogenous α-synuclein in acceptor cells. Most importantly, we show for the first time that fibrils normally directed to the lysosomal compartment for degradation both in neuron-like cells and in primary neurons can shuttle between cells in TNTs within lysosomal vesicles. Overall, our results support TNTs as efficient means for propagation of α-synuclein fibrils between neurons, and reveal a novel role played by lysosomes in this cell-to-cell transfer process. Results α-synuclein fibrils transfer efficiently between neuron-like cells To investigate whether α-synuclein fibrils could transfer between neurons, we first assessed whether recombinant human α-synuclein fibrils of known dimension and molecular mass (on average 4,000 monomers for one fibril, Pieri et al, 2012) (Fig EV1A) were capable of entering catecholaminergic mouse (CAD) neuron-like cells. In time course experiments, we quantified the percentage of cells containing the fluorescent α-synuclein fibrils using flow cytometry and found a time-dependent internalization of fibrils with maximum fluorescence reached after 6 h (Figs 1A and EV1B). Time course measurements of lactate dehydrogenase (LDH) release after exposure to α-synuclein fibrils (up to 1 μM) showed no change compared to control cells (Fig EV1C). This suggested that cells were viable, thus validating their use as donor cells in our transfer assay (see below and schematic in Fig EV2). Click here to expand this figure. Figure EV1. α-synuclein fibril characterization and internalization in neuron-like cells and cortical primary neurons Electron micrographs of α-synuclein fibrils used throughout this study. The scale bar represents 100 nm. Fibrillar α-synuclein was adsorbed to a carbon-coated copper grid and stained with freshly prepared 1% uranyl acetate. Samples were imaged using a JEOL 1400 electron microscope equipped with an LaB6 filament and operated at 80 kV, and 10,000× magnification, images were recorded with a Gatan Orius CCD camera (Gatan). The left panel shows representative Z-stack projection of confocal images showing neuron-like CAD cells after loading with α-synuclein fibrils for 16 h. Cells were then trypsin-washed, fixed and labelled with HCS CellMask Blue; scale bar is 10 μm. The right panel depicts the (z, y) three-dimensional reconstruction (3D) of a cell loaded with fibrils shown in the confocal image (left panel); scale bar is 5 μm. As seen on the three-dimensional images, α-synuclein fibrils are present only in the cytosol and not at the plasma membrane. Cell toxicity was measured by LDH release in neuron-like cells after 10, 24 and 48 h of loading increasing concentrations of sonicated α-synuclein fibrils. The bar graph represents the percentage of cytotoxicity normalized to control values. There were no significant differences between control and α-synuclein-loaded cells at any of the time points or concentrations evaluated. Ns, not significant by two-way Student's t-test. Representative images showing intracellular ROS levels in neuron-like cells upon addition of 1 μM of α-synuclein fibrils for up to 9 h. Intracellular ROS was measured by CellRox Green fluorescence in control and α-synuclein-loaded cells (red). Scale bars are 10 μm. The graph shows the percentage of the relative fluorescence intensity of intracellular ROS at different time points in α-synuclein-loaded CAD cells and controls. The values show ˜50% increase in ROS production at all time points. ***P < 0.001 compared to the control condition. Ns, not significant by two-way Student's t-test. Quantification of the percentage of primary cortical neurons containing α-synuclein puncta after 16 h of incubation with 0.5 μM (blue bar) and 1 μM (red bar) of α-synuclein fibrils. **P < 0.001 by two-way Student's t-test. Cell toxicity was measured by LDH release in primary neurons on addition of 1 μM of sonicated α-synuclein fibrils for 16, 24, 48 and 72 h. The bar graph represents the percentage of cytotoxicity normalized to control values. There were no significant differences between control (blue bar) and α-synuclein-loaded (red bar) cells at any of the time points evaluated. Ns, not significant by two-way Student's t-test. Data information: Data in (C and E–G) represent the mean ± s.e.m. of three independent experiments. Whereas at least 100 cells were scored for each experiment of internalization and ROS, values for toxicity experiments are derived from triplicates of three different experiments. Download figure Download PowerPoint Figure 1. α-synuclein fibrils transfer efficiently between neuron-like CAD cells Time course of rapid internalization of fluorescent α-synuclein fibrils by CAD cells (for characterization of α-synuclein assemblies, see Fig EV1A). Internalization was measured by recording ATTO-550-positive neuron-like cells by flow cytometry. Percentage of ATTO-550-positive cells was quantified (mean ± s.e.m.) (left panel) and representative histograms of ATTO-550-positive cells are shown on the right panel (a.u., arbitrary units). n = 3 independent experiments. Similarly, α-synuclein fibril internalization was also confirmed by fluorescent microscopy (see Fig EV1B). Representative images of donor (upper panel) and acceptor cells (lower panel) after 24-h co-culture. Donor cells were loaded with α-synuclein fibrils prior to co-culture with GFP-transfected acceptor cells: in red, α-synuclein fibrils; in green, acceptor cells; and in blue, nuclei. Scale bars represent 10 μm. n = 3 independent experiments. A larger field where donor and acceptor cells are shown is presented in Fig EV3A. Percentage of donor and acceptor cells containing α-synuclein fibrils after co-culture as in (B): all acceptor cells received α-synuclein fibrils. Quantification of the number of α-synuclein fibrils in donor and acceptor cells after co-culture as in (B). Donor cells contain around 70 α-synuclein fibril puncta (median), while acceptor cells contain 38 α-synuclein fibril puncta, respectively (****P < 0.0001 by two-tailed Mann–Whitney U-test). Quantification of the average size of α-synuclein fibrillar foci in donor and acceptor cells after co-culture as in (B) (****P < 0.0001 by two-tailed Mann–Whitney U-test). See also Fig EV3 for an example of α-synuclein fibrillar puncta detection in an acceptor cell. After detection, the number and the size of foci were determined using the ICY software. Data information: In the box and whisker plots in (D and E), boxes extend from the first to the third quartile, the line inside the boxes shows the median and the whiskers represent the min/max value of at least 100 cells scored for each independent experiment (n = 3). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Schematic of the experimental design of co-culture experiments Experimental set-up used for the co-culture experiment (also referred to as a transfer experiment). CAD neuron-like cells are loaded for 16 h with human fluorescent ATTO-550 α-synuclein fibrils. Cells are trypsin-washed and are used as “Donor cells” since their cytosol is loaded with α-synuclein fibrils. Donor cells are mixed with GFP-transfected cells referred to as “Acceptor cells” for 24 h. Then, the co-culture is fixed and imaged and (i) the percentage of cells containing ATTO-550 α-synuclein fibrils and (ii) the average number and size of ATTO-550 α-synuclein fibrils per cells are quantified using ICY software. Experimental set-up used for the conditioned medium experiment. This experiment allows investigating the contribution of secretion to cell-to-cell α-synuclein fibril transfer. Here, donor cells are obtained as described in (A) (i.e. loading followed by trypsin wash) and then cultured for 24 h. The medium of donor cells referred to as conditioned medium (CM) is entirely collected and used as is to culture GFP-transfected acceptor cells for 24 h. The same analysis described in (A) is performed (i.e. percentage of cells containing α-synuclein fibrils, number and size of α-synuclein fibrils per cells) but also quantitative analysis of the amount of fibrils within donor cells and the culture medium by filter trapping on cellulose acetate membranes. Experimental set-up used for the filter experiment. This set-up was designed to separate donor and acceptor cells to investigate the contribution of (i) secretion or/and (ii) cell contact to transfer. The co-culture is performed similarly as described in (A) with the exception that donor cells are plated in the well, and then a transwell filter is placed on top of which acceptor cells are plated. After 24-h co-culture, the same analysis is performed (see A). Experimental set-up used for the seeding experiment. Here, donor cells loaded with α-synuclein fibrils Alexa-488 (and trypsin-washed as described in A) were co-cultured with acceptor cells overexpressing ChFP-α-synuclein for 72 h. The number of ChFP-α-synuclein fibril puncta as well as the co-localization rate between α-synuclein fibrils Alexa-488 and ChFP α-synuclein fibril puncta was quantified. Schematic of the experimental design of exogenous α-synuclein fibril internalization in co-cultured cells. Donor cells previously loaded with ATTO-550 α-synuclein fibrils were co-cultured with untransfected acceptor cells for 24 h. After 12 h of co-culture, cells were challenged with α-synuclein fibrils Alexa-488 (i.e. exogenously added α-synuclein fibrils) for an additional 12 h. Schematic of the experimental design of α-synuclein fibril internalization and transfer between primary neurons. Donor neurons pre-loaded with ATTO-550 α-synuclein fibrils were co-cultured with CTG-labelled acceptor neurons for 72 h. Acceptor neurons were prepared from a different dissection and labelled in suspension before adding them on top of the donor neurons. After 72 h, the cells are fixed and imaged and (i) the percentage of cells containing α-synuclein puncta and (ii) the number and average size of α-synuclein puncta per cell are quantified using ICY software. Download figure Download PowerPoint Although transfer of α-synuclein fibrils between neuronal cells has been shown, quantification (i.e. percentage of cells containing fibrils, number and size of α-synuclein foci) and intracellular localization of the transferred aggregates have not been thoroughly characterized. We set up a new co-culture assay where “donor” CAD cells (loaded overnight with α-synuclein fibrils) were co-cultured for 24 h at the ratio of 1:1 with naïve “acceptor” CAD cells expressing GFP. In order to remove any membrane-bound extracellular α-synuclein fibrils, donor cells were trypsin-washed before co-culture (Fig EV2A). To automatically detect and quantify the number and size of fibrillar α-synuclein puncta in the donor and acceptor cell populations separately, we used confocal microscopy and developed a specific script of the ICY software (see Appendix and Fig EV3). By this method, we found that 100% of acceptor cells contained fibrillar α-synuclein (Fig 1B and C). Furthermore, to determine the efficiency of transfer, we quantified the number of fluorescent puncta in the donor and acceptor cell populations. While donor cells contained about 70 puncta per cell, acceptor cells contained 38 puncta (Fig 1D) indicating that 35% of the puncta transferred from donor to acceptor cells. Together with the fact that 100% acceptor cells contained fibrillar α-synuclein puncta, this shows that in our model cell-to-cell transfer of fibrillar α-synuclein occurs and is very efficient. Click here to expand this figure. Figure EV3. α-synuclein fibril detection in acceptor cells A. Representative confocal picture of donor cells loaded with α-synuclein fibrils (D, in red), co-cultured with GFP vector-transfected acceptor cells (A, in green) and stained with DAPI (in blue). Scale bar represents 10 μm. B, C. Representative confocal picture of an acceptor cell from (A) (top left cell) showing cell segmentation (large yellow ROI) and detection of α-synuclein fibrils in small green square ROIs (left panel). The green square ROIs change in size according to the size of the fibrils. Thereby the large fibrils are seen in green squares and the smaller fibrils appear in small green spots which when the magnification is increased resolve into squares as seen in the inset in (C). Note in (B) the correlation between α-synuclein fibrils (in white, right panel) and the spots detected by the software (in green, left panel). Download figure Download PowerPoint We next analysed the size of α-synuclein puncta in donor and acceptor cells and found that the transferred puncta in acceptor cells correspond to smaller puncta (the median value is 0.15 μm3) than the ones within the donor cells (the median value is 0.42 μm3) (Fig 1E). This finding suggests size limitation of fibrillar α-synuclein puncta upon cell-to-cell transfer. α-synuclein fibrils induce the aggregation of soluble α-synuclein after transfer in acceptor cells We have previously demonstrated that α-synuclein fibrils identical to those used in this study efficiently seed the aggregation of soluble reporter α-synuclein (ChFP-α-synuclein) when exogenously added to neuron-like cells in culture or in rodent (Bousset et al, 2013; Peelaerts et al, 2015). However, a key question is whether these fibrils would seed cytoplasmic reporter α-synuclein, following their transfer from donor to acceptor cells. To address this question, we performed our co-culture experiment for 72 h using as acceptor CAD cells stably overexpressing soluble ChFP-α-synuclein (see schematic Fig EV2D). As expected for soluble α-synuclein, in control conditions, ChFP-α-synuclein cells cultured alone displayed a nucleocytoplasmic diffuse α-synuclein signal with a few ChFP-α-synuclein in discrete puncta, revealing low aggregation state of ChFP-α-synuclein (Fig 2A and B). In contrast, quantitative analysis revealed that after co-culture with donor cells loaded with α-synuclein fibrils, acceptor cells contained a significantly higher number of endogenous ChFP-α-synuclein in discrete puncta (4 and 15 α-synuclein-ChFP puncta per acceptor cell in control and co-culture conditions, respectively) (Fig 2A and B). In addition, 11% of the transferred Alexa-488 α-synuclein co-localized with ChFP-α-synuclein in discrete puncta (Fig 2A and C). These results indicate that α-synuclein fibrils are able to recruit soluble ChFP-α-synuclein, suggesting that they retain their seeding activity after being transferred from donor to acceptor cells. Figure 2. α-synuclein fibrils induce the aggregation of the soluble α-synuclein protein after transfer in acceptor cells Representative confocal images showing ChFP-α-syn-transfected cells (in red) cultured alone (upper) or co-cultured with donor cells containing fluorescent Alexa-488 α-synuclein fibrils (in green) for 72 h (bottom). The arrow is pointing out representative α-synuclein fibrils co-localized with ChFP-α-syn puncta. Scale bars represent 10 μm. Nuclei are stained with DAPI (blue). n = 3 independent experiments. Quantification of the number of ChFP-α-synuclein puncta in ChFP-α-syn acceptor cells showed a significant increase in ChFP-α-synuclein puncta number when cells were co-cultured with donor cells containing α-synuclein fibrils (***P < 0.001 by two-tailed Mann–Whitney U-test). In the box and whisker plot, boxes extend from the first to the third quartile, the line inside the boxes shows the median and the whiskers represent the min/max value of at least 100 cells scored for each independent experiment (n = 3). Quantification of the percentage of co-localization of α-synuclein fibrils Alexa-488 and ChFP-α-syn puncta. Data are shown as mean ± s.e.m. After co-culture, 11% of the transferred α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta. n = 3 independent experiments. Download figure Download PowerPoint α-synuclein fibril transfer is favoured by cell-to-cell contact To further characterize the mechanism of transfer, we first determined whether transferred α-synuclein fibrils propagating from cell to cell co-localize with freshly taken up fibrils. Overnight co-cultures of donor (loaded with ATTO-550 α-synuclein fibrils) and acceptor cells were exposed for additional 12 h to α-synuclein fibrils labelled with Alexa-488. As expected, exogenously added Alexa-488 α-synuclein fibrils were taken up by both donor and acceptor cells with similar efficiency (Fig 3A). Alexa-488 fibrils co-localized with the ATTO-550 fibrils in donor cells indicating that fibrils taken up initially (ATTO-550) and freshly taken up fibrils (Alexa-488) are directed to the same sub-cellular compartment (Fig 3B). Intriguingly, however, while in donor cells we observed high co-localization percentage between ATTO-550- and Alexa-488-labelled fibrils (and reverse) (66 ± 0.66%), indicating that the two fibril batches were taken up following the same pathway (Fig 3B), in acceptor cells the taken up (Alexa-488) and transferred fibrils (ATTO-550) from the donor population co-localized much less (16 ± 0.9%) (Fig 3B). These data suggest that taken up and transferred fibrils in the acceptor cells may not undertake the same pathway. Figure 3. α-synuclein fibril transfer is favoured by cell-to-cell contact Representative images of donor cells (upper panel) and acceptor cells (bottom panel) of the co-culture system explained in Fig EV2E: in red, ATTO-550 α-synuclein fibrils; in green, α-synuclein fibrils Alexa-488; and in blue, nuclei. Scale bar represents 10 μm (n = 3 independent experiments). In insets, arrows point to co-localized ATTO-550 and Alexa-488 α-synuclein fibril puncta, whereas arrowheads point to puncta of α-synuclein Alexa-488 fibrils that do not co-localize with fibrillar ATTO-550 α-synuclein. Quantification of the percentage of Alexa-488 α-synuclein fibrils co-localizing with ATTO-550 α-synuclein fibrils and the reverse co-localization in discrete puncta in donor and acceptor cells as in (A). Both measurements revealed high co-localization of the two fluorophores in donor cells (white bar), but less in acceptor cells. Data are shown as mean ± s.e.m. of 3 independent experiments. The amount of fibrils taken up by CAD cells exposed to 1 μM fibrils, remaining in the cells and exported into the medium after 24-h incubation was quantified by a filter trap assay. n = 3 independent measurements, filtered in duplicate. The standard fluorescence curve for increasing ATTO-550 α-synuclein fibril concentrations is given. Representative images of GFP-transfected acceptor cells that were either (i) co-cultured with donor cells (upper panel, Co-culture), (ii) cultured with the conditioned medium of donor cells (middle panel, CM) or (iii) physically separated from donor cells using a filter (bottom panel, Filter). Prior to culture, donor cells were loaded with ATTO-550 α-synuclein fibrils. In red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bars represent 10 μm. n = 3 independent experiments. Quantification of the percentage of acceptor cells containing α-synuclein fibrils from images such as those presented in (C). When acceptor cells were cultured with the conditioned medium from donor cells previously treated with α-synuclein fibrils (not diluted, concentrated or diluted) or co-cultured with a filter, the percentage of acceptor cells containing α-synuclein puncta was low. Data are shown as mean ± s.e.m. of 3 independent experiments. ***P < 0.001 by two-tailed Student's t-test. Quantification of the number of puncta of α-synuclein fibrils per acceptor cell from (D). While in co-culture conditions the number of α-synuclein puncta in acceptor cells is on average 35 (median), this number was on average 1 puncta per acceptor cells in both CM and filter conditions (***P < 0.001 by two-tailed Mann–Whitney U-test). In the box and whisker plot, boxes extend from the fi
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