Clustering of Tau fibrils impairs the synaptic composition of α3‐Na + /K + ‐ ATP ase and AMPA receptors
2019; Springer Nature; Volume: 38; Issue: 3 Linguagem: Inglês
10.15252/embj.201899871
ISSN1460-2075
AutoresAmulya Nidhi Shrivastava, Virginie Redeker, Laura Pieri, Luc Bousset, Marianne Renner, Karine Madiona, Caroline Mailhes‐Hamon, Audrey Coens, Luc Buée, Philippe Hantraye, Antoine Triller, Ronald Melki,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoArticle10 January 2019Open Access Transparent process Clustering of Tau fibrils impairs the synaptic composition of α3-Na+/K+-ATPase and AMPA receptors Amulya Nidhi Shrivastava Amulya Nidhi Shrivastava orcid.org/0000-0002-1322-8018 CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France Search for more papers by this author Virginie Redeker Virginie Redeker orcid.org/0000-0003-2694-8388 CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Laura Pieri Laura Pieri CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Luc Bousset Luc Bousset CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Marianne Renner Marianne Renner INSERM, UMR – S 839 Institut du Fer à Moulin (IFM), Sorbonne Université, Paris, France Search for more papers by this author Karine Madiona Karine Madiona CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Caroline Mailhes-Hamon Caroline Mailhes-Hamon Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France Search for more papers by this author Audrey Coens Audrey Coens CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Luc Buée Luc Buée CHU Lille, INSERM UMR-S 1172, JPArc, "Alzheimer & Tauopathies", Universite Lille, Lille, France Search for more papers by this author Philippe Hantraye Philippe Hantraye CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Antoine Triller Corresponding Author Antoine Triller [email protected] orcid.org/0000-0002-7530-1233 Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France Search for more papers by this author Ronald Melki Corresponding Author Ronald Melki [email protected] orcid.org/0000-0003-0000-7096 CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Amulya Nidhi Shrivastava Amulya Nidhi Shrivastava orcid.org/0000-0002-1322-8018 CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France Search for more papers by this author Virginie Redeker Virginie Redeker orcid.org/0000-0003-2694-8388 CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Laura Pieri Laura Pieri CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Luc Bousset Luc Bousset CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Marianne Renner Marianne Renner INSERM, UMR – S 839 Institut du Fer à Moulin (IFM), Sorbonne Université, Paris, France Search for more papers by this author Karine Madiona Karine Madiona CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Caroline Mailhes-Hamon Caroline Mailhes-Hamon Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France Search for more papers by this author Audrey Coens Audrey Coens CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Luc Buée Luc Buée CHU Lille, INSERM UMR-S 1172, JPArc, "Alzheimer & Tauopathies", Universite Lille, Lille, France Search for more papers by this author Philippe Hantraye Philippe Hantraye CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Antoine Triller Corresponding Author Antoine Triller [email protected] orcid.org/0000-0002-7530-1233 Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France Search for more papers by this author Ronald Melki Corresponding Author Ronald Melki [email protected] orcid.org/0000-0003-0000-7096 CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France Search for more papers by this author Author Information Amulya Nidhi Shrivastava1,2, Virginie Redeker1, Laura Pieri1, Luc Bousset1, Marianne Renner3, Karine Madiona1, Caroline Mailhes-Hamon2, Audrey Coens1, Luc Buée4, Philippe Hantraye1, Antoine Triller *,2 and Ronald Melki *,1 1CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France 2Institut de Biologie de l'ENS (IBENS), École Normale Supérieure, INSERM, CNRS, PSL Research University, Paris, France 3INSERM, UMR – S 839 Institut du Fer à Moulin (IFM), Sorbonne Université, Paris, France 4CHU Lille, INSERM UMR-S 1172, JPArc, "Alzheimer & Tauopathies", Universite Lille, Lille, France *Corresponding author. Tel: +33 1 44 32 35 47; E-mail: [email protected] *Corresponding author. Tel: +33 1 46 54 93 78; E-mail: [email protected] The EMBO Journal (2019)38:e99871https://doi.org/10.15252/embj.201899871 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 assemblies have prion-like properties: they propagate from one neuron to another and amplify by seeding the aggregation of endogenous Tau. Although key in prion-like propagation, the binding of exogenous Tau assemblies to the plasma membrane of naïve neurons is not understood. We report that fibrillar Tau forms clusters at the plasma membrane following lateral diffusion. We found that the fibrils interact with the Na+/K+-ATPase (NKA) and AMPA receptors. The consequence of the clustering is a reduction in the amount of α3-NKA and an increase in the amount of GluA2-AMPA receptor at synapses. Furthermore, fibrillar Tau destabilizes functional NKA complexes. Tau and α-synuclein aggregates often co-exist in patients' brains. We now show evidences for cross-talk between these pathogenic aggregates with α-synuclein fibrils dramatically enhancing fibrillar Tau clustering and synaptic localization. Our results suggest that fibrillar α-synuclein and Tau cross-talk at the plasma membrane imbalance neuronal homeostasis. Synopsis Pathogenic fibrillar Tau remodel excitatory synaptic protein composition and imbalance neuronal homeostasis. Exogenous fibrillar-Tau clusters at excitatory synapses. The membrane interactome of fibrillar Tau is identified. Fibrillar-Tau interacts with Na+/K+-ATPase and GluA2-AMPA receptor. Fibrillar Tau reduces Na+/K+-ATPase and increases GluA2-AMPA receptor at synapses. Synuclein fibrils cross-talk with fibrillar-Tau at neuronal membrane. Introduction The six different isoforms of the microtubule-associated protein Tau (MAPT) neurons express have various functions, the main ones being in microtubules stabilization, cell morphogenesis, and axonal transport (Kovacs, 2015). These isoforms are the products of the alternative splicing of the pre-mRNA encoded by the 16 exons of MAPT gene. The six isoforms differ from each other by the presence or absence of one or two inserts (26 or 58 amino acid residues) in the N-terminal part of the protein and by the presence of either three (3R) or four (4R) repeated sequences (31–32 amino acid residues long) microtubule-binding motifs in the protein C-terminal part (Goedert et al, 2017). The phosphorylation of Tau is intimately linked to its assembly into high molecular weight oligomeric species (Buée & Delacourte, 1999). The latter, together with characteristic phosphorylation patterns, are hallmarks of several tauopathies including Alzheimer's disease (AD), Pick's disease, Tangle-only dementia, progressive supranuclear palsy, frontotemporal dementia, corticobasal neurodegeneration, argyrophilic grain disease, and frontotemporal dementia with parkinsonism linked to chromosome 17 (Kovacs, 2015; Goedert et al, 2017). Experiments performed in vitro and using animal models support the notion that high molecular weight assemblies of Tau have prion-like properties. They are released by projections of affected neuronal cells, are taken up by unaffected cells, and are amplified by seeding the aggregation of endogenous Tau (Clavaguera et al, 2009; Brundin et al, 2010; Holmes et al, 2013; Pooler et al, 2013; Sanders et al, 2014; Iba et al, 2015; Takeda et al, 2015; Guo et al, 2016; Kaufman et al, 2016; Wegmann et al, 2016; Woerman et al, 2016; Narasimhan et al, 2017). Although key in the prion-like propagation of Tau assemblies, the binding to and molecular interactions of exogenous Tau assemblies with the plasma membrane of neurons are not fully characterized. In this study, we document the interaction of exogenous fibrillar Tau (Fib-Tau) assemblies with neuron plasma membrane. We report that Fib-Tau clusters at the plasma membrane in a concentration- and time-dependent manner following lateral diffusion. We show that Fib-Tau resides within the clusters briefly. We identify 29 neuronal membrane proteins with extracellular domains that interact with Fib-Tau 3R and 4R assemblies. Of particular interest were Na+/K+-ATPase (NKA) complex α1, α3 and β1 subunits, AMPA receptor GluA1 and GluA2 subunits, and NMDA receptor GluN1 and GluN2B subunits. We show that Fib-Tau clustering alters synaptic protein composition at excitatory synapses with a reduction in the amount of α3-NKA and an increase in the amount of AMPA receptor GluA2 subunit. The NMDA receptor subunit distribution was unaffected by Fib-Tau clustering. Single molecule trajectories of α3-NKA revealed displacement of this subunit from functional NKA pump. Our data suggest that Fib-Tau clusters destabilize functional NKA complexes, thus, reducing neuron's capacity to control membrane depolarization (Azarias et al, 2013). Therefore, we postulate that neuronal function is compromised by Fib-Tau clusters effect on NKA α3 subunit half-life and turnover. We further postulate that α3-NKA plays a role in Tau fibrils endocytosis and possibly in their subsequent amplification within neuron cytosol. This, together with GluA2-AMPA receptor redistribution and trapping within Fib-Tau clusters at excitatory synapses, may initiate deleterious signaling cascades. Aβ, α-Syn, and Tau deposits are often observed in the brain of patients at late stages of PD and AD (Eisenberg & Jucker, 2012; Jellinger, 2012; Irwin et al, 2013; Morales et al, 2013; Moussaud et al, 2014). Here, we show that pathogenic α-Syn dramatically enhanced Fib-Tau clustering on neuronal plasma membrane. Ultimately, our results suggest that fibrillar α-Syn and Tau cross-talk at neuron plasma membrane may contribute to Alzheimer, Parkinson's, and related diseases onset or progression. Results Rapid uptake and redistribution of preformed Tau fibrils following injection in the CA1 region of the hippocampus The injection of Tau assemblies into rodents' hippocampus was shown to trigger widespread deposition of pathological phosphorylated Tau within weeks (Sanders et al, 2014; Takeda et al, 2015; Guo et al, 2016; Kaufman et al, 2016; Narasimhan et al, 2017). We first assessed the fate over 24 h of exogenous Tau fibrils. Preformed Tau-1N3R fibrils (hereafter referred to as Fib-Tau) were labeled with ATTO-550 dye and fragmented (see Materials and Methods and Appendix Figs S1, and S2A and B). Fib-Tau (3.9 μg in 1 μl) was injected in the right CA1-hippocampal cell body layer (Fig 1A, with permission from The Mouse Brain Library) of 1-month-old adult mice (C57BL6J strain; Rosen et al, 2003). Mice were sacrificed 8 or 24 h after injection. Low magnification (10×) fluorescence imaging revealed the presence of Fib-Tau (red) in the right corpus callosum region that is located between the hippocampus CA1 and cortex (Fig 1B, left) for all animals. The corpus callosum is rich in oligodendrocytes and myelinated axons facilitating communication between the two hemispheres of the brain (Meyer et al, 2018). High magnification (63×) images indicate that exogenous Fib-Tau binds along the processes (Fig 1B, right). Some accumulation within cells, possibly oligodendrocytes, of the corpus callosum could also be seen. No exogenous Fib-Tau could be detected in the corpus callosum of the left hemisphere of the brain within the time frame we explored. Furthermore, no differences in Fib-Tau distribution could be observed within 24 h in the right corpus callosum of the injected mice. Figure 1. Rapid uptake and redistribution of preformed Fib-Tau following injection in the CA1 region of the hippocampus A, B. Coronal brain section, bregma: −2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). Eight hours post-injection, majority of the injected Fib-Tau-1N3R-ATTO-550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10×) and high (right, 63×) magnification. C–H. Representative images of the distribution of Fib-Tau (red) 8 h (C, E) and 24 h (D, F) after injection. Dendrites are immuno-labeled using MAP2 antibody (green). Eight hours after injection, both diffused and clustered distribution of exogenous Fib-Tau are observed in stratum oriens and CA1 cell body region (C, E, Appendix Fig S3). No exogenous Fib-Tau is detected in the adjoining cortex, stratum radiatum, and entorhinal cortex (see Appendix Fig S3). Twenty-four hours after injection, clustered distribution is primarily restricted to the pyramidal (CA) cell body layer (D). Uptake of Fib-Tau is seen in some cells in both stratum oriens and pyramidal cell body layer (D). The number of Fib-Tau clusters per μm2 within stratum oriens decreases between 8 and 24 h post-injection (G). A similar decrease in the total fluorescence (clusters + diffused) is observed indicating the diffusion and/or clearance of exogenous Fib-Tau (H). Paired t-test performed between "8/24 h-right" and "8/24 h-left"; unpaired t-test performed between "8 h-left" and "24 h-left" columns (n is number of animals: 6 for 8 h and 5 for 24 h); ***P < 0.001, **P < 0.01, *P < 0.05, ns = not significant. Note: The images presented in panels (C and D) were independently acquired images under non-identical exposure setting at low magnification for display purpose; panels (E and F) are shown for same exposure and are used for quantification. Data information: Scale bar, 100 μm in the left panel in (B), 10 μm everywhere else. Download figure Download PowerPoint We assessed Fib-Tau distribution within regions adjacent to the injection site. Exogenous fibrillar Tau exhibited a diffused and clustered distribution in the stratum oriens and a pre-dominantly clustered distribution in the CA1 cell body layer 8 h after injection (Fig 1C and E, Appendix Fig S3). A weaker staining was observed 24 h after injection (Fig 1D and F, note the images presented in panel C and D were independently acquired images under non-identical exposure setting for display purpose) suggesting diffusion and potential clearance from stratum oriens. Background equivalent signal was detected in the cortex, stratum radiatum, and entorhinal cortex at all time points (Appendix Fig S3). To assess these changes, the density (number per μm2) of exogenous Fib-Tau clusters (Fig 1G, obtained after thresholding, see Materials and Methods section) and total Fib-Tau fluorescence (Fig 1H, without thresholding) was quantified. Our measurements show a decrease in Fib-Tau clusters and total fluorescence over 24 h suggesting diffusion and/or the clearance of exogenous Tau fibrils. Time-, concentration-, and lateral diffusion-dependent clustering of Fib-Tau on neuronal plasma membrane Exogenous fibrillar Tau binding and clustering in vivo (Fig 1) are reminiscent of that of amyloid-β (Aβ) oligomers (Renner et al, 2010) and fibrillar α-Syn (Shrivastava et al, 2015). To assess Fib-Tau clustering in details, we used mouse primary neuronal cultures. Unless otherwise stated, the experiments were performed on densely mature (DIV 21–24) primary hippocampal neuronal cultures with well-developed synapses and network activity (Ivenshitz & Segal, 2010). Neurons were exposed for 1 h to exogenous Fib-Tau (red, 0.36 nM expressed in particle concentration—see Appendix Fig S1) and labeled with anti-MAP2 (dendrites, blue) and anti-endogenous Tau (axons, green) antibodies (Appendix Fig S4A). Our observations reveal the presence of clusters of exogenous Fib-Tau, with some background diffused labeling, both on axons and on dendrites (Appendix Fig S4A). This indicates that there is no preferential binding to either dendrites or axons. Similar clustering was observed on HEK cell membranes upon exposure (10 min) to Fib-Tau (Appendix Fig S4B). We next assessed the clustering of exogenous ATTO-488-labeled Fib-Tau (Appendix Fig S2D), in time-dependent (0.5 s to 4 h at 0.36 nM concentration, a concentration where low-density labeling of Tau was achieved and neurons could be identified) and concentration-dependent (0.36 nM to 1,000 nM for 1-h exposure) manners. Representative images are shown in Fig 2A, and quantification is displayed in Fig 2B and C. Within the concentration range 0.36–0.72 nM, only the density (number per μm2) of exogenous Fib-Tau clusters increased with time (Fig 2A, top panel, Fig 2B). The size of the clusters (measured by fluorescence intensity) did not increase in a significant manner (Fig 2C). At higher concentrations (1.8 nM and higher), both the density and size of Fib-Tau clusters increased with time. Figure 2. Time-, concentration-, and lateral diffusion-dependent clustering of Fib-Tau on neuronal plasma membrane A–C. Time- and concentration-dependent clustering of Fib-Tau in primary neurons. Representative images are shown for certain conditions to illustrate Fib-Tau clustering time dependence (A, top row) and concentration dependence (A, bottom row). Quantification of the number of Fib-Tau clusters per μm2 (B) or fluorescence intensity of clusters (C, indicating size, refer to Materials and Methods). At low concentrations (up to 0.72 nM), the density of clusters increased with time (between 10 and 60 min) but the increase in intensity was small. At high concentrations of Fib-Tau (≥ 1.8 nM), both density and size increased with increasing time. Box-plot represents median, interquartile range, and 10–90% distribution; one-way ANOVA with Dunnett's post hoc test, number of images analyzed from three cultures (from left to right: 25, 25, 25, 25, 70, 45, 45, 45, 45, 45, 30, 40, 40, 40, 40, and 40 images). D–F. Single-particle tracking using quantum dots (SPT-QD) of biotin-tagged Fib-Tau. Representative single molecule trajectories of Fib-Tau following 10- or 60-min exposure are shown (D). Note after 60-min exposure (0.36 nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see Materials and Methods) shows that both these parameters decrease after 60-min exposure to Fib-Tau. Unpaired t-test, n is averaged value per cells imaged in three experiments (10 min: 22, 60 min: 23). C. Neurons were exposed for 60 min to Fib-Tau (0.36 nM) labeled with both biotin and ATTO-488 (red). Cell surface-exposed biotin was labeled using streptavidin-550 (green) followed by live imaging. Note that most of the clusters of ATTO-488 (red) are co-labeled with streptavidin-550 (green) indicating that the clusters are at the cell surface. H–J. Clearance of Tau clusters from neurons. Neurons were exposed (0.36 nM) to ATTO-550-labeled Fib-Tau for 10 min, and the unbound fibrils were washed. Cells were fixed immediately (10 min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60-min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box-plot represents median, interquartile range, and 10–90% distribution; unpaired t-test, n is number of images analyzed from three cultures (49 images). Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant. Scale bar, 5 μm in (G), 2 μm everywhere else. Download figure Download PowerPoint The clustering of exogenous Fib-Tau was next assessed by single-particle tracking using quantum dots (SPT-QD) approach. Neurons were exposed (0.36 nM) to biotin-labeled Fib-Tau (0.36 nM, Appendix Fig S2E) for 10 or 60 min followed by labeling a small fraction of the exogenous biotin-labeled fibrils with streptavidin-QD-655. Representative single molecule trajectories (red) of biotin-labeled Fib-Tau (Fig 2D, red) show they explore large region area after 10 min, while their diffusion is confined after 60 min. This global slowdown in the diffusion coefficient (Fig 2E) and decrease in explored area (Fig 2F, indicative of mean squared displacement, MSD) suggest that Fib-Tau experience molecular interactions leading to the formation of clusters on the plasma membrane. To confirm that Fib-Tau clusters are located on the outer side of the plasma membrane, neurons were exposed (60 and 240 min) to preform Fib-Tau labeled simultaneously with both biotin and ATTO-488 (0.36 nM, Fig 2G, Appendix Fig S5, red). Labeling of the biotin with streptavidin-550 in live neurons (Fig 2G, green) shows that most, if not all, Fib-Tau clusters are at the cell surface (overlay, Fig 2G, Appendix Fig S5). In vivo experiments (Fig 1E–H) suggest a substantial diffusion/clearance of exogenous Fib-Tau 24 h after injection within the hippocampus. To determine whether this also occurs in vitro, primary neurons were exposed (0.36 nM) for 10 min to ATTO-550-labeled Fib-Tau, the excess of fibrils was washed away, and the cells were imaged immediately or allowed to recover for 60 min. A significant number of Fib-Tau clusters were observed on the surface of neurons upon imaging immediately (10 min) after removing the excess of exogenous fibrils (Fig 2H, left). In contrast, few clusters were detected when the cells were allowed to recover for 60 min (Fig 2H, right). A quantitative assessment of Fib-Tau cluster density and fluorescence confirms these observations (Fig 2I and J). This suggests either that Fib-Tau clusters dissociate from neuronal membranes over time or that they are taken up by the cells and processed. We conclude from these observations that (i) Fib-Tau clusters in a time- and concentration-dependent manner at the surface of neurons, (ii) clustering slows down the diffusion of Fib-Tau, and (iii) Fib-Tau either dissociates from neuronal membrane with time or are taken up and processed within the cells. Dynamic assessment of the clustering of exogenous Fib-Tau on neuron plasma membranes The diffusion-dependent clustering of exogenous Fib-Tau at the surface of neurons exhibits similarities with that of Aβ oligomers and α-Syn assemblies with two exceptions. The time-dependent increase in cluster size for Fib-Tau (Fig 2B) is much slower than that previously measured for Aβ oligomers (Renner et al, 2010) and α-Syn assemblies (Shrivastava et al, 2015), and the dissociation and/or clearance of Fib-Tau, both in vitro and in vivo, is much faster than that observed for the other two assemblies (Figs 1E and F, and 2H). These data suggest that the interaction of Fib-Tau with the plasma membrane is highly dynamic and the clusters the fibrils form are unstable. To assess in a quantitative manner the amounts of clustered and non-clustered Fib-Tau, we performed super-resolution STORM (stochastic optical reconstruction microscopy) analysis on neurons exposed to ATTO-647-labeled Fib-Tau (0.36 nM, Appendix Fig S2F) for increasing time (10, 60, 120, and 240 min). Rendered images obtained with pixel size of 5 nm revealed a large proportion of non-clustered ATTO-647-labeled Fib-Tau at all time points (Fig 3A). Fib-Tau distribution was non-uniform over the entire surface of neurons. The total number of "single-particle detection events" per μm2 of neuronal surface increased with time (Fig 3B). The total number of clusters identified using DBSCAN (density-based spatial clustering of applications with noise; Ester et al, 1996; Malkusch et al, 2012) approach increased with time (Fig 3C) in agreement with the data presented in Fig 2B. The radius of Fib-Tau clusters (Fig 3D) and the number of events per cluster (Fig 3E), however, were unchanged, in agreement with the observation presented in Fig 2C. We also assessed the proportion of single-particle detection events within Fib-Tau clusters as a function of time. The data (Fig 3F) show that 50% of Fib-Tau reside within the clusters at any given time point and concentration of membrane-bound Fib-Tau. This contrasts with the behavior of fibrillar α-Syn, where about 90% of single fibrils were within clusters (Shrivastava et al, 2015). Figure 3. Equilibrium between clustered and free Fib-Tau on neuronal plasma membrane A. Super-resolution STORM images of neuronal membrane after exposure to ATTO-647N-labeled Fib-Tau (0.36 nM). Primary neurons were fixed 10, 60, 120, and 240 min after exposure to ATTO-647N-labeled Fib-Tau. Images rendered with a pixel size of 5 nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1 μm. B–F. Quantification of various parameters from the super-resolution images. Averaged values per image are plotted. Increased "total detections per μm2" (B) and "cluster density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (˜50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min and n is number of images (eight images). *P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant. Download figure Download PowerPoint Identification of membrane proteins that interact with extracellularly applied Fib-Tau assemblies A proteomic screening was performed to identify membrane proteins interacting with extracellularly applied Fib-Tau. Preformed biotin-labeled Fib-Tau (Appendix Fig S2E) was used as described in the Materials and Methods section to identify specific neuronal membrane protein partners as illustrated (Fig 4A). Pure cultures of cortical neurons were exposed to biotin-Fib-Tau (14.4 nM, to achieve rapid binding and saturate the binding sites) for 10 min. Biotin-Fib-Tau and associated proteins were pulled down from whole-cell lysates using streptavidin immobilized on magnetic beads, eluted with Laemmli buffer and in-gel trypsin digested. The resulting peptides were identified by nanoLC-MS/MS (LC: liquid chromatography; MS/MS: tandem mass spectrometry). Control samples were prepared from neurons unexposed to Fib-Tau. Protein abundance was assessed by a label-free quantitative proteomic method using spectral counting. We identified 103 and 957 neuronal protein partners from cells used as control or exposed to exogenous Fib-Tau, respectively (Fig 4B). Several intracellular proteins were identified possibly following their interaction with endocytosed Fib-Tau (Flavin et al, 2017) and/or interaction following cell disruption during protein extraction. A total of 372 synaptic and membrane proteins were identified (Fig 4C, Appendix Table S1). Twenty-nine proteins had extracellular domains. The rest of the proteins we identified are scaffolding and signaling proteins. Indeed, pull-down experiments identify not only the direct partners of Fib-Tau but also the protein complexes that interact with Fib-Tau binders. The list of membrane proteins with extracellular domains that interact with Fib-Tau is given in Fig 4D. Figure 4. Identification of intrinsic neuronal membrane proteins interacting with extracellularly applied Fib-Tau-1N3R Strategy used to purify and identify neuron intrinsic membrane proteins with extracellular domain that interact specifically with Fib-Tau-1N3R. Fib-Tau was labeled 1 h with 10 molar equivalents of NHS-S-S-Biotin. Mouse cortical neuron cultures were exposed for 10 min to biotinylated Fib-Tau (14.4 nM). Fresh protein extracts from those neurons were incubated with streptavidin magnetic beads to pull down Fib-Tau together
Referência(s)