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Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies - PubMed

  • ️Fri Jan 01 2016

Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies

Meryem Tardivel et al. Acta Neuropathol Commun. 2016.

Abstract

A given cell makes exchanges with its neighbors through a variety of means ranging from diffusible factors to vesicles. Cells use also tunneling nanotubes (TNTs), filamentous-actin-containing membranous structures that bridge and connect cells. First described in immune cells, TNTs facilitate HIV-1 transfer and are found in various cell types, including neurons. We show that the microtubule-associated protein Tau, a key player in Alzheimer's disease, is a bona fide constituent of TNTs. This is important because Tau appears beside filamentous actin and myosin 10 as a specific marker of these fine protrusions of membranes and cytosol that are difficult to visualize. Furthermore, we observed that exogenous Tau species increase the number of TNTs established between primary neurons, thereby facilitating the intercellular transfer of Tau fibrils. In conclusion, Tau may contribute to the formation and function of the highly dynamic TNTs that may be involved in the prion-like propagation of Tau assemblies.

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Figures

Fig. 1
Fig. 1

Tau is associated with TNTs in neuronal CAD cells. Cells were plated in Lab-Tek chamber slides and co-infected with LVs encoding mCherry-Actin (red) and V5-hTau1N4R (green). Neuronal CAD cells were processed for immunocytochemistry analysis using anti-V5 antibodies visualized with an Alexa 488-labeled secondary antibody (green) and anti-acetylated-tubulin visualized with an Alexa 647-labeled secondary antibody (white). Nuclei were labeled with DAPI (blue). Cells were imaged with an inverted laser-scanning confocal microscope using a 40× oil-immersion lens (NA 1.3), and the images were processed using ZEN and ImageJ. A focal plane was collected for each specimen. Scale bars: 10 μm. TNTs (white arrows), which are not always bound to the dish, are shown in enlargements

Fig. 2
Fig. 2

Extracellular Tau species activate TNT formation in neuronal CAD cells. a Characterization of Tau fibrils. Electron micrographs of recombinant hTau1N4R (40 μM) fibrils assembled in the presence of heparin (10 μM), left panel, Sup35NM (20 μM) fibrils, right panel). Scale bars: 0.1 μm. b Confocal imaging of TNTs in neuronal CAD cells in basal conditions. Cells were infected with LVs encoding GFP-Actin (red) and mCherry-Tubulin (green), and confocal imaging was performed 24 h later. Inset: high magnification of TNTs (white arrows). c Confocal imaging of TNTs in neuronal CAD cells after activation with Tau. Cells were infected with LVs encoding GFP-Actin (red) and mCherry-Tubulin (green). At 24 h later, non-labeled hTau1N4R fibrils were added to the extracellular medium, and cells were immediately imaged. Inset: high magnification of TNTs (white arrows). d Quantification of CAD cells with TNTs under basal conditions (CTL) and after activation with 1 μM hTau1N4R fibrils (Tau); n = 4 independent experiments; 200 cells per experiment (*, p < 0.1; Mann-Whitney test). e Quantification of CAD cells with TNTs under basal conditions (CTL) and after activation with the amyloid fibril prion Sup35NM (Sup35); n = 3 independent experiments; 200 cells per experiment (NS, non significant; Mann-Whitney test). For (b), (c), (d) and (e), cells were observed via laser-scanning confocal microscopy using a 40× oil-immersion lens (NA 1.3) and processed with ZEN and ImageJ software. A focal plane was collected for each specimen. Scale bars: 10 μm

Fig. 3
Fig. 3

Extracellular hTau1N4R species favor the establishment of TNTs between primary neurons. a Imaging of TNTs in primary neurons. Cells were infected with LVs encoding mCherry-Actin (red). At 72 h post-infection, cells were processed for immunostaining analysis using anti-acetylated tubulin antibodies visualized with an Alexa 647-labeled secondary antibody (green, polymerized tubulin). Nuclei were labeled with DAPI (blue). b Real-time snapshots of TNTs in primary neurons co-infected with LVs encoding GFP-Actin (red) and mCherry-Tubulin (green, monomeric and polymerized tubulin). (c) Myo10 is present in TNTs in primary neurons. Neurons were plated in Lab-Tek chambers, infected with LVs encoding mCherry-Actin (red) and processed for immunocytochemistry analysis using anti-myosin 10 antibodies visualized with an Alexa 488-labeled secondary antibody (green) and anti-acetylated tubulin visualized with an Alexa 647-labeled secondary antibody (white). Images for (a), (b) and (c) were acquired via laser-scanning confocal microscopy using a 40× oil-immersion lens (NA 1.3) and processed with ZEN and ImageJ software. A focal plane was collected for each specimen. TNTs are shown in enlargements. For (a), (b) and (c), to observe TNTs in primary neurons, 1 μM hTau1N4R fibrils were added in extracellular medium. TNTs were never observed in primary neurons without addition of exogenous Tau fibrils. Scale bars: 10 μm

Fig. 4
Fig. 4

Tau is found in TNTs in primary neurons. a Neurons were infected with LVs encoding mCherry-Actin and V5-hTau1N4R. At 72 h post-infection, cells were processed for immunostaining analysis using anti-V5 antibodies visualized with an Alexa 488-labeled secondary antibody (green) and anti-acetylated tubulin visualized with an Alexa 647-labeled secondary antibody (white). b Acquisition of TNTs connecting primary neurons and containing endogenous Tau protein. Cells were infected with LVs encoding mCherry-Actin. At 72 h post-infection, neurons were processed for immunostaining analysis using anti-C-Terminal Tau antibodies (Tau-Cter) visualized with an Alexa 488-labeled secondary antibody (green) and anti-acetylated tubulin visualized with an Alexa 647-labeled secondary antibody (white). For (a) and (b), TNTs containing Tau are shown in enlargements (white arrows). c Cells were infected with LVs encoding mCherry-Actin. At 72 h post-infection, neurons were processed for immunocytochemistry using anti-acetylated tubulin visualized with an Alexa 647-labeled secondary antibody (white) and anti C-Terminal Tau antibodies (Tau-Cter) visualized with an Alexa 488-labeled secondary antibody (green). The C-Terminal Tau antibodies used were saturated with Tau proteins for 24 h at 4 °C to block the specific fluorescence signal of tau. Nuclei were labeled with DAPI (blue). For (a), (b) and (c), 1 μM hTau1N4R fibrils were added in the extracellular medium. (d) Cells were infected with LVs encoding mCherry-Actin. TNT formation was activated by ATTO 647-hTau1N4R fibrils. At 72 h later, neurons were processed for immunocytochemistry using anti C-Terminal Tau antibody (Tau-Cter) visualized with an Alexa 488-labeled secondary antibody (green). For (a), (b), (c) and (d), images were acquired using laser-scanning confocal microscopy with a 40× oil-immersion lens (NA 1.3) and processed with ZEN and ImageJ software. Images in (b) and (c) were acquired with the same optical settings and a focal plane was collected. Nuclei were labeled with DAPI (blue). Scale bars: 10 μm

Fig. 5
Fig. 5

Exogenous Tau fibrils are found within TNTs. CAD neuronal cells (a) or primary neurons (b) were plated in Lab-Tek chamber slides and infected with LVs encoding mCherry-Actin (red). At 48 h post-infection, cells were incubated for six hours with extracellular ATTO 488-hTau1N4R fibrils/liposomes preparation (green). Cells were processed for immunostaining analysis using an anti-acetylated tubulin visualized with an Alexa 647-labeled secondary antibody (white). Nuclei were labeled with DAPI (blue). TNTs containing Tau are shown in enlargements (white arrows). For acquisition, a focal plane was collected for specimen. Images were acquired with an inverted laser-scanning confocal microscope using a 40× oil-immersion lens (NA 1.3) and processed using the programs ZEN and ImageJ. Scale bars: 10 μm

Fig. 6
Fig. 6

Neuron-to-neuron transfer of Tau fibrils through TNTs. For (a) and (b), cells were infected with LVs encoding GFP-Actin (red). At 48 h post-infection, cells were incubated for six hours with 1 μM ATTO 568-hTau1N4R fibrils (green). a Snapshots selected from a time-lapse video and placed in a gallery to visualize neuron-to-neuron transfer of extracellular Tau fibrils by TNTs in neuronal CAD cells. Time-lapse videos were acquired during 5 h and 33 min with an inter-image interval of 6 s. b Images selected from a video to visualize transfer of extracellular hTau1N4R fibrils by TNTs in primary neurons. Neurons were imaged every 6 s for 51 min. For (a) and (b), cells were filmed with an inverted spinning disk microscope using a 63× oil-immersion lens (NA 1.4) and processed with ZEN blue and ImageJ software. For time-lapse acquisition, a focal plane was collected for each specimen. Scale bars: 10 μm

Fig. 7
Fig. 7

Characteristics of Tau fibrils trafficking in TNTs. For (a) and (b), cells were infected with LVs encoding GFP-Actin (red). At 48 h post-infection, cells were incubated for six hours with 1 μM ATTO 568-hTau1N4R fibrils (green). a The speed of Tau fibrils in TNTs in CAD neuronal cells was calculated from 17 independent videos. b Co-localization analysis between Tau fibrils and actin in TNTs. Significant positive correlation was found between Tau fibrils and actin (Pearson correlation coefficient: 0.727). For (a) and (b), cells were filmed with an inverted spinning disk microscope using a 63× oil-immersion lens (NA 1.4) and processed with ZEN blue and ImageJ software. A focal plane was collected for each specimen Scale bar: 10 μm

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