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, 284 (19), 12845-52

Propagation of Tau Misfolding From the Outside to the Inside of a Cell

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Propagation of Tau Misfolding From the Outside to the Inside of a Cell

Bess Frost et al. J Biol Chem.

Abstract

Tauopathies are neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau in neurons and glia. Although Tau is normally considered an intracellular protein, Tau aggregates are observed in the extracellular space, and Tau peptide is readily detected in the cerebrospinal fluid of patients. Tau aggregation occurs in many diseases, including Alzheimer disease and frontotemporal dementia. Tau pathology begins in discrete, disease-specific regions but eventually involves much larger areas of the brain. It is unknown how this propagation of Tau misfolding occurs. We hypothesize that extracellular Tau aggregates can transmit a misfolded state from the outside to the inside of a cell, similar to prions. Here we show that extracellular Tau aggregates, but not monomer, are taken up by cultured cells. Internalized Tau aggregates displace tubulin, co-localize with dextran, a marker of fluid-phase endocytosis, and induce fibrillization of intracellular full-length Tau. These intracellular fibrils are competent to seed fibril formation of recombinant Tau monomer in vitro. Finally, we observed that newly aggregated intracellular Tau transfers between co-cultured cells. Our data indicate that Tau aggregates can propagate a fibrillar, misfolded state from the outside to the inside of a cell. This may have important implications for understanding how protein misfolding spreads through the brains of tauopathy patients, and it is potentially relevant to myriad neurodegenerative diseases associated with protein misfolding.

Figures

FIGURE 1.
FIGURE 1.
Full-length Tau-YFP binds microtubules while MTBR-YFP aggregates in C17.2 neural cells. A, Tau constructs. YFP fusion proteins were created for expression in mammalian cells as follows: full-length Tau (441 amino acids), Tau-YFP, the microtubule-binding region, MTBR-YFP. For bacterial expression, we used either MTBR alone or with a C-terminal hemagglutinin tag, MTBR-HA. B, Tau-YFP co-localizes with tubulin when transfected into C17.2 cells. C, MTBR-YFP does not co-localize with tubulin and spontaneously aggregates when transfected into C17.2 cells. D, Western blot showing similar expression levels of Tau-YFP and MTBR-YFP. Blots were probed with GFP and actin antibodies. E, based on counting transfected cells, 6% of cells have spontaneous aggregation of Tau-YFP, whereas 79% of cells have spontaneous aggregation of MTBR-YFP (n = 4, 100 transfected cells counted per experiment). F, 83% of cells have Tau-YFP that co-localizes with tubulin, whereas 3.5% of cells have MTBR-YFP that co-localizes with tubulin (n = 4, 100 transfected cells counted per experiment). Scale bars, 10 μm.
FIGURE 2.
FIGURE 2.
C17.2 cells take up aggregated Tau. A, recombinant MTBR Tau was prepared in vitro and induced to fibrillize using arachidonic acid. Tau monomer is not detectable via AFM. After 24 h of incubation with arachidonic acid, however, Tau is highly aggregated, forming many oligomeric and fibrillar species. Scale bars, 600 nm. B, aggregated Tau was treated with buffer or 0.125% trypsin for 1 min and resolved by SDS-PAGE 4–15% gradient gel, followed by Coomassie stain. Aggregated Tau is very sensitive to trypsin digestion. C, C17.2 cells were exposed to AF488-containing buffer, AF488-labeled monomer, or aggregates. After 3 and 9 h, cells were harvested by 0.25% trypsin treatment. Intracellular AF488 fluorescence was then quantified by flow cytometry. After 3 h, 2.0% of monomer-treated cells scored positive, versus 18% for aggregate-treated cells. After 9 h, 3.0% of monomer-treated cells scored positive, versus 22% for aggregate-treated cells. *, p < 10-6 (unpaired t test, n = 4, 10,000 cells counted per experiment). D, MTBR-AF488 aggregate-treated C17.2 cells with or without trypsin treatment and visualized by confocal microscopy. Scale bar, 30 μm. E, MTBR-AF488 aggregate-treated C17.2 cell stained for tubulin and visualized via confocal microscopy after treatment with 0.25% trypsin illustrates internalized aggregates. Arrows indicate displacement of tubulin. An apical-to-distal slice (bar) obtained from a three-dimensional image rendered from Z-stacks shows MTBR-AF488 in the same plane as tubulin. Scale bar, 10 μm.
FIGURE 3.
FIGURE 3.
Multiple images of C17.2 cells treated with MTBR-AF488 aggregates and rhodamine-dextran contain co-localizing and non-co-localizing aggregates. Scale bars, 5 μm.
FIGURE 4.
FIGURE 4.
Extracellular Tau enters cells and induces aggregation of full-length Tau-YFP in HEK293 and C17. 2 cells. HEK293 cells (A) or C17.2 cells (B) expressing Tau-YFP were treated for 15 h with buffer (lane B), MTBR monomer (lane M), or MTBR aggregates (lane A) followed by 1% Triton detergent fractionation. Control cells (lane C) were lysed and treated with aggregates. Soluble (Sol.) and insoluble (Insol.) Tau-YFP bands were detected using the Tau5 antibody, with actin as a loading control. Treatment with Tau aggregates increased insoluble Tau-YFP in both cell types. C, C17.2 cells expressing Tau-YFP were treated for 15 h with buffer, MTBR monomer, or MTBR aggregates, followed by syringe lysis in PBS. The whole cell lysate was loaded onto a 4–15% SDS-polyacrylamide gel, and blotted with a GFP antibody or an actin antibody to control for loading. Full-length aggregated Tau-YFP appears in the well when cells are treated with MTBR aggregates. D, C17.2 cells expressing Tau-YFP were exposed to MTBR-HA aggregates, and double label confocal microscopy was performed using an HA antibody (red channel) and direct visualization of YFP (green channel). A representative example of co-localization between MTBR-HA (exogenous) and Tau-YFP (endogenous) aggregates is shown. Scale bar = 10 μm. E, 3% of aggregates were composed of MTBR-HA in the absence of Tau-YFP aggregates; 23% of aggregates were composed of Tau-YFP in the absence of MTBR-HA aggregates; 74% of aggregates were dual-fluorescent, composed of Tau-YFP and MTBR-HA (n = 3, 100 aggregates counted per experiment).
FIGURE 5.
FIGURE 5.
Induced Tau-YFP aggregates in C17.2 cells are fibrillar and seed MTBR fibrillization in vitro. C17.2 cells, in which Tau-YFP aggregation was induced by treatment with MTBR aggregates, were extracted with Sarkosyl. Scale bars, 0.2 μm. A, insoluble material was visualized by AFM, which demonstrates fibrils. B, Tau5 antibody labeling increases the diameter of the observed fibrils, indicating their principle constituent is Tau-YFP. C, Sarkosyl-extracted Tau-YFP fibrils from aggregate-treated cells were used to seed the fibrillization of recombinant MTBR in vitro. The resultant fibrils were visualized by AFM. D, MTBR fibrils seeded by Tau-YFP were exposed to Tau5 antibody, which labels the Tau-YFP seeds within the MTBR fibrils.
FIGURE 6.
FIGURE 6.
MTBR-YFP aggregates transfer between co-cultured cells. A, cells were transfected separately with mCherry or MTBR-YFP. The two cell populations were either mixed immediately prior to analysis or co-cultured for 24 h. Flow cytometry was used to quantify dual positive cells (10,000 cells sorted per condition). B, quantification of flow cytometry revealed that 0.5% of cells scored positive for mCherry and YFP (upper right quadrant of cell plot) when cells were simply mixed prior to sorting, versus 1.5% of cells that scored dual positive when cells were co-cultured for 24 h, indicating transfer of MTBR-YFP between cells. *, p < 10-5 (unpaired t test, n = 4, 10,000 cells sorted per experiment). C, dual positive cells were collected via FACS, fixed, and mounted. A MTBR-YFP inclusion (white arrowhead) is visible within an mCherry-expressing cell. Scale bar, 10 μm.
FIGURE 7.
FIGURE 7.
Tau-YFP aggregates transfer between co-cultured cells. A, cells were transfected separately with mCherry or Tau-YFP. The two cell populations were either mixed immediately prior to analysis or co-cultured for 48 h. Co-cultured cells were treated with buffer, monomer, or aggregates, followed by flow cytometry (10,000 cells sorted per condition). tx, treatment. B, quantification of flow cytometry revealed that 0.15% of cells score positive for mCherry and YFP (upper right quadrant of cell plot) when cells were mixed immediately prior to counting. 0.3 and 0.25% of cells are dual positive when cells are treated with buffer (B) or monomer (M), versus 1% when treated with Tau aggregates (A), indicating transfer of aggregated full-length Tau-YFP between cells. *, p < 10-7 (unpaired Student's t test, n = 4, 10,000 cells counted per experiment). C, dual positive cells were collected, fixed, and mounted. Direct visualization indicates a Tau-YFP inclusion (white arrowhead) within an mCherry-expressing cell. Scale bar, 10 μm.

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