Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds

Abstract

Recent experimental evidence suggests that transcellular propagation of fibrillar protein aggregates drives the progression of neurodegenerative diseases in a prion-like manner. This phenomenon is now well described in cell and animal models and involves the release of protein aggregates into the extracellular space. Free aggregates then enter neighboring cells to seed further fibrillization. The mechanism by which aggregated extracellular proteins such as tau and α-synuclein bind and enter cells to trigger intracellular fibril formation is unknown. Prior work indicates that prion protein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface to transmit pathologic processes. Here, we find that tau fibril uptake also occurs via HSPG binding. This is blocked in cultured cells and primary neurons by heparin, chlorate, heparinase, and genetic knockdown of a key HSPG synthetic enzyme, Ext1. Interference with tau binding to HSPGs prevents recombinant tau fibrils from inducing intracellular aggregation and blocks transcellular aggregate propagation. In vivo, a heparin mimetic, F6, blocks neuronal uptake of stereotactically injected tau fibrils. Finally, uptake and seeding by α-synuclein fibrils, but not huntingtin fibrils, occurs by the same mechanism as tau. This work suggests a unifying mechanism of cell uptake and propagation for tauopathy and synucleinopathy.

Keywords: Alzheimer's disease; macropinocytosis; neurodegeneration; prion-like mechanisms.

Fig. 1.

Tau RD fibril internalization is mediated by macropinocytosis. (A) Internalized tau RD fibrils are associated with filamentous actin as demonstrated by colocalization with rhodamine-phalloidin. (Scale bar: 10 µm.) (B) EM ultrastructure of tau fibrils and their association with the plasma membrane. (i) Scanning EM image of tau RD fibrils. (ii) Tau RD fibrils near the plasma membrane of a C17.2 cell. (iii) Tau RD fibrils adherent to the membrane of a cell. (iv) Top-down view of tau fibrils engulfed in lamellipodia-like membrane protrusion. (v) Cross-sectional view of tau fibrils surrounded by lamellipodia-like membrane protrusion. (vi) Large tau fibril-containing vesicle within a cell. (C) Inhibition of macropinocytosis reduces tau fibril uptake as measured by flow cytometry after exposure of cells to tau RD-488 fibrils for 90 min. Data are expressed relative to the untreated control group. A total of 25,000 cells were analyzed per group in each experiment, and the graph represents the average of two independent experiments. (D) Tau fibrils colocalize with macropinosome marker TAT-TAMRA. (E) Tau RD fibrils stimulate fluid-phase endocytosis. A total of 100 µg/mL of dextran-fluorescein was applied to cells in the presence of increasing concentrations of unlabeled tau RD fibrils for 90 min before analysis by automated microscopy. NT, not treated.

Fig. 2.

HSPGs mediate binding of tau RD fibrils to C17.2 cells. (A) Tau RD-488 fibrils colocalize with anti-HSPG antibody (10E4). (Scale bar: 10 µm.) (B) At 4 °C, tau RD-546 fibrils bind to the plasma membrane but are not internalized, a process that is inhibited by pretreatment with heparin and chlorate. HSPG inhibition abolishes the association between tau RD fibrils and C17.2 cells observed by confocal microscopy. (C and D) Flow cytometry quantification of tau fibril binding to the cell membrane in the presence of chlorate or heparin. Cells were treated with 50 nM tau RD-488 fibrils at 4 °C for 1 h. A total of 25,000 cells were analyzed for each condition, which was run in triplicate. Error bars show SEM.

Fig. 3.

HSPGs mediate tau RD fibril uptake. (A) At 37 °C, tau RD-546 fibrils are internalized by the cell, a process that is inhibited by chlorate and heparin. (Scale bar: 20 µm.) (B and C) Automated microscopy analysis of tau fibril internalization in the presence of chlorate or heparin. Cells were treated with 50 nM tau RD-488 fibrils and chlorate or heparin at 37 °C for 3 h and trypsinized before imaging. The left y-axis (blue) depicts percentage of positive cells and the right y-axis (red) depicts the average number of tau aggregates per cell. Approximately 40,000 cells were analyzed for each condition, run in quadruplicate. (D) Percent cells positive for tau fibril internalization in the presence of heparinase III or chondroitinase AC as measured by automated microscopy analysis. Approximately 40,000 cells were analyzed for each condition, run in duplicate. (E) HSPG inhibition does not affect clathrin-mediated transferrin endocytosis. Internalization of Tfn-488 (25 µg/mL) was unaltered in the presence of chlorate (63.2 mM), heparin (200 µg/mL), heparinase III, or chondroitinase AC (10² IU/mL) as measured by flow cytometry mean fluorescence intensity. A total of 25,000 cells were analyzed for each condition in duplicate. The nontreated group (NT) received no inhibitor, and data reflect uptake relative to this group. (Error bars: B, C, and E, SEM; D, range.)

Fig. 4.

HSPGs mediate internalization of FL tau fibrils in C17.2 cells and primary hippocampal neurons. (A and B) Automated microscopy analysis of FL tau-488 fibril internalization into C17.2 cells in the presence of chlorate or heparin. Cells were treated with 50 nM FL tau-488 fibrils at 37 °C for 3 h and trypsinized before imaging. Approximately 40,000 cells were analyzed for each condition and run in quadruplicate. The left y-axis (blue) depicts percentage of positive cells and the right y axis (red) depicts the average number of tau aggregates per cell. (C) Lentivirus encoding Ext1 shRNA reduces Ext1 transcript in C17.2 cells by quantitative PCR relative to GAPDH (n = 3). (D) Knockdown of murine Ext1 by shRNA, but not luciferase shRNA, reduces the internalization of FL tau-488 fibrils into primary hippocampal neurons by mean fluorescence intensity measurements. The nontreated (NT) group received no shRNA (n = 9). Error bars show SEM. (E) Knockdown of murine Ext1 by shRNA does not reduce Tfn-488 internalization into primary hippocampal neurons (n = 4, error bars show SEM; ***P < 0.001 by one-way ANOVA).

Fig. 5.

Inhibition of HSPGs blocks seeded aggregation and transcellular propagation of tau aggregation. (A) Heparin (Hep) and heparinase (h’ase) inhibit intracellular seeding by recombinant tau RD fibrils in a cell-based FRET assay. HEK293 cells cotransfected with tau RD(ΔK)-CFP/YFP were pretreated with heparinase (0.01 IU/mL) for 3 h before treatment with tau fibrils or treated with tau RD fibrils plus vehicle or heparin (6 µg/mL) for 24 h before reading FRET measurements on a plate reader. The FRET signal is shown as a percentage relative to the vehicle treated group. (B) Neither heparin nor heparinase affect cell-autonomous tau aggregation. Cells expressing RD(ΔK)-CFP/YFP were treated with vehicle or heparin (6 µg/mL) for 24 h or heparinase (0.01 IU/mL) for 27 h. A value of 100% represents baseline aggregation signal for the vehicle-treated group. (C) Heparin and heparinase block transcellular propagation. HEK293 cells expressing RD(ΔK)-CFP/YFP were cocultured with an equivalent number of cells expressing tau RD(LM)-HA for 48 h to monitor transcellular propagation of tau protein misfolding. Heparin dose dependently inhibited transcellular aggregate propagation, as did 0.01IU/mL of heparinase. The FRET signal is shown as a percentage relative to the vehicle-treated group. Error bars show SEM from four biological replicates per experiment for heparin and from six biological replicates per experiment for heparinase (***P < 0.001, **P < 0.01, Student t test).

Fig. 6.

Heparin mimetic F6 inhibits tau fibril uptake and induction of misfolding. (A) F6 inhibits tau RD fibril internalization into C17.2 cells whereas PD has no effect. Cells were treated with 50 nM tau RD-AF488 fibrils at 37 °C for 3 h and trypsinized before replating and imaging with automated microscopy. Approximately 40,000 cells were analyzed for each condition and run in quadruplicate. (B) F6 (6 µg/mL) and heparin (6 µg/mL) equivalently inhibit seeded aggregation in a FRET-based assay. (C) F6 and heparin each block transcellular propagation between cells expressing tau RD(LM) and cells expressing RD(∆K)-CFP/YFP. (B and C) FRET signal is shown as a percentage relative to the vehicle treated group. (D) F6 does not inhibit cell-autonomous tau aggregation. A value of 100% represents baseline aggregation signal of the vehicle-treated group. Error bars show SEM from four biological replicates per experiment (***P < 0.001, Student t test).

Fig. 7.

HSPGs mediate internalization of FL tau fibrils in vivo. (A) Schematic representation of stereotactic injection site. (B) Injection of FL tau fibrils with heparin mimetic F6 leads to a reduction of fibril internalization by cortical neurons. Mice (n = 3 per group) were injected with 472 ng of FL tau-488 fibrils (green) and 1 µg of PD or F6. Brain sections were immunostained with anti-NeuN antibody to label neurons (red) and counterstained with DAPI (blue). Arrowheads designate FL tau fibrils associated with neurons. (Scale bar: 10 µm.) (C and D) Heparin mimetic F6 blocks FL tau fibril uptake, but not Tfn-488. Mice (n = 4 per group) were injected with 472 ng of FL tau-647 fibrils (white), 5 µg of Tfn-488 (green), and 1 µg of PD or F6. Injection of tau fibrils and transferrin with PD resulted in entry of both proteins into neurons, whereas coinjection with F6 resulted in the selective inhibition of tau fibril entry. The grayscale images are single channel fluorescence images corresponding to the multichannel images labeled as “merged.” Arrowheads designate FL tau fibrils; long arrows designate transferrin. (E) Quantification of tau-positive neurons from experiment C and D. A total of 494 and 660 neurons were counted and averaged in the PD and F6 conditions, respectively, from four male mice per cohort. Error bars show SEM (**P < 0.01, Student t test). (F and G) Quantification of neuronal fluorescence intensity from experiment C and D. Mean fluorescence intensity of tau fibrils and transferrin were calculated per neuron and averaged from individual mice, each represented by a single data point (P = 0.028, Mann–Whitney U test, two-tailed exact significance) comparing treatment effects on tau fibril uptake.

Fig. 8.

HSPGs mediate internalization and seeding of α-synuclein but not huntingtin fibrils. (A and B) Uptake of α-synuclein-488 and fluorescein-Htt exon1(Q50) fibrils (green) into C17.2 cells counterstained with anti-HSPG antibody (red) and DAPI (blue). (A) α-Synuclein-488 (200 nM) colocalizes with HSPGs. (B) Htt(Q50) fibrils (5 µM) do not colocalize with HSPGs. (Scale bar: 10 µM.) (C and D) α-Synuclein fibrils (blue) colocalize with TAT (red) and tau (green), whereas Htt(Q50) fibrils (blue) do not. (E and F) Heparin and chlorate dose dependently decrease the internalization of α-synuclein, but not Htt(Q50) fibrils, into C17.2 cells, as measured by automated microscopy analysis. α-Synuclein fibrils (100 nM) and Htt(Q50) fibrils (1 µM) were applied to cells for 5 h before harvesting for microscopy or flow cytometry. Approximately 40,000 cells were analyzed for each α-synuclein condition; ∼12,000 cells were analyzed for each Htt(Q50) condition, and all were run in triplicate. (G and H) Heparin blocks seeding by α-synuclein fibrils, but not Htt(Q50) fibrils. HEK293 cells cotransfected with α-synuclein-CFP/YFP or Htt exon1(Q25)-CFP/YFP were cotreated with unlabeled α-synuclein fibrils (100 nM) or Htt exon1(Q50) fibrils (1 µM), along with heparin or PD for 24 h before reading FRET measurements. FRET values reflect subtraction of signal from the nontreated (NT) group.