Heparan Sulfate Proteoglycans (HSPGs) Serve as the Mediator Between Monomeric Tau and Its Subsequent Intracellular ERK1/2 Pathway Activation

Abstract

The conversion of soluble tau protein to insoluble, hyperphosphorylated neurofibrillary tangles (NFTs) is a major hallmark leading to neuronal death observed in neurodegenerative tauopathies. Unlike NFTs, the involvement of monomeric tau in the progression of tau pathology has been less investigated. Using live-cell confocal microscopy and flow cytometry, we demonstrate that soluble 0N4R monomers were rapidly endocytosed by SH-SY5Y and C6 glioma cells via actin-dependent macropinocytosis. Further, cellular endocytosis of monomeric tau has been demonstrated to be HSPG-dependent, as shown in C6 glial cells with genetic knockouts of xylosyltransferase-1-a key enzyme in HSPG synthesis-with a reduced level of tau uptake. Tau internalization subsequently triggers ERK1/2 activation and therefore, the upregulation of IL-6 and IL-1β. The role of ERK1/2 in regulating the levels of pro-inflammatory gene transcripts was confirmed by inhibiting the MEK-ERK1/2 signaling pathway, which led to the attenuated IL-6 and IL-1β expressions but not that of TNF-α. Moreover, as a key regulator of tau internalization, LRP1 (low-density lipoprotein receptor-related protein 1) levels were downregulated in response to monomeric tau added to C6 cells, while it was upregulated in HSPG-deficient cells, suggesting that the involvement of LRP1 in tau uptake depends on the presence of HSPGs on the cell surface. The subsequent LRP1 knockdown experiment we performed shows that LRP1 deficiency leads to an attenuated propensity for tau uptake and further elevated IL-6 gene expression. Collectively, our data suggest that tau has multiple extracellular binding partners that mediate its internalization through distinct mechanisms. Additionally, this study demonstrates the important role of both HSPGs and LRP1 in regulating cellular immune responses to tau protein monomers, providing a novel target for alleviating the neuroinflammatory environment before the formation of neurofibrillary tangles.

Keywords: 0N4R tau; Alzheimer’s disease; Endocytosis; Heparan sulfate; Lipoprotein receptor; Tauopathies.

Fig. 1

Cell type differences in tau uptake. A Representative confocal microscopy images for HEK293, SH-SY5Y, and C6 glioma following addition of 1-µM 0N4R tau monomer labeled with Alexa Fluro 488. Scale bar = 10 µM. B Quantification of live-cell confocal microscopy: C6 glioma cells (triangles) endocytose 0N4R monomer about three to four times faster compared to SH-SY5Y cells (circles) and HEK293 cells (squares) (* < 0.05 between C6 and the other two cell types) C–E The relative uptake of tau-AF488 at indicated concentrations by different cell lines was quantified by flow cytometry, where control (untreated) cells were subtracted from those subjected to incubation with tau-AF488. Percent positive indicates the fraction of the cells incubated with a fluorescence above background. Figure 6C(ii) shows typical histograms obtained. For HEK293 cells (C) reducing the concentration of tau-AF488 to 25 nM (open squares) significantly reduced the tau internalization. For SH-SY5Y cells (D), the internalization was reduced at 25 nM (open circles), while for C6 glioma cells (E), there was no observed difference between the concentrations

Fig. 2

Monomeric tau is internalized via actin-mediated endocytosis. A Representative confocal microscopy images of HEK293 cells incubated with 1 μM tau-488. Scale bar = 10 µM. B Pre-incubating HEK293 cells on wet ice (4 °C) limits tau uptake as measured by confocal microscopy analysis of mean fluorescence intensity (**p < 0.005, unpaired t-test of 37 °C compared with 4 °C, n > 10/image) at 15 min of incubation. Individual points represent individual cells where bars show mean and SEM. C Similarly, as measured by mean peak intensity via flow cytometry, pre-incubating HEK293 cells on wet ice (4 °C) decreases tau uptake compared with cells at 37 °C (*p < 0.05 for the 37 °C compared to 4 °C; n = 3 independent biological replicates). D 1 μM cytochalasin-D (open symbols, cyto-D) decreases the rate of tau-488 internalization in SH-SY5Y (circles) and C6 glioma cells (triangles) compared with untreated cells (filled symbols; p < 0.05 for 1 µM compared to untreated cells) observed by confocal microscopy

Fig. 3

Tau monomer uptake depends on HSPG levels. A When characterized by the number of vesicles per cell observed via live-cell imaging, CHO745 cells had significantly fewer tau-containing vesicles per cell compared with wild-type CHO cells (*p < 0.05 for CHO745 cells compared with wild-type cells; n = 9, where points are the average, and error bars represent the standard error). B Heparan sulfate detection on the cell surface was determined via immunolabeling with anti-heparan sulfate antibodies and detected by flow cytometry, as described in the “Materials and methods” section. (Bars represent the mean for n = 2 independent replicates performed in duplicate, and error bars represent the standard error; *p < 0.05 based on t-test, ***p < 0.001 based on one-way ANOVA)

Fig. 4

Construction of a Xylt1 knockdown C6 glioma cell line with CRISPR/Cas9 genome editing technology. A Schematic illustration of Xylt-1- gene of Rattus norvegicus, containing two functional domains and 10 exons. Single-guide RNA (sgRNA) was designed to target Exon7 of Xylt1 within the xylosyltransferase domain as indicated by the red arrow. The sgRNA sequence is highlighted in green. B Overlay of phase-contrast images with fluorescent images of transfected C6 cells with either the control or the pre-constructed CRISPR/Cas9 + sgRNA plasmid which uses GFP as a selection marker to enrich the cell population with higher levels of Cas9 and sgRNA expressions. Scale bar = 100 μm. C Quantification of heparan sulfate (HS) expression by flow cytometry of non-edited C6 (C6), clone 5 (CL5), or clone 3 (CL3), showing highest knockdown for CL5. Shaded histogram: negative control; black curve: parental C6 cells; red curve: either C6 single-cell clone (CL) 5 or 3, as indicated. Xylt1 gene knockdown in clone 5 (CL5) was verified by both quantitative reverse transcription-polymerase reaction (RT-qPCR) (D), where * indicates p < 0.05 between different test conditions, and western blot analysis using Xylt1 antibody (E). F The relative uptake of tau in C6 parental cells (filled circles) compared with CL5 cells (filled triangles) was quantified by flow cytometry after a 30-min incubation at the indicated concentrations. Error bars reflect the standard error, where * represents p < 0.05 for CL5 cells compared with parental (n = 2 independent biological replicates performed in duplicate)

Fig. 5

The role of HSPGs in regulating the immune response of glial cells to tau endocytosis is mediated by intracellular ERK1/2 signaling. A C6 cells (i and ii) and HSPG-deficient (Xylt-1-) CL5 cells (iii) were incubated with different tau variants for 24 h. RNA was isolated and then subjected to quantitative reverse transcription-polymerase reaction (RT-qPCR) for relative mRNA expression quantification of IL-6 (open bars) and TNF-α (darkly shaded bars). In A.ii, C6 cells were incubated with 1 μM VPR-0N4R along with MEK-ERK1/2 inhibitor or vehicle for 24 h. RNA was extracted and the relative mRNA levels of IL-1β (lightly shaded bars in ii), IL-6, and TNF-α were determined by RT-qPCR. *p < 0.05 between different test conditions; n = 2 independent biological samples performed in replicates. B and C Cell lysates were analyzed by western blotting for p-ERK1/2, total ERK (t-ERK), and β-actin in C6 (B.i) and CL5 (C.i), with a representative western blot shown. Quantification of p-ERK/β-actin ratios in C6 (B.ii) and CL5 (C.ii) from three biological replicates. Dashed line serves as a guide to the eye (*p < 0.05 between different test conditions, ns = not significant; n = 3)

Fig. 6

HSPGs are relevant to LRP-mediated tau endocytosis. A Reduction of LRP1 mRNA was observed for C6 cells but not for CL5 (Xylt1-) after incubation with 1 μM VPR (darkly shaded bars) for 24 h, as analyzed by RT-qPCR (n = 2, *p < 0.05 between different conditions). B Rat C6 glioma cells were transiently transfected with non-targeting siRNA (NC; open bars) or LRP1-specific siRNA1, siRNA2, and siRNA3 for 48 h. LRP1 knockdown was then confirmed by RT-PCR analysis. Darkly shaded bars indicate LRP1-specific siRNA, compared with NC-siRNA (open bars) (n = 2, *p < 0.05 between different conditions). C.i The propensity of AlexaFluor488-labeled monomeric VPR-0N4R (VPR-AF488, green) uptake was examined in C6 cells post-transfection with non-targeting siRNA (NC) or LRP1-specific siRNA3 at 1 μM for 30 min. Scale bar = 100 μm. C.ii Flow cytometry histogram of VPR-AF488 uptake in NC siRNA cells (black curve) compared with LRP1-specific siRNA3 (red curve) shows quantitative decrease in tau uptake following knockdown. Gray filled curve shows distribution with vehicle only. Rat C6 glioma cells were transiently transfected with either non-targeting siRNA (NC) or LRP1-specific siRNA3 for 48, followed by incubation with 1 μM VPR-AF488 or vehicle for 24 h. D RT-qPCR analysis was performed to detect the expression level of IL-6 in C6 cells post-transfection as in C, with β-actin used as an internal control. Open bars, NC-siRNA transfected; gray shaded bars, LRP1-siRNA3 (n = 2, *p < 0.05 between different conditions) E Cell lysates were then analyzed by immunoblotting (i) against p-ERK1/2, total ERK (t-ERK), and β-actin, followed by quantitation (ii) of p-ERK/β-actin ratios from a representative western blot experiment (n = 3, *p < 0.05 between different test conditions)

Fig. 7

Rat primary astrocytes readily take up tau in an HPSG-dependent manner. A The amount of VPR-0N4R labeled with AlexaFluor488 (+ VPR, green) internalized by primary astrocytes increases over time from 1 to 6 h, while the presence of heparin has attenuated the tau uptake rate in astrocytes, as observed by fluorescence microscopy. Scale bar = 100 μm B Representative fluorescent images of GFAP (red) and Hoechst (blue) for primary astrocytes under different conditions, as indicated, where VPR indicates the addition of VPR-488 with (middle panel) or without (bottom panel) heparin to the cell as observed via fluorescence microscopy. Scale bar = 100 μm. C To determine population behavior, VPR-AF488 endocytosed by primary astrocytes was determined by flow cytometry with and without heparin addition. Grey filled line: negative control, black line: cells without heparin, red line: cells with heparin. D The propensity of VPR-0N4R uptake was quantified in primary astrocytes by flow cytometry (n = 2) E An RT-qPCR analysis was performed to detect the expression levels of IL-1β (lightly shaded bars), IL-6 (open bars), and TNF-α (filled bars) in primary astrocytes affected by incubation with VPR and heparin, as indicated. β-Actin was used as an internal control. *p < 0.05 between different test conditions; n = 2

Fig. 8

Schematic illustration of the intracellular responses affected by monomeric tau endocytosis. Monomeric tau is rapidly internalized by both neuronal and glial cells, mediated by the actin-dependent macropinocytosis pathway. In addition, an important role of HSPG has been demonstrated in regulating inflammatory gene expression, including IL-6 and IL-1β, partially via ERK1/2 activation following tau uptake. LRP1 modulates IL-6 responses to tau endocytosis, and this interaction is HSPG-dependent but not ERK-dependent. Solid arrows represent the pathways that have been demonstrated in this study. Dashed arrows represent the pathways speculated to be responsible for subsequent cellular responses based on other relevant publications, as described in the “Discussion” section