Alzheimer disease-associated tau post-translational modification mimics impact tau propagation and uptake

Abstract

As Alzheimer disease (AD) progresses, pathological tau spreads by cell-to-cell propagation of tau. This study aims to elucidate the impact of AD-associated post-translational modifications of tau-on-tau propagation. Tau propagation reporter constructs distinguishing donor cells from recipient cells were developed, and additional constructs were made with tau residues mutated from serine or threonine to aspartate to mimic the negative charge of a phosphorylation and/or from lysine to glutamine to mimic the charge-neutralizing effect of acetylation. Flow cytometry was used to quantify donor and recipient cells. This revealed that the mutations generally tended to reduce tau propagation compared to wildtype tau. Recombinant tau containing either wildtype or posttranslational modification mimicking mutations were used to treat Chinese hamster ovary cells or human induced pluripotent stem cell-derived neurons to quantify tau uptake, revealing that the mutations generally resulted in reduced uptake compared to wildtype tau. Surface plasmon resonance revealed that the mutations had a reduced affinity for lipoprotein receptor-related protein 1 (LRP1), a tau uptake receptor, compared to wildtype tau. Overall, these results suggest that AD-associated posttranslational modification mimicking mutations reduce the cell-to-cell propagation of tau by reducing tau uptake by recipient cells, which may be in part due to reduced binding affinity to LRP1.

Keywords: Alzheimer disease; acetylation; low-density lipoprotein receptor-related protein 1; phosphorylation; post-translational modification; propagation; tau.

Figure 1.

A cellular model of tau propagation. A model of tau propagation was developed for use in CHO cell culture. (A) Schematic of tau propagation construct expressing a polypeptide containing fluorescent protein TagRFP-T, a self-cleaving 2A peptide sequence, and a tau 2N4R tau sequence with or without an amino-terminal V5 tag, which is then cleaved at the 2A site into separate TagRFP-T and tau fragments. Created with biorender.com. (B) Schematic of the tau propagation experiment in the cell culture model. A transfected donor cell expresses both the TagRFP-T (magenta) and tau (green) proteins. The tau is propagated to an untransfected recipient cell, which is identified as containing tau but not TagRFP-T. Created with biorender.com. (C) Experimental overview of the tau propagation assay with either immunocytochemistry or flow cytometry as readouts. (D) Immunocytochemistry of CHO cells transfected with a tau propagation construct. Donor cells are identified by staining for TagRFP-T and V5 (example shown by magenta arrow). Recipient cells are identified by staining for V5 only (green arrow). Scale bar: 50 µm. (E) Example flow cytometry dot plot with green fluorescence intensity (V5) on the x-axis and red fluorescence intensity (TagRFP-T) on the y-axis. Each dot represents a cell. Quadrants corresponding to donor (green⁺red⁺) and recipient cells (green⁺red^-) are indicated. (F) Comparison of tau propagation constructs with or without the amino-terminal V5 tag using flow cytometry readout demonstrating no difference with or without a V5-tag. Data analyzed by unpaired t test.

Figure 2.

Alzheimer disease-associated tau PTM mimics influence tau propagation. Tau propagation constructs containing AD-associated tau PTM mimics or the wildtype tau sequence were compared in the cellular model of tau propagation using a flow cytometry readout. (A) Diagram of mutations in the 8D2Q construct that mimics the PTMs found in LMW tau in AD patients. Diagram made with IBS. (B) Diagram of mutations in the 20D3Q construct that mimics the PTMs found in HMW tau in AD patients. Diagram made with IBS. (C) Comparison of wildtype tau, AD-associated tau PTM mimics, or mutations that impair AD-associated PTMs demonstrating the tau PTM mimics reduced tau propagation, while the mutations that impair AD-associated PTMs enhanced tau propagation compared to wildtype. Data analyzed by one-way ANOVA. (D) Comparison of wildtype tau and individual residue PTM mimics corresponding to the 8D2Q construct. The mutations with significantly increased or decreased tau propagation relative to wildtype are demonstrated with the corresponding P-value. Data analyzed by one-way ANOVA.

Figure 3.

Alzheimer disease-associated tau PTM mimics do not impact tau release from donor cells. Tau propagation constructs AD-associated tau PTM mimics or the wildtype tau sequence were compared in the cellular model of tau propagation with a tau immunoassay as the readout. (A) Schematic of the experimental design. (B) Comparison of wildtype tau or AD-associated tau PTM mimics demonstrating no significant differences in release from wildtype or mutant constructs. Data analyzed by one-way ANOVA.

Figure 4.

Alzheimer disease-associated tau PTM mimics reduce tau uptake in CHO cells. Tau uptake was assessed in CHO cells using an immunocytochemistry assay. (A) Schematic of the experimental design. (B) Diagram of protein expression construct open reading frame used to express wildtype or mutant tau proteins. (C) Representative images of immunocytochemistry of CHO cells treated with wildtype or AD-associate tau PTM mimic proteins. Scale bar: 50 µm. The green arrows indicate areas of qualitatively bright signal in the HiBiT (tau) channel. (D) Comparison of tau uptake of wildtype or AD-associate tau PTM mimic proteins in CHO cells demonstrating a trend toward reduced uptake of mutant proteins compared to wildtype. Data analyzed by Kruskal-Wallis test.

Figure 5.

Alzheimer disease-associated tau PTM mimics reduce tau uptake in iPSC-derived neurons. Tau uptake was assessed in iPSC-derived neurons using an immunocytochemistry assay. (A) Representative images of immunocytochemistry of iPSC-derived neurons treated with wildtype or AD-associate tau PTM mimic proteins. Scale bar: 10 µm. The green arrows indicate areas of qualitatively bright signal in the HiBiT (tau) channel. (B) Comparison of tau uptake of wildtype or AD-associate tau PTM mimic proteins in iPSC-derived neurons demonstrating significantly reduced uptake of mutant proteins compared to wildtype. Data from each iPSC line are represented by a different symbol. Data analyzed by one-way ANOVA.

Figure 6.

Alzheimer disease-associated tau PTM mimics have reduced affinity for LRP1. Determination of the affinity of AD-associated tau PTM mimic proteins for LRP1. (A) Diagram of protein expression construct open reading frame used to express wildtype or mutant tau proteins. (B) Gel electrophoresis of expressed proteins with or without the his-tag removed by 3C cleavage stained with Coomassie stain. (C) Western blot of expressed proteins with or without the his-tag removed by 3C cleavage using an anti-tau antibody. (D) Western blot of expressed proteins with or without the his-tag removed by 3C cleavage using an anti-his-tag antibody. (E) Representative images of a single surface plasmon resonance experiment. (F) Nonlinear regression analysis of R_max vs concentration of surface plasmon resonance experiments demonstrating reduced affinity of tau mutants for LRP1 compared to wildtype tau.