Tubulin-binding region alters tau-lipid interactions and changes toxicity of tau fibrils formed in the presence of phosphatidylserine lipids

Abstract

Alzheimer's disease is the fastest-growing neurodegenerative disease that affects over six million Americans. The abnormal aggregation of amyloid β peptide and Tau protein is the expected molecular cause of the loss of neurons in brains of AD patients. A growing body of evidence indicates that lipids can alter the aggregation rate of amyloid β peptide and modify the toxicity of amyloid β aggregates. However, the role of lipids in Tau aggregation remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which phospatidylserine (PS) altered the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N terminal inserts that enhance the binding of Tau to tubulin. We found that the length and saturation of fatty acids (FAs) in PS altered the aggregation rate of 2N4R isoform, while no changes in the aggregation rate of 1N4R were observed. These results indicate that N terminal inserts play an important role in protein-lipid interactions. We also found that PS could change the toxicity of 1N4R and 2N4R Tau fibrils, as well as alter molecular mechanisms by which these aggregates exert cytotoxicity to neurons. Finally, we found that although Tau fibrils formed in the presence and absence of PS endocytosed by cells, only fibril species that were formed in the presence of PS exert strong impairment of the cell mitochondria.

Keywords: 1N4R tau; 2N4R tau; AFM‐IR; fibrils; oligomers; phosphatidylserine; toxicity.

SCHEME 1

The amino acid sequence of 2N4R Tau (top) with basic amino acids shown in blue, polar in green, acidic in red and nonpolar in brown. The N2 region missing in 1N4R is shown with a red frame. Molecular structures (bottom) of POPS, DOPS, DMPS and DSPS.

FIGURE 1

Kinetics of protein aggregation. ThT kinetics (a and c) together with corresponding histograms of t _lag and t _1/2 (b and d) of 2N4R and 1N4R aggregation in lipid‐free environment (red), as well as in the presence of POPS (yellow), DOPS (green), DMPS (blue) and DSPS (purple). Gray vertical lines indicate t _lag of 2N4R and 1N4R Tau aggregation. According to one‐way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS shows the absence of statistically significant differences.

FIGURE 2

Morphology of protein aggregates. AFM images (a) together with the corresponding height histograms (b) of 2N4R and 1N4R aggregates formed in lipid‐free environment (red), as well as in the presence of POPS (yellow), DOPS (green), DMPS (blue) and DSPS (purple).

FIGURE 3

Structural characterization of amyloids. CD (a‐b) and AFM‐IR (c and e) spectra of 2N4R and 1N4R aggregation in lipid‐free environment (red), as well as in the presence of POPS (yellow), DOPS (green), DMPS (blue) and DSPS (purple). Histograms (d and f) that summarize distribution of parallel β‐sheet, α‐helix, random coil and β‐turn, as well as anti‐parallel β‐sheet in the secondary structure of analyzed protein aggregates. According to one‐way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS shows the absence of statistically significant differences.

FIGURE 4

Toxicity of Tau aggregates. Histograms of LDH (a) and ROS (b) assays reveal differences between cell toxicity of 2N4R, 2N4R:POPS, 2N4R:DOPS, 2N4R:DMPS, and 2N4R:DSPS (left) and 1N4R, 1N4R:POPS, 1N4R:DOPS, 1N4R:DMPS, and 1N4R:DSPS (right). Black asterisks (*) show significance level of differences between protein aggregates and the control; blue and green asterisks show significance level of difference between protein aggregates formed in the lipid‐free environment and in the presence of lipids. According to one‐way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS shows absence of statistical significance. Fluorescence microscopy images (c) show ROS response (red) in the exposed to amyloids N27 cells. The blue fluorescence represents the nuclear fluorescence dye.

FIGURE 5

Molecular mechanism of amyloid toxicity. Schematic (a) illustration of endosomal damage induced by Tau aggregates that activate Chmp1b, Gal3, and TFEB factors involved in endosomal repair, clearance of damaged endosomes by autophagy, and de novo biogenesis of organelles, respectively. Histograms (b) of fluorescent puncta per cell, as well as the sum of pixels from fluorescent puncta observed in HEK 293 T cells after their incubation with 2N4R and 1N4R Tau aggregates grown in the presence of POPS, DOPS, DMPS and DSPS, as well as in the lipid‐free environment. For each of the presented results, at least 15 individual images were analyzed. Schematic (c) illustration of mitochondrial unfolded protein response (mt‐UPR) that activates CLPP, YME1A, UBL‐5, PINK1, and PRKN kinases. Heat‐map (d) of relative mt‐UPR gene expression in N27 rat dopaminergic cells exposed to 2N4R, 2N4R:POPS, 2N4R:DOPS, 2N4R:DMPS, 2N4R:DSPS, 1N4R, 1N4R:POPS, 1N4R:DOPS, 1N4R:DMPS, and 1N4R:DSPS aggregates for 24 h showing a suppression (green), activation (red) or no changes (yellow) in the mt‐UPR gene expression.