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. 2025 Jan;169(1):e16294.
doi: 10.1111/jnc.16294.

Tubulin-Binding Region Modulates Cholesterol-Triggered Aggregation of Tau Proteins

Abid Ali  1 , Mikhail Matveyenka  1 , Davis N Pickett  1 , Axell Rodriguez  1 , Dmitry Kurouski  1
Affiliations

Tubulin-Binding Region Modulates Cholesterol-Triggered Aggregation of Tau Proteins

Abid Ali et al. J Neurochem. 2025 Jan.

Abstract

A hallmark of Alzheimer disease (AD) and tauopathies, severe neurodegenerative diseases, is the progressive aggregation of Tau, also known as microtubule-associated Tau protein. Full-length Tau1-441, also known as 2N4R, contains two N-terminal inserts that bind to tubulin. This facilitates the self-assembly of tubulin simultaneously enhancing stability of cell microtubules. Other Tau isoforms have one (1N4R) or zero (0N4R) N-terminal inserts, which makes 2N4R Tau more and 0N4R less effective in promoting microtubule self-assembly. A growing body of evidence indicates that lipids can alter the aggregation rate of Tau isoforms. However, the role of N-terminal inserts in Tau-lipid interactions remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which N-terminal inserts alter interactions of Tau isoforms with cholesterol, one of the most important lipids in plasma membranes. Our results showed that 2 N insert prevents amyloid-driven aggregation of Tau at the physiological concentration of cholesterol, while the absence of this N-terminal repeat (1N4R and 0N4R Tau) resulted in the self-assembly of Tau into toxic amyloid fibrils. We also found that the presence of cholesterol in the lipid bilayers caused a significant increase in the cytotoxicity of 1N4R and 0N4R Tau to neurons. This effect was not observed for 2N4R Tau fibrils formed in the presence of lipid membranes with low, physiological, and elevated concentrations of cholesterol. Using molecular assays, we found that Tau aggregates primarily exert cytotoxicity by damaging cell endosomes, endoplasmic reticulum, and mitochondria.

Keywords: Tau; amyloids; cholesterol; fibrils.

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Conflict of interest statement

The authors declare no competing financial interests

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CONFLICT OF INTEREST STATEMENT

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Figures

Figure 1.
Figure 1.
Cholesterol alters the rate of Tau aggregation. ThT kinetics (top row) and corresponding histograms (bottom row) of tlag and t1/2 for 0N4R (A), 1N4R (B) and 2N4R (C) aggregation in the lipid-free environment (red), as well as in the presence of 10:90 Cho:PC (light blue), 45:55 Cho:PC (blue), 60:40 Cho:PC (purple) and PC (orange). Each curve shown in panels A-C is the average of three independent replicates (n=3). The graphical data are presented as the mean ± SEM. According to one-way ANOVA followed by Tukey’s HSD test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 2.
Figure 2.
Cholesterol changes morphology of protein aggregates. AFM images of amyloid aggregates formed by different Tau isoforms in the lipid free environment (0N4R, 1N4R, and 2N4R), as well as in the presence of PC, 10:90 Cho:PC, 45:55 Cho:PC, and 60:40 Cho:PC.
Figure 3.
Figure 3.
Cholesterol changes the height of protein aggregates. Histograms of height distribution of amyloid aggregates formed by different Tau isoforms in the lipid free environment (red), as well as in the presence of 10:90 Cho:PC (light blue), 45:55 Cho:PC (blue), 60:40 Cho:PC (purple) and PC (orange).
Figure 4.
Figure 4.
Secondary structure of protein aggregates. FTIR (top panels) and CD (bottom panels) spectra acquired from amyloid aggregates formed by 0N4R (A), 1N4R (B) and 2N4R (C) in the lipid free environment (red), as well as in the presence of 10:90 Cho:PC (light blue), 45:55 Cho:PC (blue), 60:40 Cho:PC (purple) and PC (orange). Each curve shown in panels A-C is the average of three sample replicates (n=3).
Figure 5.
Figure 5.
AFM-IR spectra (left) and histograms (right) showing the amount of parallel β-sheet (blue), α-helix, random coil and β-turn (orange), as well as anti-parallel β-sheet (grey) in the secondary structure of 0N4R (A), 1N4R (B), and 2N4R (C) Tau aggregates grown in the lipid free environment (red), as well as in the presence of 10:90 Cho:PC (light blue), 45:55 Cho:PC (blue), 60:40 Cho:PC (purple) and PC (orange). Each curve shown in panels A-C is the average of three sample replicates (n=3). The graphical data are presented as the mean ± SEM. According to one-way ANOVA followed by Tukey’s HSD test, * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. NS shows the absence of statistically significant differences.
Figure 6.
Figure 6.
Cholesterol alters toxicity of Tau fibrils. Histograms of LDH assay revealing cytotoxicity of 0N4R (A), 1N4R (B) and 2N4R (C) Tau fibrils grown in the lipid free environment and in the presence of LUVs composed of 10:90 Cho:PC, 45:55 Cho:PC, 60:40 Cho:PC, and PC, as well as lipids themselves (D). Each curve shown in panels A-C is the average of three biological replicates (n=3); each well contained 50,000 cells. The graphical data are presented as the mean ± SEM. According to one-way ANOVA followed by Tukey’s HSD test, black asterisk (*) show differences in the cytotoxicity between samples and control (ctr), whereas green asterisk show differences in the cytotoxicity between 0N4R, 1N4R and 2N4R grown in the lipid free environment and in the presence of LUVs; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS- non-significant difference.
Figure 7.
Figure 7.
Molecular mechanism of amyloid toxicity. Histograms of fluorescent puncta per cell observed in N27 rat dopaminergic cells after their incubation with 0N4R, 1N4R and 2N4R Tau fibrils grown in the lipid free environment and in the presence of LUVs composed of 10:90 Cho:PC, 45:55 Cho:PC, 60:40 Cho:PC, and PC, as well as lipids themselves. For each of the presented results, at least 15 individual images were analyzed. Each curve shown in panels A-C is the average of three biological replicates (n=3); each well contained 50,000 cells. The graphical data are presented as the mean ± SEM. According to one-way ANOVA followed by Tukey’s HSD test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS- non-significant difference.
Figure 8.
Figure 8.
Cholesterol alters the extent to which Tau aggregates cause UPR in ER of N27 rat dopaminergic neurons. Histograms of relative expression of PERK, ATF6, and XBP1 in N27 rat dopaminergic neurons after cell exposition to 0N4R, 1N4R and 2N4R Tau fibrils grown in the lipid free environment and in the presence of LUVs composed of 10:90 Cho:PC, 45:55 Cho:PC, 60:40 Cho:PC, and PC. Each curve shown in panels A-C is the average of three biological replicates (n=3); each well contained 50,000 cells.
Figure 9.
Figure 9.
Cholesterol alters the extent to which Tau aggregates damage mitochondria in N27 rat dopaminergic neurons. Histograms of JC-1 assay revealing mitochondria damage exerted by 0N4R (A), 1N4R (B) and 2N4R (C) fibrils grown in the lipid free environment and in the presence of LUVs composed of 10:90 Cho:PC, 45:55 Cho:PC, 60:40 Cho:PC, and PC, as well as lipids themselves (D). Each curve shown in panels A-C is the average of three biological replicates (n=3); each well contained 50,000 cells. The graphical data are presented as the mean ± SEM. According to one-way ANOVA followed by Tukey’s HSD test, black asterisks (*) show differences in the cytotoxicity between samples and control (ctr), whereas green asterisks show differences in the cytotoxicity between 0N4R, 1N4R and 2N4R grown in the lipid free environment and in the presence of LUVs; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS- non-significant difference.
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