Understanding the kinetic roles of the inducer heparin and of rod-like protofibrils during amyloid fibril formation by Tau protein

Abstract

The aggregation of the natively disordered protein, Tau, to form lesions called neurofibrillary tangles is a characteristic feature of several neurodegenerative tauopathies. The polyanion, heparin, is commonly used as an inducer in studies of Tau aggregation in vitro, but there is surprisingly no comprehensive model describing, quantitatively, all aspects of the heparin-induced aggregation reaction. In this study, rate constants and extents of fibril formation by the four repeat domain of Tau (Tau4RD) have been reproducibly determined over a full range of heparin and protein concentrations. The kinetic role of heparin in the nucleation-dependent fibril formation reaction is shown to be limited to participation in the initial rate-limiting steps; a single heparin molecule binds two Tau4RD molecules, forming an aggregation-competent protein dimer, which then serves as a building block for further fibril growth. Importantly, the minimal kinetic model that is proposed can quantitatively account for the characteristic bell-shaped dependence of the aggregation kinetics on the stoichiometry of protein to heparin. Very importantly, this study also identifies for the first time short and thin, rod-like protofibrils that are populated transiently, early during the time course of fibril formation. The identification of these protofibrils as bona fide off-pathway species has implications for the development of therapies for tauopathies based on driving fibril formation as a means of protecting the cell from smaller, putatively toxic aggregates.

FIGURE 1.

Formation of amyloid fibrils by Tau4RD in the presence of heparin monitored by multiple probes. a, ThT fluorescence monitored kinetics of 50 μm Tau4RD in the presence of 37.5 μm heparin at 37 °C in 25 mm Tris buffer, 50 mm NaCl, 1 mm DTT, pH 7. The inset shows a plot of ThT fluorescence-monitored kinetics in 25 mm Tris buffer, 50 mm NaCl, 1 mm DTT, pH 7.5. The continuous line through the data points in the main figure and in the inset is a least square fit to a single-exponential equation. The error bars represent the spread in the data calculated from two or more independent experiments and across different protein preparations. a.u., arbitrary units. b, the extent of amyloid fibril formation at the end of the aggregation reaction is linear with respect to protein concentration as monitored by ThT fluorescence (■), light scattering at 800 nm (○), and a sedimentation assay (▴). The continuous line through the data points was drawn by inspection.

FIGURE 2.

Tau4RD forms a mixture of structures including characteristic PHFs in the presence of heparin in Tris buffer, pH 7, at 37 °C. a–c, AFM images in the amplitude format of PHFs formed by 50 μm Tau4RD in the presence of 37.5 μm heparin display the expected cross-over repeat of ∼80 nm as indicated by the white arrows. The green arrow in c point to a straight filament. The blue arrow in c points to an instance of a single filament emerging from a multi-stranded fibril. d, negatively stained TEM image of fibrils formed in the same conditions. The white arrows point to a PHF, and the green arrow points to a straight filament. The scale bar in all images corresponds to 200 nm. The Z scale for all AFM images corresponds to 8 nm.

FIGURE 3.

Formation of protofibrils and fibrils by 50 μm Tau4RD in the presence of 37.5 μm heparin in Tris buffer, pH 7, at 37 °C. a, an AFM image demonstrates the presence of short, rod-like protofibrils at 1 h of aggregation. The inset shows the beaded appearance of the protofibrils. The scale bar for the image corresponds to 600 nm, whereas that for the inset corresponds to 200 nm. The Z scale for the image corresponds to 8 nm, and that for the inset corresponds to 15 nm. b, distribution of heights of protofibrils is shown. The mean height calculated from the fit is 2.6 ± 0.5 nm. The inset shows the length distribution of the protofibrils is shown. The mean length calculated from the fit is 51 ± 16 nm. The solid line in both the image and the inset represents a fit to a Gaussian equation. c, an AFM image demonstrates the presence of fibrils at 1 h of aggregation. The scale bar for the image corresponds to 600 nm. The Z scale for the image corresponds to 50 nm. d, distribution of heights of fibrils is shown. The mean height calculated from the fit is 10.2 ± 3.9 nm. The solid line represents a fit to a Gaussian equation.

FIGURE 4.

The appearance and disappearance of protofibrils when 50 μm Tau4RD is aggregated in the presence of 37.5 μm heparin in Tris buffer, pH 7, at 37 °C. a–c, AFM images of protofibrils at 20 min, 40 min, and 1 h of aggregation are shown. d, an AFM image demonstrates the absence of protofibrils at 2 h of aggregation. The inset is an image demonstrating the presence of fibrils at the same time point. The scale bar in all images as well as the inset corresponds to 600 nm. The Z scale for the main images corresponds to 8 nm, and the color scale applies to the main images. The Z scale for the inset corresponds to 50 nm.

FIGURE 5.

Dependence on heparin concentration of ThT fluorescence-monitored kinetics in Tris buffer, pH 7. a, kinetics of fibril formation by 50 μm Tau4RD in the presence of 8.3 μm (○), 16.6 μm (△), and 56.2 μm (♢) heparin are shown. The continuous lines through the data points are least-squares fits to a single-exponential equation. a.u., arbitrary units. b, the apparent rate constant of ThT-monitored kinetics is plotted against heparin concentration. c, the amplitude of change in ThT fluorescence is plotted against heparin concentration. The error bars in a–c represent the spread in the data calculated from two or more independent experiments and across protein preparations. The continuous lines through the data points in b and c were drawn by inspection to guide the eye.

FIGURE 6.

Dependence on protein concentration of ThT fluorescence-monitored kinetics in Tris buffer, pH 7. a, kinetics of fibril formation by 10 μm (○), 25 μm (△) and 50 μm Tau4RD (♢) in the presence of 37.5 μm heparin are shown. The continuous lines through the data points represent least-squares fits to a single-exponential equation. a.u., arbitrary units. b, the apparent rate constant of ThT-monitored kinetics is plotted against protein concentration. The continuous line through the data points was drawn by inspection. c, the amplitude of the change in ThT fluorescence when protein is added to 5 μm heparin (●) and when protein is added to 37.5 μm heparin (□) as a function of the molar ratio of protein:heparin is shown. The straight line through the data points is a least-square fit. The continuous line through the data points was drawn by inspection to guide the eye. The error bars in a–c represent the spread in the data calculated from two or more independent experiments.

FIGURE 7.

Dependence of the apparent rate constant of ThT fluorescence-monitored kinetics on the molar ratio of protein to heparin. The apparent rate constant of fibril formation is plotted as a function of molar ratio from aggregation experiments wherein the protein concentration is held constant and the heparin concentration is varied (white symbols) and wherein the heparin concentration is held constant and the protein concentration is varied (black symbols).

FIGURE 8.

Model for the formation of fibrils by the repeat domain of Tau in the presence of heparin. Protein binds heparin and undergoes a conformational change to form an on-pathway intermediate, PH. When a second protein molecule binds the same heparin molecule, an aggregation-competent dimer (PHP) is formed. Elongation leading to fibril formation occurs by the process of monomer addition to this building block. An off-pathway intermediate in the form of a tight binding, P*H complex also forms and modulates the aggregation kinetics. It appears that this off-pathway intermediate can aggregate to form the rod-like protofibrils that are observed to accumulate transiently.