Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-beta structure

Abstract

Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimer's disease, Down's syndrome, and various dementing tauopathies. In Alzheimer's disease and Down's syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-beta structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-beta structure.

Fig. 1.

Electron micrograph of a negatively stained preparation of abnormal tau-containing filaments from the frontal cortex of a patient with Down's syndrome. Examples of the characteristic PHF (P) and SF (S) are indicated. (Bar = 100 nm.)

Fig. 2.

Selected area electron diffraction patterns from an unstained frozen preparation of tau filaments from a patient with Down's syndrome. A, C, E, and G show the areas from which the corresponding diffraction patterns B, D, F, and H were recorded. In A, the clump of filaments is partially dehydrated but shows clearly the morphology of PHFs. In C, the filaments are fully embedded in ice. In E, the scattering arises predominantly from one PHF and in G from one SF. The diffraction patterns (B, D, F, and H), which have been aligned relative to the images (A, C, E, and G) and which have had the strong central scattering removed, all show a clear arc at 0.47-nm spacing (arrowed) in the axial direction of the filaments, indicative of cross-β structure.

Fig. 3.

Selected area electron diffraction patterns from tau filaments assembled in vitro. A, C, and E show images of areas from which diffraction patterns B, D, and F were recorded. (A and B) Wild-type four-repeat human tau. (C and D) P301S mutant 4-repeat tau. (E and F) K257T mutant three-repeat tau. The diffraction patterns show strong scattering in the 0.47-nm region with partial orientation indicative of cross-β structure.

Fig. 4.

X-ray diffraction pattern from a partially aligned fiber of tau filaments assembled in vitro. The filaments were assembled from P301S mutant four-repeat human tau. There is strong meridional scattering at a spacing of 0.47-nm and a weak ring, shown with enhanced contrast in the central inset, at a spacing of 1.3 nm.

Fig. 5.

FTIR spectra of soluble and filamentous P301S mutant four-repeat human tau. The spectrum of soluble tau (dashed line) shows no indication of β-structure, but the filamentous sample (solid line) shows a strong peak at 1,628 cm^-1, indicating that the assembled filaments have significant β-structure.