Three- and four-repeat Tau coassemble into heterogeneous filaments: an implication for Alzheimer disease

Abstract

Tau filaments are the pathological hallmark of numerous neurodegenerative diseases including Alzheimer disease, Pick disease, and progressive supranuclear palsy. In the adult human brain, six isoforms are expressed that differ by the presence or absence of the second of four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half of the isoforms have four repeats (4R tau). Tauopathies can be characterized based on the isoform composition of their filaments. Alzheimer disease filamentous inclusions contain all isoforms. Pick disease filaments contain 3R tau. Progressive supranuclear palsy filaments contain 4R tau. Here, we used site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy to obtain structural insights into these filaments. We find that filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of β-strands. This structure is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassemble into heterogeneous filaments. These filaments share the highly ordered core in the third repeat; however, they differ in their overall composition. Our findings indicate that at least three distinct types of filaments exist: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. These results suggest that individual filaments found in Alzheimer disease are structurally distinct from those in the 3R and 4R tauopathies.

FIGURE 1.

Bar diagrams of tau isoforms and constructs. A, tau isoforms are defined by the presence or absence of two inserts in the N-terminal half (striped) and the inclusion or exclusion of the second microtubule binding repeat in the C-terminal half (marked as 2). Isoform sizes range from 352 amino acids (aa) to 441 amino acids and are provided on the right. B, the truncated constructs K18 and K19 contain the repeats (four for K18 and three for K19) plus three additional residues at the C termini.

FIGURE 2.

Structural analysis of htau40 spin-labeled at position 322. A, labeling of a cysteine in the protein with the paramagnetic label (1-oxy-2,2,5,5-tetramethyl-d-pyrroline-Δ3-methyl)-methanethiosulfonate results in the side chain R1. B, monomeric tau, labeled at position 322, produces a spectrum with three sharp lines (left) characteristic for random structure (left inset). Formation of filaments results in a spectrum with a single line indicative of crystal-type order with parallel, in-register arrangement of β-strands (right inset). C, negative stain electron microscopy reveals twisted filaments with 15–20 Å diameter. Bar = 100 nm.

FIGURE 3.

EPR analysis of spin-labeled htau40 filaments. A, residues 321–358 were individually replaced by the side chain R1. All spectra were taken at 150 G and normalized to the same number of spins. Note that for completeness, spectrum C322R1 is reproduced from Fig. 2. The dotted line between the left and middle columns marks the boundary between repeats 3 and 4. B, the amplitudes of all EPR spectra are plotted against respective residue numbers. All amplitudes are normalized to spectrum F346R1, which has the highest amplitude.

FIGURE 4.

EPR spectra of filaments from htau23, K18, and K19. Residues in the third repeat of htau23, K18, and K19 were replaced by the paramagnetic side chain R1. Spectra of all filaments were recorded at 150 G scan width and normalized to the same number of spins.

FIGURE 5.

Coassembly of 3R and 4R tau. A, EPR spectra of tau filaments coassembled from K18R1 (paramagnetic) and K19R1′ (nonparamagnetic). The following mole percentages were used: 1) 10% K18R1/90% K19R1′ (red trace), 2) 60% K18R1/40% K19R1′ (green trace), and 3) 100% K18R1 (black trace). In both constructs, labels were attached to position 310. B, the amplitudes of the central lines of EPR spectra in A together with those of additional dilutions are plotted as a function of the mole percent of K18R1. Regions in the plot in which spectra are dominated by no spin interactions, by dipolar coupling, or by spin exchange are interpreted in terms of different β-strand combinations (insets I–III). K18R1 = green arrow, yellow dot; K19R1′ = red arrow, black dot. C, spectrum of filaments formed from 50% K18R1 and 50% K19R1 (labels attached to position 310). The inset provides a structural interpretation. K18R1 = green arrow, yellow dot; K19R1 = red arrow, yellow dot. D, electron micrograph of heterogeneous filaments (K18R1/K19R1). Bar = 100 nm. E, EPR spectra of filaments formed from htau40R1 and htau23R1′ (all labels attached at position 310). Dilutions have same color coding as in A. F, amplitudes of central lines from filaments formed at different dilutions of htau40R1 and htau23R1′. G, spectrum of filaments formed from 50% htau40R1 and 50% htau23R1. All spectra were taken at 150 G and normalized to the same number of spins. H, electron micrograph of htau40R1/htau23R1 filaments. Bar = 400 nm.

FIGURE 6.

Types of tau filaments. 3R tau (red) and 4R tau (green) assemble into at least three different types of filaments: 1) homogeneous filaments of 3R tau, 2) homogeneous filaments of 4R tau, and 3) heterogeneous filaments of 3R and 4R tau. All filaments have in common a parallel, in-register arrangement of β-strands in repeat 3.