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By Aurelio J. Dregni, Pu Duan, Hong Xu, Lakshmi Changolkar, Nadia El Mammeri, Virginia M.-Y. Lee, and Mei Hong

Excerpt from the article published in Nature Communications 13, 2967 (2022). https://doi.org/10.1038/s41467-022-30585-0

Editor’s Highlights

• Alzheimer’s disease (AD) is defined by intracellular neurofibrillary tangles formed by the microtubule-associated protein tau and extracellular plaques formed by the β-amyloid peptide.
• Extensive biochemical data have established that misfolded tau proteins in AD and other tauopathies spread by recruiting soluble tau protein into the aggregate, and distinct isoforms might be expected to interfere with the propagation of the amyloid structure.
• In the human brain, the microtubule-associated protein tau exists in six isoforms, which are mainly distinguished by the presence or absence of the second microtubule-binding repeat, R2.
• Tau isoforms that contain four microtubule-binding repeats (R1, R2, R3, and R4) are called four-repeat (4R) tau, whereas those that contain three microtubule-binding repeats (R1, R3, and R4) are called three-repeat (3R) tau.
• The AD tau tangles contain a mixture of 4R and 3R tau isoforms, whereas other tauopathies such as corticobasal degeneration and Pick’s disease are characterized by either aggregated 4R tau or aggregated 3R tau.
• In AD tau filaments recruit 4R and 3R tau monomers with a ratio of 60:40, and with a small preference for homotypic contacts.
• The AD tau fold may have evolved over its slow replication phase to adapt to the environment of the AD brain.
• The fluent molecular mixing of both 4R and 3R tau isoforms and the preservation of the β-sheet conformation regardless of the isoform could potentially be an adaptive trait to optimize prion growth and propagation.
• The significant amplification of the morphology and toxicity of AD tau filaments shown here opens new avenues for future characterization of the molecular structure of tau in different tauopathies using a wide range of biophysical methods.
• The most relevant tauopathies are: Behavioral variant of frontotemporal dementia (bvFTD), non-fluent agrammatic variant of primary progressive aphasia (nfavPPA), semantic variant of primary-progressive aphasia (svPPA), and amnestic syndrome of hippocampal type (AS), Richardson syndrome (RS), corticobasal syndrome (CBS), primary gait freezing (PGF), cerebellar syndrome (C), primary lateral sclerosis (PLS), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and familial FTD with parkinsonism linked to MAPT (FTDP-17).

Abstract

Alzheimer’s disease (AD) is defined by intracellular neurofibrillary tangles formed by the microtubule-associated protein tau and extracellular plaques formed by the β-amyloid peptide. AD tau tangles contain a mixture of tau isoforms with either four (4R) or three (3R) microtubule-binding repeats. Here we use solid-state NMR to determine how 4R and 3R tau isoforms mix at the molecular level in AD tau aggregates. By seeding differentially isotopically labeled 4R and 3R tau monomers with AD brain-derived tau, we measured intermolecular contacts of the two isoforms. The NMR data indicate that 4R and 3R tau are well mixed in the AD-tau seeded fibrils, with a 60:40 incorporation ratio of 4R to 3R tau and a small homotypic preference. The AD-tau templated 4R tau, 3R tau, and mixed 4R and 3R tau fibrils exhibit no structural differences in the rigid β-sheet core or the mobile domains. Therefore, 4R and 3R tau are fluently recruited into the pathological fold of AD tau aggregates, which may explain the predominance of AD among neurodegenerative disorders.

Introduction

In the human brain, the microtubule-associated protein tau exists in six isoforms, which are mainly distinguished by the presence or absence of the second microtubule-binding repeat, R2 (ref. ¹). Tau isoforms that contain four microtubule-binding repeats (R1, R2, R3, and R4) are called four-repeat (4R) tau, whereas those that contain three microtubule-binding repeats (R1, R3, and R4) are called three-repeat (3R) tau. In a number of neurodegenerative diseases, these intrinsically disordered tau proteins become hyperphosphorylated and form intraneuronal aggregates. Alzheimer’s disease (AD) is defined by these tau neurofibrillary tangles as well as the extracellular plaques formed by the β-amyloid peptide^2,3. Interestingly, the AD tau tangles contain a mixture of 4R and 3R tau isoforms, whereas other tauopathies such as corticobasal degeneration and Pick’s disease are characterized by either aggregated 4R tau or aggregated 3R tau. Recent cryo-electron microscopy (cryoEM) studies^4,5,6 revealed that there is a single well-defined molecular conformation for the β-sheet core of AD tau filaments, but did not determine how 4R tau and 3R tau are mixed in the aggregate. The rigid core of the AD tau filaments spans the R3, R4, and part of the R′ repeats, which are present in both 4R and 3R tau isoforms. Another mixed-tau disease, chronic traumatic encephalopathy, also exhibits a single rigid core structure for its tau filaments⁷, with no discernible differentiation between 4R and 3R tau in the fibril. Understanding how different tau isoforms are mixed in these filaments is important, because extensive biochemical data have established that misfolded tau proteins in AD and other tauopathies spread by recruiting soluble tau protein into the aggregate, and distinct isoforms might be expected to interfere with the propagation of the amyloid structure^{8,9,10,11,12,13,14,15,16,17}. To determine how 4R and 3R tau isoforms are mixed in the neurofibrillary tangles in AD brains, here we use solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate AD-tau seeded ¹⁵N-labeled and ¹³C-labeled tau proteins. We show that the 4R and 3R tau isoforms are fluently mixed in AD tau filaments, with no detectable structural differences among pure 4R tau fibrils, pure 3R tau fibrils, and mixed 4R and 3R tau fibrils.

Results

To determine the mode of mixing of 4R and 3R tau in AD tau aggregates, we seeded ¹⁵N or ¹³C-labeled recombinant tau monomers with 10% AD brain-derived sarkosyl-insoluble tau (AD tau) (Fig. 1a) and characterized the resulting fibrils using solid-state NMR. AD tau was pooled from ten neuropathologically confirmed diagnosis of AD (Supplementary Tables 1 and 2). The use of multiple AD brains ensures that the molecular structural results we obtain reflect the average properties of AD brain tau filaments instead of the property of one AD patient. We chose brains with short postmortem intervals and high tau-burdens at late stages of the disease^18,19. AD tau seeds were sequentially extracted from these brains. To these AD tau extracts we added ninefold excess of either a 1:1 mixture of 0N4R and 0N3R tau monomers or one of the two monomers. After 7 days of incubation under shaking, about half of the added monomers became insoluble (Fig. 1b and Supplementary Fig. 1), indicating a fivefold increase in insoluble mass^18,19. Unseeded recombinant tau remained largely soluble. Transmission electron microscopy (TEM) images reveal that the monomer-added samples formed much longer filaments (200–300 nm) than the original AD tau seeds (40–70 nm) (Fig. 2 and Supplementary Fig. 2a). Most of these filaments have a twisted morphology whereby an ~10 nm width alternates with an ~25 nm width. A small number of relatively straight fibrils with ~16-nm width is also observed (Supplementary Table 3). These two morphologies are similar to the paired helical filaments (PHF) and straight filaments (SF) in AD tau²⁰, but differ from the long, straight and 14–23 nm wide fibrils assembled using heparin^21,22,23 (Fig. 2c and Supplementary Fig. 2b). The AD-tau templated fibrils can seed mouse tau in wild-type primary neurons into morphologically identical aggregates, as detected by immunofluorescence using a mouse-tau specific antibody (Fig. 1c). Recent studies showed that these AD-tau-seeded mouse tau aggregates are localized to dendrites and axons^19,24. Quantification of the fluorescence intensities shows that the amount of aggregated mouse tau increased in the presence of templated fibrils compared to the 10% AD-tau control (Fig. 1d). Therefore, the templated recombinant tau fibrils are pathologically similar to AD tau; moreover, the templated fibrils have higher overall pathogenicity compared the original 10% AD tau seeds. A similar increase in pathogenicity were observed whether 4R tau alone, 3R tau alone, or a mixture of both isoforms were added to the AD tau seeds²⁵. Together, these data indicate that the ultrastructural morphology and pathological activities of AD tau are faithfully propagated by recombinant tau, regardless of whether 4R tau, 3R tau or both isoforms were added.

To determine how 4R and 3R tau monomers are organized in AD-tau templated fibrils, we conducted a ¹⁵N–¹³C rotational-echo-double-resonance (REDOR) NMR experiment²⁶ (Fig. 3). This experiment measures the proportion of ¹³C-labeled tau that is one strand away from an ¹⁵N-labeled tau in the hydrogen-bonded cross-β fibril. Because two hydrogen-bonded β-strand backbones are separated by ~4.8 Å, the distance-dependent intermolecular ¹⁵N–¹³C dipolar coupling depends only on how two differentially labeled tau monomers are mixed (Fig. 3a, b). ¹³C dipolar dephasing of an ¹⁵N-labeled tau is manifested as intensity loss between a spectrum measured with the ¹³C pulses off (S₀) and the ¹³C pulses on (S). Greater ¹⁵N–¹³C dipolar dephasing, or lower S/S₀ values, indicates a higher probability of ¹³C-labeled tau following an ¹⁵N-labeled tau. To increase the spectral sensitivity, we observed this ¹⁵N intensity loss through the signals of the directly bonded amide proton. No ¹H spectral resolution is required, as the REDOR experiment detects all rigid amides in the rigid β-sheet fibril core (Fig. 3c, d). To simplify the ¹H spectra and facilitate the comparison of 2D ¹⁵N–¹H correlation spectra, we perdeuterated the protein monomers and back-exchanged them with water. The resulting recombinant tau contains only NH and OH protons.

We mixed 50% ¹⁵N-labeled 4R tau and 50% ¹³C-labeled 3R tau in two independently prepared AD-tau seeded samples ([AD]4R^N3R^C) (Supplementary Table 4). A second pair of AD-tau seeded samples has the reverse mixed labeling motif, containing 50% ¹⁵N-labeled 3R tau and 50% ¹³C-labeled 4R tau ([AD]3R^N4R^C). We first measured the REDOR dephasing (S/S₀) of heparin-fibrillized 0N4R tau samples^21,22 containing varying molar ratios of ¹⁵N-labeled and ¹³C-labeled monomers. These samples allow direct experimental calibration of the REDOR dephasing of different statistical mixtures of ¹⁵N-labeled protein and ¹³C-labeled protein. For these 0N4R tau samples, the probability of a ¹³C-labeled monomer following an ¹⁵N-labeled monomer (p_N→C) in the fibril is equal to the fraction of ¹³C-labeled monomers in the mixture. The fact that in vitro-fibrilized tau and AD-seeded tau do not have the same rigid core structure does not affect the use of intermolecular REDOR for determining the statistics of mixing, because the REDOR experiment only depends on the existence of parallel-in-register cross-β packing, which is true for both heparin-fibrillized tau and AD tau.

Figure 3e shows the REDOR dephasing of mixed ¹³C-labeled and ¹⁵N-labeled 0N4R tau samples, whose ¹³C:¹⁵N molar ratios range from 0:100% to 70%:30%. The dephasing curves show the expected trend: the higher the fraction of ¹³C-labeled tau, the faster the dipolar dephasing, and the lower the S/S₀ values. The measured REDOR decay curves can be simulated using the known intermolecular distances in a cross-β amyloid fibril (Supplementary Fig. 3). To do this, we conducted both explicit spin simulations²⁷ and second-moment analysis²⁸. Both methods of simulation reproduce the trend of the mixing-ratio-dependent dipolar dephasing, but do not match quantitatively the measured REDOR dephasing of the calibration samples. This is not surprising, because each simulation method has shortcomings that limit its accuracy (see “Methods”). The explicit spin simulation is restricted to a maximum of about 10 spins, while the second-moment analysis neglects nuclear spin interactions other than ¹⁵N–¹³C dipolar coupling. Moreover, neither simulation captures the effects of natural abundance ¹³C spins in the ¹⁵N-labeled monomer. For these reasons, we rely on the more accurate experimental REDOR results of the calibration samples to extract the mixing probabilities of AD tau filaments.

The spectra of the [AD]4R^N3R^C samples yielded a REDOR dephasing curve that falls between the calibration curves for 30:70 and 40:60 ¹³C:¹⁵N labeled 0N4R tau (Fig. 3e). Interpolation of the measured S/S₀ values between the two calibration curves yielded a p_4→3 value of 0.37 ± 0.03 (Supplementary Figs. 3 and 4). Thus, in AD-seeded tau filaments, a 4R tau is followed by a 3R tau with 37% probability, and hence by a 4R tau with 63% probability. The reversely mixed labeled [AD]3R^N4R^C samples showed faster REDOR dephasing: the measured REDOR curve falls between the calibration curves for 50:50 and 60:40 ¹³C:¹⁵N 0N4R tau. Interpolation of the two measured calibration curves to match the REDOR dephasing of the AD-seeded sample yielded a p_3→4 value of 0.56 ± 0.02. Thus, in the AD-seeded tau filaments, a 3R tau is followed by a 4R tau with 56% probability, and hence by a 3R tau with 44% probability. These data together indicate that each 4R tau monomer is flanked by 63% 4R tau, while each 3R tau monomer is flanked by 56% 4R tau. Therefore, the AD tau filaments, when offered equal amounts of 4R tau and 3R tau monomers, neither completely separate the two isoforms, which would correspond to p_4→3 = p_3→4 = 0, nor strictly alternate the two isoforms, which would correspond to p_4→3 = p_3→4 = 1. Moreover, both 4R tau and 3R tau have a small preference for contacting 4R tau. A two-state Markov model (see “Methods”) shows that these probabilities can be converted to the mole fraction of 4R tau in the filament according to 𝜒4=𝑝3→4/(𝑝3→4+𝑝4→3)χ4=p3→4/(p3→4+p4→3), giving a 4R tau mole fraction of 60 ± 2%. Thus, the AD tau filaments incorporate the two isoforms with a 60: 40 molar ratio in favor of 4R tau. This composition is consistent with the estimated 4R and 3R tau incorporation levels from the ¹⁵N and ¹³C spectral intensities (Supplementary Fig. 5 and Supplementary Table 4). Furthermore, because p_4→4 (63%) is larger than the overall 4R tau incorporation level (60%) while p_3→4 (56%) is smaller than it, there is a small preference for homotypic 4R–4R and 3R–3R contacts over heterotypic 3R–4R contacts. We can alternatively express this preference for homotypic contact in terms of a mixing quotient, 𝑄=𝑝3→4𝑝4→3/𝑝3→3𝑝4→4Q=p3→4p4→3/p3→3p4→4, defined in analogy to the equilibrium constant of mixing. Large Q values indicate a preference for heterotypic mixing while small Q values indicate a preference for block copolymerization. When there is no homotypic or heterotypic preference, i.e., when the mixing is agnostic of the present isoform, then p_3→4 = p_4→4 = χ₄, and Q = 1. The measured p_4→3 and p_3→4 values correspond to a Q value of 0.75 ± 0.11, indicating a small preference for homotypic contacts, independent of the overall isoform incorporation level. To provide a molecular view of this isoform mixing, we replicated the cryoEM structure of AD PHF tau⁴ into a long filament containing 360 monomers in each protofilament, and simulated the 4R and 3R tau alternation to match the measured p_4→3 and p_3→4 values (Fig. 3f, g). The simulation depicts both the larger abundance of 4R tau over 3R tau in the filament and the intimate and fluent molecular mixing of the two tau isoforms.

To assess whether the AD-templated recombinant tau fibrils show isoform-dependent conformation and dynamics, we measured 2D ¹⁵N–¹H correlation NMR spectra. Dipolar hNH spectra selectively detect rigid β-sheet residues (Fig. 4a–e) whereas J-hNH INEPT spectra selectively detect nearly isotropically mobile residues (Fig. 4f–i). In addition to the mixed isoform samples, we also prepared AD-tau seeded 4R-only tau and 3R-only tau samples. All four seeded tau fibrils show very similar dipolar hNH spectral patterns. For example, the chemical shifts of the resolved Gly peaks are the same. This AD-tau seeded spectral fingerprint is distinct from the peak pattern of heparin-fibrillized 4R tau. For example, the heparin-fibrillized sample shows resolved signals of the only two Cys residues in the protein and their sequential Gly residues (C291–G292 and C322–G323)²². These peaks are absent from the AD-tau seeded spectra. Therefore, the AD tau fold can recruit 3R tau monomers, 4R tau monomers, or a mixture of both, without changing the conformation of the rigid β-sheet core.

The 2D INEPT spectra (Fig. 4f–i) also show similar chemical shifts for the four AD-tau-seeded samples^22,23,29, indicating that the fuzzy coat has the same motionally averaged conformation for different tau isoforms. N-terminal residues to G207 at the beginning of the P2 domain exhibit the same chemical shifts and relative intensities. In comparison, C-terminal residues show weaker intensities and larger intensity variations, indicating that the C-terminus is less mobile compared to the N-terminus. In contrast to the dipolar spectra, the INEPT spectra of AD-tau-seeded samples are similar to the INEPT spectrum of heparin-fibrillized tau. Therefore, the mobile part of the fuzzy coat has similar dynamic conformations for all isoforms in both AD tau aggregates and in vitro fibrillized tau.

Discussion

These data provide direct structural evidence of the nature of molecular mixing of 4R and 3R tau in AD brain tau filaments. The ¹H-detected ¹⁵N-¹³C REDOR experiment probes the molecular packing of tau isoforms along the fibril axis of AD tau, perpendicular to the C-shaped β-sheet structure⁴. We find that AD tau filaments recruit 4R and 3R tau monomers with a ratio of 60:40, and with a small preference for homotypic contacts. Moreover, the AD tau fibril incorporates 4R tau, 3R tau, or a mixture of the two isoforms without changing the β-sheet core structure, as seen by the similarity of the 2D dipolar hNH spectra (Fig. 4). Thus the AD tau prion incorporates either tau isoform into its pathological fold, without cross-seeding barrier between them, in contrast to in vitro assembled tau fibrils^30,31,32. This fluent molecular mixing explains the biochemical observation that AD tau fibrils amplified with 4R tau, 3R tau, or both are equally capable of seeding both 4R and 3R mouse tau^18,19,25. We hypothesize that this fluent mixing may promote fibril growth in vivo and play a role in making AD the most prevalent neurodegenerative disorder in humans.

Because the NMR data were obtained from AD tau seeds pooled from multiple patient brains, the 60:40 mixing and the probabilities we measured reflect the general properties of AD brain tau fibrils instead of the property of a single patient brain. The AD-seeded tau fibrils show highly reproducible 2D NMR spectra as well as reproducible REDOR dephasing, suggesting that there is no detectable molecular structural variation among the multiple AD brains. Although some biochemical studies have reported certain levels of heterogeneity in AD brain tau, these observations were all made based on total tau, including both pathogenic and non-pathogenic tau, in AD patients^33,34. Our study instead focuses on sarkosyl-insoluble tau, which has been shown to have highly consistent fibril core structures across multiple AD patient cases⁵, and which exhibits consistent seeding abilities in vitro and in vivo¹⁹. Therefore, these prior biochemical and biophysical data provide additional support for the isoform-independent molecular structure observed here.

The 4R and 3R tau mixing probabilities we measured here are based on the assumption that the number of residues that contributes to the β-sheet core is the same for 4R and 3R tau in the AD-tau seeded fibrils. If the number of residues in the core differs between 4R and 3R tau, then the mixing probabilities would differ from the reported value. For example, if 4R tau incorporates more β-sheet residues into the fibril core than 3R tau, then the [AD]4R^N3R^CREDOR S/S₀ intensities would be biased high, and the true p_4->3 value would be larger than the measured value. Similarly, the true p_3->4 would be smaller than the measured value. However, three observations suggest that it is unlikely for the two isoforms to have different fibril core sizes. First, tau filaments obtained from multiple AD brains have been shown by cryoEM to have the same β-sheet core, without signs of structural heterogeneity^4,5. Second, the 2D hNH dipolar and INEPT spectra measured here (Fig. 4) show similar spectral peak patterns for the mixed isoform samples and the single isoform samples. Thus, the AD tau seeds are able to propagate by either isoform to develop the same structure. Third, the AD tau seeds are well amplified in our current study, which would be difficult to achieve if a monomer of the opposite isoform is added to the growing fibril with a different core size than the current isoform.

The moderate over-incorporation of 4R over 3R tau into the AD tau filament is consistent with fluorescence data of mammalian cells expressing 4R and 3R tau repeat domains. These data showed that both 4R and 3R tau are aggregated by AD brain extracts, but overexpression of 4R tau increased the total amount of the aggregates more than 3D tau did¹⁶. It is also consistent with the observation that 4R tau isoforms have faster fibril nucleation rates and elongation rates than 3R tau isoforms in vitro^35,36.

The isoform-independent propagation of the AD tau structure and pathological properties is noteworthy. The R2 domain contains a fibrillary hexapeptide motif like the R3 domain^37,38,39. Thus, if a series of 4R tau monomers is added to the elongating filament, we would expect their R2 domains to begin to form cross-β hydrogen bonds and join the rigid core. This should gradually lead to a new fibril structure that prefers to incorporate 4R tau instead of 3R tau. The 2D NMR spectra measured here indicate that the AD tau is able to avoid this fate, even when only 4R tau monomers are added to the seed. The molecular mechanism for this avoidance is still unclear. It is possible that the posttranslational modification pattern of AD tau favors 4R tau incorporation³⁶ as well as predisposing the seeded structure to prevent the incorporation of the R2 domain into the β-sheet core. The AD tau fold may have evolved over its slow replication phase⁴⁰ to adapt to the environment of the AD brain⁴¹. The fluent molecular mixing of both 4R and 3R tau isoforms and the preservation of the β-sheet conformation regardless of the isoform could potentially be an adaptive trait to optimize prion growth and propagation. The significant amplification of the morphology and toxicity of AD tau filaments shown here opens new avenues for future characterization of the molecular structure of AD tau using a wide range of biophysical methods.