In healthy brain, the intrinsically disordered protein tau functions to assemble and stabilize microtubules,1, 2 which regulate the shape and polarity of cells and serve as the track for cellular transport.3 When tau undergoes posttranslational modifications (PTMs) such as phosphorylation, acetylation and truncation, its microtubule association is impaired.4 In Alzheimer’s disease (AD), the failure of tau to associate with microtubules disrupts axonal integrity, mis-sorts tau to the synapses5 and correlates with tau aggregation into paired helical filaments (PHFs) and straight filaments, which make up the neurofibrillary tangles in AD.6, 7 Understanding the molecular mechanisms by which PTMs alter the microtubule interaction of tau and induce self-assembly of the protein into pathological filaments is crucial for developing better diagnostics and therapeutics for AD.

Among tau PTMs, abnormal hyperphosphorylation is the best studied.8, 9 About 45 phosphorylation sites have been identified in AD tau, many at proline (Pro)-preceding Ser and Thr residues. Some of the most widely used tau antibodies for postmortem diagnosis of AD target pSer-Pro and pThr-Pro motifs. For example, the antibody AT8 recognizes pS202, pT205, and pS208 in the P2 domain,10, 11 whereas the antibody PHF-1 recognizes pS396, pS400, pT403, and pS404 at the junction of R’ and the C-terminal domain.12, 13 In addition to phosphorylation, truncation also occurs in pathological tau. Tau can be truncated by several caspases, including caspases 3, 7, and 8. Caspase-cleaved tau is known for its high neurotoxicity and is associated with AD and other tauopathies. Among the many cleaved tau species found in AD, truncation after D421 has received the most attention.14, 15 Treatment of primary neurons by Aβ42 fibrils caused caspase cleavage of tau at D421, and the truncation product is present in AD neurofibrillary tangles, recognized by the antibody Tau-C3.14 Comparison of mRNA levels of multiple caspases in AD versus healthy brains found caspase 8 to be the most important for ΔD421 truncation.16In vitro, ΔD421 tau fibrillizes more rapidly and to a higher level compared to full-length tau14. Similarly, E391 truncated tau is also found in AD PHF tau and is recognized by the antibody mAb 423.17, 18, 19 When tau constructs that start from R3 and end at varying positions of the C-terminal domain were tested for their abilities to form fibrils, it was found that the intact C-terminal domain inhibited fibril formation, ΔD421 truncation promoted fibrilization, whereas E391 truncation allowed the formation of AD-fold fibrils under certain ionic and pH conditions.20

Although both phosphorylation and truncation occur in AD brain, the chronology of these events during disease progression is not well understood. In transgenic mice models, immunoblotting data showed that hyperphosphorylation started at 3 months, preceding ΔD421 truncation at 6 months.21 In Braak stage III–V AD brains, immunohistochemical and confocal microscopy data found higher levels of phosphorylated tau compared to ΔD421 tau, also suggesting that phosphorylation precedes truncation. On the other hand, ΔD421 truncation induces a conformational epitope that is recognized by the early tau marker MC1,15 which labels tau in Braak stage I and II AD brains.22 Moreover, ΔD421 tau is phosphorylated by the glycogen synthase kinase 3β and subsequently recognized by PHF-1,15 suggesting that truncation precedes or concurs with hyperphosphorylation. A proteomics study of tau PTMs in AD and control subjects23 reported the frequency of tau modifications at different stages of AD, concluding that the earliest events of tau aggregation include not only phosphorylation in the proline-rich region and at the PHF-1 epitope but also C-terminal cleavage.

An important approach for investigating the impact of PTMs on tau aggregation is to determine the structure of in vitro assembled tau fibrils that contain mutations mimicking the posttranslational modifications in diseased brain. By comparing the structures of these in vitro assembled tau fibrils with the ex vivo structures, we can gain insights into the PTM code of tau. Unmodified wild-type tau is highly soluble and net positively charged at neutral pH; thus aggregation of wild-type full-length tau has traditionally required polyanionic cofactors such as heparin and RNA.24 PTMs that reduce the positive charges of tau are expected to increase the aggregation potential of tau and may obviate the need for anionic cofactors. Indeed, we recently showed that two phospho-mimetic full-length tau constructs self-assembled into ordered amyloid fibrils without anionic cofactors.25 We introduced three Glu mutations at the AT8 epitope (S202E, T205E, and S208E) in one construct and four Glu mutations at the PHF-1 epitope (S396E, S400E, T403E, and S404E) in the other construct. Using solid-state NMR and cryoelectron microscopy (cryoEM), we found that AT8-3E tau fibrillized into a multi-layered rigid core that extends from the R3 repeat to the C-terminus of the protein. In comparison, PHF1-4E tau formed a three-layered β-sheet core comprising R2, R3 and R4 repeats. This fold qualitatively resembles the structure of progressive supranuclear palsy (PSP) tau.26 When both AT8 and PHF-1 epitopes were mutated to Glu, the resulting 7E mutant adopted the same structure as PHF1-4E tau.

Here we investigate the impact of D421 truncation on tau aggregation by examining two ΔD421 fibrils designed from full-length 0N4R tau. One construct, ΔD421-WT tau, has no further modifications other than truncating the last 20 residues of the protein. The second construct, ΔD421-3E tau, additionally contains the three AT8 phospho-mimetic mutations. We show that both proteins self-assemble into ordered filaments in the absence of anionic cofactors. Using solid-state NMR and cryoEM, we determined the atomic structure of the ΔD421-WT tau fibril and characterized the conformation of the ΔD421-3E fibril. Remarkably, ΔD421-WT tau adopts the same rigid core structure as PHF1-4E tau, revealing another layer of redundancy in the PTM code of tau amyloid formation.