Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses

ARTICLE https://doi.org/10.1038/s41467-022-30461-x OPEN 1234567890():,; Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses Jonathan X. Meng 1,2,12, Yu Zhang1,3,4,12, Dominik Saman 5, Arshad M. Haider2,6, Suman De 1,7, Jason C. Sang1,2, Karen Brown3,4, Kun Jiang1, Jane Humphrey 1, Linda Julian1,8, Eric Hidari 2, Steven F. Lee Gabriel Balmus 2,6, R. Andres Floto 3,4, Clare E. Bryant 9, Justin L. P. Benesch 5, Yu Ye 1,10,11 & David Klenerman 1,2 ✉ 1, Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies. 1 Department of Chemistry, University of Cambridge, Cambridge, UK. 2 UK Dementia Research Institute at Cambridge, Cambridge, UK. 3 Molecular Immunity Unit, Department of Medicine, MRC Laboratory of Molecular Biology, University of Cambridge, Cambridge, UK. 4 Cambridge Centre for AI in Medicine, University of Cambridge, Cambridge, UK. 5 Department of Chemistry, University of Oxford, Oxford, UK. 6 Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. 7 Department of Neuroscience Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, UK. 8 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK. 9 Medicine and Veterinary Medicine, University of Cambridge, Cambridge, UK. 10 Department of Brain Sciences, Imperial College London, London, UK. 11 UK Dementia Research Institute at Imperial College London, London, UK. 12 These authors contributed equally: Jonathan X. Meng, Yu Zhang. ✉email: NATURE COMMUNICATIONS | (2022)13:2692 | https://doi.org/10.1038/s41467-022-30461-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30461-x I Most of these potential sites are located in the vicinity of the microtubule-binding domains (R1–4) in the proline-rich region and in the C-terminus of the molecule except for Ser262, Ser293, Ser324, and Ser356 (motif KXGS) in R1- 4 domains8 (Fig. 1b). Out of these potential phosphorylation sites about 30 sites have been reported to be aberrantly phosphorylated in the AD brain but not in the healthy control9 and commonly associated with tau aggregation processes such as incomplete binding and destabilization of microtubules, causing the transition from pretangles to NFTs10–12. Therefore, to examine the pathological consequences of tau (hyper)phosphorylation, we need to be able to reproducibly generate disease-relevant phospho-tau in quantities that meet the demand for subsequent structural and toxicity characterization studies. An early study has demonstrated that prior phosphorylation with cAMP-dependent protein kinase A (PKA) can prime the tau proteins for other kinases by inducing conformational changes to allow further phosphorylation events13. Moreover, a recent study that screens for the effect of 352 human kinases has shown that glycogen synthase kinase 3β (GSK-3β) and stress-activated protein kinase 4 (SAPK4) were the most active protein kinases phosphorylating tau at AD-specific epitopes that were recognized by phospho-tau specific antibodies including AT8 (pSer202 and ntracellular neurofibrillary tangles (NFTs) constituting hyperphosphorylated tau proteins are a pathological hallmark of several neurodegenerative diseases including Alzheimer’s disease (AD)1, Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration2, collectively called “tauopathies”. On the contrary, monomeric tau is highly soluble and intrinsically disordered, thereby showing little tendency in its native form for aggregation3. Thus, it is generally believed that tau proteins must undergo a sequence of biochemical and conformational changes before turning into misfolded substrates. Phosphorylation is the most common form of tau post-translational modifications found in vivo4 and has been suggested to be a pathological switch, leading to the formation of cytotoxic tau aggregates in NFTs5. On the other hand, when tau phosphorylation is inhibited, insoluble tau load and neurodegeneration are attenuated in vivo6, further implicating phosphorylation as a possible mechanism for the pathogenesis and progression of AD and other tauopathies. However, the identification of phosphorylation sites responsible for neurotoxicity remains elusive, and the challenge mainly lies in the heterogeneous and combinatorial nature of tau phosphorylation. For instance, a 0N4R tau isoform harbors up to 71 potential phosphorylation sites, all of which can be modified by a multitude of kinases as well as phosphatases7. a phosphate group WT 37 ˚C + heparin (1:1 ratio) heparin + PKA g-tau or s-tau + GSK-3E or SAPK4 37 ˚C pka-tau b WT KDa pk a-t au aggregates taken for structural characterization via fluorescence microscopy monomer samples taken for mass spectrometry phosphorylation stablizes the opening of the transient folding au g-t s-t d au pka-tau g-tau s-tau 0.16 0.14 62 Relative intensity 0.12 49 38 c 137 SGYSSPGSPGTPGS150 151 RSRTPSLPTPPT162 AT8 93 R1 R2 186 AT180 166 0.08 0.06 0.04 AT100 Proline-rich domain 1 0.10 KKVAVVRTPPKSPSS R3 0.02 R4 311 383 PHF-1 180 336 YKSPVVSGDTSPR 348 0.00 0 5 10 15 20 25 30 35 40 Number of phosphate moieties per tau molecule Fig. 1 Sequential hyperphosphorylation of tau in vitro generates AD-specific epitopes. a A pictorial representation of the experimental design: WT tau was sequentially hyperphosphorylated, first by PKA and then by either GSK-3β or SAPK4 kinase. Hyperphosphorylation is shown to be able to cause an opening of such transient paperclip conformation50 and simultaneously stabilizes the α-helical structures, which is associated to the aggregation process51 Recent cryo-EM structures and computational study reveals heparin can stabilize the interaction between R2 and R3 (r (...truncated)