Site-Specific Hyperphosphorylation Inhibits, Rather than Promotes, Tau Fibrillization, Seeding Capacity, and Its Microtubule Binding

Abstract

The consistent observation of phosphorylated tau in the pathology of Alzheimer's disease has contributed to the emergence of a model where hyperphosphorylation triggers both tau disassociation from microtubules and its subsequent aggregation. Herein, we applied a total chemical synthetic approach to site-specifically phosphorylate the microtubule binding repeat domain of tau (K18) at single (pS356) or multiple (pS356/pS262 and pS356/pS262/pS258) residues. We show that hyperphosphorylation of K18 inhibits 1) its aggregation in vitro, 2) its seeding activity in cells, 3) its binding to microtubules, and 4) its ability to promote microtubule polymerization. The inhibition increased with increasing the number of phosphorylated sites, with phosphorylation at S262 having the strongest effect. Our results argue against the hyperphosphorylation hypothesis and underscore the importance of revisiting the role of site-specific hyperphosphorylation in regulating tau functions in health and disease.

Keywords: aggregation; hyperphosphorylation; native state stabilization; protein modifications; tau protein.

Figure 1

A) Schematic representation of the longest tau isoform (2N4R), which contains an N‐terminal projection domain, MTBD, and a C‐terminal domain (PTMs highlighted). The MTBD is composed of four microtubule binding repeats, R1, R2, R3, and R4. Two hexapeptides, PHF6* and PHF6 in the R2 and R3 repeats, play critical roles in tau fibrillization. Among the 85 putative phosphorylation sites (80 serine/threonine and 5 tyrosine residues) on tau, 28 sites were identified to be exclusively phosphorylated in AD brains. In the case of MTBD, the sites S258, S262, S289, and S356, which occur in the MTBD, are heavily phosphorylated in AD pathology. B) Schematic depiction illustrating the different experimental approaches used in this study to elucidate the effect of single and multiple phosphorylation states on the functional (tubulin assembly) and pathological (aggregation and seeding) aspects of K18.

Scheme 1

Strategy for the total chemical synthesis of the WT and mono‐, di‐, and triphosphorylated K18. A) K18 amino acid sequence with the three fragments shown in green, red, and orange and the ligation sites highlighted by gray boxes. B) Schematic representation of the synthesis of hyperphosphorylated K18 from the three synthetic fragments. C) Analytical RP‐HPLC traces and mass spectrometry (ESI‐MS) analysis of the ligation reactions between the different fragments of K18. Ligation of fragments 1 and 2 i) at 0 h and ii) at 4.5 h followed by in situ thiazolidine deprotection. Ligation of fragments 3 and 4 iii) at 0 h and iv) 2 h. D) Characterization of the purified K18 proteins by analytical RP‐HPLC, ESI‐MS, and SDS‐PAGE.

Figure 2

Characterization of synthetic and recombinant WT K18. A) SDS‐PAGE analysis (top) of WT recombinant and synthetic K18 as well as a western blot using the tau specific antibody (polyclonal rabbit anti‐total tau antibody, generated in house; bottom). B) Circular dichroism spectra of the WT recombinant (10 μm, black) and synthetic (10 μm, red) K18. C) Tubulin polymerization assay in the presence of synthetic and recombinant WT K18.

Figure 3

In vitro aggregation of WT and phosphorylated K18 proteins. A) Aggregation kinetics measured by ThS fluorescence at 490 nm (mean±SEM, n=3). B) TEM images of WT and K18_pS356 after 7 h (scale bars are 100 nm (upper panels) and 500 nm (lower panels)). C) TEM images of K18_pS356, pS262 and K18_pS356, pS262, pS258 after 3 h (scale bar is 100 nm) and D) 48 h of incubation (scale bar is 500 nm).

Figure 4

A) Schematic representation of the FRET‐based seeding assay using HEK293T biosensor cell reporter lines. Heparin‐induced aggregates are transfected into the biosensor cells using Lipofectamine. Aggregation seeded by the K18 proteins is detected as FRET emission and quantified using IFD. B) IFD measured following transduction of WT, mono‐, di‐, tri‐, and tetraphosphorylated K18 previously incubated with or without heparin for 24 h at 37 °C (N=3 repeats). C) Effect of different heparin exposure times with K18 proteins prior to transfection of the aggregates (N=3 repeats).

Figure 5

K18 protein binding to MT. A) MT assembly in the presence of K18 proteins (K18, K18_pS356, K18_pS356, pS262, and K18_pS356, pS262, pS258) was evaluated by measuring light scattering at 350 nm over time. B) The percentage of MT formed after 60 min of incubation with K18 proteins determined by measuring the absorbance at 350 nm (3 repeats, represented as the mean±SD).

Figure 6

The residue S262 plays a vital role in stabilizing the binding of tau to tubulin of MTs. Phosphorylation at S262 disrupts the tau–MT interaction (top), leading to tau accumulation and pathology formation. Inhibiting the phosphorylation at S262 or other sites that directly or indirectly disrupt the tau–MT interaction by targeting the kinases involved could be an effective therapeutic strategy (bottom) to prevent tau dissociation from MTs by stabilizing the native state of the tau–tubulin complex.